selective hdac6 inhibition in systemic lupus erythematosus miranda diane … · 2020-01-16 ·...

Selective HDAC6 Inhibition in Systemic Lupus Erythematosus

Miranda Diane Vieson

Dissertation submitted to the faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of

Doctor of Philosophy

In Biomedical and Veterinary Sciences

Xin M. Luo, Chair David L. Caudell

William R. Huckle Tanya LeRoith

November 28, 2016 Blacksburg, VA

Keywords: Systemic lupus erythematosus, histone deacetylase, lupus nephritis, B cell

development



ACADEMIC ABSTRACT Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease characterized by

abnormalities in multiple components of the immune system resulting in progressive damage to

multiple organs. Current treatments for SLE are often intensive and result in side effects and the

potential for continued flares and progression of disease. Histone deacetylase (HDAC) enzymes

control multiple cellular functions by removing acetyl groups from lysine residues in various

proteins. HDAC inhibitors have been investigated as a potential treatment for SLE with

promising results, however selective HDAC6 inhibition (HDAC6i) has become a leading

candidate for pharmacologic inhibition to reduce the potential for side effects. We hypothesize

that HDAC6i will decrease SLE disease by targeting substrates of HDAC6 in multiple

components of immunity and organ systems. NZB/W mice were treated with ACY-738 or

ACY-1083, followed by evaluation of multiple disease parameters and mechanisms involved in

disease pathogenesis within the kidney, bone marrow, and spleen. Within the kidney, HDAC6i

decreased glomerular pathology scores, proteinuria, and IgG and C3 deposition. Within

glomerular cells, HDAC6i increased alpha-tubulin acetylation and decreased nuclear NF-κB.

Within the spleen, there was a dose-dependent decrease in the frequency of Th17 cells and a

mild decrease in the frequency of Treg cells. Concurrently, there were decreased levels of IL-

12/IL-23 and minimal decreases in TGF-β in the serum. Within the bone marrow, B cell

development through Hardy fractions exhibited accelerated progression through later stages as

NZB/W mice aged. This accelerated progression may allow B cells to bypass important

regulatory checkpoints in maintaining immune tolerance and contribute to autoimmunity.

Treatment with an HDAC6i corrected the aberrant B cell development in the bone marrow and

RNAseq analysis unveiled six genes (Cebpb, Ccr9, Spib, Nfil3, Lgals1, and Pou2af1) that may

play a role in the aforementioned abnormalities. Overall, these findings show that HDAC6i

decreased disease in NZB/W mice by targeting multiple components of the immune response,

including glomerular cells, T cell subsets in the spleen, and bone marrow B cells. In conclusion,

selective HDAC6i is an excellent candidate for pharmacologic therapy for SLE because it targets

multiple immune abnormalities involved in SLE pathogenesis while remaining selective and

safe.



PUBLIC ABSTRACT Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by multiple

abnormalities in the immune system resulting in progressive immune-mediated damage to

multiple organs. Current treatment regimens are often intensive, result in side effects, and may

only provide temporary relief of disease. Histone deacetylase (HDAC) inhibition is currently

being investigated as a new treatment modality for SLE with aims for improved efficacy and

decreased potential for unwanted side effects. HDAC enzymes remove acetyl groups from

multiple proteins (substrates) and subsequently regulate their function. HDAC6 is a specific

HDAC enzyme that is of particular interest and are the subject of the following studies. These

studies hypothesize that HDAC6 inhibition will decrease SLE by targeting multiple protein

targets involved in the immune-mediated pathway of disease initiation and progression. NZB/W

mice were utilized as a model of the human disease, and were treated by HDAC6 inhibitors

during various stages of disease progression. Long-term treatment initiated early in disease

decreases disease as evidenced by decreased renal pathology scores, immune complex deposition

in the kidneys, decreased T cell subtypes in the spleen, and decreased inflammatory cytokines.

HDAC6 inhibition corrects abnormal B cell development within the bone marrow of NZB/W

mice, which is otherwise altered during disease progression. Furthermore, HDAC6 inhibition

altered gene expression within the bone marrow, and deep sequencing analysis revealed multiple

genes that may be involved in the pathway of disease progression. Overall, HDAC6 inhibition

targets multiple pathways involved in SLE disease initiation and progression in various organs

including the bone marrow, spleen, and kidneys. Because SLE is a disease that is multi-factorial

and effects multiple organs, it would be ideal that a potential drug therapy also targets multiple

targets and organ systems while remaining safe to use. Based on these studies, HDAC6

inhibitors are excellent candidates for the treatment of SLE.

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Dedication

“The difference between school and life? In school, you’re taught a lesson and then given a test.

In life, you’re given a test that teaches you a lesson.”

- Tom Bodett

I’ve learned more from the process of completing this work than any will ever know. I owe it all

to the people in my life that have supported me, inspired me to be a better version of myself

while accepting that I will never and shouldn’t be perfect, and that I shouldn’t take things so

seriously all the time.

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ACKNOWLEDGEMENTS

This work was supported by grants from Acetylon Pharmaceuticals (www.acetylon.com) and

National Institutes of Health/National Institute of Allergy and Infectious Diseases (R15

AR062883)

viii

ATTRIBUTIONS

Christopher M. Reilly, PhD was the principle investigator and is currently a professor at Edward Via College of Osteopathic Medicine (VCOM). He is a co-author on chapters 2, 3, and 4 and assisted with project ideas, writing, and editing. He also contributed with his expertise in lupus pathophysiology, lupus mouse models, and histone deacetylase inhibitors (HDACs). Xin M. Luo, PhD is the chair of the research committee and currently working in the Department of Biomedical Sciences and Pathobiology (DBSP) at Virginia-Maryland College of Veterinary Medicine (VMCVM) as an assistant professor. She assisted with project ideas, writing, editing of chapters 3 and 4 and provided her expertise in B cell biology and development for chapter 4. David L. Caudell, DVM, PhD was an assistant professor in DBSP at VMCVM and is currently working at Wake Forest School of Medicine as an Associate Professor. He contributed with his expertise in histopathological evaluation of lupus nephritis and assisted and provided training in grading kidney pathology in chapters 3 and 4. Alexander M. Gojmerac was an undergraduate student working in the lab and is currently a graduate student at University of Connecticut in Storrs, CT working on a Master’s degree. He assisted with experiments in the laboratory and with the care of the mice utilized in chapters 3 and 4. Deena Khan, PhD was a postdoctoral fellow in DBSP at VMCVM and is currently working at Cincinnati Children’s Hospital, in Cincinnati, OH as a research fellow. She contributed with her expertise and technical knowledge in Th17 cell biology in chapter 3. Rujuan Dai, PhD is currently a research assistant professor in DBSP at VMCVM. She contributed with her expertise and technical knowledge of miRNA biology in chapter 3. John H. van Duzer, PhD is currently the vice president in chemistry at Acetylon Pharmaceuticals, Inc and Ralph Mazitschek, PhD is currently an assistant professor at Harvard Medical School. They both assisted by providing pharmacokinetic data in chapter 3 and their expertise with the HDAC6 inhibitors utilized in all the studies. Xiaofeng Liao is currently a graduate student in DBSP at VMCVM and provided technical assistance in some experiments utilized for chapter 3. Song Li, PhD is currently an assistant professor in Crop and Soil Environmental Sciences at Virginia Tech and contributed his expertise in bioinformatics and performed analysis on the RNAseq data set in chapter 4. Adrian Castaneda was a graduate student in DBSP at VMCVM and assisted with experiments in the laboratory and with the care of the mice utilized in chapter 4.

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TABLE OF CONTENTS

Page Abstracts ……………………………………………………………………………...…. ii Dedication ………………………………………………………………………………. vi Acknowledgements …………………………………………………………………...…vii Attributions ……………………………………………………………………………. viii Table of Contents ……………………………………………………………………….. ix List of Tables …………………………………………………….………….…….…… xii List of Figures ………………………………………………………………………….. xii

Chapter 1. Introduction Introduction ……………………………………………………………………………… 1 References ……………………………………………………………………………….. 3 Chapter 2. (Literature Review) HDAC6 Regulation of Non-Histone Proteins in Systemic Lupus Erythematosus Title Page ………………………………………………………………………………... 5 Abstract ……………………………………………………………………………….…. 6 Introduction ……………………………………………………………………………… 7 Non-Histone Substrates of HDAC6 Tubulin ………………………………………………………………………..... 10 β-Catenin …………………………………………………………………...…... 12 Heat Shock Protein 90 ………………………………………………………..... 13 Smad7 ……………………………………………………………………….…. 15 Forkhead Box P3 …………………………………………………………….…. 17 Ku70 ………………………………………………………………………....…. 18 Conclusions ………………………………………………………………………….…. 19 References ……………………………………………………………………………… 21 Chapter 3. Treatment With a Selective Histone Deacetylase 6 Inhibitor Decreases Lupus Nephritis in NZB/W Mice Title page ……………………………………………………………………….……… 37 Abstract ………………………………………………………………………………… 38 Introduction ……………………………………………………………………………. 39 Materials and Methods Mice ……………………………………………………………………………. 42 In vivo treatments and monitoring …………………………………...………… 42 Measurement of autoantibodies ……………………………………………...… 43 Measurement of serum cytokines and immunoglobulin isoforms …….….….… 44 Splenocyte isolation and flow cytometric analysis ……………………….….… 44 Splenocyte miRNA and mRNA expression assays ………………………….… 45 Renal histopathology ………………………………………………………...… 45 Renal IgG and C3 accumulation ………………………………………….……. 46

Acetylated alpha-tubulin, histone 3, and NF-κB immunofluorescence in glomerular cells ………………………………………………………...…. 46

x

Cytoplasmic and nuclear NF-κB in mesangial cell, in vitro …………………… 47 Statistics ………………………………………………………………………... 48 Results ACY-1083 inhibits HDAC6, selectively ………………………………………. 49

ACY-1083 treatment had no effects on mouse survival and body weight, and decreased proteinuria and splenic weight ………………………….……… 49

Treatment with ACY-1083 had no effect on autoantibody levels and maintained lower levels of total IgG and IgG2a in the serum …….……………………. 52

ACY-1083 treatment decreased Th17 cells and Treg cells in the spleen ……… 53 Treatment of NZB/W F1 female mice with ACY-1083 decreased serum IL-12/IL-

23 p40 levels in a dose-dependent manner and decreased serum TGF-β …. 55 Treatment with ACY-1083 had no effect on relative expression levels of lupus-

associated miRNAs in the spleen …………………………………………. 57 Treatment with ACY-1083 decreased lupus nephritis as well as glomerular IgG

and C3 deposition in a dose-dependent manner …………………………... 58 Treatment with ACY-1083 increased alpha-tubulin acetylation and decreased NF-

KB in glomerular cells ………………………………………………….…. 60 ACY-1083 decreased nuclear NF-κB protein in mensangial cells, in vitro …… 60

Discussion ……………………………………………………………………………… 63 Conclusions ………………………………………………………………………….…. 68 References ……………………………………………………………………………… 69 Chapter 4. Selective HDAC6 Inhibition Corrects Aberrant B Cell Development in the Bone Marrow of NZB/W F1 Mice Title Page ………………………………………………………………………………. 78 Abstract ………………………………………………………………………………… 79 Introduction ………………………………………………………………………….…. 80 Materials and Methods Mice ……………………………………………………………………………. 84 In vivo treatments with HDAC6 inhibitors ………………………………….…. 84 Flow cytometric analysis …………………………………………………….… 85 RNAseq and analysis ………………………………………………………...… 85 Real-time PCR …………………………………………………………………. 86 Renal histopathology …………………………………………………………... 86 Statistics ……………………………………………………………………...… 87 Results and Discussion

Decreases in percentages of developing B cells in the bone marrow occur mostly within Hardy fractions B and D with a concurrent increase in Fraction F as NZB/W mice age ……………………………………………………….…. 88

Initiation of HDAC6 inhibition during early disease increases the percentage of bone marrow cells within Hardy fractions B, D, and E …………………… 90

Oral administration of a selective HDAC6 inhibitor decreases spleen size and lupus nephritis when initiated during early disease ...................................... 93

High-dose HDAC6i initiated during late-stage disease did not alter proportions of bone marrow B cells in developmental Hardy fractions .............................. 95

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Genes related to B cell development and differentiation are differentially expressed in the bone marrow after HDAC6i in NZB/W mice …………… 96

Summary and Conclusions …………………………………………………………… 103 References …………………………………………………………………………….. 104 Chapter 5. Future Directions Future directions …………………………………………………………………….... 109 References …………………………………………………………………………….. 115 Appendix A. (Table) Genes differentially expressed in the bone marrow of NZB/W mice after HDAC6 inhibition based on RNAseq analysis ………………………………………. 119

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LIST OF TABLES Page

Chapter 4. Selective HDAC6 Inhibition Corrects Aberrant B Cell Development in the Bone Marrow of NZB/W F1 Mice

Table 1. Differentially expressed genes in the bone marrow related to B cell development and differentiation after HDAC6 inhibition based on RNAseq analysis ……….…. 98

LIST OF FIGURES Page

Chapter 3. Treatment With a Selective Histone Deacetylase 6 Inhibitor Decreases Lupus Nephritis in NZB/W Mice Figure 1. ACY-1083 is selective for HDAC6 ……………………………………….…. 49 Figure 2. Progression of disease in NZB/W F1 female mice …………………….….… 51 Figure 3. Sera autoantibodies targeting dsDNA and Immunoglobulin (Ig) isoforms …. 52 Figure 4. Splenic T cell subsets ………………………………………………………... 54 Figure 5. Serum levels of TGF-β and IL-12/IL-23 ………………………………….…. 56 Figure 6. Evaluation of splenic miRNAs associated with lupus …………………….…. 57 Figure 7. Glomerular pathology ………………………………………………….….…. 59 Figure 8. Alpha-tubulin and histone 3 acetylation in glomerular cells ………………… 61 Figure 9. NF-κB in glomerular and mesangial cells …………………………………… 62 Chapter 4. Selective HDAC6 Inhibition Corrects Aberrant B Cell Development in the Bone Marrow of NZB/W F1 Mice Figure 1. B cell development stages in the bone marrow ……………………………… 81

Figure 2. Progression of B cells through developmental fraction in the bone marrow of aging NZB/W mice ………………………………………………………………... 89

Figure 3. Development and differentiation of B cells in the bone marrow of NZB/W F1 female mice after HDAC6 inhibition initiated early in disease …………………… 92

Figure 4. Disease progression and Hardy fraction analysis in NZB/W mice following oral administration of an HDAC6 inhibitor (ACY-738) …………………………….…. 94

Figure 5. B cell development and differentiation in the bone marrow of diseased NZB/W mice treated with a high-dose 2-week course of HDAC6 inhibition ……………… 95

Figure 6. Differential expression of genes in the bone marrow of diseased NZB/W mice treated with HDAC6 inhibition …………………………………………………… 97

Figure 7. Genes related to B cell development and differentiation in the bone marrow ………………………………………………………………………. 99

Chapter 5. Future Directions Figure 1. Smad7 and renal inflammation ……………………………………………... 111

1

CHAPTER 1. INTRODUCTION

Miranda D. Vieson

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease with an

estimated prevalence of 20-150 cases per 100,000 individuals (1-4). Prevalence rates are higher

among women when compared to men and in African Americans compared to Caucasians (4).

Other ethnicities with a higher prevalence rate include Asians, Hispanics, and Native Americans

(4). The economic burden of SLE is substantial not only for patients, but also for health care

providers with mean direct cost estimates for the treatment of SLE per patient in the US ranging

from $2,214 – $16,875 and mean annual indirect cost estimates ranging from $2,239 – $35,540

per year (5).

While the etiology is unknown, there are multiple genetic/heritable (6-9) and

environmental factors (10) implicated in increasing the risk of developing SLE. Disease results

from abnormalities in multiple components of immunity and inflammation including, but not

limited to: breaks in immune tolerance, irregular functions and signaling in multiple immune cell

types, aberrant cytokine and chemokine production, disrupted clearance of apoptotic debris, and

formation of autoantibodies (11, 12).

Treatment for SLE is primarily based on broad-spectrum immunosuppressive and

cytotoxic agents (13-15) that are often intensive, associated with side effects, and carry the

potential for relapse and progression of disease (14). More targeted biological agents, like

monoclonal antibodies, have been developed however clinical trials have uncovered several

disappointments (16). Therefore, continued research and development of a safe and effective

drug for the treatment of all facets of SLE is paramount.

2

Histone deacetylase (HDAC) enzymes can regulate multiple cellular functions by

removing acetyl groups from lysine residues in histones and other proteins (17) and have been

investigated as regulators of immunity and inflammation (18). Of the 18 mammalian HDACs,

HDAC6 has become a leading candidate for pharmacologic inhibition for the treatment of SLE.

A review of non-histone protein targets of HDAC6 and their potential role in the pathogenesis of

SLE are reviewed in Chapter 2. It is hypothesized that HDAC6 inhibition will decrease lupus

disease by targeting multiple non-histone protein targets in multiple cell types within the bone

marrow, spleen, and kidney. The following studies aim to investigate the efficacy of HDAC6

inhibition in treating SLE and the potential targets of HDAC6 regulation involved in the

progression of lupus nephritis and B cell development in lupus-prone NZB/W F1 female mice.

3

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systemic lupus erythematosus in California and Pennsylvania in 2000: estimates obtained

using hospitalization data. Arthritis Rheum 56: 2092-4

2. Lawrence RC, Helmick CG, Arnett FC, Deyo RA, Felson DT, Giannini EH, Heyse SP,

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4. Feldman CH, Hiraki LT, Liu J, Fischer MA, Solomon DH, Alarcón GS, Winkelmayer

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RP, Harley JB, Sivils KL, Vyse TJ, Gaffney PM, Langefeld CD, Jacob CO. 2014. GWAS

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GWAS. J Autoimmun 41: 25-33

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8. Graham RR, Hom G, Ortmann W, Behrens TW. 2009. Review of recent genome-wide

association scans in lupus. J Intern Med 265: 680-8

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erythematosus. Ann Rheum Dis 72 Suppl 2: ii56-61

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Hormonal, environmental, and infectious risk factors for developing systemic lupus

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Emerging and critical issues in the pathogenesis of lupus. Autoimmun Rev 12: 523-36

13. Hahn BH. 2011. Targeted therapies in systemic lupus erythematosus: successes, failures

and future. Ann Rheum Dis 70 Suppl 1: i64-i6

14. Ponticelli C, Moroni G. 1998. Flares in lupus nephritis: Incidence, impact on renal

survival and management. Lupus 7: 635-8

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erythematosus. Int J Rheumatol 2012: 578641

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deacetylases as regulators of inflammation and immunity. Trends Immunol 32: 335-43

5

CHAPTER 2: LITERATURE REVIEW

HDAC6 Regulation of Non-Histone Proteins in Systemic Lupus Erythematosus

Miranda D. Vieson1

Christopher M. Reilly1,2

1. Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of

Veterinary Medicine, Blacksburg, VA, United States.

2. Edward Via College of Osteopathic Medicine, Blacksburg, VA, United States.

Published in Current Trends in Immunology. 2015;16:93-103

6

ABSTRACT

Therapeutic inhibition of histone deacetylase (HDAC) enzymes has been widely reported for the

treatment of many cancers. Recently, increasing evidence of HDACs playing a role in regulating

inflammation and immunity has triggered more in-depth investigations of how pharmacologic

HDAC inhibitors could be beneficial for inflammatory and autoimmune diseases. Initial

investigations of HDAC enzymes focused on their ability to regulate gene transcription by

removing acetyl groups from lysine residues of core histone proteins. Current research indicates

a broad repertoire of non-histone proteins that could also act as substrates for HDAC enzymes,

further expanding their regulatory potential in cell processes. There are 18 known HDAC

enzymes classified based on structure and function into classes I-IV. As pan-selective HDAC

inhibitors have been reported to show adverse side effects, isoform-selective inhibitors are

becoming more desirable as pharmacologic agents. In this review, we discuss the current

understanding of how HDAC6 contributes to the pathogenesis of systemic lupus erythematosus

(SLE), therefore making it a suitable candidate for selective pharmacologic inhibition.

7

INTRODUCTION

Systemic lupus erythematosus (SLE) is a prototypic autoimmune disease that affects an

estimated 140 per 100,000 individuals in the United States (1). The underlying etiology of SLE

is unknown, but multiple influences and predispositions from genetic abnormalities as well as

environmental and hormonal factors have been attributed to the development of disease (2, 3).

Ultimately, patients exhibit abnormalities in immune tolerance, B and T cell signaling and

function, innate immune responses, cytokine and chemokine production, apoptosis and

subsequent clearance of debris, and autoantibody formation (4, 5). These abnormalities

culminate in progressive, relapsing damage of multiple organs including the kidneys, joints, skin,

heart, lungs, blood vessels, and brain (6).

Although genome-wide association studies have identified many genes that may play a

role in the initiation or progression of SLE (7-9), these studies do not account for risk attributed

to heritable factors (10), and have failed to identify a unifying switch. This has led researchers to

investigate other factors involved in disease pathogenesis. Alterations in gene expression and

phenotype which are heritable but do not alter the DNA sequence comprise “epigenetics” (11).

There is increasing evidence that epigenetics may play a key role in SLE pathogenesis, and

epigenetic-targeted therapies may be efficacious (12, 13). Of particular interest for this review

are interactions between DNA and core histone proteins, which are important epigenetic

mechanisms regulating the exposure and binding of promoter regions of genes to regulate

transcription (14). Histone acetyltransferases (HATs) and histone deacetylases (HDACs) can

alter the charge and subsequent binding affinity of core histone proteins through removal or

addition of acetyl groups on lysine residues and thus alter gene transcription (15-17).

Furthermore, investigations have revealed that HATs and HDACs are also capable of modifying

8

lysine residues on numerous non-histone nuclear and cytosolic proteins (17, 18), which has

driven some researchers to alternatively refer to the enzymes as lysine (K) acetyltransferases

(KATs) and lysine deacetylases (KDACs).

There are 18 mammalian HDACs, which remove acetyl groups from lysine residues in

histones and other proteins to control multiple cellular functions including transcription, cell

cycle kinetics, cell signaling, and cellular transport processes (19). HDACs are classified based

on structure, homology to yeast HDACs, and function into classes I-IV (20, 21). Class I HDACs

(HDAC1, -2, -3, and -8) are nuclear exclusive enzymes found in a wide range of tissues and cell

lines where they are known for histone modification and repression of transcription (22, 23).

Class II HDACs are further subdivided into class IIa (HDAC4, -5, -7, and -9) and class IIb

(HDAC6, and -10) based on domain organization (24), and exhibit selective tissue expression,

nucleocytoplasmic shuttling, and function through recruitment of distinct cofactors (23). Class

III comprises the sirtuins, which act through a distinct NAD+-dependent mechanism and are not

considered “classical” HDACs (22). HDAC11 is the sole member of class IV as phylogenetic

analysis revealed very low similarity to HDACs in the other classes (20).

In addition to their initial relevance in cancer biology (25), HDAC enzymes are now

increasingly being investigated as regulators of inflammation and immunity (22). As reviewed

by Shakespear et al., HDACs are documented to play a role in myeloid development, Toll-like

receptor (TLR) and interferon (IFN) signaling in innate immune cells, antigen presentation, and

development and function of B and T lymphocytes (22). Subsequently, pharmacologic inhibition

of HDACs has been evaluated as a possible treatment modality in a wide spectrum of diseases,

including inflammatory and autoimmune diseases (26).

9

The use of non-selective HDAC inhibitors has been shown to decrease disease in lupus-

prone MRL/lpr and NZB/W mice (27-30). Mechanisms by which HDAC inhibition decrease

SLE disease have previously been reviewed by Reilly et al (16). Some of the highlights include:

corrected hypoacetylation states of histones H3 and H4 (31), increased CD4+CD25+Foxp3+ T

regulatory (Treg) cells (28, 30), reduced Th1- and Th17-inducing cytokines (IL-12 and IL-23) as

well as Th1-attracting chemokines (16), and inhibition of germline and post-switch

immunoglobulin transcripts in splenic B cells (32). Most importantly, decreased renal disease

(glomerulonephritis and proteinuria) has been consistently reported in studies investigating the

use of non-selective HDAC inhibitors to treat lupus in various mouse models (27-30). However

cytotoxicity remains a concern with long-term treatment (29, 33). Indeed, pan-selective HDAC

inhibitors available in the clinic have been associated with abnormalities such as fatigue, nausea,

vomiting, diarrhea, thrombocytopenia, neutropenia, and cardiac irregularities (34). Investigation

of specific functions for each HDAC isoform in knockout mice revealed that elimination of class

I and class IIa HDACs resulted in embryonic lethal phenotypes or fatal cardiac, vascular,

musculoskeletal, or neural crest defects (34, 35). Therefore, it would be desirable to produce

HDAC-inhibiting compounds that are time-, cell-, tissue-, and/or isoform-specific to improve

safety, while still effectively reducing disease.

HDAC6

HDAC6 is a class IIb HDAC that localizes within the cytoplasm due to its inclusion of

both a nuclear export signal and Ser-Glu-containing tetrapeptide domain (21, 36). Therefore,

HDAC6 predominantly contributes to cell functions within the cytoplasm, including cell

signaling, activation, survival, motility, and protein degradation (37), which can all contribute to

10

inflammation and immunity. HDAC6 knockout mice exhibit a viable phenotype, develop

normally, and have no life-limiting defects. Interestingly, lymphocyte development and numbers

in these mice are normal, and there is a mild decrease in the immune response after antigenic

stimulation (38). Documentation of HDAC6 playing a role in the formation of the immune

synapse in T cells (39) and chemotaxis in lymphocytes (40) provides supportive evidence for

immunomodulatory effects. In regards to SLE, we have observed increased expression and

activity of HDAC6 within B cells, T cells, and glomerular cells of diseased lupus-prone mice

(41). Additionally, selective HDAC6 inhibition in lupus mice ameloriates disease pathogenesis

by decreasing renal histopathology scores, IgG and C3 immune complex deposition, and

proteinuria (42). To further understand how HDAC6 contributes to disease, the remainder of this

review discusses the roles of non-histone protein targets of HDAC6 in the context of SLE.

NON-HISTONE SUBSTRATES OF HDAC6

Tubulin

Tubulin heterodimers are the building blocks for microtubules and serve as a target for

HDAC6 (37). Microtubules are vital in maintaining morphology and structure of the cell and

many subcellular structures creating a diverse repertoire of functions that can be regulated by

post-transcriptional modifications like acetylation (43). As reviewed by Li et al., multiple

studies have linked tubulin acetylation in immune responses (43), however the role of tubulin

acetylation in SLE pathogenesis has yet to be defined.

SLE patients with lupus nephritis almost always exhibit podocyte pathology on renal

biopsy (44), the degree of which correlates with proteinuria (45). Microtubules, and therefore

tubulin, are important for the structural integrity and physiology of podocytes (46). Increased

11

acetylation of tubulin serves as a marker for improved microtubule stability (43). As previously

mentioned, HDAC6 knockout mice exhibit hyperacetylation of tubulin in most tissues (38). Our

laboratory has documented increased alpha-tubulin acetylation in mesangial cells treated with a

selective HDAC6 inhibitor in vitro (41). Furthermore, diseased lupus-prone mice had increased

HDAC6 expression and activity in glomerular cells, which was reduced after pharmacologic

inhibition of HDAC6 (41). More recently, we have observed decreased lupus nephritis and

proteinuria in murine models after pharmacologic HDA6 inhibition in conjunction with

increased acetylation of tubulin in glomerular cells (publication under review). Overall,

inhibition of HDAC6 increases acetylation of tubulin and subsequently improves microtubule

stability and podocyte structural integrity, which may help alleviate renal damage in SLE.

Another possible mechanism for decreased nephritis observed in lupus-prone mice after

selective HDAC6 inhibition may be due to its inhibition of nuclear factor kappa B (NF-κB). NF-

κB is a transcription factor that regulates the expression of numerous genes that contribute to the

inflammatory response in the kidney (47) and is constitutively activated in many autoimmune

diseases, including SLE (48). In addition to increased tubulin acetylation, HDAC6 inhibition

also reduced nuclear NF-κB protein in immune-stimulated mesangial cells in vitro (41).

Nephrin, a key protein involved in the slit diaphragm, is downregulated during podocyte injury,

and when deficient activates NF-κB, which promotes glomerular injury (49). In human

podocytes cultured in vitro, promotion of foot process formation and maturation are associated

with increased expression of both tubulin and nephrin (50). The underlying connection between

acetylated tubulin and NF-κB in the kidney is uncertain. However, given the current data, it is

possible that nephrin in conjunction with tubulin acetylation acts to inhibit NF-κB in glomerular

cells.

12

β-catenin

The function of β-catenin is dependent on its intracellular localization. On the cell

membrane, β-catenin plays a role in junctional domains and adherence between epithelial cells.

While in the cytoplasm, β-catenin participates in the canonical Wnt/β-catenin signaling cascade

resulting in regulation of genes involved in cell proliferation, survival, and differentiation (51).

Elevated β-catenin has been documented from kidney biopsies of SLE patients and in the kidney

of NZB/W mice with lupus nephritis suggesting increased Wnt/β-catenin activation (52, 53).

Hyperactivation of the Wnt/β-catenin cascade has been implicated in podocyte dysfunction with

subsequent albuminuria as well as in renal interstitial fibrosis (54, 55). HDAC6 deacetylates β-

catenin, which regulates Wnt/β-catenin signaling (37). When HDAC6 is inhibited, β-catenin

nuclear translocation and downstream transcription factor expression are decreased (56, 57).

Therefore, inhibition of HDAC6 may help to diminish hyperactive Wnt/β-catenin signaling in

lupus nephritis by increasing the acetylation and nuclear translocation of β-catenin.

Bone marrow transplantation studies in SLE patients and lupus-prone mice revealed

abnormalities in mesenchymal stem cells (MSCs), which were further determined to be increased

senescence related to hyperactivation of Wnt/β-catenin signaling. It is thought that this increased

senescence in bone marrow MSCs contributes to the failure of syngeneic bone marrow MSC

transplantation (58). The mechanism underlying altered Wnt/β-catentin in bone marrow MSCs

in SLE is currently unknown; alteration in the acetylation of β-catenin could be a possibility and

warrants further investigation.

13

Heat Shock Protein 90

Heat shock protein (HSP) 90 is one of many heat shock proteins, which contribute to

housekeeping functions and act as chaperones, which play an important role in mediating normal

protein folding, prevention of damaging protein aggregation, and transportation of proteins

through various cell compartments (59-61). HSPs are intracellular proteins that may also be

released extracellularly, are upregulated in relation to various cell stressors, and contribute to the

physiology of inflammation and immune responses (62, 63). Inhibition of HDAC6 results in

hyperacetylation of HSP90 leading to a subsequent loss of HSP90 chaperone activity (64, 65).

Elevated levels of HSP90 in peripheral blood mononuclear and lymphoid cells of SLE

patients (66-68) and within the spleen of lupus-prone MRL/lpr mice (69, 70) is attributed to

increased IL-6 and enhanced expression of the hsp90β gene (66, 67). Further studies in IL-6

transgenic mice support the notion that elevated IL-6 results in higher HSP90 levels and also

correlates with the production of anti-HSP90 autoantibodies (70). Anti-HSP90 autoantibodies

are primarily of the IgG isotype (71) and SLE patients with elevated levels are more likely to

have low levels of C3 and renal disease (72). Autoantibodies to HSP90 have been detected in

glomerular and mesangial deposits in SLE patients with glomerulonephritis (73) implying a

pathogenic nature of these autoantibodies.

In lupus-prone MRL/lpr mice, treatment with the HSP90 inhibitor ganetespib decreased

proteinuria, total number of IgG-positive glomeruli, and glomerular pathology scores (74).

Within mesangial cells, expression of nitric oxide (NO), IL-6, and IL-12 in response to

inflammatory stimuli was decreased after inhibition of HSP90 (75). The decrease in these

inflammatory cytokines is likely related to a reduction in the expression of inhibitor of κB (IκB)

kinase and decreased nuclear factor-κB (NF-κB) translocation to the nucleus, as we have

14

observed in J774 macrophages (76). Further, HSP90 inhibition in macrophages also prevents

HSP90 chaperoning of newly synthesized cytokines (77). While HSP90 inhibition results in

decreased inflammatory cytokine expression, we found that there were no differences in IgG or

C3 deposition in glomeruli or glomerular pathology scores in MRL/lpr mice despite reductions

in proteinuria following treatment with 17-DMAG when compared to controls (75). However,

pharmacologic inhibition of HDAC6 results in increased HSP90 acetylation and decreased

nuclear translocation of NF-κB in immune stimulated mesangial cells (41) as well as decreased

glomerular pathology scores and deposition of IgG and C3 in NZB/W mice (42).

Abnormalities in T cell signaling, phenotype, activation, and function all play a role in

the pathogenesis of SLE (78). HSP90 plays a role in the activation of T cells by stabilizing

lymphocyte-specific protein tyrosine kinase (Lck)(79) and being an essential regulator for the

expression of LAT (linker for activation of T cells) (80). In our laboratory, inhibition of HSP90

in MRL/lpr mice decreased the number of double negative T cells and increased CD8+ T cells

within the spleen culminating in a reduced CD4/CD8 ratio (75). Further, reductions in CD4+ T

cells in the lymph node and spleen have been documented after inhibition of the HSP90

homologue, gp96, in mice with lupus-like disease (81). In both of these studies, alterations in T

cell populations occurred in conjunction with ameliorated lupus-like disease in mice (76, 81)

suggesting therapeutic potential of HSP90 inhibition in T cell-mediated diseases. Because

HSP90 is a substrate of HDAC6 (64, 65), HDAC6 inhibition also carries the potential to exert

similar results in treating autoimmune diseases, like SLE. In fact, selective HDAC6 inhibition

with ACY-738 in NZB/W mice decreased double negative T cells in the thymus in addition to

increasing Treg cells in the spleen (42). Additionally, in our current studies, we have found that

selective HDAC6 inhibition in NZB/W mice decreased the number of Th17 cells in the spleen

15

(unpublished data). Whether these alterations in T cell subtypes are attributed to modulation of

HSP90 or other HDAC6 substrates warrants further investigation.

Plasmacytoid dendritic cells (pDCs) are the primary secretors of type I interferons (82) in

response to engagement of toll-like receptors (TLRs) 7 and 9 by nucleic acids (83). Increased

stimulation of pDCs subsequently increases the secretion of interferon (IFN)-α and is implicated

in the maintenance and progression of disease in SLE (84). Recently, HSP90 has been shown to

be crucial in TLR 7/9-mediated INF-α production by pDCs through associating with and

delivering TLR7/9 from the endoplasmic reticulum to early endosomes and mediating self-

nucleic acid recognition in SLE (85).

Smad7

Transforming growth factor-beta (TGF-β) is an important mediator of fibrosis in multiple

chronic kidney diseases, including lupus nephritis (86). Interestingly, reduced levels of TGF-β in

immune cells coincides with increased levels in target organs (87), including kidneys in SLE

patients with lupus nephritis (88). These imbalances predispose to autoantibody production and

contribute to tissue inflammation and extracellular matrix production.

Smad7 is an inhibitory molecule involved in the TGF-β signaling cascade and acts by

promoting ubiquitination and degradation of receptor complexes (89). Additionally, Smad7

contributes to the suppression of renal inflammation by inducing IκB and therefore inhibiting

NF-κB-driven inflammatory responses (90). Gene therapy to increase the expression of Smad7

in kidneys has been documented to decrease inflammation, histologic damage, and ameliorate

chronic kidney diseases, including autoimmune crescentic glomerulonephritis in mice (91, 92).

Smad7 synthesis is increased in podocytes, but not mesangial cells, treated in vitro with TGF-β

16

and in NZB/W mice with immune-mediated glomerular injury (93). While overexpression of

Smad7 inhibits profibrotic Smad3-dependent TGF-β signaling in podocytes, it may alternatively

shift TGF-β signaling activities towards apoptotic responses (93). TGF-β also enhances

transcription of Smad7 in peripheral blood mononuclear cells (PBMCs). However in one study,

PBMCs from 50% of lupus patients failed to transcribe Smad7 in response to TGF-β (94).

Whether this resistance is also found in renal cells of SLE patients, and how this resistance in

PBMCs plays a role in the propagation of lupus nephritis is uncertain. HDACs have been found

to interact with and deacetylate Smad7 resulting in its decreased stability (95). Our laboratory

has previously documented increased expression and activity of HDAC6 in glomerular cells of

diseased MRL/lpr mice (41), which may be contributing to progression of lupus nephritis due to

the decreased stability of Smad7. While currently unproven, it is possible that this mechanism is

partly responsible for the decreased lupus nephritis we have documented in NZB/W mice treated

with HDAC6 inhibitors (42).

Interstitial inflammation and scarring in lupus nephritis is more reliable in identifying

SLE patients that are at the greatest risk of developing renal failure (96). TGF-β is a crucial

cytokine that triggers myofibroblastic differentiation, which contributes to chronic fibrotic

diseases (97). Immunohistochemical studies using alpha-smooth muscle actin (α-SMA) showed

myofibroblastic differentiation in the interstitium of diseased kidneys (98), and in experimental

renal scarring experiments alpha-SMA-positive interstitial cells increased over time as

tubulointerstitial fibrosis progressed (99). In one study, silencing of HDAC6 by RNA

interference impaired TGF-β induced α-SMA expression in fibroblasts (100). Therefore,

HDAC6 inhibition carries the potential to suppress the progression of renal fibrosis by blocking

α-SMA expression.

17

Forkhead Box P3 (Foxp3)

An important down-stream molecule in the TGF-β cascade is Foxp3, a transcription

factor for regulatory T cells (Tregs); TGF-β promotes expression of Foxp3 and differentiation of

Tregs from naïve CD4+ T cells (101). Complete loss of Foxp3 protein results in a lack of Tregs

and Foxp3-deficient (scurfy) mice develop a severe and fatal autoimmune disease (102, 103).

Tregs comprise approximately 2% of the CD4+ T cell population in humans (104) and function

to maintain immune tolerance to self-antigens and to suppress excessive and deleterious immune

responses (105). Reduced numbers and function of circulating Tregs have been reported in

human SLE patients (106-109) as well as resistance of lupus effector cells to Treg-cell

suppression (110). In regards to lupus-prone mice, Treg cell frequencies are reduced in NZB/W

mice before disease onset, while frequencies are mainly reduced in diseased MRL/lpr mice and

continue to decline as disease progresses (111). Importantly, Treg cells suppress inflammation

in the kidney, as depletion of CD4+CD25+ (Treg) cells in NZB/W mice results in accelerated

development of lupus glomerulonephritis (112).

Treatment with non-selective HDAC inhibitors or a selective HDAC6 inhibitor has been

shown to increase splenic Treg cell percentages in conjunction with decreased disease

parameters in lupus-prone mice (28, 30, 42). Our laboratory has also recently documented

increased HDAC6 expression and activity in splenic CD4+CD25+ cells from diseased MRL/lpr

mice (41). Two studies have reported decreased suppressive functions of Tregs from diseased

MRL/lpr mice (113, 114), which may be related to this elevated HDAC6. Tregs from HDAC6

knockout mice express more Foxp3 and exhibit enhanced suppressive function in vitro and in

vivo (115). Furthermore, there is more acetylated Foxp3 in Tregs in the absence of HDAC6,

18

implying that HDAC6 deacetylates Foxp3 (116). Collectively, HDAC6 inhibition results in

Foxp3 acetylation, which increases Foxp3 stability and leads to increased Treg cell

differentiation (117), development, and function (118).

Ku70

Ku70 is a component of DNA repair machinery responsible for non-homologous end

joining (NHEJ) of double strand breaks (119) and is also a substrate for HDAC6 (37). HDAC6

deacetylates ku70, which plays a role in apoptosis through regulating cytoplasmic ku70

interactions with pro-apoptotic protein, Bax, or anti-apoptotic protein, FLIP. In both

mechanisms, inhibition of HDAC6 leads to increased apoptosis (120-122). Abnormalities in

apoptosis and clearance of apoptotic cells have been implicated in the etiopathogenesis of SLE.

Defective apoptosis may contribute to the breakdown of tolerance by allowing autoreactive T

and B lymphocytes to survive, allow exposure of autoantigens to the immune system, and

contribute to cell damage as an effector mechanism (123). Studies have shown abnormalities in

early checkpoints regulating B cell development and removal of autoreactive B cells within the

bone marrow in SLE (124). We have also recently identified alterations in the proportions of B

cells in various stages of development and differentiation in the bone marrow of diseased

NZB/W mice suggestive of a possible apoptotic defect (42). Furthermore, HDAC6 inhibition

applied to pre-B cells in vitro increased Bax protein, which was associated with decreased cell

growth (41). Lastly, a recent genomic admixture mapping and molecular modeling study

discovered an intronic single nucleotide polymorphism (SNP) that disrupts the activity of

ku70/80 binding at a newly discovered SLE susceptibility locus (125). This abnormality could

contribute to autoantibody production and interaction (125) since the ku70/80 complex mediates

19

the predominant pathway of NHEJ during immunoglobulin class switch recombination (126).

Based on these results, further studies are warranted to establish a possible link between ku70,

Bax protein, and HDAC6 inhibition in the regulation of bone marrow B cell development in

SLE.

CONCLUSIONS

Treatment for SLE has relied on the administration of nonsteroidal anti-inflammatory

drugs, anti-malarial agents, glucocorticoids, and immunosuppressants (cyclophosphamide,

methotrexate, mycophenolate mofetil) (2, 127). These treatment regimens are often intensive,

associated with side effects, and still carry the potential for relapse and progression of disease

flares (128). Continued research of the molecular mechanisms involved in SLE pathogenesis has

led to the development of biological agents, like monoclonal antibodies, that target B cells, T

cells, cytokines, and complement (129). However, there have been several disappointments in

clinical trials involving these approaches (129). Within the past 50 years Belimumab has been

the only therapy approved by the US Food and Drug Administration for non-renal SLE (130).

Lupus nephritis is one of the most important manifestations of disease in SLE, contributing

significantly to morbidity and mortality (131). While recent prognostic studies have documented

improvement in survival rates in SLE patients (132), the incidence of end-stage renal disease

attributed to lupus nephritis has not changed (133) and several questions remain unanswered.

Therefore, the investigation for effective and safe treatments is still paramount in SLE research.

The multifactorial etiology and involvement of multiple branches of immunity and

inflammation in SLE creates a difficult disease to effectively and safely treat and manage. The

biggest challenge in developing a compound for treatment is finding a balance between its

20

specificity for SLE-associated aberrations and minimizing unwanted and deleterious side effects.

As this review highlights, HDAC6 carries the potential to play a role in multiple target areas

involved in SLE pathogenesis by controlling the acetylation status of its many substrates.

Furthermore, when HDAC6 is knocked out, mice exhibit a viable phenotype with few alterations

in the immune response (38), suggesting that inhibition of HDAC6 carries a certain level of

safety. In addition to the potential of being a safe and efficacious treatment modality for SLE,

HDAC6 inhibition has unveiled additional molecular pathways and abnormalities that will

enhance our knowledge of SLE pathogenesis as they are investigated further.

21

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37

CHAPTER 3

Treatment with a Selective Histone Deacetylase 6 Inhibitor Decreases Lupus Nephritis in

NZB/W Mice

Miranda D. Vieson1

Alexander M. Gojmerac2

Deena Khan1

Rujuan Dai1

John H. van Duzer3

Ralph Mazitschek4

David L. Caudell5

Xiaofeng Liao1

Xin M. Luo1



Veterinary Medicine, Blacksburg, VA, United States.

2. Virginia Polytechnic Institute and State University, Blacksburg, VA, United States.

3. Acetylon Pharmaceuticals, Boston, MA, United States.

4. Center for Systems Biology, Massachusetts General Hospital, Boston, MA, United States.

5. Department of Pathology/Comparative Medicine, Wake Forest School of Medicine, Winton-

Salem, NC, United States.

6. Edward Via College of Osteopathic Medicine, Blacksburg, VA, United States.

38

ABSTRACT

To date, there are 18 histone deacetylase (HDAC) enzymes, divided into four classes,

which alter protein function by removing acetyl groups from lysine residues. Prior studies report

that non-selective HDAC inhibitors decrease disease in lupus mouse models. Concern for

adverse side effects of non-selective HDAC inhibition supports investigation of selective-HDAC

inhibition. We hypothesized that a selective HDAC-6 inhibitor (HDAC6i) will alleviate disease

in a mouse model of lupus by increasing acetylation of alpha-tubulin. Intraperitoneal injections

of the selective HDAC6i ACY-1083 (0.3 mg/kg, 1 mg/kg, or 3 mg/kg), vehicle control, or

dexamethasone were administered to 21-week-old, female NZB/W mice, 5 days a week, for 13

weeks. Disease progression was evaluated by proteinuria, serum levels of anti-dsDNA antibody,

cytokines and immunoglobulins, and post mortem evaluation of nephritis and T cell populations

in the spleen. HDAC6i treatment decreased proteinuria, glomerular histopathology, IgG, and C3

scores when compared to vehicle-treated mice. Within glomeruli of HDAC6i-treated mice, there

was increased acetylation of alpha-tubulin and decreased NF-κB. Additionally, HDAC6i

decreased serum IL-12/IL-23 and Th17 cells in the spleen. Taken together, these results suggest

HDAC-6 inhibition may decrease lupus nephritis in NZB/W mice via mechanisms involving

acetylation of alpha-tubulin and decreased NF-κB in glomeruli as well as inhibition of Th17

cells.

39

INTRODUCTION

Urowitz and Gladman (1999) state that understanding the clinical presentation of

systemic lupus erythematosus (SLE) now focuses on late stage manifestations that contribute to

morbidity and mortality, including chronic renal insufficiency (1). Lupus nephritis is one of the

most costly (2) and important manifestations of disease in SLE patients, contributing

significantly to morbidity and mortality (3). The development of autoantibodies is a mainstay

feature of SLE, and contributes to lupus nephritis by cross-reacting with renal antigens,

indirectly binding to nuclear material in glomerular basement membranes, or forming immune

complexes that circulate and become deposited in kidney glomeruli (4). Deposition of immune

complexes (IC) in the kidneys induces an inflammatory response that disrupts glomerular

filtration eventually leading to proteinuria and if not treated can result in end-stage renal disease

(5). Over time, changes in the kidneys due to repeated flares of inflammation and damage will

ultimately lead to chronic renal failure.

Th17 cells have been implicated in playing a role in the pathogenesis of lupus nephritis.

Circulating Th17 cells are increased in human SLE patients and correlate with SLE disease

activity index scores (6, 7). Additionally, infiltrating T cells within the kidneys of SLE patients

exhibit higher expression levels of IL-17, which correlates with multiple disease parameters

including glomerular and interstitial disease activity scores, urine protein levels, and elevated

blood urea nitrogen levels (8). Abnormal IL-17 production in SLE may promote autoimmune

disease in at least two ways: (1) increased IL-17 in sites of inflammation (kidney) increases the

influx of effector cells therefore amplifying the immune response, and (2) IL-17 can contribute

to the excessive activation of the B cell compartment leading to further antibody production (9).

40

Guidelines for the treatment of lupus nephritis, established by The American College of

Rheumatology, is based on administration of either cyclophosphamide or mycofenolate in

conjunction with glucocorticoids in varying regimens depending on the histological classification

(10). Pharmacological intervention is necessary to reverse the unabated inflammatory response,

but the use of current therapies is often accompanied by severe side effects and the possibility

that continual renal flares and progression to end-stage renal disease may still occur (11). The

investigation for effective treatments that do not elicit severe side effects is still paramount in

SLE research.

There are at least 18 mammalian histone deacetylase (HDAC) enzymes, which remove

acetyl groups from lysine residues in histones and other proteins to control multiple cellular

functions including transcription, cell cycle kinetics, cell signaling, and cellular transport

processes (12). HDACs are classified based on structural and functional similarities into classes

I-IV, of which classes I and II are most widely studied. Class I HDACs include HDAC1, -2, 3, -

6, and -8, and class II HDACs are further subdivided into class IIa (HDAC4, -5, -7, and -9) and

class IIb (HDAC6, and -10) (13, 14). In addition to their initial relevance in cancer biology (15),

HDACs have been reported to play a key role in inflammation and immunity (13).

Subsequently, pharmacologic inhibition of HDACs has been evaluated as a possible treatment

modality in a wide spectrum of diseases unrelated to cancer, including inflammatory and

autoimmune diseases (16). In regard to SLE, decreased disease and renal pathology has been

reported in various mouse models using pharmacologic inhibition of HDACs or by gene deletion

(17-20). The majority of HDAC inhibitors are considered “pan-inhibitors” or non-selective

inhibitors due to their broad action on multiple enzymes of both class I and II HDACs. Because

HDAC I enzymes are ubiquitously distributed throughout the body, pharmacologic inhibition of

41

these enzymes may result in unwanted side effects. Additionally, phenotypes associated with

knocking-out class I and class IIa HDACs are often embryonic lethal or have life-limiting

developmental abnormalities (21). Therefore, selective inhibition of particular HDAC enzymes is

desired (22). HDAC6 knock-out mice exhibit a viable phenotype, develop normally, and have

no life-limiting defects. More importantly, while lymphocytes in these mice develop normally,

there is a mild decrease in the immune response after antigenic stimulation (23). Based on these

findings, we have chosen to focus on specific inhibition of HDAC6 for our studies.

The following studies evaluate the ability of a selective HDAC6i (ACY-1083) to

decrease lupus nephritis in a murine model of SLE (NZB/W F1 female mice). In association

with lupus nephritis, we will also evaluate multiple SLE disease parameters, alpha-tubulin

acetylation in the kidneys, and the status of Th17 cells. We hypothesize that treatment of

NZB/W F1 female mice with ACY-1083 will decrease lupus nephritis concurrently with

decreased Th17 kinetics related to HDAC6i-mediated acetylation of alpha-tubulin.

42

MATERIALS AND METHODS

Mice

Female NZB/W F1 and C57BL/6 mice were purchased from Jackson Laboratories (Bar

Harbor, ME, USA). All mice were used in accordance with the Institutional Animal Care and

Use Committee (IACUC) after protocol approval by the IACUC of Virginia Tech University and

housed in the animal facility at the Virginia-Maryland College of Veterinary Medicine

(Blacksburg, VA, USA). Mice were humanely euthanized with inhalation of isoflurane followed

by cervical dislocation and thoracotomy.

In vivo treatments and monitoring

Hydroxy-propyl-methyl cellulose (HPMC, Sigma, St. Louis, MO, USA) was diluted in

sterile deionized water at a concentration of 0.05%, autoclaved, then used as the vehicle for all

drug solutions. ACY-1083, a histone deacetylase 6 inhibitor (HDAC6i), was courtesy of a

generous donation from Acetylon Pharmaceuticals (Boston, MA, USA) for use in all studies. A

pharmacokinetic analysis of ACY-1083 was used to determine the optimum doses for the study.

Male C67Bl/6 mice were injected with ACY-1083 in 20% hydroxypropyl beta

cyclodextrin/0.5% HPMC by intravenous (IV) route at 1 mg/kg or by intraperitoneal (IP) route at

10 mg/kg. Plasma levels of ACY-1083 were determined by LC/MS/MS and quantitated by a

standard curve made in matching plasma. Based on these results, ten mice were included in each

of 5 treatment groups: (1) vehicle control (HPMC), (2) 0.3 mg/kg ACY-1083, (3) 1 mg/kg ACY-

1083, (4) 3 mg/kg ACY-1083, (5) 2 mg/kg dexamethasone (DEX, positive control). Mice were

injected IP 5 days/week with a 50uL volume of their respective treatments beginning at 21-

weeks-of-age, and treatments continued until euthanasia during late stage clinical disease at 34

43

weeks-of-age. Proteinuria and body weight were measured bi-weekly before treatment, then

weekly after treatment began. Proteinuria was determined by a standard semi-quantitative test

using Siemens Uristix dipsticks (Siemens Healthcare, Deerfield, IL, USA). Results were

quantified according to the manufacturer’s instructions and scored as follows: dipstick reading of

0 mg/dL= 0, trace = 1, 30-100 mg/dL = 2, 100-300 mg/dL = 3, 300-2000 mg/dL = 4, and 2000+

= 5.

Measurement of autoantibodies

Sera were collected prior to treatment at 20-weeks-of-age, and then once every 4 weeks

until euthanasia. The mice were anesthetized using isoflurane (Piramal Healthcare, Mumbai,

Maharashtra, India) and bled from the retro-orbital sinus. Blood was allowed to clot for 2 h and

then centrifuged for 15 min at 10,000x g. The levels of autoantibodies to dsDNA were measured

by semi-quantitative ELISA. High-binding EIA plates were coated with 100uL of a 5ug/mL

Calf Thymus DNA (Sigma, St. Louis, MO, USA) in saline-sodium citrate (SCC) buffer solution

and incubated overnight at 37C. Plates were washed with 0.05% Tween-20 in PBS, then blocked

with 1% Bovine Serum Albumin (BSA, Sigma, St. Louis, MO, USA) in PBS for 1h. Sera

samples were added to the plate at a 1:100 dilution followed by a two-fold serial dilution, then

allowed to incubate for 45 min at room temperature. Plates were washed, then incubated with an

HRP-conjugated goat anti-mouse IgG antibody (1:4000, Southern Biotech, Birmingham, AL,

USA) for 45 minutes at room temperature. Plates were washed once more. SureBlue Reserve

TMB substrate was added to wells, and then plates were read at 380 nm on a Spectramax 340PC

microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). Serum from a

C57BL/6 mouse and a diseased MRL/lpr mouse was used as negative and positive controls,

44

respectively. Values on each plate were normalized to the positive control (set as 1) and a final

dilution of 1:3600 was reported.

Measurement of serum cytokines and immunoglobulin isoforms

Total IgG, IgG2a, IL-12/IL-23, and TGF-β protein levels were measured in the sera by

quantitative ELISA according to manufacturer’s protocol (eBioscience, San Diego, CA, USA).

Plates were read on a microplate spectrophotometer at 450 nm.

Splenocyte isolation and flow cytometric analysis

Spleens were aseptically removed from the mice after euthanasia and dissociated into

single cell suspensions by gentle compression and traction between two frosted glass microscope

slides. Cells were collected in RPMI 1640 (phenol red free) media supplemented with 10%

charcoal-stripped FBS (Atlanta Biologicals), 2 mM L-glutamine, 100 IU/ml penicillin, 100

μg/ml streptomycin, and 1% nonessential amino acids (Mediatech, Inc, Tewksbury, MA,

USA). Cell suspensions were incubated in ACK lysis buffer (Lonza, Alpharetta, GA, USA) to

remove red blood cells, then washed three times with culture media. A subset of isolated

splenocytes from treated mice were washed with cold DPBS containing magnesium and calcium

(HyClone, Logan, UT, USA) followed by staining of phenotyping antigens with directly

conjugated fluorescent-labeled murine monoclonal antibodies (eBiosciences, San Diego, CA,

USA). For evaluation of T cell populations, two sets of splenocytes were washed with cold flow

cytometry buffer followed by staining of cell surface antigens by either CD4-FITC and CD25-

PerCP-Cy5.5 (Treg) or CD4-FITC only (Th17). Next, both sets of T cells (Treg and Th17) were

fixed and permeabilized with Foxp3/Transcription factor Fix/Perm buffer (eBiosciences, San

45

Diego, CA, USA) followed by intracellular antigen staining with Foxp3-PE (Treg) or RORγ-

APC and IL-17-PE (Th17). Both sets of T cells were sorted by in a FACS Aria 1 flow cytometer

(BD Biosciences, San Jose, CA, USA) then analyzed by FlowJo Software (Tree Star, Ashland,

OR, USA).

Splenocyte miRNA and mRNA expression assays

Total RNA was isolated from whole splenocytes using the miRNeasy Mini Kit (Qiagen,

Germantown, MD, USA). To remove residual amounts of DNA contamination in isolated RNA,

on-column DNase digestion with RNase-Free DNase was performed. The RNA concentration

was quantified using a NanoDrop 2000. As we described previously (24), the Taqman miRNA

assay system (Applied Biosystems, Foster City, CA, USA) was used to quantitatively detect the

expression of miRNAs (miR-127, miR-148a, miR-182, miR-31, miR-379, miR-155, and miR-

27) following the manufacturer's instructions. Expression levels of miRNA were normalized to

the small RNA housekeeping control snoRNA 202, and data are shown as relative expression

levels compared to the vehicle control (HPMC) group by calculating with the formula: 2-

DDCt (Livak method).

Renal histopathology

At the time of euthanasia, both kidneys were removed. One kidney was fixed in 10%

neutral buffered formalin for 24 hours, then routinely processed, embedded in paraffin, sectioned

at 4-5μm, and stained with Periodic acid-Schiff (PAS). Kidney sections were scored (0-4) for

glomerular proliferation, inflammation, crescent formation, necrosis, and fibrosis by a

pathologist (D.L. Caudell) in a blinded manner.

46

Renal IgG and C3 accumulation

One kidney was placed in OCT media and snap-frozen in a slurry containing dry ice and

2-methylbutane (Fisher Scientific, Hampton, NH, USA). Frozen kidney sections were cut into 4

μm sections, fixed in acetone for 10 min, then washed 3 times with PBS for 5 min each. Next,

the sections were incubated with goat anti-mouse IgG conjugated to FITC (1:100, Sigma, St.

Louis, MO, USA) or goat anti-mouse C3 conjugated to FITC (1:100, Cederalane, Burlington,

NC, USA) antibodies in a humid chamber for 1 h. Slides were mounted using Vectashield

mounting media (Vector Labs, Burlingame, CA, USA) and examined by an Olympus IX73

fluorescent microscope (Olympus America Inc., Center Valley, PA, USA). Deposition of IgG

and C3 within glomeruli was scored (0-3) by a board-certified veterinary anatomic pathologist

(M.D. Vieson).

Acetylated alpha-tubulin, Histone 3, and NF-κB immunofluorescence in glomerular cells

One kidney was placed in OCT media and snap-frozen in a slurry containing dry ice and

2-methylbutane (Fisher Scientific, Hampton, NH, USA). Frozen kidney sections were cut into 5-

7 μm sections, fixed in 4% formaldehyde for 15 min, then washed 3 times with PBS for 5 min

each. Next, the sections were blocked with normal goat serum for 1 hour then incubated with

rabbit anti-mouse acetylated-alpha-tubulin (K40) or rabbit anti-mouse nuclear factor – kappa B

(NF-κB) (Cell Signaling Technologies, Danvers, MA, USA) overnight at 4C. Sections were

washed with PBS 3 times for 5 min each, then incubated with either goat anti-rabbit IgG

conjugated to R-Phycoerythrin (PE) or goat anti-rabbit IgG conjugated to FITC (Sigma, St.

Louis, MO, USA) for 2 hours. Sections stained for acetylated alpha-tubulin were washed thrice

47

with PBS, then incubated with rabbit anti-mouse acetylated-histone H3 (Lys9) conjugated with

AlexaFluor488 (Cell Signaling Technologies, Danvers, MA, USA) overnight at 4C. Sections

were washed thrice with PBS, then mounted with Vectashield mounting media with DAPI

(Vector Labs, Burlingame, CA, USA) and examined by a Zeiss LSM800 confocal microscope

(Carl Zeiss Microscopy, Thornwood, NY, USA).

Cytoplasmic and nuclear NF-κB in mesangial cells, in vitro

SV40-Mes13 mesangial cells (ATCC, Manassas, VA, USA) were cultured in complete

media composed of Dulbecco’s Modified Eagle’s Medium (DMEM)/Ham’s F12 Medium

(75/25) with 14 mM HEPES (Corning Cellgro, Manassas, VA, USA) supplemented with 5%

fetal bovine serum (HyClone, Logan, UT, USA) and 1% Penicillin/Streptomycin (Corning

Cellgro, Manassas, VA, USA). Cells were incubated in a humidified, 5% CO2 incubator

(Thermo Fisher Scientific, Waltham, MA, USA) at 37°C and treated with ACY-1083 for 2 hours

before being stimulated by 1 ug/mL LPS (Sigma, St. Louis, MO, USA) and 100 ng/mL IFN-γ

(Cedarlane Laboratories Limited, Burlington, NC, USA) or DI water for 24 hours. HPMC was

used as the vehicle control, similar to in vivo studies.

Cytoplasmic and nuclear protein fractions were isolated utilizing NE-PER Nuclear and

Cytoplasmic Extraction Reagents (Thermo Fisher Scientific, Waltham, MA, USA) according to

the manufacturer’s protocol. Protein lysates were quantitated and normalized by Bradford assay,

then subjected to electrophoresis in 7.5% Criterion TGX Precast gels (Bio-Rad, Hercules, CA,

USA) followed by protein transfer to PVDF membranes (Bio-Rad, Hercules, CA, USA).

Membranes were thoroughly washed, blocked with TBS with 5% non-fat milk, then incubated

with primary antibodies against NF-κB and beta-actin (Cell Signaling Technologies, Danvers,

48

MA, USA) overnight. Membranes were then thoroughly washed and incubated with horseradish

peroxidase (HRP)-linked anti-mouse or anti-rabbit IgG (secondary) antibodies (Cell Signaling

Technologies, Danvers, MA, USA) followed by thorough washing then addition of Amersham

ECL Detection Reagents (GE Healthcare Life Sciences, Logan, UT, USA). Membranes were

then exposed and images acquired with a Kodak 4000MM imaging station (Carestream,

Rochester, NY, USA).

Statistics

Statistical analysis was performed using GraphPad Prism software (La Jolla, CA, USA).

Linear regression was utilized to analyze differences in urine protein levels, body weight,

dsDNA autoantibodies, IgG isotypes, and serum cytokine levels over time (age). Differences

between the slopes and intercepts of different treatment groups were determined by analysis of

covariance (ANCOVA). Differences among groups with more than two conditions were

analyzed using one-way ANOVA followed by further analysis using Tukey’s multiple

comparison tests. Differences between two means were assessed with unpaired two-tailed t-tests.

A p-value < 0.05 was considered statistically significant.

49

RESULTS

ACY-1083 inhibits HDAC6, selectively

ACY-1083 was generated as a result of a drug discovery effort at Acetylon

Pharmaceuticals to produce a highly selective inhibitor of HDAC6 (Fig. 1A). The compound

was tested against recombinant HDACs in a biochemical assay as previously described (25).

ACY-1083 inhibits HDAC6 with a potency of 4 nM and HDAC1 (the next most affected target)

with a potency of 961 nM (Fig. 1B). ACY-1083 does not potently inhibit HDAC8 or any class

IIa or Class III HDAC (not shown). In a pharmacokinetic study ACY-1083 has a plasma half-life

of 3.8 hours when injected intraperitonealy (IP). Dose levels for the in vivo experiments were

chosen to achieve a plasma level high enough to inhibit HDAC6 but not high enough to inhibit

HDAC1.

Fig. 1. ACY-1083 is selective for HDAC6. (A). Basic chemical structure of ACY-1083. (B) ACY-1083 selectively decreased HDAC6 activity with a potency of 4 nM. ACY-1083 treatment had no effects on mouse survival and body weight, and decreased

proteinuria and splenic weight.

Body weight was monitored every other week before the initiation of treatment, then

weekly until euthanasia at 34 weeks-of-age. Eight to ten mice were analyzed in each treatment

group. Body weight decreased in all treatment groups between 20 and 22 weeks-of-age (Fig.

50

2A). During this same time point, urine protein levels also began to increase (Fig. 2B). Because

these changes are associated with disease development, treatments were initiated for all

treatment groups. After initiation of treatment at 21 weeks-of-age, mice in the vehicle control

(HPMC) and 0.3 mg/kg ACY-1083 groups showed no significant weight changes (Fig. 2A). An

increase in body weight over time is noted in groups treated with 0.3 mg/kg ACY-1083 (r2 =

0.057; p = 0.007), 1 mg/kg ACY-1083 (r2 = 0.039; p = 0.037), and 2 mg/kg dexamethasone

(DEX, r2 = 0.311; p < 0.0001). After 13 weeks of treatment, there were no significant

differences in body weight between treatment groups.

Proteinuria was monitored weekly in all treatment groups as they aged. The urine protein

score increased over time in NZB/W F1 mice despite treatment with ACY-1083 at 0.3 mg/kg (r2

= 0.204; p < 0.0001), 1 mg/kg (r2 = 0.090; p = 0.0012), and 3 mg/kg (r2 = 0.108; p = 0.0004)

doses, or HPMC (r2 = 0.276; p < 0.0001) (Fig. 2B). Of the ACY-1083-treated mice, the urine

protein scores of the 0.3 mg/kg ACY-1083 group increased the most (F = 8.88; p = 0.0008),

while the 1 mg/kg ACY-1083 group increased the least over time (F = 11.57; p = 0.0032) when

compared to the HPMC group. After 13 weeks of treatment, HPMC-treated mice had the highest

degree of proteinuria compared to DEX-treated mice (p < 0.05).

Spleen weight was evaluated in all treatment groups after euthanasia at 34 weeks-of-age.

A mild dose-response of decreasing splenic weight associated with increasing doses of ACY-

1083 is noted, however it was not statistically significant (Fig. 2C). Spleens from the DEX-

treated mice were significantly smaller than the other treatment groups (p < 0.001).

51

Fig. 2. Progression of Disease in NZB/W F1 Female Mice. (A) Body weight, measured weekly after initiation of treatments at 21 weeks-of-age, was not affected by treatment with ACY-1083 (0.3 mg/kg, 1 mg/kg, and 3 mg/kg in HPMC) and mildly increased over time by dexamethasone (DEX, 2 mg/kg). (B) Treatment with ACY-1083 significantly decreased the rate of elevation in proteinuria over time at 1 mg/kg and 3 mg/kg doses when compared to mice treated with HPMC. Mice treated with dexamethasone (2 mg/kg) maintained low urine protein scores throughout the study (34 weeks). (C) Spleens were weighed after euthanasia at 34 weeks-of-age, then the spleen weight:body weight ratio was calculated. A mild dose-dependent decrease in size of spleens is noted with increasing doses of ACY-1083 but does not reach statistical significance. Dexamethasone (2 mg/kg) significantly decreased spleen size. (n ≥ 8; ** p < 0.01, ***p < 0.001, ****p < 0.0001)

52

Treatment with ACY-1083 had no effect on autoantibody levels and maintained lower

levels of total IgG and IgG2a in the serum

Serum was collected every 4 weeks beginning at 16 weeks-of-age and at the time of

euthanization at 34 weeks-of-age for measurement of anti-dsDNA antibody levels. Similar to the

vehicle control (HPMC) group (r2 = 0.02, p < 0.001), serum anti-dsDNA antibody levels

increased as the mice aged in each treatment group (r2 = 0.21-0.38, p < 0.001). Mice treated

with DEX had significantly lower anti-dsDNA antibodies levels (p < 0.001) at 28, 32, and 34

weeks-of-age (Fig. 3A) compared to those treated with HPMC.

Levels of total IgG and IgG2a were evaluated in the sera of mice beginning at 20 weeks-

of-age, then every 4 weeks, and again at the time of euthanasia at 34 weeks-of-age. There was

no significant increase or decrease in the levels of total IgG or IgG2a over time in all treatment

groups evaluated. After 13 weeks of treatment, serum levels of total IgG and IgG2a were lower in

mice treated with ACY-1083 or DEX when compared to the HPMC group (Figs. 3B and 3C),

which was statistically significant for IgG2a levels in mice treated with 1 or 3 mg/kg ACY-1083

or DEX (p < 0.05).

53

Fig 3. Sera Autoantibodies Targeting dsDNA and Immunoglobulin (Ig) Isoforms. Anti-dsDNA and Ig isoforms were measured in the sera of NZB/W F1 female mice treated with ACY-1083 (0.3 mg/kg, 1 mg/kg, or 3 mg/kg), dexamethasone (DEX, 2 mg/kg) or vehicle control (HPMC). (A) Autoantibodies continued to increase over time in mice treated with all doses of ACY-1083 and HPMC. Mice treated with DEX maintained low serum autoantibody levels for the entirety of the study (n ≥ 8, *p < 0.05). (B) By the time of euthanasia at 34 weeks-of-age, mice treated with all doses of ACY-1083 and DEX had lower levels of total IgG in the serum. (C) Serum IgG2a was decreased in a dose-dependent manner in mice treated with ACY-1083 compared to the HPMC group, which reached statistical significance at 1 and 3 mg/kg doses. Mice treated with DEX had significantly lower serum IgG2a compared to controls. (n = 4, *p < 0.05)

ACY-1083 treatment decreased Th17 cells and Treg cells in the spleen

Following euthanasia at 34 weeks-of-age, spleens were removed and single cell

suspensions were obtained. Splenocytes were roughly quantitated by hemocytometer then

evaluated by flow cytometric analysis for percentages of T cell subsets. There was a similar

number of total splenocytes isolated from mice treated with HPMC (M = 177.3 x 106, SEM =

32.07 x 106) or ACY-1083 at 0.3 mg/kg (M - 142.8 x 106, SEM = 19.45 x 106), 1 mg/kg (M =

114.6 x 106, SEM = 18.87 x 106), or 3 mg/kg (M = 126.0 x 106, SEM = 24.09 x 106) doses.

However, dexamethasone treatment significantly decreased the total number of splenocytes (M =

4.88 x 106, SEM = 0.52 x 106) compared to HPMC (p < 0.001).

There is a mild dose-dependent decrease in the percentage of CD4+IL-17+RORy+ (Th17)

cells in the spleen with increasing doses of ACY-1083 (Figs. 4A and 4B), which was

statistically significant at the 3 mg/kg dose (p < 0.05). Mice treated with DEX had significantly

54

lower percentages of Th17 cells in the spleen (p < 0.01) compared to vehicle control (HPMC)

mice.

Treatment with ACY-1083, at all doses investigated mildly decreased the number of

CD4+CD25+Foxp3+ (Treg) cells in the spleen (Figs. 4C and 4D) in comparison to the vehicle

control group, which reached statistical significance in the 3 mg/kg group (p < 0.05).

Furthermore, DEX treated animals showed a significant decrease in the percentage of Treg cells

in the spleen (p < 0.0001).

55

Fig 4. Splenic T Cell Subsets. Subsets of T cells from the spleen of NZB/W F1 mice were assessed after 13 weeks of treatment with ACY-1083 (0.3, 1, or 3 mg/kg), dexamethasone (DEX, 2 mg/kg), or vehicle (HPMC). (A and B) Treatment with ACY-1083 for 13 weeks resulted in a dose-dependent decrease in the percentage of Th17 (CD4+IL-17+RORy+) cells in the spleen, which reached statistical significance in the mice treated with 3 mg/kg ACY-1083. There is also a significant depletion of Th17 cells in the spleen after dexamethasone treatment. (C and D). There was no significant alteration in the percentage of Treg (CD4+CD25+Foxp3+) cells in the spleen of mice after 0.3 or 1 mg/kg ACY-1083 treatment, and a decrease in the percentage of Treg cells in mice treated with 3 mg/kg ACY-1083. Dexamethasone treatment significantly depleted the number of Treg cells in the spleen. (n ≥ 8; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001) Treatment of NZB/W F1 female mice with ACY-1083 decreased serum IL-12/IL-23 p40

levels in a dose-dependent manner and decreased serum TGF-β

Serum was collected from mice every 4 weeks starting at 20 weeks-of-age and again at

the time of euthanasia (34 weeks-of-age) for evaluation of cytokines involved in polarization of

T cell subsets. Over time, serum TGF-β levels decreased in mice treated with ACY-1083 at

either the 0.3 mg/kg (r2 = 0.26, p = 0.02) or 3 mg/kg (r2 = 0.36, p = 0.005) doses (Fig. 5A).

TGF-β levels were maintained in mice treated with 1 mg/kg ACY-1083, vehicle control, or

dexamethasone. After 13 weeks of treatment (Fig. 5B), TGF-β decreased in the serum of mice

treated with ACY-1083 at 0.3, 1 or 3 mg/kg doses (not significant), and significantly increased in

mice treated with dexamethasone (p < 0.05).

Serum IL-12/IL-23 p40 levels progressively increased over time (Fig. 5C) in mice treated

with vehicle control (r2 = 0.30, p = 0.01) and 0.3 mg/kg ACY-1083 (r2 = 0.25, p = 0.03). Mice

56

treated with 1 mg/kg or 3 mg/kg ACY-1083 also experienced increasing serum levels of IL-

12/IL-23 p40 over time (r2 = 0.30 – 0.36 , p < 0.02). However, the rate of increase was reduced

in both treatment groups, which reached statistical significance in the 3 mg/kg group (F = 5.21, p

= 0.03). In contrast, IL-12/IL-23 p40 levels decreased over time in mice treated with

dexamethasone (r2 = 0.54, p < 0.001). At the time of euthanasia (Fig. 5D), there was a dose-

dependent decrease in serum IL-12/IL-23 p40 levels in mice treated with ACY-1083, although

not statistically significant. Dexamethasone treatment, however, significantly reduced IL-12/-

IL23 p40 levels in the serum of NZB/W F1 mice by the end of the study (p < 0.05).

Fig 5. Serum Levels of TGF-β and IL-12/IL-23. Serum cytokine levels were evaluated every 4 weeks from NZB/W F1 female mice treated with ACY-1083 (0.3, 1, or 3 mg/kg), vehicle (HPMC), or dexamethasone (DEX, 2 mg/kg). (A) Serum TGF-β levels decreased over time in mice treated with 0.3, 1, or 3 mg/kg ACY-1083 and remained unchanged in mice treated with vehicle (HPMC) and dexamethasone (2 mg/kg). (B) At the time of euthanasia (34 weeks-of-age), serum TGF-β levels were lower in mice treated with ACY-1083 (not statistically significant), and significantly increased in mice treated with dexamethasone (2 mg/kg). (C) Serum IL-12/IL-23 p40 increased over time in mice treated with vehicle control and 0.3 mg/kg ACY-1083. Serum levels were maintained in mice treated with 1 mg/kg or 3 mg/kg ACY-1083, and serum

57

levels decreased in mice treated with dexamathasone (2 mg/kg). (D) By the end of the study (34 weeks-of-age), there was a dose-dependent decrease in serum levels of IL-12/IL-23 p40 in mice treated with ACY-1083, and a significant decrease mice treated with dexamethasone (2 mg/kg). (n ≥ 3, *p < 0.05, ****p < 0.0001)

Treatment with ACY-1083 had no effect on relative expression levels of lupus-associated

miRNAs in the spleen.

After euthanasia, splenocytes were isolated from spleens and evaluated by real-time RT-

PCR for the relative expression levels of miRNAs associated with SLE pathogenesis (24, 26, 27)

– miR-127, miR-148a, miR-182, miR-31, miR-379, miR-155, and miR-21. There were no

significant changes in the expression of any of the evaluated miRNAs after 13 weeks of ACY-

1083 treatment, at all doses investigated (0.3, 1, and 3 mg/kg), when compared to the vehicle

control (HPMC) group (Fig. 6). On the other hand, treatment with dexamethasone (DEX, 2

mg/kg) significantly decreased expression of miR-182 (p < 0.001), miR-155 (p < 0.0001), and

miR-21 (p < 0.05). Treatment with DEX also increased expression of miR-127 (p < 0.0001) and

miR-148a (not statistically significant) compared to the HPMC group.

58

Fig 6. Evaluation of Splenic miRNAs Associated with Lupus. Total RNA was extracted from splenocytes of 34-week-old NZB/W F1 mice treated with ACY-1083 (0.3, 1, or 3 mg/kg), vehicle control (HPMC), or dexamethasone (DEX, 2mg/kg). Expression levels of select miRNAs were evaluated by RT-PCR. There were no significant differences in the relative expression levels of miR-127 (A), miR-148a (B), miR-182 (C), miR-31 (D), miR-379 (E), miR-155 (F), and miR-21 (G) from splenocytes of ACY-1083-treated mice and vehicle-treated mice. Splenocytes from dexamethasone-treated mice exhibited significantly increased relative expression of miR-127 (A), and significantly decreased relative expression of miR-182 (C), miR-155 (F), and miR-21 (G). (n ≥ 8; *p < 0.05, ***p < 0.001, ****p < 0.0001)

Treatment with ACY-1083 decreased lupus nephritis as well as glomerular IgG and C3

deposition in a dose-dependent manner

After euthanasia, kidneys were evaluated by histopathology and immunofluorescent

microscopy for severity of lupus nephritis and accumulation of immune complexes within the

glomerulus. Evaluation of PAS-stained sections showed a dose-dependent decrease in the

glomerular histopathology score with increasing doses of ACY-1083 (Figs. 7A and 7B), which

reached statistical significance in the 3 mg/kg ACY-1083 group (p < 0.05). Mice treated with

dexamethasone (DEX, 2 mg/kg) had the lowest glomerular histopathology scores (p < 0.01)

compared to the other treatment groups. Frozen kidney sections stained with fluorescent-tagged

antibodies revealed dose-dependent decreases in the deposition of IgG (Figs. 7C and 7D) and

59

C3 (Figs. 7E and 7F) within the glomeruli of ACY-1083-treated. Deposition of IgG and C3

were significantly decreased in mice treated with 3 mg/kg ACY-1083 (p < 0.05) when compared

to mice treated with the vehicle. Dexamethasone treatment also decreased glomerular deposition

of IgG (p < 0.05) and C3 (not statistically significant).

60

Fig 7. Glomerular Pathology. Kidneys from 34-week-old NZB/W F1 female mice after 13 weeks of treatment with either ACY-1083 (0.3, 1, or 3 mg/kg), dexamethasone (DEX, 2 mg/kg), or vehicle control (HPMC) were removed, sectioned, then stained with Periodic Acid-Schiff (PAS), anti-IgG-FITC, or anti-C3-FITC. (A and B) Glomerular histopathology scores on PAS-stained sections decreased in a dose-dependent manner in mice treated with ACY-1083. Glomerular deposition of IgG (C and D) and C3 (E and F) similarly decreased in a dose-dependent manner in mice treated with ACY-1083. (n ≥ 8; *p < 0.05, **p < 0.01)

Treatment with ACY-1083 increased alpha-tubulin acetylation and decreased NF-κB in

glomerular cells

After 17 weeks of treatment, mice were euthanized and kidneys were collected and snap-

frozen for immunofluorescent investigation of alpha-tubulin (α-Tub) and histone H3 (H3)

acetylation at lysine residues 40 and 9, respectively (Fig. 8), and NF-κB (Fig. 9A). In all

treatment groups, staining for acetylated-H3 exhibited diffuse nuclear reactivity in all the

glomerular cells, and there were no significant changes in either the staining pattern or amount of

positive-staining nuclei between treatment groups. There was no specific positive staining for

acetylated α-Tub within the glomeruli of mice treated with HPMC, DEX, or 0.3 mg/kg ACY-

1083. Minimal, punctate, multifocal cytoplasmic staining is noted in glomerular cells in mice

treated with 1 mg/kg ACY-1083, and more numerous and prominent cytoplasmic reactivity is

noted in mice treated with 3 mg/kg ACY-1083. Glomerular cells exhibit diffuse, granular,

cytoplasmic immunoreactivity to NF-κB, which decreases in a dose-dependent manner with

increasing doses of ACY-1083. There is minimal staining in glomerular cells of mice treated

with DEX.

ACY-1083 decreased nuclear NF-κB protein in mesangial cells, in vitro

An SV40/Mes13 murine mesangial cell line was utilized to determine how mesangial

cells contribute to the changes noted in vivo. The cells were cultured with varying

61

concentrations of ACY-1083 (0, 1, 2.5, 5, 10, or 100 nM) then stimulated with

lipopolysaccharide (LPS) and interferon (IFN)-γ or DI water for 24 hours. Nuclear and

cytoplasmic protein fractions were then extracted and evaluated by western blot to investigate

NF-kB levels (Fig. 9B). The levels of NF-κB within cytoplasmic protein fractions remained

unchanged in all treatment groups. However, nuclear NF-κB increased in mesangial cells

stimulated with LPS/IFN-γ and decreased in a concentration-dependent manner in LPS/IFN-γ-

stimulated cells treated with ACY-1083.

Fig 8. Alpha-Tubulin and Histone 3 Acetylation in Glomerular Cells. Kidneys from NZB/W F1 female mice treated with ACY-1083 (0.3, 1, or 3 mg/kg), dexamethasone (DEX, 2 mg/kg), or vehicle (HPMC) for 13 weeks were removed, snap-frozen, sections, then evaluated for acetylation of histone 3 (H3) and alpha-tubulin (α-Tub) by immunofluorescence. Treatment with ACY-1083 increased the acetylation of α-Tub (R-Phycoerythrin) within glomerular cells. In comparison, there was no increase in α-Tub acetylation after treatments with HPMC or DEX. No differences are noted in H3 acetylation (AlexaFluor488) between all treatment groups. (n=5)

62

Fig. 9. NF-κB in glomerular and mesangial cells. (A) Snap-frozen sections of kidneys from NZB/W mice treated with ACY-1083 (0.3, 1, or 3 mg/kg), dexamethasone (DEX, 2 mg/kg), or vehicle (HPMC) were immunofluorescently evaluated for NF-κB (p65). Glomerular cells from HPMC-treated mice exhibited the strongest and most abundant NF-κB staining, which decreased in a dose-dependent manner in mice treated with ACY-1083. There is limited NF-κB staining in glomerular cells from DEX-treated mice. (n=3) (B) Mesangial (SV40/Mes13) cells were treated with varying concentrations of ACY-1083 for 2 hours, then stimulated with 1 ug/mL LPS and 100 ng/mL IFN-γ or DI water for 24 hours. Cytoplasmic and nuclear protein fractions were extracted and evaluated by western blot for NF-kB. Treatment with ACY-1083 decreased nuclear NF-κB after LPS/INF-γ stimulation in a concentration-dependent manner.

63

DISCUSSION

Histone deacetylases (HDACs) are increasingly being investigated for their roles in

modulating inflammation and immunity (13), and therefore becoming therapeutic targets

candidates for various inflammatory and autoimmune diseases (16). Due to the potential of

adverse side effects associated with non-selective HDAC inhibition (21), we sought to determine

the effects of specific HDAC6 inhibition on SLE disease in NZB/W F1 female mice.

Glomerular cells from lupus-prone mice exhibit increased expression and activity of HDAC6,

particularly in the cytoplasm, which is reduced when treated with a selective HDAC6i (ACY-

738) (28). One of the major functions of HDAC6, a cytoplasmic, class IIb HDAC, is controlling

the acetylation status of alpha-tubulin (29). Overexpression of HDAC6 results in

hypoacetylation of tubulin, while inhibition of HDAC6 results in hyperacetylation (29). In the

current study, ACY-1083 increased acetylation of alpha-tubulin and did not alter the acetylation

status of histone 3 within glomerular cells of NZB/W F1 mice confirming specific inhibition of

HDAC6. Further, there was a concurrent decrease in SLE disease parameters after treatment

with ACY-1083, including decreased proteinuria, lupus nephritis, and splenic weight. These

data suggest that the acetylation status of alpha-tubulin plays a role in the progression of SLE

disease in NZB/W F1 female mice.

To determine the contribution of glomerular changes from mesangial cells, SV40/Mes13

mesangial cells were cultured with ACY-1083 then stimulated with LPS and IFN-γ. Treatment

with ACY-1083 increased acetylated alpha-tubulin and concurrently decreased nuclear NF-κB

protein in mesangial cells. Another HDAC6-selective inhibitor, ACY-738, has also been shown

to increase alpha-tubulin acetylation and decrease nuclear NF-κB in mesangial cells with a

similar treatment and stimulation protocol (28). NF-κB is constitutively activated in many

64

autoimmune diseases, including SLE (30), and contributes to the inflammatory response in the

kidney by regulating the expression of numerous important contributory genes (31). The

underlying connection between acetylated tubulin and NF-κB in the kidney is uncertain. One

theory is that acetylated tubulin acts in concert with nephrin, a key protein involved in the slit

diaphragm, to inhibit NF-κB in glomerular cells (32). Other mechanisms related to HDAC

inhibition that may contribute to decreased NF-κB in glomeruli, independently of alpha-tubulin

acetylation, include inhibition of heat shock protein (Hsp) 90 (28, 33-35), and modulation of

Smad7 stability (32, 36, 37).

Increased serum immunoglobulin titers are some of the more reliable indicators of SLE

activity in human SLE patients. Immunoglobulins of the IgG isotype directed against dsDNA,

C1q, and nucleosomes (autoantibodies) are commonly analyzed titers because they correlate

significantly with disease manifestations of SLE, particularly lupus nephritis (38-40). The

NZB/W F1 mouse model shares many of these clinical manifestations, including markedly

elevated serum levels of IgG antibodies that bind dsDNA and histones (41, 42). Deposition of

these autoantibodies, particularly IgG anti-dsDNA and IgG2a in glomeruli is one of the major

contributors to the pathogenesis of lupus nephritis in both human patients (43) and in lupus

mouse models (42, 44). Importantly, IgG2a is considered one of the more pathogenic subclasses

of immunoglobulin due to its unique ability to bind and activate the Fcγ receptor FcγIV (45).

We have previously shown that treatment with a class I and II HDAC inhibitor (ITF2357)

significantly decreased anti-dsDNA antibodies, as well as total IgG and IgG2a in the serum of

NZB/W mice(18). In our current study, inhibition of HDAC6 with ACY-1083 had no significant

effect on the serum levels of anti-dsDNA antibodies in NZB/W F1 mice. In contrast, serum

65

levels of total IgG and IgG2a did not significantly increase over time, and were lower in mice

treated with ACY-1083.

The development of autoantibodies is a mainstay feature of SLE, and contributes to lupus

nephritis by cross-reacting with renal antigens, indirectly binding to nuclear material in

glomerular basement membranes, or forming immune complexes that will circulate and become

entrapped within glomeruli (4). However, there is a discordance between serum levels of anti-

dsDNA antibodies and lupus nephritis (46), which has been reported in humans (47) and various

lupus-prone mouse strains (48, 49). Similarly, we saw a decrease in lupus nephritis and

glomerular IgG deposition with increasing doses of ACY-1083 despite increasing levels of anti-

dsDNA in the serum of NZB/W F1 female mice. This discrepancy may be due to the differential

ability of different subclasses of these antibodies to deposit in the kidney and incite an

inflammatory response (50). Therefore, determining the subclass, cross-reactivity with different

cellular antigens, and the ability to deposit in the kidneys is more important in determining the

pathogenic potential of anti-dsDNA antibodies than the overall serum titer, alone (46).

Elevated IL-17 production has been reported to be instrumental in the inflammatory

cascade in SLE patients (51) and also plays a role in the pathogenesis of lupus in various mouse

models including NZB/W F1 mice (52). Elevated Th17 cells have been detected in the peripheral

blood of human patients with lupus nephritis and correlated with disease activity index scores (6,

7). Our laboratory has previously shown decreased splenic Th17 cells with concomitant

reduction in SLE disease parameters in MRL/lpr mice after treatment with the non-selective

HDACi, ITF2357 (18). In the current studies, we observed a similar dose-dependent decrease in

the number of Th17 cells in the spleen. This decrease mirrors the dose-dependent decrease in

66

lupus nephritis pathology in ACY-1083-treated mice suggesting that HDAC6 inhibition

decreases lupus nephritis in NZB/W F1 mice by decreasing Th17 cells in the spleen.

In addition to the decreases in splenic Th17 cells and lupus nephritis, pharmacologic

HDAC 6 inhibition in NZB/W F1 mice resulted in a dose-dependent decrease in serum IL-12/IL-

23. IL-23 is a heterodimer that shares a common p40 subunit with IL-12 as well as some similar

signal-transduction components (53). However, IL-23 is different from IL-12 because it is

paramount in the production of Th17 cells and the production of pro-inflammatory cytokines IL-

22 and IL-17 (54). In human SLE patients, serum IL-23 levels and the number of Th17 cells are

significantly elevated compared to controlled subjects suggesting that the IL-23/IL-17 axis is

important in the inflammatory status in SLE (55). Additionally, glomerular IL-17 and IL-23

expression levels positively correlate with SLE activity and renal histopathology scores in lupus

nephritis patients (6). Treatment of dendritic cells isolated from the peripheral blood of humans

with the non-specific HDAC inhibitors trichostatin A or suberoylanilide hydroxamic acid

(SAHA) reduced IL-23 and IL-12 production after stimulation with LPS and INF-y (56).

Multiple reports have shown decreased numbers and function of CD4+ regulatory T

(Treg) cells in patients with active SLE (57-59). Also, restoring or increasing these Treg

populations has decreased the severity of disease in SLE patients(60) and in NZB/W mice (20).

In particular, we have previously reported that HDAC inhibitors (Trichostatin A and ITF2357)

increased regulatory T cell populations, which correlated with decreased disease in NZB/W mice

(18, 20). In the current study, we did not see an increase in the Treg population despite reduction

in disease severity in NZB/W mice. Instead, CD4+CD25+Foxp3+ Tregs were slightly decreased

in mice treated with the HDAC6 inhibitor. Similarly, treatment with the HDAC inhibitor

Trichostatin has also been reported to decrease the number of Tregs in C57BL6 mice (61).

67

Along with the decrease in splenic Tregs, we also saw a decrease in serum TGF-β levels. TGF-β

promotes expression of Foxp3 (62, 63) and naïve CD4+ T cell differentiation into Treg cells (62).

Based on our findings, reduction of disease in NZB/W F1 female mice after treatment with the

selective HDAC6 inhibitor ACY-1083 may be attributed to alterations in the number and

function of cell populations other than regulatory T cells.

Epigenetic regulation of the immune system by microRNAs (miRNA) has been reported

to play a critical role in the pathogenesis of SLE. Dai and colleagues have identified a common

lupus-associated microRNA expression pattern in 3 widely utilized lupus mouse models

(NZB/W, MRL/lpr, and B6/lpr), which includes increased expression of miR-182, miR-31, miR-

379, and miR-146a (24). Additionally, miR-21 and miR-148a are overexpressed in CD4+ T

cells of MRL/lpr mice and human patients (27, 64), and miR-31 is decreased in T cells from

human patients (26). We sought to investigate if alterations in miRNA expression could be

attributed to HDAC6. Interestingly, we did not reveal any differences in the relative expression

levels of these lupus-associated miRNAs from the splenocytes of NZB/W mice after treatment

with the HDAC6 inhibitor, ACY-1083, when compared to vehicle controls. This suggests that

these particular miRNAs were not involved in the mechanism responsible for decreasing lupus

disease after treatment with ACY-1083.

68

CONCLUSIONS

Lupus nephritis is a major contributor to the morbidity and mortality in SLE patients (3)

for which a specific, safe, and effective treatment regimen is not currently available.

Pharmacologic inhibition of HDACs offers these desired treatment characteristics, and has

consistently decreased renal disease in lupus-prone mice (17-20). Concerns for adverse side

effects associated with non-selective HDAC inhibition (21, 22) directed these studies towards

specifically targeting HDAC6. Similar to non-specific HDAC inhibition, these studies show that

selective inhibition of HDAC6 decreases SLE disease in lupus-prone mice by decreasing lupus

nephritis. Within the glomeruli, HDAC6i increased acetylation of alpha-tubulin and decreased

nuclear NF-κB. A connection between alpha-tubulin and NF-κB has not been clearly elucidated

and renders further investigation. In comparison to other studies in our laboratory utilizing

HDAC inhibitors (17-20), the current study shows no significant alterations in the amount of

Treg cells and a decrease in Th17 cells. These data suggest that ACY-1083 may decrease

disease by mechanisms targeting predominantly Th17 cells, instead of Tregs. Not only do Th17

cells play a role in the pathogenesis in SLE (51), they are also implicated in multiple

inflammatory and autoimmune diseases (65). Therefore, further studies are warranted to further

investigate the role ACY-1083 plays, as a specific HDAC6 inhibitor, in Th17 cell pathobiology.

69

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78

CHAPTER 4

Selective HDAC6 inhibition corrects aberrant B cell development in the bone marrow of

NZB/W F1 mice

Miranda D. Vieson1

Xin M. Luo1

Song Li2

Alexander M. Gojmerac3

Adrian Castaneda1



Veterinary Medicine, Blacksburg, VA

2. College of Agricultural and Life Sciences, Virginia Tech, Blacksburg, VA

3. Department of Biological Sciences, Virginia Tech, Blacksburg, VA

4. Edward Via College of Osteopathic Medicine, Blacksburg, VA

Published in Cellular and Molecular Medicine: Open Access. 2016;2(3):11

79

ABSTRACT

B cell development in the bone marrow is highly complex and includes vital regulatory

checkpoints to maintain central tolerance. Defects in central tolerance are implicated in systemic

lupus erythematosus (SLE) and aberrant B cell development has been reported in NZB/W mice.

We hypothesized that altered B cell development in the bone marrow of lupus-prone NZB/W

mice would be corrected after HDAC6 inhibition. B cell development was evaluated by flow

cytometric analysis of Hardy fractions from bone marrow cells of NZB/W mice treated with an

HDAC6 inhibitor or vehicle control. Additionally, deep sequence analysis of RNA from the

bone marrow was utilized to identify potential targets of HDAC6. As NZB/W mice aged, there

was an apparent shift in later stages of B cell development suggesting accelerated progression

through maturation and potential to bypass key regulatory checkpoints. After HDAC6

inhibition, the alteration was corrected and RNAseq analysis revealed differential expression of

849 genes in the bone marrow. We focused on 6 genes related to B cell development and

differentiation (ccr9, spib, pou2af1, nfil3, cebpb, and lgals1) and found that HDAC6 appears to

have the most impact on expression of spiB, an early regulator of B cell development, and

pou2af1, a regulator during later stages of B cell development. We conclude that HDAC6

inhibition helps correct aberrant B cell development and differentiation in the bone marrow of

lupus-prone NZB/W mice. These results also identified new potential targets for HDAC6

regulation within the bone marrow, particularly spiB and pou2af1.

80

INTRODUCTION

Mechanisms regulating the development of autoreactive B cells in the bone marrow are

referred to as central tolerance. The three main mechanisms of self-tolerance are deletion by

apoptosis, secondary recombination events of the B cell receptor (BCR), and anergy. Defects in

any of these mechanisms may result in the production of autoreactive B cells that enter

circulation and contribute to autoimmune diseases like (SLE) (1). Central tolerance as a factor

inducing autoimmunity was demonstrated after transplantation of bone marrow cells from New

Zealand Black (NZB) mice into irradiated DBA/2 mice incited manifestations of autoimmune

disease (2). In regard to human SLE, there is a high frequency of auto-reactivity in new

emigrant and mature naïve B cells in SLE patients, as well as BCR signaling abnormalities and

altered receptor editing by secondary recombination in SLE B cells (3).

B cells originate from pluripotent hematopoietic stem cells (HSCs) within the bone

marrow, a subset of which develop into common lymphoid progenitor cells (CLPs) that can

further develop into early B lineage cells (4). Beyond this point, development and maturation of

B cells progresses through phenotypic stages classified by three systems of nomenclature

(Philadelphia, Basel, and Hardy) (5, 6), which are summarized in FIGURE 1. Within the Hardy

classification scheme, B cells first progress through pro B cell stages A, B, C, then C’ followed

by progression through pre B cell stages D, E, then F. At least two checkpoints have been

postulated during B cell development in the bone marrow where central tolerance likely

regulates auto-reactive B cells. The first checkpoint occurs within the C and C’ Fractions. At

this point in development, it appears that proliferation of large pre B cells is over least twice the

number of cells that progress to the small pre B cell population in its steady state, suggesting that

over half the large B cells are prevented from differentiating further and subsequently deleted (7,

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8). A second checkpoint occurs between the D and E fractions. At this stage of development,

many early immature B cells express auto-reactive antibodies including antinuclear antibodies

(ANAs) and are subsequently removed before development into immature B cells (Fraction E)

(9).

Figure 1. B cell developmental stages in the bone marrow. The first phenotypic stage of B cell development is composed of cells that are B220+CD43+CD24-BP1-, which are included in Fraction A and referred to as Pre-Pro B cells (Philadelphia) or Pro-B cells (Basel). The Basel classification scheme is primarily based on the status of Ig gene rearrangements; cells in the germline configuration are referred to as Pro-B cells. Cells in Fractions B (B220+CD43+CD24+BP-1-) and C (B220+CD43+CD24lowBP-1+) are grouped together in the Philadelphia and Basel systems as Pro-B cells or Pre-BI cells, respectively. At this stage, Pro-B cells are defined as those expressing a unique intranuclear enzyme, TdT, that is active during VH-gene rearrangement. Pre-BI cells are those that have undergone partial Ig H-gene rearrangements of diversity (D) and joining (J) segments. Cells then progress into the C’ Fraction where cells are B220+CD43+CD24highBP-1+ and now referred to as early Pre-B cells in the Philadelphia system and Pre-BII cells in the Basel system. Pre-B cells are phenotypically defined by the expression Ig μ H chains within the cytoplasm (cμ) and then further subdivided into two populations based on their size and mitotic activity. The large and mitotically active (early Pre-

82

B) cells are included in the C’ fraction. These cells also have a fully rearranged H-chain locus at this point, and thus referred to as large Pre-BII cells. The small, non-dividing (late Pre-B) cells are included in the next developmental Hardy fraction, D, where cells are B220lowCD43-IgM-

IgD-. At this stage, cells now have fully rearranged H- and L-chain loci and may also be referred to as small Pre-BII cells (Basel). Lastly, the cells go through Fractions E then F. Cells in Fraction E, or new B cells (Philadelphia) begin expressing IgM on the cell surface (B220lowCD43-IgM+IgD-), which is a universally accepted criterion of immature B cells. Cells in Fraction F express both IgM and IgD (B220highCD43-IgM+IgD+) and can also be referred to as mature B cells. † = Total percent of cells in within Hardy Fractions from the bone marrow of BALB/c mice (3) * = First regulatory checkpoint ** = Second regulatory checkpoint

Our laboratory has recently documented alterations in proportions of B cells within

Hardy fractions in the bone marrow of diseased NZB/W mice. This suggests abnormalities in B

cell development contribute to disease progression. Specifically, we noted a significantly

decreased percentage of cells in the C, C’, D and E fractions, and a significant increase in cells

within the F fraction in 38 week-old (diseased) mice compared to pre-diseased (8 week-old)

mice. These results led us to believe that B cells may progress through developmental stages

more rapidly and bypass central tolerance checkpoints allowing increased numbers of auto-

reactive B cells to be released into circulation and contribute to lupus disease (10).

Histone deacetylase (HDAC) 6 is a class IIb HDAC enzyme that predominantly localizes

in the cytoplasm and modulates the acetylation status and subsequent function of multiple

proteins that play a role in SLE (11). Expression and activity of HDAC6 is increased in B cells

from the bone marrow of diseased lupus-prone mice (12). Furthermore, selective HDAC6

inhibition in NZB/W mice decreased lupus nephritis and increased proportions of bone marrow

B cells within the C, D, and E fractions with a corresponding decrease in the F fraction (10).

In our current studies, we sought to understand further the progression of B cells through

developmental fractions in the bone marrow of NZB/W mice as they age and develop disease.

83

Additionally, we investigated the role of HDAC6 in contributing to altering the proportions of B

cells in bone marrow developmental fractions.

84

MATERIALS AND METHODS

Mice

Female NZB/W F1 mice were purchased from Jackson Laboratories (Bar Harbor, ME,

USA). All mice were used in accordance with the Institutional Animal Care and Use Committee

(IACUC) after protocol approval by the IACUC of Virginia Tech University and housed in the

animal facility at the Virginia-Maryland College of Veterinary Medicine (Blacksburg, VA,

USA).

In vivo treatments with HDAC6 inhibitors

ACY-1083 and ACY-738, selective histone deacetylase (HDAC) 6 inhibitors, were

courtesy of a generous donation from Acetylon Pharmaceuticals (Boston, MA, USA) for use in

all studies. ACY-1083 was dissolved in 0.05% hydroxy-propyl-methyl cellulose (HPMC, Sigma,

St. Louis, MO, USA) and ACY-738 was dissolved in dimethyl sulfoxide (DMSO). All mice

were injected intraperitoneally (IP) 5 days/week with a 50uL volume of their respective

treatments. Beginning at 21-weeks-of-age, ten mice were included in each of 5 treatment

groups: 1) vehicle control (HPMC), 2) 0.3 mg/kg ACY-1083, 3) 1 mg/kg ACY-1083, 4) 3 mg/kg

ACY-1083 and 5) 2 mg/kg dexamethasone (TCI America, Portland, OR, USA). Treatments in

these groups continued until euthanasia during late stage clinical disease at 34 weeks-of-age. A

separate group of 10 mice were not treated until 35 weeks-of-age at which point they were

separated into 2 treatment groups and received either 40 mg/kg ACY-738 or vehicle control

(DMSO) treatments for 2 weeks followed by euthanasia. In a pilot study, ACY-738 was

formulated in rodent chow at 100 mg/kg and fed to 10 mice ad libitum. For control groups, 10

mice were fed standard rodent chow (negative control) and 10 mice were treated with

85

dexamethasone (2 mg/kg) IP (positive control). Treatment was initiated at 16 weeks-of-age and

continued until euthanasia at 34 weeks-of-age.

Flow cytometric analysis

After euthanasia, femurs and tibias were isolated, then bone marrows were flushed out

and collected with cold PBS containing 1% BSA to create a single cell suspension. The cells

were washed with cold DPBS followed by staining of cell surface antigens with directly

conjugated fluorescent-labeled murine monoclonal antibodies (eBiosciences, San Diego, CA,

USA). Cells were sorted by a FACS Aria 1 flow cytometer (BD Biosciences, San Jose, CA,

USA) and analyzed by FlowJo Software (Tree Star, Ashland, OR, USA). Cells from the bone

marrow were first gated by CD43-fluorescein isothiocyanate (FITC) and B220/CD45R-

allophycocyanin (APC) staining into Pro B cell (B220+CD43+) and Pre B cell (B220+CD43-)

fractions. The Pro B cell fractions were further gated by CD24-eFlour450 and BP1-

phycoerythrin (PE) staining into Fractions A (CD24-BP1-), B (CD24+BP1-), C (CD24loBP1+),

and C’ (CD24hiBP1+). The Pre B cell fraction was further gated by IgM-eFluor450 and B220-

APC staining into Fractions D (IgM-B220lo), E (IgM+B220lo), and F (IgM+B220hi).

RNAseq and analysis

Total RNA was isolated from primary bone marrow cells using the RNeasy Mini Kit

(Qiagen, Germantown, MD, USA) per manufacturer’s instructions. Total RNA was sent to

Beckman Coulter (Danvers, MA, USA) for 2x100bp paired-end Illumina RNA sequencing with

an average of 40 million reads per sample. Sequencing data (FASTQ files) was trimmed for

both adaptor sequences and quality using a combination of ea-utils and Btrim (13, 14).

86

Sequencing reads were then aligned to the genome (Ensembl.org 38.74) using Bowtie2/Tophat2

(15, 16) and counted via HTSeq (17). Following successful alignment and counting, statistical

analysis of mRNA differential expression was determined using the Benjamini-Hochberg

corrected Wald Test (FDR<0.1) in the R-package DESeq2 (R version 3.1.1, DESeq2 version

1.4.5)(18)

Real-time PCR

Reverse transcription was first performed on isolated total RNA using a high-capacity

cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA) per manufacturer’s

instructions. Real-time PCR was then performed utilizing the TaqMan Gene Expression Assay

system (Applied Biosystems, Foster City, CA, USA) per manufacturer’s instructions.

Expression levels were normalized to an endogenous control, GAPDH. Data are reported as

relative expression levels compared to the vehicle control group by calculating with formula 2-

ΔΔCt (Livak method).

Renal histopathology

At the time of euthanasia, both kidneys were removed. One kidney was fixed in 10%

neutral buffered formalin for 24 hours, then routinely processed, embedded in paraffin, sectioned

at 4-5μm, and stained with Periodic acid-Schiff (PAS). Kidney sections were scored (0-4) for

glomerular proliferation, inflammation, crescent formation, necrosis, and fibrosis by a board

certified veterinary pathologist in a blinded manner.

87

Statistics

Statistical analysis was performed using GraphPad Prism Version 6 software (La Jolla,

CA, USA). Differences among groups with more than two conditions were analyzed using one-

way ANOVA followed by further analysis using Tukey’s multiple comparison tests. Differences

between two means were assessed with unpaired two-tailed t-tests. A p-value < 0.05 was

considered statistically significant.

88

RESULTS AND DISCUSSION

Decreases in percentages of developing B cells in the bone marrow occur mostly within

Hardy fractions B and D with a concurrent increase in fraction F as NZB/W mice age

Hardy fractions organize B cells into sequential stages of development and differentiation

based on their phenotypic expression of select progenitor and B cell markers (5). Previous

studies have shown aberrant development and differentiation of B cells in the bone marrow of

NZB/W mice (10), although the point in disease progression where abnormalities in the bone

marrow arise is uncertain. Therefore, we sought to characterize further B cell development and

differentiation in the bone marrow by investigating the percentage of cells within Hardy

Fractions in female NZB/W mice at 23, 27, 32, and 35 weeks-of-age (Figure 2). The sorting and

gating scheme for Hardy Fractions (5) is characterized (Figure 2A) by first gating into Pro B cell

(B220+CD43+) and Pre B cell (B220+CD43-) fractions. The Pro B cell fractions were further

gated into Fractions A (CD24-BP1-), B (CD24+BP1-), C (CD24loBP1+), and C’ (CD24hiBP1+).

The Pre B cell fraction was further gated into Fractions D (IgM-B220lo), E (IgM+B220lo), and F

(IgM+B220hi). Data throughout is presented as total percentages of all nucleated cells in the bone

marrow.

There was a decrease in the percentage of Pro and Pre B cells within the bone marrow as

the mice aged, although this was not statistically significant (Figure 2B). The percentage of

cells within the B fraction progressively decreased in NZB/W mice as they aged and reached

statistical significance at 37 weeks-of-age (Figure 2C). A significant decrease in the A and C

fractions was also noted at 27 weeks-of-age. After this time point, the percentage of cells in the

A fraction remained decreased and the percentage of cells in the C fraction recovered. The

reason for the recovery and Fraction C is currently unknown. While not statistically significant,

89

there appeared to be a decrease in the percentage of cells within the D fraction at 37 weeks-of-

age (Figure 2D). Cells within the F fraction progressively increased as the mice aged and

reached statistical significance at 37 weeks-of-age.

Overall, we have shown that alterations in the development and differentiation of B cells

within the bone marrow progressively occur during mid- to late-stage disease in NZB/W mice.

These data together with our previous results suggest that B cells are transitioning through later

stages of development faster and allowing autoreactive B cells to bypass regulatory checkpoints

with subsequent release into the circulation (10).

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Figure 2. Progression of B cells through developmental fractions in the bone marrow of aging NZB/W mice. (A) Flow cytometry gating scheme for organizing bone marrow cells into Hardy fractions. Control mice were euthanized at progressing ages to evaluate B cell developmental through Hardy fractions in the bone marrow over time. (B) Percentages of B cells in both the Pro and Pre B cell fractions decreased as the mice aged, however this trend was not statistically significant. (C) Of the Pro B cells, there was a decrease in the percentage of cells within the B (CD24+BP1-) fraction as mice aged, which reached statistical significance at 37 weeks-of-age. (D) Cells within the D fraction were significantly decreased in 37-week-old mice compared to younger age groups. There was an increasing percentage of cells within the F (IgM+B220hi) fraction as the mice aged, however this trend was not statistically significant. (n = 3. *p < 0.05)

Initiation of HDAC6 inhibition during early disease increases the percentage of bone

marrow cells within Hardy fractions B, D, and E

In our previous studies, we have shown correction of aberrant development and

differentiation of B cells in the bone marrow of NZB/W mice after selective HDAC6 inhibition

(10). In our present studies, we investigated when initiation of selective HDAC6 inhibition

would be most effective in correcting B cell development in the bone marrow. We first

evaluated selective HDAC6 inhibition in early disease by treating NZB/W mice with increasing

doses of ACY-1083 (0.3 mg/kg, 1 mg/kg, and 3 mg/kg) at 21 weeks-of-age (Figure 3). Another

group of mice were treated with dexamethasone to serve as a positive treatment control group.

After 13 weeks of treatment, there was a significant dose-dependent decrease in lupus nephritis

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and spleen size was decreased in mice treated with ACY-1083 compared to controls

(unpublished data).

To evaluate B cell development and differentiation bone marrow cells were collected

then sorted into Hardy fractions as previously described (10). Treatment with ACY-1083 at all

investigated doses had no effect on the percentage of Pro B (B220+CD43+) cells in the bone

marrow (Figure 3A). A dose-dependent increase in the percentage of Pre B (B220+CD43-) cells

in the bone marrow was noted after ACY-1083 treatment, which reached statistical significance

in the 3 mg/kg treatment group (p > 0.001). Dexamethasone treatment (DEX, 2 mg/kg)

significantly reduced the percentages of Pro and Pre B cells in the bone marrow (p < 0.0001).

Treatment with 0.3 mg/kg ACY-1083 and 1 mg/kg ACY-1083 increased the amount of

Pro B cells in the B fraction (p > 0.05) compared to vehicle (HPMC) treated mice (Figure 3B).

In contrast, DEX treatment significantly reduced the amount of Pro B cells in the B fraction (p >

0.001). Percentages of cells in the A, C, and C’ fractions were not significantly altered by

treatment with all doses of ACY-1083 (0.3, 1, and 3 mg/kg) and DEX.

There was a dose-dependent increase in the percentage of cells within the E fraction,

which reaches statistical significance in mice treated with 3 mg/kg ACY-1083 compared to

vehicle controls (Figure 3C). There was also an increase in the percentages of cells in the D and

F fractions after ACY-1083 treatment, although not statistically significant. Treatment with DEX

significantly depleted cells in fractions D, E, and F (p < 0.0001).

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Figure 3. Development and differentiation of B cells in the bone marrow of NZB/W F1 female mice after HDAC6 inhibition initiated early in disease. Mice were treated with ACY-1083 (0.3 mg/kg, 1 mg/kg or 3 mg/kg), or 2 mg/kg dexamethasone (DEX) for 13 weeks, then progression of bone marrow cells through Hardy fractions was evaluated by flow cytometry. (A) There was a dose-dependent increase in the percentage of B220+CD43+ (Pre B) cells after treatment with ACY-1083. Treatment with DEX significantly decreased the percentages of B220+CD43- (Pro B) and B220+CD43+ (Pre B) cells in the bone marrow. (B) Treatment with 1 mg/kg or 3 mg/kg ACY-1083 increased percentages of Pro B cells in the B (CD24+BP-) fraction. Treatment with DEX significantly decreased percentages in the B fraction. (C). There was an increase in the percentages of Pre B cells in the D (IgM-B220Lo), E (IgM+B220Lo), and F (IgM+B220Hi) fractions after treatment with ACY-1083, which reached statistical significance in the group treated with 3 mg/kg for the E fraction. Treatment with DEX significantly decreased the percentages of cells in fractions D, E, and F. (n ≥ 8; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

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Oral administration of a selective HDAC6 inhibitor decreases spleen size and lupus

nephritis when initiated during early disease

A similar selective HDAC6 inhibitor, ACY-738 was formulated in rodent chow and

utilized in a pilot study where mice were fed ad libitum with either the ACY-738-formulated

chow or standard rodent chow for 18 weeks starting at 16 weeks-of-age (Figure 4). At the same

time, another group of NZB/W mice were treated with DEX to serve as a positive treatment

control group. The ACY-738 rodent chow was formulated to deliver 100 mg/kg/day to achieve

an estimated plasma concentration of 100nM and generously provided by Acetylon

Pharmaceuticals. The mean plasma concentration of ACY-738 at different time intervals was

57.3 ng/mL as determined by mass spectrometry (LC/MS) by Agilux (Worcester, MA, USA).

After euthanasia, spleens and kidneys were removed to evaluate disease progression. In

mice fed ACY-738, there was a significant decrease in spleen weight (p < 0.0001) and lupus

nephritis (p < 0.0001) when compared to mice fed standard chow, which was similar to the

decreased parameters in mice treated with DEX (Figures 4A and 4B).

B cell development and differentiation was also evaluated in these mice by flow

cytometric analysis of Hardy fractions in the bone marrow. Following treatment with ACY-738,

there was no change in the percentage of Pro B cells but there was an increase in the percentage

of Pre B cells in the bone marrow, although not statistically significant (Figure 4C).

Furthermore, there was a significant increase in the percentage of cells within fraction E (p <

0.01), and an increase in the percentage of cells in fraction B (not statistically significant). These

shifts in percentages of B cells within Hardy fractions were similar to those found after NZB/W

mice were treated with ACY-1083 (Figure 3).

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Figure 4. Disease progression and Hardy fraction analysis in NZB/W mice following oral administration of an HDAC6 inhibitor (ACY-738). Mice were treated with ACY-738-formulated chow, standard rodent chow, or dexamethasone (DEX, 2 mg/kg) IP injections for 18 weeks followed by euthanasia at 34 weeks-of-age. (A) Spleens from mice treated with ACY-738 and DEX weighed significantly less than those from controls. (B) Glomerular histopathology evaluated on PAS-stained sections revealed decreased lupus nephritis in mice treated with ACY-738 and DEX compared to controls. (C) Evaluation of B cell development and differentiation in the bone marrow was evaluated by flow cytometric analysis of Hardy fractions. ACY-738 treatment significantly increased the amount of cells in fraction E. DEX significantly depleted cells within fractions A, B, C’, E, and F. (n ≥ 9; **p < 0.01, ***p < 0.001, ****p < 0.0001)

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High-dose HDAC6i initiated during late-stage disease did not alter proportions of bone

marrow B cells in developmental Hardy fractions

To determine if HDAC6 inhibition would alter bone marrow development in late-stage

disease in NZB/W mice, we treated 35-week-old NZB/W mice with 40 mg/kg of the HDAC6

inhibitor ACY-738 (Figure 5). The mice were treated for two weeks after which they were

euthanized and bone marrow cells collected and sorted into Hardy fractions. There were no

significant differences between mice treated with the HDAC6 inhibitor and those treated with

vehicle (DMSO) alone in regards to percentages of bone marrow cells within any of the Hardy

fractions (Figure 5). Of particular interest, the percentage of cells within fraction D (Figure 5C)

was extremely low in mice treated with either the HDAC6 inhibitor (mean = 0.087%) or vehicle

(mean = 0.091%).

In addition to no alterations in proportions of bone marrow cells in Hardy fractions after

HDAC6 inhibition, there were also no changes in clinical disease parameters or lupus nephritis

in these mice (data not shown). Our results showed that treatment initiated during late-stage

disease (35 weeks-of-age) was not able to decrease lupus nephritis, which had progressed to

irreversible stages of damage.

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Figure 5. B cell development and differentiation in the bone marrow of diseased NZB/W treated with a high-dose 2-week course of HDAC6 inhibition. NZB/W mice in late-stage disease (35 weeks old) were treated with 40 mg/kg ACY-738 by IP injection for 2 weeks followed by euthanasia and investigation of Hardy fractions by flow cytometry. There were no differences in the distribution of B cells in Hardy fractions within the bone marrow between the control and treatment groups. (n=3)

Genes related to B cell development and differentiation are differentially expressed in the

bone marrow after HDAC6i in NZB/W mice

Substrates of HDAC6 related to B cell development and differentiation in the bone

marrow are unknown. To identify potential targets of HDAC6 regulation, total RNA was

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extracted from bone marrow cells of the NZB/W mice treated with a high-dose (40 mg/kg) of

ACY-738 for two weeks. The RNA was analyzed by RNAseq to unveil genes involved in B cell

development and differentiation that may be regulated by HDAC6. A total of 849 protein-

coding mRNA sequences were differentially expressed in mice treated with ACY-738 compared

to those treated with vehicle alone (Figure 6). Of these mRNA sequences, 601 were

Figure 6. Differential expression of genes in the bone marrow of diseased NZB/W mice treated with HDAC6 inhibition. After 2 weeks of high-dose HDAC6i treatment, bone marrow cells were isolated and total RNA was extracted and analyzed by RNAseq. There was differential expression of 849 protein coding mRNA sequences in HDAC6i treated mice compared to controls. Of these differentially expressed genes, 70% were down-regulated in mice treated with ACY-738 (HDAC6i). (n=3)

significantly down-regulated and 248 were significantly up-regulated (Appendix A). Of the

genes differentially regulated, six transcripts involved in the regulation of B cell development

and differentiation in the bone marrow were selected for further investigation: cebpb, ccr9, spib,

nfil3, lgals1, and pou2af1 (Table 1 and Figure 7).

CCAAT/enhancer-binding protein β (C/EBPβ) is a transcription factor that is crucial for

the differentiation of a variety of cell types and also plays a role in hematopoiesis (19). C/EBPβ

knockout mice exhibited a significant reduction in the number of B220+ cells in the bone marrow

(20, 21). Specifically, this reduction was attributed to lower percentages of cells in Hardy

fractions A, B, C, and C’ (21). Ultimately, the reduction in B220+CD43+ pro B cells in the bone

98

marrow of C/EBPβ-deficient mice was due to the impaired ability of bone marrow mesenchymal

stromal cells to support differentiation of hematopoietic stem cells into precursor B cells (21).

Table 1. Differentially expressed genes in the bone marrow related to B cell development and differentiation after HDAC6 inhibition based on RNAseq analysis.

Gene Mean Expression Fold-Change p-value Adjusted

p-value

Ccr9 259.69655 2.1538 0.00075 0.03178 Chemokine (C-C

motif) receptor 9 Spib

5150.14736 2.14819 0.00467 0.07917 Spi-B transcription factor (spi-1/PU.1) Pou2af1

4348.51165 1.71947 0.00062 0.02801 Pou domain, class 2, associating factor (BOB.1/0) Nfil3

1439.04321 -1.76353 0.00017 0.01308 Nuclear factor, interleukin 3, regulated Cebpb

4501.85776 -2.45591 0.00074 0.03143 CCAAT/enhancer binding protein (C/EBP), beta Lgals1

10559.35381 -2.85919 2.93E-15 7.64E-12 Lectin, galactose binding, soluble 1

After selective HDAC6 inhibition, there was a 2.46-fold decrease in cebpb expression according

to RNAseq analysis. According to the literature, this should lead to decreased percentages of

cells within Fractions A-C’. We saw decreased cell percentages in Fractions A, C, and C’ after

HDAC6 inhibition, however they were not statistically significant.

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Figure 7. Genes related to B cell development and differentiation in the bone marrow. RNA from the bone marrow of NZB/W mice treated with either ACY-738 (40 mg/kg) or vehicle control (DMSO) was analyzed by real-time PCR. Mice treated with HDAC6i (ACY-738) exhibited higher relative expression levels of ccr9, spib and pou2af which was statistically significant for spib and pou2af. In contrast, relative expression levels of cebpb, nfil3, and lgals1 were lower in HDAC6-treated mice compared to controls, although not statistically significant. (*p < 0.05, n=3)

CC chemokine receptor 9 (CCR9) is a receptor for CCL25 (thymus-expressed

chemokine, TECK). In mice, bone marrow cells phenotypically residing in Hardy Fraction A

(pre-pro B cells) migrate in response to TECK and can further generate pro B colony forming

units in the presence of IL-7 and Flt-3. Beyond the pre-pro stage, B lineage cells in the bone

marrow and periphery lack a migratory response to TECK. It is postulated that responsiveness to

TECK may direct pre-pro B cells into specialized niches that are supportive to their

developmental stage. Additionally, TECK may play a role in total B cell output by regulating

the total number of B lineage cells allowed to develop (22). In support of this theory, when

CCR9 is knocked out in mice, there is a 3-fold decrease in the number of pre-pro B cells in the

bone marrow compared to wild type. However, these CCR9-/- mice exhibit a normal

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complement of mature B cells, suggesting a homeostatic adjustment occurring within

downstream developmental stages (23). When treated with the selective HDAC6 inhibitor

ACY-738, there is a 2.15-fold increase in ccr9. Based on the literature, we might expect an

increase in the percentage of cells in early B cell fractions. While amounts of cells in Fraction A

were relatively unchanged, we saw an increase in the percentage of cells within Fraction B after

selective HDAC6 inhibition. Additionally, as the mice age and develop disease, the percentage

of cells within Fractions A and B decrease. Whether or not ccr9 is involved in altering B cell

development and differentiation in the bone marrow and subsequently contributes to SLE disease

is unknown.

Of the 3 genes involved in early B cell differentiation and development in the bone

marrow (cebpb, ccr9, and spib), the most differentially regulated gene after HDAC6 inhibition

was spib. Spi-B is an Ets-family transcription factor that shares a high degree of identity with

PU.1 including an indistinguishable DNA binding specificity (24, 25). Spi-B can substitute for

PU.1 and promote differentiation of hematopoietic progenitor cells into pro B cells, however it is

not required (26). Furthermore, mice deficient in Spi-B do not exhibit any abnormalities in

lymphoid development (27). After HDAC6 inhibition in NZB/W mice, spib was upregulated by

2.15-fold according to RNAseq analysis and 3.62-fold by qPCR. The consequences of increased

Spi-B in the bone marrow and whether or not there are altered levels of Spi-B as SLE progresses

are both unknown.

Nuclear factor, interleukin-3 regulated (NFIL3) protein, also known as E4BP4, is a basic

leucine zipper transcription factor that has roles in diverse hematopoietic lineages including NK

cells, T cells, and dendritic cells (28). In regards to B cells, E4BP4 has been implicated in

controlling IgE production and class switching in response to IL-4 (29, 30). More interestingly,

101

NFIL3/E4BP4 is also involved in a group of IL-3-dependent signaling pathways that regulate the

survival of murine pro B lymphocytes (31). More specifically, IL-3-dependent suppression of

apoptosis may be accomplished through induction of NFIL3 (31). If NFIL3 is elevated in SLE,

it may be contributing to decreased apoptosis of autoreactive B cells at regulatory checkpoints in

the bone marrow. If this were the case, it would be ideal to decrease NFIL3 to allow apoptosis to

resume. After HDAC6 inhibition, RNAseq analysis reveals a 2.86-fold decrease in nfil3

expression.

Galectin-1 (GAL1), encoded by the lgals1 gene, is expressed in the bone marrow by

osteoblasts and reticular cells and creates a stromal cell niche for pre-BII cells (Fraction D) (32).

Within this niche, interactions between GAL1 and pre B cells create an immune developmental

synapse promoting pre-BCR clustering, signaling, and activation (33, 34). Inhibition of pre-

BCR/GAL1/integrin interactions in normal pre-BII cells impairs in vitro B cell differentiation,

and pre-BII cells from GAL1-deficient mice also exhibit decreased differentiation and

proliferation (35). Following HDAC6 inhibition, RNAseq analysis showed decreased lgals1 in

the bone marrow, which should lead to decreased differentiation and proliferation. Instead, we

saw an increased percentage of cells in Fraction D. As NZB/W mice age there is a decrease in

the percentage of cells in Fraction D raising the possibility that decreased lgals1 may play a role

in later stages of SLE disease. However, GAL1-deficient mice do not exhibit a B cell phenotype

(35).

Pou domain, class 2, associating factor (Pou2af1), also known as BOB.1/OBF.1, is a

transcriptional coactivator that works in concert with Oct1 and Oct2 in developing B cells. In

BOB.1/OBF.1-deficient mice there is a reduction in transitional immature B cells (Fractions E

and F) and an increase in B cell apoptosis in the bone marrow suggesting an important role for

102

BOB.1/OBF.1 in early B cell differentiation and survival (36). After HDAC6 inhibition,

RNAseq analysis showed a 1.72-fold increase in pou2af1 in the bone marrow and the percentage

of cells in Fraction E increased. Furthermore, qPCR showed a 1.55-fold increase in pou2af1 in

the bone marrow after HDAC6 inhibition, implying HDAC6 plays a role in regulating pou2af1.

Total RNA from the bone marrow was also reverse transcribed into cDNA, then analyzed

by real-time PCR to quantitate relative expression levels of cebpb, ccr9, spib, nfil3, lgals1, and

pou2af1. The relative expression levels of pou2af and spiB were significantly higher in mice

treated with ACY-738 compared to vehicle controls (p < 0.05) (Figure 7). There was also

higher ccr9 expression in HDAC6i-treated mice, however it was not statistically significant. In

contrast, the expression levels of cebpb, lgals1, and nfil3 were lower in HDAC6i-treated mice

compared to controls, albeit not statistically significant.

103

SUMMARY AND CONCLUSIONS

Autoreactive B cells are a major contributor to disease in SLE. The first regulatory control

mechanism where autoreactive B cells should be removed or rendered anergic is within the bone

marrow (1). Previous studies have shown aberrant development and differentiation of B cells in

the bone marrow of NZB/W mice and correction after selective HDAC6 inhibition (10). We

have shown that alterations in the development and differentiation of B cells within the bone

marrow progressively occur during mid- to late-stage disease in NZB/W mice. More

specifically, B cells within the bone marrow appear to transition more quickly into the later

stages of development (Fraction F) and bypassing important regulatory checkpoints to maintain

central tolerance. Treatment with a selective HDAC6 inhibitor is able to correct these changes

when initiated during the early stages of disease by apparently slowing progression through

developmental stages. Therefore, the cells are able progress through regulatory checkpoints more

efficiently. After deep sequencing analysis, we investigated the differential expression of 6

genes related to B cell development and differentiation and whether they could be regulated by

HDAC6. HDAC6 appears to have the most impact on expression of spiB, an early regulator of B

cell development, and pou2af1, a regulator during later stages of B cell development.

Differential expression of these 6 genes and their contribution to the progression of SLE disease

is uncertain at this point and warrants further investigation.

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CHAPTER 5. FUTURE DIRECTIONS

Miranda D. Vieson

Overall, the current studies show that HDAC6 inhibition in lupus-prone NZB/W F1

female mice decreases lupus nephritis, which is a critical component of SLE that significantly

contributes to morbidity and mortality in humans (1). These studies also showed that HDAC6

inhibition corrected aberrant B cell development within the bone marrow and decreased the

percentages important T cell subtypes (Th17 and Treg) within the spleen. These findings

support the notion that while inhibition of HDAC6 is selective, that it is broad-spectrum enough

to have effects on multiple components of immunity. The broad-spectrum effect is important in

management and treatment of SLE because multiple components of the immune response are

abnormal (2, 3) and may act as targets for pharmacologic therapy. This aspect of drug

development in the realm of SLE is important because current treatment modalities, both broad-

spectrum and selective, either result in undesirable side effects, or are only partially or

temporarily effective. While HDAC6 inhibition showed efficacy in reducing disease in lupus-

prone mice, it is also imperative to understand the mechanism(s) involved, not only to be sure

side effects are avoided, but also to further understand the pathogenesis of SLE. Therefore,

further investigations are warranted, particularly within the kidney, development of B cells, and

T cell physiology

In regards to lupus nephritis, selective HDAC6 inhibition decreased lupus nephritis in a

dose-dependent manner in NZB/W mice. In the mice treated with the HDAC6i, there was

decreased NF-κB within glomerular cells. More specifically, within mesangial cells treated with

the HDAC6i in vitro, nuclear NF-κB levels decreased. Smad7 is an inhibitory molecule involved

in the TGF-β signaling cascade (4) and contributes to suppression of renal inflammation by

110

inducing IκB and therefore inhibiting NF-κB-driven inflammatory responses (5). HDACs have

been found to interact with and deacetylate Smad7 resulting in its decreased stability (6). In

lupus-prone MRL/lpr mice, HDAC6 expression and activity are increased (7), which would

theoretically result in decreased acetylation and stability of Smad7. We hypothesize that

inhibition of HDAC6 increases the acetylation of Smad7, which will restore its stability and

decrease lupus nephritis by decreasing NF-κB (Figure 1). This theory may be investigated by in

vitro studies with cultured mesangial cells stimulated with IFN-γ/LPS (8, 9) or treated with TGF-

β with and without HDAC6 inhibition followed by immunoprecipitation of Smad7 and

immunoblotting for acetylated-lysine. HDAC6 activity could be evaluated by similar

immunoprecipitation of HDAC6 followed by an HDAC activity assay, and HDAC6 and Smad7

expression may be evaluated by PCR analysis. In vivo, mesangial cells may be isolated from

HDAC6i-treated and non-treated MRL/lpr mice followed by similar protein isolation techniques

and immunoblotting, HDAC activity assay, and PCR. In one study, Smad7 synthesis increased

in podocytes treated in vitro with TGF-β, but not in mesangial cells (10). Therefore, it may be

warranted to perform similar in vitro studies in a podocyte cell line as well. While investigating

this theory in a podocyte cell line, it may also be interesting to investigate a potential link

between alpha-tubulin acetylation, NF-κB, and nephrin. Nephrin is a key protein involved in the

slit diaphragm (11). Nephrin is downregulated during podocyte injury, which results in NF-kB

activation that subsequently promotes glomerular injury (11). It is possible that nephrin, in

conjunction with tubulin acetylation after HDAC6i acts to inhibit NF-κB. In addition to

investigating Smad7 in HDAC6i-treated podocytes, it would also be interesting to investigate

nephrin expression and protein levels by immunoblotting, PCR, and immunofluorescence

studies.

111

Figure 1. Smad7 and renal inflammation. (A) In a study by Wang et al. 2005, a transgenic mouse that overexpressed latent TGF-β had decreased renal inflammation post renal obstruction attributed to increased Smad7 and IκBα resulting in suppression of NF-κB in the kidney. A similar increase in IκBα and decreased NF-κB activity were also appreciated in a renal tubular cell line that overexpressed Smad7 and was stimulated by TNFα and IL-1β. (B). We hypothesize that administration of and HDAC6i will increase acetylation of Smad7, increasing its stability and resulting in increased IκBα and decreased NF-κB activity. Another component of the immune response that was evaluated in the NZB/W mice treated

with HDAC6i was the development of B cells within the bone marrow. This was evaluated by

tracing the proportion of B cells through Hardy fractions by flow cytometric analysis. The

results suggest that there may be accelerated progression of developing B cells through later

stages of development creating the potential of bypassing key regulatory checkpoints involved in

tolerance. The aberration was corrected after HDAC6 inhibition and deep sequencing analysis of

RNA revealed multiple potential genes that are likely to be involved (cebpb, ccr9, spib, nfil3,

lgals1, and pou2af1). Given the preliminary nature of this study, there are lots of questions

regarding the impact of the differentially expressed genes of interest on B cell development both

in health and in disease states like SLE. Some light may be shed utilizing in vitro culture

systems to simulate B cell development in the bone marrow and use siRNA to silence or dampen

112

the genes of interest to evaluate how they affect B cell development. Another part of this study

that would be interesting, and important to determine, is how these genes impact B cell

development as the lupus-prone mice age. In our study, significant changes in Hardy fraction

proportions developed as the mice aged. Our laboratory reported similar changes in Hardy

fractions when comparing NZB/W mice before disease onset and mice in late-stage disease (12).

Determining the point at which B cell development aberration begins and concurrently

evaluating the differentially expressed genes of interest may help to determine which gene(s)

is/are responsible for the onset of altered B cell development in SLE. In addition to investigating

differential expression of genes involved in B cell development, the RNAseq data set from this

study may also be utilized to see differential expression of genes related to other immune cells

and their functions as they related to SLE, like dendritic cells.

In addition to determining how these genes of interest impact SLE disease pathogenesis,

the underlying mechanism of how these genes are altered by HDAC6i is also important.

HDAC6 functions within the cytoplasm of cells (13), therefore the enzyme should not have a

direct effect on gene transcription within the nucleus. Instead, HDAC6 likely contributes in an

indirect manner by altering proteins involved in regulatory signaling cascades upstream of gene

transcription. Expression of both spib and pou2af1 were significantly upregulated in bone

marrow cells after HDAC6i. NF-kB is a transcription factor that binds to the BOB.1 promoter

and activates both basal and inducible pou2af1/BOB.1 expression (14). The study by Kilzheimer

et al., utilized various inducers of canonical and non-canonical NF-κB signaling in combination

with various silencing mutations in the BOB.1 promoter region to determine if NF-κB played a

role in controlling the expression of BOB.1 (14). It would be interesting to utilize these studies

to determine if NF-κB can similarly induce expression of spib in developing B cells, which

113

would serve as a common mechanism that may be regulated by HDAC6. Further experiments to

evaluate how the components of the canonical and non-canonical NF-κB signaling cascade are

affected by HDAC6 would also be warranted. Another potential transcription factor that may be

investigated Pax5, which has been found to directly regulate genes required during B cell

differentiation, including the gene encoding Spi-B (15).

The last component involved in SLE pathogenesis that was briefly investigated in these

studies were T cells, particularly Th17 and Treg cells. Treg cells function to maintain immune

tolerance to self-antigens and suppress excessive and deleterious immune responses (16). Treg

cell function and frequency are reduced in human SLE patients (17-20) and in lupus-prone mice

during disease (21-23). In contrast to previous studies in our laboratory, the current studies have

shown decreased frequencies of Tregs in the spleen after HDAC6i. More important than the

number and proportion of Tregs, is their function. The suppressive activity of Foxp3+ Treg cells

has improved after HDAC6 inhibition in models of inflammation and autoimmunity (24).

Therefore Treg suppression assays (25) on cells isolated from HDAC6i-treated and diseased

mice would be imperative to determine how HDAC6i affects Treg function. Th17 cells play an

opposite role in SLE and are implicated in contributing to lupus pathogenesis. In these studies,

we found that HDAC6i decreased Th17 cells in the spleen and decreased IL-12/IL-23 levels in

the serum. Another study that would provide important information is a Th17 polarization study

(26), where naïve T cells from healthy and diseased lupus mice are isolated, then stimulated in

vitro to differentiate into Th17 cells, to see if HDAC6i decreases Th17 cells by decreasing their

polarization. As an extension of the B cell development project, spleens from HDAC6i-treated

and non-treated mice were similarly processed and analyzed by RNAseq analysis. Results from

114

this study are currently pending, and may shed light on how genes involved in Treg and Th17

function may be altered by HDAC6i in diseased lupus mice.

There are many components of the pathogenesis of lupus that are outside the realm of the

investigations in these studies. Collaboration with colleagues that focus on these other areas

would be interesting to further support the notion that HDAC6i addresses multiple aspects of the

dysregulated immune response in SLE while avoiding undesirable side effects.

115

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Immunol 180: 1719-28

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Gene IDGene Name

Mean Expression

Fold-change

log2Fold-Change

log2Fold- Change

Standard Error

stat p -valueAdjusted p -

valueGene Function

ENSMUSG00000069792 Gm11428 1765.1250 3.4192 -1.7737 0.1670 -10.6228 2.334E-26 2.434E-22

WAP four-disulfide core domain 17 [Source:MGI Symbol;Acc:MGI:3649773]

ENSMUSG00000001020 S100a4 6636.0288 2.4493 -1.2924 0.1529 -8.4515 2.875E-17 1.499E-13

S100 calcium binding protein A4 [Source:MGI Symbol;Acc:MGI:1330282]

ENSMUSG00000022769 Sdf2l1 3275.1328 2.2864 -1.1931 0.1433 -8.3272 8.278E-17 2.878E-13

stromal cell-derived factor 2-like 1 [Source:MGI Symbol;Acc:MGI:2149842]

ENSMUSG00000068220 Lgals1 10559.3538 2.8592 -1.5156 0.1920 -7.8938 2.931E-15 7.642E-12

lectin, galactose binding, soluble 1 [Source:MGI Symbol;Acc:MGI:96777]

ENSMUSG00000059325 Hopx 1100.0807 2.6143 -1.3864 0.1889 -7.3402 2.133E-13 4.45E-10

HOP homeobox [Source:MGI Symbol;Acc:MGI:1916782]

ENSMUSG00000018500 Adora2b 266.4642 3.1964 -1.6765 0.2314 -7.2452 4.317E-13 7.504E-10

adenosine A2b receptor [Source:MGI Symbol;Acc:MGI:99403]

ENSMUSG00000005054 Cstb 1617.7612 2.3176 -1.2127 0.1788 -6.7816 1.189E-11 1.771E-08

cystatin B [Source:MGI Symbol;Acc:MGI:109514]

ENSMUSG00000030793 Pycard 2338.9188 2.5640 -1.3584 0.2100 -6.4691 9.857E-11 1.285E-07

PYD and CARD domain containing [Source:MGI Symbol;Acc:MGI:1931465]

ENSMUSG00000003541 Ier3 1216.9266 2.0314 -1.0225 0.1692 -6.0412 1.53E-09 1.773E-06

immediate early response 3 [Source:MGI Symbol;Acc:MGI:104814]

ENSMUSG00000016256 Ctsz 8966.9255 1.8627 -0.8974 0.1516 -5.9213 3.194E-09 3.331E-06

cathepsin Z [Source:MGI Symbol;Acc:MGI:1891190]

Genes differentially expressed in the bone marrow of NZB/W mice after HDAC6 inhibition based on RNAseq analysis

APPENDIX A

119

ENSMUSG00000041736 Tspo 7297.1320 2.5759 -1.3651 0.2319 -5.8870 3.932E-09 3.728E-06translocator protein [Source:MGI Symbol;Acc:MGI:88222]

ENSMUSG00000050335 Lgals3 16469.8178 2.0424 -1.0303 0.1802 -5.7185 1.074E-08 9.338E-06

lectin, galactose binding, soluble 3 [Source:MGI Symbol;Acc:MGI:96778]

ENSMUSG00000020571 Pdia6 9472.7698 1.9704 -0.9785 0.1740 -5.6227 1.88E-08 1.307E-05

protein disulfide isomerase associated 6 [Source:MGI Symbol;Acc:MGI:1919103]

ENSMUSG00000032966 Fkbp1a 2133.9305 1.9385 -0.9549 0.1698 -5.6227 1.88E-08 1.307E-05

FK506 binding protein 1a [Source:MGI Symbol;Acc:MGI:95541]

ENSMUSG00000041959 S100a10 7128.7097 2.1734 -1.1200 0.1986 -5.6385 1.716E-08 1.307E-05

S100 calcium binding protein A10 (calpactin) [Source:MGI Symbol;Acc:MGI:1339468]

ENSMUSG00000028041 Adam15 2083.5392 2.1227 -1.0859 0.1938 -5.6018 2.122E-08 1.383E-05

a disintegrin and metallopeptidase domain 15 (metargidin) [Source:MGI Symbol;Acc:MGI:1333882]

ENSMUSG00000034659 Tmem109 2152.4338 1.7828 -0.8342 0.1497 -5.5733 2.5E-08 1.534E-05

transmembrane protein 109 [Source:MGI Symbol;Acc:MGI:1915789]

ENSMUSG00000023367 Tmem176a 1092.4774 2.3834 -1.2530 0.2273 -5.5137 3.514E-08 2.036E-05

transmembrane protein 176A [Source:MGI Symbol;Acc:MGI:1913308]

ENSMUSG00000026385 Dbi 3402.4910 2.6772 -1.4207 0.2599 -5.4670 4.578E-08 2.513E-05

diazepam binding inhibitor [Source:MGI Symbol;Acc:MGI:94865]

ENSMUSG00000062070 Pgk1 12460.8214 1.9918 -0.9941 0.1824 -5.4496 5.048E-08 2.583E-05

phosphoglycerate kinase 1 [Source:MGI Symbol;Acc:MGI:97555]

ENSMUSG00000072620 Slfn2 7904.8482 2.1174 -1.0823 0.1988 -5.4443 5.202E-08 2.583E-05

schlafen 2 [Source:MGI Symbol;Acc:MGI:1313258]

120

ENSMUSG00000029322 Plac8 25370.6166 1.9600 -0.9708 0.1793 -5.4154 6.116E-08 2.687E-05

placenta-specific 8 [Source:MGI Symbol;Acc:MGI:2445289]

ENSMUSG00000032231 Anxa2 13594.1322 1.8876 -0.9165 0.1693 -5.4148 6.137E-08 2.687E-05

annexin A2 [Source:MGI Symbol;Acc:MGI:88246]

ENSMUSG00000049881 2810025M1 584.4795 2.1102 -1.0774 0.1990 -5.4134 6.183E-08 2.687E-05

RIKEN cDNA 2810025M15 gene [Source:MGI Symbol;Acc:MGI:1917203]

ENSMUSG00000053175 Bcl3 831.1488 1.8336 -0.8746 0.1619 -5.4025 6.571E-08 2.741E-05

B cell leukemia/lymphoma 3 [Source:MGI Symbol;Acc:MGI:88140]

ENSMUSG00000028691 Prdx1 10328.2922 1.9094 -0.9332 0.1768 -5.2767 1.315E-07 5.276E-05

peroxiredoxin 1 [Source:MGI Symbol;Acc:MGI:99523]

ENSMUSG00000038650 Rnh1 4678.6342 1.8199 -0.8638 0.1679 -5.1456 2.666E-07 0.000103

ribonuclease/angiogenin inhibitor 1 [Source:MGI Symbol;Acc:MGI:1195456]

ENSMUSG00000073987 Ggh 730.0293 1.8912 -0.9193 0.1796 -5.1175 3.096E-07 0.0001153

gamma-glutamyl hydrolase [Source:MGI Symbol;Acc:MGI:1329035]

ENSMUSG00000030142 Clec4e 2084.4462 2.0615 -1.0437 0.2042 -5.1103 3.216E-07 0.0001157

C-type lectin domain family 4, member e [Source:MGI Symbol;Acc:MGI:1861232]

ENSMUSG00000043252 Tmem64 988.3239 0.6098 0.7136 0.1401 5.0918 3.547E-07 0.0001233


ENSMUSG00000048970 C1galt1c1 2007.5335 2.1532 -1.1065 0.2188 -5.0577 4.244E-07 0.0001428

C1GALT1-specific chaperone 1 [Source:MGI Symbol;Acc:MGI:1913493]

ENSMUSG00000006378 Gcat 239.8126 2.6342 -1.3974 0.2822 -4.9515 7.365E-07 0.00024

glycine C-acetyltransferase (2-amino-3-ketobutyrate-coenzyme A ligase) [Source:MGI Symbol;Acc:MGI:1349389]

121

ENSMUSG00000025492 Ifitm3 7602.5311 2.0788 -1.0558 0.2140 -4.9327 8.109E-07 0.0002563

interferon induced transmembrane protein 3 [Source:MGI Symbol;Acc:MGI:1913391]

ENSMUSG00000063694 Cycs 5477.9672 1.8537 -0.8904 0.1817 -4.9016 9.507E-07 0.0002916cytochrome c, somatic [Source:MGI Symbol;Acc:MGI:88578]

ENSMUSG00000021996 Esd 4245.6085 1.6989 -0.7646 0.1572 -4.8642 1.149E-06 0.0003425

esterase D/formylglutathione hydrolase [Source:MGI Symbol;Acc:MGI:95421]

ENSMUSG00000033386 Frrs1 554.3773 1.8848 -0.9144 0.1919 -4.7645 1.893E-06 0.0005335

ferric-chelate reductase 1 [Source:MGI Symbol;Acc:MGI:108076]

ENSMUSG00000060962 Dmkn 102.2364 3.3389 -1.7394 0.3650 -4.7648 1.89E-06 0.0005335

dermokine [Source:MGI Symbol;Acc:MGI:1920962]

ENSMUSG00000018821 Avpi1 115.5000 2.5601 -1.3562 0.2871 -4.7240 2.312E-06 0.0006183

arginine vasopressin-induced 1 [Source:MGI Symbol;Acc:MGI:1916784]

ENSMUSG00000025512 Chid1 1124.3659 1.7248 -0.7864 0.1664 -4.7252 2.299E-06 0.0006183

chitinase domain containing 1 [Source:MGI Symbol;Acc:MGI:1915288]

ENSMUSG00000032381 Fam96a 5446.9583 1.7499 -0.8073 0.1721 -4.6903 2.728E-06 0.0007112

family with sequence similarity 96, member A [Source:MGI Symbol;Acc:MGI:1915500]

ENSMUSG00000022218 Tgm1 222.5313 3.0640 -1.6154 0.3468 -4.6579 3.194E-06 0.0008124

transglutaminase 1, K polypeptide [Source:MGI Symbol;Acc:MGI:98730]

ENSMUSG00000022584 Ly6c2 14141.5951 1.8526 -0.8896 0.1928 -4.6146 3.938E-06 0.0009778

lymphocyte antigen 6 complex, locus C2 [Source:MGI Symbol;Acc:MGI:3712069]

122

ENSMUSG00000023992 Trem2 308.4668 3.3442 -1.7417 0.3812 -4.5691 4.899E-06 0.0011883

triggering receptor expressed on myeloid cells 2 [Source:MGI Symbol;Acc:MGI:1913150]

ENSMUSG00000001227 Sema6b 394.5468 3.1546 -1.6574 0.3649 -4.5418 5.578E-06 0.001322

sema domain, transmembrane domain (TM), and cytoplasmic domain, (semaphorin) 6B [Source:MGI Symbol;Acc:MGI:1202889]

ENSMUSG00000052310 Slc39a1 1911.7880 1.6877 -0.7551 0.1666 -4.5330 5.816E-06 0.0013478

solute carrier family 39 (zinc transporter), member 1 [Source:MGI Symbol;Acc:MGI:1353474]

ENSMUSG00000030717 Nupr1 314.3949 2.4795 -1.3100 0.2894 -4.5264 5.998E-06 0.0013599

nuclear protein transcription regulator 1 [Source:MGI Symbol;Acc:MGI:1891834]

ENSMUSG00000021477 Ctsl 2097.7032 1.8470 -0.8852 0.1959 -4.5182 6.238E-06 0.0013763

cathepsin L [Source:MGI Symbol;Acc:MGI:88564]

ENSMUSG00000038034 Igsf8 1691.0036 1.6936 -0.7601 0.1685 -4.5109 6.457E-06 0.0013763

immunoglobulin superfamily, member 8 [Source:MGI Symbol;Acc:MGI:2154090]

ENSMUSG00000060703 Cd302 782.5768 2.4904 -1.3164 0.2918 -4.5105 6.466E-06 0.0013763

CD302 antigen [Source:MGI Symbol;Acc:MGI:1913455]

ENSMUSG00000044060 A830010M2 111.9440 0.4521 1.1453 0.2549 4.4937 6.999E-06 0.0014599

RIKEN cDNA A830010M20 gene [Source:MGI Symbol;Acc:MGI:2445097]

ENSMUSG00000006574 Slc4a1 1869.9618 0.5804 0.7850 0.1751 4.4835 7.343E-06 0.0015015

solute carrier family 4 (anion exchanger), member 1 [Source:MGI Symbol;Acc:MGI:109393]

123

ENSMUSG00000024644 Cndp2 3096.4055 1.5695 -0.6503 0.1454 -4.4736 7.692E-06 0.0015427

CNDP dipeptidase 2 (metallopeptidase M20 family) [Source:MGI Symbol;Acc:MGI:1913304]

ENSMUSG00000021951 N6amt2 559.4373 1.8461 -0.8845 0.1982 -4.4615 8.14E-06 0.0016018

N-6 adenine-specific DNA methyltransferase 2 (putative) [Source:MGI Symbol;Acc:MGI:1915293]

ENSMUSG00000019579 D17Wsu104 2337.1022 1.9637 -0.9735 0.2186 -4.4526 8.483E-06 0.0016086

myeloid derived growth factor [Source:MGI Symbol;Acc:MGI:2156020]

ENSMUSG00000030339 Ltbr 1931.1044 1.7483 -0.8059 0.1809 -4.4557 8.363E-06 0.0016086

lymphotoxin B receptor [Source:MGI Symbol;Acc:MGI:104875]

ENSMUSG00000030539 Sema4b 1444.2884 0.5542 0.8514 0.1917 4.4414 8.937E-06 0.0016644

sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 4B [Source:MGI Symbol;Acc:MGI:107559]

ENSMUSG00000028214 Gem 198.3792 0.4053 1.3030 0.2938 4.4342 9.243E-06 0.0016912

GTP binding protein (gene overexpressed in skeletal muscle) [Source:MGI Symbol;Acc:MGI:99844]

ENSMUSG00000029438 Bcl7a 2263.5184 0.4466 1.1631 0.2636 4.4125 1.022E-05 0.0018376B cell CLL/lymphoma 7A [Source:MGI Symbol;Acc:MGI:1924295]

ENSMUSG00000049299 Trappc1 1569.7778 1.7514 -0.8085 0.1838 -4.3979 1.093E-05 0.0019317

trafficking protein particle complex 1 [Source:MGI Symbol;Acc:MGI:1098727]

ENSMUSG00000019122 Ccl9 8979.4795 2.0599 -1.0426 0.2377 -4.3866 1.151E-05 0.0020009

chemokine (C-C motif) ligand 9 [Source:MGI Symbol;Acc:MGI:104533]

124

ENSMUSG00000031556 Tm2d2 1229.5760 1.6495 -0.7220 0.1648 -4.3801 1.186E-05 0.0020009

TM2 domain containing 2 [Source:MGI Symbol;Acc:MGI:1916992]

ENSMUSG00000079523 Tmsb10 14347.5982 1.8552 -0.8916 0.2036 -4.3795 1.19E-05 0.0020009

thymosin, beta 10 [Source:MGI Symbol;Acc:MGI:109146]

ENSMUSG00000000562 Adora3 141.5248 2.3946 -1.2598 0.2896 -4.3499 1.362E-05 0.0021526

adenosine A3 receptor [Source:MGI Symbol;Acc:MGI:104847]

ENSMUSG00000023272 Creld2 3041.8907 1.7462 -0.8042 0.1847 -4.3538 1.338E-05 0.0021526

cysteine-rich with EGF-like domains 2 [Source:MGI Symbol;Acc:MGI:1923987]

ENSMUSG00000049577 Zfpm1 2220.8204 0.4795 1.0604 0.2437 4.3504 1.359E-05 0.0021526

zinc finger protein, multitype 1 [Source:MGI Symbol;Acc:MGI:1095400]

ENSMUSG00000060512 0610040J01 126.0015 3.0901 -1.6276 0.3739 -4.3533 1.341E-05 0.0021526

RIKEN cDNA 0610040J01 gene [Source:MGI Symbol;Acc:MGI:1923511]

ENSMUSG00000031444 F10 3878.6694 2.4544 -1.2954 0.2993 -4.3274 1.509E-05 0.002349

coagulation factor X [Source:MGI Symbol;Acc:MGI:103107]

ENSMUSG00000048807 Slc35e4 246.9252 1.9536 -0.9662 0.2241 -4.3117 1.62E-05 0.0024487

solute carrier family 35, member E4 [Source:MGI Symbol;Acc:MGI:2144150]

ENSMUSG00000050106 Tmc8 597.8040 0.5949 0.7492 0.1736 4.3147 1.598E-05 0.0024487

transmembrane channel-like gene family 8 [Source:MGI Symbol;Acc:MGI:2669037]

ENSMUSG00000041842 Fhdc1 216.4258 0.4750 1.0739 0.2496 4.3024 1.689E-05 0.002517

FH2 domain containing 1 [Source:MGI Symbol;Acc:MGI:2684972]

ENSMUSG00000031168 Ebp 1633.0831 1.7216 -0.7838 0.1826 -4.2929 1.763E-05 0.0025901

phenylalkylamine Ca2+ antagonist (emopamil) binding protein [Source:MGI Symbol;Acc:MGI:107822]

125

ENSMUSG00000029413 Naaa 3536.5251 2.2150 -1.1473 0.2676 -4.2868 1.812E-05 0.0026253

N-acylethanolamine acid amidase [Source:MGI Symbol;Acc:MGI:1914361]

ENSMUSG00000022844 Pdia5 319.1673 1.8936 -0.9211 0.2159 -4.2665 1.986E-05 0.0027999

protein disulfide isomerase associated 5 [Source:MGI Symbol;Acc:MGI:1919849]

ENSMUSG00000032575 Manf 6582.2659 1.9901 -0.9929 0.2327 -4.2664 1.987E-05 0.0027999

mesencephalic astrocyte-derived neurotrophic factor [Source:MGI Symbol;Acc:MGI:1922090]

ENSMUSG00000022587 Ly6e 27692.4716 1.5372 -0.6203 0.1459 -4.2505 2.133E-05 0.0029659

lymphocyte antigen 6 complex, locus E [Source:MGI Symbol;Acc:MGI:106651]

ENSMUSG00000029304 Spp1 625.8452 2.6872 -1.4261 0.3361 -4.2435 2.201E-05 0.0030202

secreted phosphoprotein 1 [Source:MGI Symbol;Acc:MGI:98389]

ENSMUSG00000027566 Psma7 7321.3992 1.7798 -0.8317 0.1962 -4.2394 2.241E-05 0.0030353

proteasome (prosome, macropain) subunit, alpha type 7 [Source:MGI Symbol;Acc:MGI:1347070]

ENSMUSG00000038732 Mboat1 383.6969 2.5613 -1.3569 0.3209 -4.2283 2.355E-05 0.0031485

membrane bound O-acyltransferase domain containing 1 [Source:MGI Symbol;Acc:MGI:2387184]

ENSMUSG00000021484 Lman2 7452.9732 1.4794 -0.5650 0.1347 -4.1958 2.719E-05 0.0035888

lectin, mannose-binding 2 [Source:MGI Symbol;Acc:MGI:1914140]

ENSMUSG00000030062 Rpn1 8536.8058 1.5986 -0.6768 0.1627 -4.1590 3.197E-05 0.0041673

ribophorin I [Source:MGI Symbol;Acc:MGI:98084]

126

ENSMUSG00000021486 Prelid1 6848.3451 1.7598 -0.8154 0.1968 -4.1438 3.416E-05 0.0042499

PRELI domain containing 1 [Source:MGI Symbol;Acc:MGI:1913744]

ENSMUSG00000028248 Sfrs18 1378.8411 0.6545 0.6115 0.1475 4.1457 3.388E-05 0.0042499

PNN interacting serine/arginine-rich [Source:MGI Symbol;Acc:MGI:1913875]

ENSMUSG00000063229 Ldha 21435.8265 1.6828 -0.7509 0.1812 -4.1433 3.423E-05 0.0042499

lactate dehydrogenase A [Source:MGI Symbol;Acc:MGI:96759]

ENSMUSG00000075702 Selm 409.9103 2.8135 -1.4924 0.3599 -4.1468 3.372E-05 0.0042499

selenoprotein M [Source:MGI Symbol;Acc:MGI:2149786]

ENSMUSG00000041355 Ssr2 5364.9371 1.7975 -0.8460 0.2043 -4.1402 3.469E-05 0.0042567

signal sequence receptor, beta [Source:MGI Symbol;Acc:MGI:1913506]

ENSMUSG00000024579 Pcyox1l 818.2137 1.7365 -0.7962 0.1925 -4.1353 3.544E-05 0.0042983

prenylcysteine oxidase 1 like [Source:MGI Symbol;Acc:MGI:3606062]

ENSMUSG00000021871 Pnp 3541.0342 1.6453 -0.7184 0.1741 -4.1267 3.68E-05 0.0044112

purine-nucleoside phosphorylase [Source:MGI Symbol;Acc:MGI:97365]

ENSMUSG00000025465 Echs1 2032.0524 1.5200 -0.6041 0.1466 -4.1202 3.785E-05 0.0044858

enoyl Coenzyme A hydratase, short chain, 1, mitochondrial [Source:MGI Symbol;Acc:MGI:2136460]

ENSMUSG00000035678 Tnfsf9 97.8469 2.4090 -1.2684 0.3082 -4.1155 3.864E-05 0.0045275

tumor necrosis factor (ligand) superfamily, member 9 [Source:MGI Symbol;Acc:MGI:1101058]

ENSMUSG00000070369 Itgad 397.8620 0.4730 1.0802 0.2626 4.1127 3.911E-05 0.0045323

integrin, alpha D [Source:MGI Symbol;Acc:MGI:3578624]

ENSMUSG00000019528 Gyg 3807.0255 1.4922 -0.5775 0.1406 -4.1061 4.023E-05 0.004611

glycogenin [Source:MGI Symbol;Acc:MGI:1351614]

127

ENSMUSG00000023067 Cdkn1a 818.7496 1.7541 -0.8108 0.1977 -4.1000 4.132E-05 0.0046836

cyclin-dependent kinase inhibitor 1A (P21) [Source:MGI Symbol;Acc:MGI:104556]

ENSMUSG00000032508 Myd88 2794.1596 1.6685 -0.7386 0.1812 -4.0767 4.569E-05 0.0049316

myeloid differentiation primary response gene 88 [Source:MGI Symbol;Acc:MGI:108005]

ENSMUSG00000037278 Tmem97 1218.0333 1.9582 -0.9695 0.2376 -4.0808 4.488E-05 0.0049316


ENSMUSG00000042453 Reln 123.9730 0.4070 1.2968 0.3179 4.0792 4.52E-05 0.0049316reelin [Source:MGI Symbol;Acc:MGI:103022]

ENSMUSG00000056413 Adap1 2229.4600 1.6342 -0.7086 0.1737 -4.0799 4.505E-05 0.0049316

ArfGAP with dual PH domains 1 [Source:MGI Symbol;Acc:MGI:2442201]

ENSMUSG00000058818 Lilrb3 5989.9830 1.6617 -0.7327 0.1798 -4.0757 4.587E-05 0.0049316

paired Ig-like receptor B [Source:MGI Symbol;Acc:MGI:894311]

ENSMUSG00000047379 B3gnt1 414.4062 1.5932 -0.6719 0.1651 -4.0694 4.714E-05 0.0050164

beta-1,4-glucuronyltransferase 1 [Source:MGI Symbol;Acc:MGI:1919680]

ENSMUSG00000024462 Gabbr1 603.4070 0.6132 0.7055 0.1735 4.0666 4.769E-05 0.0050243

gamma-aminobutyric acid (GABA) B receptor, 1 [Source:MGI Symbol;Acc:MGI:1860139]

ENSMUSG00000027994 Ccdc109b 503.6498 2.3801 -1.2510 0.3079 -4.0626 4.853E-05 0.005028

coiled-coil domain containing 109B [Source:MGI Symbol;Acc:MGI:1914065]

ENSMUSG00000035493 Tgfbi 13071.4574 2.9388 -1.5552 0.3829 -4.0618 4.869E-05 0.005028

transforming growth factor, beta induced [Source:MGI Symbol;Acc:MGI:99959]

128

ENSMUSG00000012422 Tmem167 2676.6882 1.5193 -0.6034 0.1489 -4.0511 5.097E-05 0.0052113


ENSMUSG00000019996 Mtap7 480.3479 0.5814 0.7823 0.1939 4.0347 5.467E-05 0.0054826

microtubule-associated protein 7 [Source:MGI Symbol;Acc:MGI:1328328]

ENSMUSG00000020873 Slc35b1 1568.2912 1.5519 -0.6341 0.1571 -4.0362 5.433E-05 0.0054826

solute carrier family 35, member B1 [Source:MGI Symbol;Acc:MGI:1343133]

ENSMUSG00000037759 Ptger2 326.4473 1.6409 -0.7145 0.1781 -4.0127 6.004E-05 0.0059632

prostaglandin E receptor 2 (subtype EP2) [Source:MGI Symbol;Acc:MGI:97794]

ENSMUSG00000028937 Acot7 1702.8217 1.7047 -0.7696 0.1925 -3.9982 6.382E-05 0.0062787

acyl-CoA thioesterase 7 [Source:MGI Symbol;Acc:MGI:1917275]

ENSMUSG00000029406 Pitpnm2 574.1333 0.5691 0.8131 0.2041 3.9844 6.765E-05 0.0065937

phosphatidylinositol transfer protein, membrane-associated 2 [Source:MGI Symbol;Acc:MGI:1336192]

ENSMUSG00000020089 Ppa1 1541.7838 1.6587 -0.7301 0.1834 -3.9811 6.858E-05 0.0066228

pyrophosphatase (inorganic) 1 [Source:MGI Symbol;Acc:MGI:97831]

ENSMUSG00000026696 Vamp4 1369.1278 1.4864 -0.5718 0.1444 -3.9609 7.468E-05 0.0071025vesicle-associated membrane protein 4 [Source:MGI Symbol;Acc:MGI:1858730]

ENSMUSG00000071379 Hpcal1 2742.9464 1.5594 -0.6410 0.1619 -3.9601 7.491E-05 0.0071025

hippocalcin-like 1 [Source:MGI Symbol;Acc:MGI:1855689]

129

ENSMUSG00000024730 Ms4a8a 392.8003 2.1458 -1.1015 0.2790 -3.9480 7.881E-05 0.0074049

membrane-spanning 4-domains, subfamily A, member 8A [Source:MGI Symbol;Acc:MGI:1927657]

ENSMUSG00000016528 Mapkapk2 9937.4332 1.5012 -0.5861 0.1488 -3.9381 8.214E-05 0.0076488

MAP kinase-activated protein kinase 2 [Source:MGI Symbol;Acc:MGI:109298]

ENSMUSG00000024769 Cdc42bpg 321.8156 0.5772 0.7929 0.2019 3.9278 8.573E-05 0.007912

CDC42 binding protein kinase gamma (DMPK-like) [Source:MGI Symbol;Acc:MGI:2652845]

ENSMUSG00000021178 Psmc1 4777.7765 1.5189 -0.6030 0.1536 -3.9252 8.665E-05 0.0079274

protease (prosome, macropain) 26S subunit, ATPase 1 [Source:MGI Symbol;Acc:MGI:106054]

ENSMUSG00000048164 Gm7204 146.4455 2.8296 -1.5006 0.3826 -3.9220 8.78E-05 0.0079622

predicted pseudogene 7204 [Source:MGI Symbol;Acc:MGI:3645884]

ENSMUSG00000041084 Ostc 3441.0647 1.7455 -0.8037 0.2054 -3.9136 9.093E-05 0.008175

oligosaccharyltransferase complex subunit [Source:MGI Symbol;Acc:MGI:1913607]

ENSMUSG00000011256 Adam19 1198.3477 0.5201 0.9432 0.2423 3.8926 9.918E-05 0.0087813

a disintegrin and metallopeptidase domain 19 (meltrin beta) [Source:MGI Symbol;Acc:MGI:105377]

ENSMUSG00000042747 Krtcap2 2728.4431 1.9182 -0.9398 0.2415 -3.8922 9.936E-05 0.0087813

keratinocyte associated protein 2 [Source:MGI Symbol;Acc:MGI:1913309]

130

ENSMUSG00000074825 Itpripl1 863.3876 1.4784 -0.5640 0.1452 -3.8837 0.0001029 0.0090167

inositol 1,4,5-triphosphate receptor interacting protein-like 1 [Source:MGI Symbol;Acc:MGI:1920588]

ENSMUSG00000021338 Lrrc16a 226.2988 0.4187 1.2560 0.3244 3.8721 0.0001079 0.0093029

leucine rich repeat containing 16A [Source:MGI Symbol;Acc:MGI:1915982]

ENSMUSG00000028692 Akr1a1 12329.3612 1.5699 -0.6507 0.1681 -3.8720 0.0001079 0.0093029

aldo-keto reductase family 1, member A1 (aldehyde reductase) [Source:MGI Symbol;Acc:MGI:1929955]

ENSMUSG00000022452 1500032L24 4016.2221 2.0284 -1.0203 0.2644 -3.8586 0.000114 0.0097493

single-pass membrane protein with aspartate rich tail 1 [Source:MGI Symbol;Acc:MGI:1916279]

ENSMUSG00000001473 Tubb6 291.3040 2.1878 -1.1295 0.2938 -3.8439 0.0001211 0.0102684tubulin, beta 6 class V [Source:MGI Symbol;Acc:MGI:1915201]

ENSMUSG00000026914 Psmd14 3118.0232 1.4947 -0.5799 0.1512 -3.8344 0.0001258 0.0105843

proteasome (prosome, macropain) 26S subunit, non-ATPase, 14 [Source:MGI Symbol;Acc:MGI:1913284]

ENSMUSG00000044469 Tnfaip8l1 166.8281 1.9915 -0.9938 0.2595 -3.8301 0.0001281 0.0106865

tumor necrosis factor, alpha-induced protein 8-like 1 [Source:MGI Symbol;Acc:MGI:1913693]

ENSMUSG00000026604 Ptpn14 148.7793 0.4343 1.2033 0.3149 3.8215 0.0001327 0.0109806

protein tyrosine phosphatase, non-receptor type 14 [Source:MGI Symbol;Acc:MGI:102467]

ENSMUSG00000023456 Tpi1 8536.7837 1.8270 -0.8695 0.2281 -3.8125 0.0001376 0.0112448

triosephosphate isomerase 1 [Source:MGI Symbol;Acc:MGI:98797]

131

ENSMUSG00000037692 Ahdc1 377.8702 0.5487 0.8660 0.2272 3.8117 0.000138 0.0112448

AT hook, DNA binding motif, containing 1 [Source:MGI Symbol;Acc:MGI:2444218]

ENSMUSG00000062115 Rai1 909.6987 0.5951 0.7489 0.1976 3.7908 0.0001502 0.0121408retinoic acid induced 1 [Source:MGI Symbol;Acc:MGI:103291]

ENSMUSG00000030615 Tmem126a 565.9369 1.5896 -0.6687 0.1765 -3.7888 0.0001514 0.012143

transmembrane protein 126A [Source:MGI Symbol;Acc:MGI:1913521]

ENSMUSG00000069106 Gm8910 111.5632 2.8311 -1.5014 0.3980 -3.7726 0.0001616 0.0128618 NA

ENSMUSG00000021242 Npc2 12017.0830 1.6332 -0.7077 0.1878 -3.7686 0.0001641 0.0129036Niemann-Pick type C2 [Source:MGI Symbol;Acc:MGI:1915213]

ENSMUSG00000021423 Ly86 3002.3969 1.6035 -0.6812 0.1809 -3.7661 0.0001658 0.0129036lymphocyte antigen 86 [Source:MGI Symbol;Acc:MGI:1321404]

ENSMUSG00000025270 Alas2 727.7481 0.5474 0.8694 0.2308 3.7662 0.0001657 0.0129036

aminolevulinic acid synthase 2, erythroid [Source:MGI Symbol;Acc:MGI:87990]

ENSMUSG00000031231 Cox7b 4570.7538 1.6960 -0.7621 0.2025 -3.7638 0.0001673 0.0129256

cytochrome c oxidase subunit VIIb [Source:MGI Symbol;Acc:MGI:1913392]

ENSMUSG00000056749 Nfil3 1439.0432 1.7635 -0.8185 0.2177 -3.7590 0.0001706 0.0130796

nuclear factor, interleukin 3, regulated [Source:MGI Symbol;Acc:MGI:109495]

ENSMUSG00000026347 Tmem163 236.4427 0.5571 0.8439 0.2248 3.7545 0.0001737 0.0131879


ENSMUSG00000029810 Tmem176b 2151.4575 1.8897 -0.9182 0.2447 -3.7523 0.0001752 0.0131879

transmembrane protein 176B [Source:MGI Symbol;Acc:MGI:1916348]

ENSMUSG00000057729 Prtn3 34287.4572 2.2188 -1.1498 0.3065 -3.7515 0.0001758 0.0131879

proteinase 3 [Source:MGI Symbol;Acc:MGI:893580]

132

ENSMUSG00000022371 Col14a1 173.2779 0.4535 1.1410 0.3046 3.7452 0.0001803 0.0134277collagen, type XIV, alpha 1 [Source:MGI Symbol;Acc:MGI:1341272]

ENSMUSG00000022489 Pde1b 750.0629 0.6448 0.6332 0.1693 3.7397 0.0001842 0.0136274

phosphodiesterase 1B, Ca2+-calmodulin dependent [Source:MGI Symbol;Acc:MGI:97523]

ENSMUSG00000026600 Soat1 2453.9549 1.8380 -0.8781 0.2350 -3.7361 0.0001869 0.0136308

sterol O-acyltransferase 1 [Source:MGI Symbol;Acc:MGI:104665]

ENSMUSG00000032411 Tfdp2 934.3276 0.6085 0.7167 0.1918 3.7366 0.0001865 0.0136308transcription factor Dp 2 [Source:MGI Symbol;Acc:MGI:107167]

ENSMUSG00000025289 Prdx4 2424.4603 1.8535 -0.8902 0.2386 -3.7309 0.0001908 0.0138164

peroxiredoxin 4 [Source:MGI Symbol;Acc:MGI:1859815]

ENSMUSG00000028108 Ecm1 202.8626 2.7111 -1.4389 0.3865 -3.7227 0.0001971 0.0141749

extracellular matrix protein 1 [Source:MGI Symbol;Acc:MGI:103060]

ENSMUSG00000020593 Lpin1 334.4802 0.6105 0.7120 0.1914 3.7190 0.0002 0.0142047lipin 1 [Source:MGI Symbol;Acc:MGI:1891340]

ENSMUSG00000075701 H47 2189.4260 1.6180 -0.6942 0.1867 -3.7187 0.0002002 0.0142047

VCP-interacting membrane protein [Source:MGI Symbol;Acc:MGI:95994]

ENSMUSG00000034266 Batf 585.6568 1.6563 -0.7279 0.1959 -3.7153 0.0002029 0.0142995

basic leucine zipper transcription factor, ATF-like [Source:MGI Symbol;Acc:MGI:1859147]

ENSMUSG00000028494 Plin2 4208.8751 1.8244 -0.8674 0.2342 -3.7036 0.0002125 0.0148755

perilipin 2 [Source:MGI Symbol;Acc:MGI:87920]

133

ENSMUSG00000048440 Cyp4f16 926.2999 1.8248 -0.8677 0.2346 -3.6991 0.0002164 0.0149764

cytochrome P450, family 4, subfamily f, polypeptide 16 [Source:MGI Symbol;Acc:MGI:1917351]

ENSMUSG00000072855 Gm10430 306.7777 0.5323 0.9096 0.2459 3.6985 0.0002168 0.0149764 NA

ENSMUSG00000051185 Fam174a 1368.1390 1.5543 -0.6362 0.1722 -3.6953 0.0002196 0.0150668


ENSMUSG00000036781 Rps27l 4040.9582 2.0124 -1.0089 0.2734 -3.6903 0.000224 0.0152682ribosomal protein S27-like [Source:MGI Symbol;Acc:MGI:1915191]

ENSMUSG00000022013 Dnajc15 984.2591 1.8927 -0.9205 0.2496 -3.6881 0.0002259 0.0152986

DnaJ (Hsp40) homolog, subfamily C, member 15 [Source:MGI Symbol;Acc:MGI:1913398]

ENSMUSG00000062991 Nrg1 457.4602 2.4364 -1.2847 0.3487 -3.6846 0.0002291 0.0154126

neuregulin 1 [Source:MGI Symbol;Acc:MGI:96083]

ENSMUSG00000032028 Fam55b 982.9844 0.5685 0.8148 0.2223 3.6659 0.0002465 0.016479

neurexophilin and PC-esterase domain family, member 2 [Source:MGI Symbol;Acc:MGI:1925502]

ENSMUSG00000038831 Ralgps1 262.4641 0.6112 0.7104 0.1941 3.6594 0.0002528 0.0167923

Ral GEF with PH domain and SH3 binding motif 1 [Source:MGI Symbol;Acc:MGI:1922008]

ENSMUSG00000004655 Aqp1 10944.1503 0.6227 0.6835 0.1872 3.6510 0.0002612 0.0172399

aquaporin 1 [Source:MGI Symbol;Acc:MGI:103201]

ENSMUSG00000023328 Ache 195.3728 0.5061 0.9824 0.2693 3.6479 0.0002644 0.0172598

acetylcholinesterase [Source:MGI Symbol;Acc:MGI:87876]

ENSMUSG00000059743 Fdps 1934.3711 1.6603 -0.7314 0.2005 -3.6475 0.0002648 0.0172598

farnesyl diphosphate synthetase [Source:MGI Symbol;Acc:MGI:104888]

134

ENSMUSG00000005667 Mthfd2 1712.7004 1.5865 -0.6659 0.1827 -3.6437 0.0002687 0.0174051

methylenetetrahydrofolate dehydrogenase (NAD+ dependent), methenyltetrahydrofolate cyclohydrolase [Source:MGI Symbol;Acc:MGI:1338850]

ENSMUSG00000035227 Spcs2 7493.9763 1.6433 -0.7166 0.1972 -3.6343 0.0002787 0.0179447

signal peptidase complex subunit 2 homolog (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1913874]

ENSMUSG00000070394 1810027O1 3155.2428 2.4752 -1.3075 0.3601 -3.6309 0.0002824 0.0180683


ENSMUSG00000033253 Szt2 793.9477 0.6644 0.5899 0.1627 3.6267 0.0002871 0.0182574

seizure threshold 2 [Source:MGI Symbol;Acc:MGI:3033336]

ENSMUSG00000026721 Rabgap1l 2196.1815 0.6971 0.5205 0.1436 3.6242 0.0002899 0.0183207

RAB GTPase activating protein 1-like [Source:MGI Symbol;Acc:MGI:1352507]

ENSMUSG00000019832 Rab32 2892.4896 1.6520 -0.7242 0.2003 -3.6164 0.0002988 0.018771

RAB32, member RAS oncogene family [Source:MGI Symbol;Acc:MGI:1915094]

ENSMUSG00000018362 Kpna2 7625.1747 1.4690 -0.5549 0.1536 -3.6125 0.0003033 0.0189378

karyopherin (importin) alpha 2 [Source:MGI Symbol;Acc:MGI:103561]

ENSMUSG00000022048 Dpysl2 700.6210 2.0029 -1.0021 0.2778 -3.6070 0.0003097 0.0190845dihydropyrimidinase-like 2 [Source:MGI Symbol;Acc:MGI:1349763]

ENSMUSG00000032131 Abcg4 472.4109 0.5703 0.8101 0.2247 3.6059 0.0003111 0.0190845

ATP-binding cassette, sub-family G (WHITE), member 4 [Source:MGI Symbol;Acc:MGI:1890594]

135

ENSMUSG00000036882 Arhgap33 282.7796 0.5875 0.7674 0.2127 3.6085 0.000308 0.0190845

Rho GTPase activating protein 33 [Source:MGI Symbol;Acc:MGI:2673998]

ENSMUSG00000053317 Sec61b 6299.5897 2.2083 -1.1429 0.3177 -3.5978 0.000321 0.0195751Sec61 beta subunit [Source:MGI Symbol;Acc:MGI:1913462]

ENSMUSG00000027173 Depdc7 266.2972 1.7444 -0.8027 0.2236 -3.5908 0.0003297 0.0199906

DEP domain containing 7 [Source:MGI Symbol;Acc:MGI:2139258]

ENSMUSG00000002332 Dhrs1 1317.8019 1.5435 -0.6262 0.1746 -3.5864 0.0003353 0.0201647

dehydrogenase/reductase (SDR family) member 1 [Source:MGI Symbol;Acc:MGI:1196314]

ENSMUSG00000003355 Fkbp11 343.6696 1.9966 -0.9975 0.2782 -3.5855 0.0003364 0.0201647

FK506 binding protein 11 [Source:MGI Symbol;Acc:MGI:1913370]

ENSMUSG00000004846 Plod3 3224.5295 1.5927 -0.6715 0.1879 -3.5731 0.0003528 0.0204419

procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 [Source:MGI Symbol;Acc:MGI:1347008]

ENSMUSG00000015947 Fcgr1 899.5026 2.8355 -1.5036 0.4202 -3.5785 0.0003455 0.0204419

Fc receptor, IgG, high affinity I [Source:MGI Symbol;Acc:MGI:95498]

ENSMUSG00000020474 Polm 890.9826 0.5071 0.9797 0.2741 3.5746 0.0003507 0.0204419

polymerase (DNA directed), mu [Source:MGI Symbol;Acc:MGI:1860191]

ENSMUSG00000022092 Ppp3cc 638.8690 0.5238 0.9328 0.2609 3.5758 0.0003492 0.0204419

protein phosphatase 3, catalytic subunit, gamma isoform [Source:MGI Symbol;Acc:MGI:107162]

ENSMUSG00000023830 Igf2r 1022.8483 0.6700 0.5778 0.1617 3.5733 0.0003526 0.0204419

insulin-like growth factor 2 receptor [Source:MGI Symbol;Acc:MGI:96435]

136

ENSMUSG00000060143 Gm10076 268.6257 2.9310 -1.5514 0.4333 -3.5801 0.0003434 0.0204419predicted gene 10076 [Source:MGI Symbol;Acc:MGI:3704451]

ENSMUSG00000069255 Dusp22 526.5651 1.6010 -0.6790 0.1902 -3.5703 0.0003565 0.0205417

dual specificity phosphatase 22 [Source:MGI Symbol;Acc:MGI:1915926]

ENSMUSG00000024683 Mrpl16 1288.1391 1.7197 -0.7821 0.2194 -3.5646 0.0003644 0.0205432

mitochondrial ribosomal protein L16 [Source:MGI Symbol;Acc:MGI:2137219]

ENSMUSG00000026223 Itm2c 3942.6162 1.6114 -0.6884 0.1930 -3.5659 0.0003626 0.0205432

integral membrane protein 2C [Source:MGI Symbol;Acc:MGI:1927594]

ENSMUSG00000030695 Aldoa 19972.4979 1.7021 -0.7673 0.2151 -3.5674 0.0003606 0.0205432

aldolase A, fructose-bisphosphate [Source:MGI Symbol;Acc:MGI:87994]

ENSMUSG00000036078 Sigmar1 1205.4511 1.7377 -0.7972 0.2234 -3.5688 0.0003587 0.0205432

sigma non-opioid intracellular receptor 1 [Source:MGI Symbol;Acc:MGI:1195268]

ENSMUSG00000002014 Ssr4 5019.9322 1.7463 -0.8043 0.2263 -3.5534 0.0003803 0.0212078

signal sequence receptor, delta [Source:MGI Symbol;Acc:MGI:1099464]

ENSMUSG00000078974 Sec61g 4217.5223 1.8500 -0.8875 0.2497 -3.5545 0.0003787 0.0212078SEC61, gamma subunit [Source:MGI Symbol;Acc:MGI:1202066]

ENSMUSG00000018339 Gpx3 157.5236 1.7567 -0.8129 0.2293 -3.5458 0.0003914 0.0217132

glutathione peroxidase 3 [Source:MGI Symbol;Acc:MGI:105102]

ENSMUSG00000053687 Dpep2 234.4630 2.4904 -1.3164 0.3715 -3.5434 0.000395 0.0217973

dipeptidase 2 [Source:MGI Symbol;Acc:MGI:2442042]

ENSMUSG00000029616 Erp29 9491.1817 1.8781 -0.9093 0.2568 -3.5407 0.0003991 0.0219063

endoplasmic reticulum protein 29 [Source:MGI Symbol;Acc:MGI:1914647]

137

ENSMUSG00000013033 Lphn1 250.6291 0.6211 0.6871 0.1942 3.5381 0.0004029 0.0220017

adhesion G protein-coupled receptor L1 [Source:MGI Symbol;Acc:MGI:1929461]

ENSMUSG00000030188 Magohb 653.1255 1.9838 -0.9883 0.2802 -3.5271 0.0004202 0.0227035

mago-nashi homolog B (Drosophila) [Source:MGI Symbol;Acc:MGI:1913691]

ENSMUSG00000030681 Mvp 1603.7487 1.5862 -0.6656 0.1887 -3.5271 0.0004201 0.0227035major vault protein [Source:MGI Symbol;Acc:MGI:1925638]

ENSMUSG00000021040 1810035L17 580.8277 2.1425 -1.0993 0.3119 -3.5249 0.0004237 0.0227767

SRA stem-loop interacting RNA binding protein [Source:MGI Symbol;Acc:MGI:1916394]

ENSMUSG00000022594 Lynx1 112.8253 0.4864 1.0399 0.2955 3.5191 0.0004331 0.0229264

Ly6/neurotoxin 1 [Source:MGI Symbol;Acc:MGI:1345180]

ENSMUSG00000031024 St5 300.7998 0.5847 0.7743 0.2200 3.5201 0.0004314 0.0229264

suppression of tumorigenicity 5 [Source:MGI Symbol;Acc:MGI:108517]

ENSMUSG00000057133 Chd6 610.0922 0.5942 0.7509 0.2132 3.5215 0.0004292 0.0229264

chromodomain helicase DNA binding protein 6 [Source:MGI Symbol;Acc:MGI:1918639]

ENSMUSG00000015937 H2afy 14838.2144 1.4676 -0.5534 0.1576 -3.5122 0.0004444 0.0230517

H2A histone family, member Y [Source:MGI Symbol;Acc:MGI:1349392]

ENSMUSG00000024012 Mtch1 4187.8455 1.5016 -0.5865 0.1669 -3.5139 0.0004415 0.0230517

mitochondrial carrier homolog 1 (C. elegans) [Source:MGI Symbol;Acc:MGI:1929261]

ENSMUSG00000024892 Pcx 290.6665 0.6313 0.6635 0.1892 3.5071 0.0004529 0.0230517

pyruvate carboxylase [Source:MGI Symbol;Acc:MGI:97520]

ENSMUSG00000025534 Gusb 3984.9173 1.6282 -0.7032 0.2005 -3.5075 0.0004524 0.0230517

glucuronidase, beta [Source:MGI Symbol;Acc:MGI:95872]

138

ENSMUSG00000035351 Nup37 698.2870 1.4949 -0.5801 0.1652 -3.5109 0.0004465 0.0230517

nucleoporin 37 [Source:MGI Symbol;Acc:MGI:1919964]

ENSMUSG00000040204 2810417H1 6761.3265 1.4962 -0.5813 0.1654 -3.5155 0.0004389 0.0230517

RIKEN cDNA 2810417H13 gene [Source:MGI Symbol;Acc:MGI:1915276]

ENSMUSG00000044894 Uqcrq 3729.3544 2.1796 -1.1241 0.3201 -3.5115 0.0004456 0.0230517

ubiquinol-cytochrome c reductase, complex III subunit VII [Source:MGI Symbol;Acc:MGI:107807]

ENSMUSG00000045665 Mfsd5 1510.3555 1.5739 -0.6543 0.1866 -3.5070 0.0004531 0.0230517

major facilitator superfamily domain containing 5 [Source:MGI Symbol;Acc:MGI:2145901]

ENSMUSG00000022362 9130401M0 731.8894 1.5622 -0.6435 0.1836 -3.5044 0.0004577 0.0231701predicted gene 29394 [Source:MGI Symbol;Acc:MGI:5580100]

ENSMUSG00000030751 Psma1 5738.5583 1.4787 -0.5643 0.1612 -3.5014 0.0004629 0.0233192


ENSMUSG00000028420 Tmem38b 1593.9412 1.4616 -0.5476 0.1569 -3.4906 0.0004819 0.0241609

transmembrane protein 38B [Source:MGI Symbol;Acc:MGI:1098718]

ENSMUSG00000028007 Snx7 157.1999 1.9285 -0.9475 0.2716 -3.4890 0.0004848 0.0241922

sorting nexin 7 [Source:MGI Symbol;Acc:MGI:1923811]

ENSMUSG00000033307 Mif 4713.6952 2.1464 -1.1019 0.3161 -3.4865 0.0004894 0.0243029

macrophage migration inhibitory factor [Source:MGI Symbol;Acc:MGI:96982]

ENSMUSG00000003814 Calr 27621.3835 1.5928 -0.6715 0.1927 -3.4851 0.0004919 0.0243114

calreticulin [Source:MGI Symbol;Acc:MGI:88252]

139

ENSMUSG00000000355 Mcts1 1576.5944 1.7401 -0.7992 0.2307 -3.4643 0.0005316 0.0260631

malignant T cell amplified sequence 1 [Source:MGI Symbol;Acc:MGI:1916245]

ENSMUSG00000076435 Acsf2 437.0053 0.5269 0.9245 0.2669 3.4639 0.0005323 0.0260631

acyl-CoA synthetase family member 2 [Source:MGI Symbol;Acc:MGI:2388287]

ENSMUSG00000018585 Atox1 3254.1815 2.1452 -1.1011 0.3181 -3.4610 0.0005382 0.0262285

ATX1 (antioxidant protein 1) homolog 1 (yeast) [Source:MGI Symbol;Acc:MGI:1333855]

ENSMUSG00000028671 Gale 505.4996 1.6420 -0.7154 0.2069 -3.4583 0.0005435 0.0262422

galactose-4-epimerase, UDP [Source:MGI Symbol;Acc:MGI:1921496]

ENSMUSG00000038042 Ptpdc1 144.7748 0.5956 0.7475 0.2161 3.4588 0.0005427 0.0262422

protein tyrosine phosphatase domain containing 1 [Source:MGI Symbol;Acc:MGI:2145430]

ENSMUSG00000020048 Hsp90b1 14460.4817 1.5291 -0.6127 0.1773 -3.4555 0.0005492 0.0263135

heat shock protein 90, beta (Grp94), member 1 [Source:MGI Symbol;Acc:MGI:98817]

ENSMUSG00000021728 Emb 7870.4038 1.8695 -0.9026 0.2614 -3.4524 0.0005556 0.0263135

embigin [Source:MGI Symbol;Acc:MGI:95321]

ENSMUSG00000029804 Herc3 508.2622 0.5336 0.9063 0.2625 3.4523 0.0005557 0.0263135

hect domain and RLD 3 [Source:MGI Symbol;Acc:MGI:1921248]

ENSMUSG00000035835 BC005764 1116.6229 1.7519 -0.8089 0.2344 -3.4514 0.0005576 0.0263135

lipid phosphate phosphatase-related protein type 3 [Source:MGI Symbol;Acc:MGI:2388640]

ENSMUSG00000055184 Fam72a 235.7047 1.7785 -0.8307 0.2406 -3.4531 0.0005541 0.0263135


140

ENSMUSG00000040212 Emp3 6510.9451 1.6671 -0.7373 0.2138 -3.4486 0.0005636 0.026476

epithelial membrane protein 3 [Source:MGI Symbol;Acc:MGI:1098729]

ENSMUSG00000040616 Tmem51 450.0622 1.7234 -0.7852 0.2280 -3.4437 0.0005737 0.0268308


ENSMUSG00000029553 Tfec 1148.6026 1.8480 -0.8860 0.2576 -3.4398 0.0005822 0.0271059transcription factor EC [Source:MGI Symbol;Acc:MGI:1333760]

ENSMUSG00000031634 Ufsp2 1093.0053 1.5897 -0.6687 0.1945 -3.4373 0.0005875 0.0271102

UFM1-specific peptidase 2 [Source:MGI Symbol;Acc:MGI:1913679]

ENSMUSG00000068749 Psma5 3567.1115 1.6091 -0.6863 0.1996 -3.4380 0.000586 0.0271102


ENSMUSG00000006589 Aprt 4484.5773 2.0252 -1.0181 0.2968 -3.4303 0.0006029 0.0276966

adenine phosphoribosyl transferase [Source:MGI Symbol;Acc:MGI:88061]

ENSMUSG00000020818 Mfsd11 687.7419 1.5027 -0.5876 0.1714 -3.4275 0.0006093 0.0278687

major facilitator superfamily domain containing 11 [Source:MGI Symbol;Acc:MGI:1917150]

ENSMUSG00000030930 Chst15 1671.0266 0.6085 0.7167 0.2094 3.4232 0.0006188 0.0280135

carbohydrate (N-acetylgalactosamine 4-sulfate 6-O) sulfotransferase 15 [Source:MGI Symbol;Acc:MGI:1924840]

ENSMUSG00000032053 Pou2af1 4348.5116 0.5816 0.7820 0.2285 3.4225 0.0006205 0.0280135

POU domain, class 2, associating factor 1 [Source:MGI Symbol;Acc:MGI:105086]

141

ENSMUSG00000032306 Mpi 499.9790 1.5156 -0.5999 0.1752 -3.4232 0.0006188 0.0280135

mannose phosphate isomerase [Source:MGI Symbol;Acc:MGI:97075]

ENSMUSG00000087260 Hbxip 1804.7754 1.5362 -0.6193 0.1814 -3.4136 0.000641 0.0288163

late endosomal/lysosomal adaptor, MAPK and MTOR activator 5 [Source:MGI Symbol;Acc:MGI:1915826]

ENSMUSG00000004849 Ap1s1 3027.5587 1.8692 -0.9024 0.2648 -3.4082 0.0006539 0.0291423

adaptor protein complex AP-1, sigma 1 [Source:MGI Symbol;Acc:MGI:1098244]

ENSMUSG00000079197 Psme2 5487.7694 1.7510 -0.8082 0.2371 -3.4085 0.0006531 0.0291423

proteasome (prosome, macropain) activator subunit 2 (PA28 beta) [Source:MGI Symbol;Acc:MGI:1096365]

ENSMUSG00000040732 Erg 517.3080 0.6811 0.5541 0.1628 3.4031 0.0006662 0.0295671

avian erythroblastosis virus E-26 (v-ets) oncogene related [Source:MGI Symbol;Acc:MGI:95415]

ENSMUSG00000001027 Scn4a 163.9623 0.3881 1.3657 0.4020 3.3972 0.0006809 0.0300879

sodium channel, voltage-gated, type IV, alpha [Source:MGI Symbol;Acc:MGI:98250]

ENSMUSG00000028572 Hook1 244.4894 0.6229 0.6830 0.2012 3.3941 0.0006885 0.0302971

hook homolog 1 (Drosophila) [Source:MGI Symbol;Acc:MGI:1925213]

ENSMUSG00000003812 Dnase2a 1630.5988 1.7830 -0.8343 0.2460 -3.3915 0.0006952 0.0304645deoxyribonuclease II alpha [Source:MGI Symbol;Acc:MGI:1329019]

ENSMUSG00000027506 Tpd52 6181.2225 1.5960 -0.6745 0.1991 -3.3883 0.0007033 0.0306047

tumor protein D52 [Source:MGI Symbol;Acc:MGI:107749]

142

ENSMUSG00000034424 Gcsh 677.1738 1.5745 -0.6549 0.1933 -3.3879 0.0007043 0.0306047

glycine cleavage system protein H (aminomethyl carrier) [Source:MGI Symbol;Acc:MGI:1915383]

ENSMUSG00000042351 Grap2 301.0631 0.4013 1.3171 0.3892 3.3840 0.0007143 0.0309115

GRB2-related adaptor protein 2 [Source:MGI Symbol;Acc:MGI:1333842]

ENSMUSG00000024338 Psmb8 4706.2642 1.8196 -0.8636 0.2557 -3.3778 0.0007306 0.0314262

proteasome (prosome, macropain) subunit, beta type 8 (large multifunctional peptidase 7) [Source:MGI Symbol;Acc:MGI:1346527]

ENSMUSG00000032220 Myo1e 591.6083 0.4418 1.1784 0.3489 3.3771 0.0007326 0.0314262

myosin IE [Source:MGI Symbol;Acc:MGI:106621]

ENSMUSG00000056501 Cebpb 4501.8578 2.4559 -1.2963 0.3840 -3.3761 0.0007353 0.0314262

CCAAT/enhancer binding protein (C/EBP), beta [Source:MGI Symbol;Acc:MGI:88373]

ENSMUSG00000029530 Ccr9 259.6966 0.4643 1.1069 0.3284 3.3704 0.0007507 0.0317756

chemokine (C-C motif) receptor 9 [Source:MGI Symbol;Acc:MGI:1341902]

ENSMUSG00000033918 Parl 2049.3029 1.6307 -0.7055 0.2093 -3.3712 0.0007484 0.0317756

presenilin associated, rhomboid-like [Source:MGI Symbol;Acc:MGI:1277152]

ENSMUSG00000052821 Cysltr1 277.0368 2.5870 -1.3713 0.4069 -3.3697 0.0007526 0.0317756

cysteinyl leukotriene receptor 1 [Source:MGI Symbol;Acc:MGI:1926218]

ENSMUSG00000019850 Tnfaip3 1380.8071 0.5198 0.9439 0.2803 3.3679 0.0007574 0.0318008

tumor necrosis factor, alpha-induced protein 3 [Source:MGI Symbol;Acc:MGI:1196377]

143

ENSMUSG00000024397 Aif1 918.7338 1.7782 -0.8305 0.2466 -3.3672 0.0007593 0.0318008

allograft inflammatory factor 1 [Source:MGI Symbol;Acc:MGI:1343098]

ENSMUSG00000020267 Hint1 6938.4191 2.0142 -1.0102 0.3004 -3.3633 0.0007703 0.0318675

histidine triad nucleotide binding protein 1 [Source:MGI Symbol;Acc:MGI:1321133]

ENSMUSG00000022994 Adcy6 643.8702 0.6533 0.6142 0.1826 3.3638 0.0007687 0.0318675

adenylate cyclase 6 [Source:MGI Symbol;Acc:MGI:87917]

ENSMUSG00000031633 Slc25a4 3177.1672 1.5892 -0.6683 0.1987 -3.3641 0.0007679 0.0318675

solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 4 [Source:MGI Symbol;Acc:MGI:1353495]

ENSMUSG00000032038 St3gal4 2217.5573 1.3584 -0.4419 0.1315 -3.3612 0.0007761 0.0318675

ST3 beta-galactoside alpha-2,3-sialyltransferase 4 [Source:MGI Symbol;Acc:MGI:1316743]

ENSMUSG00000033170 Card10 523.8817 0.6314 0.6634 0.1974 3.3612 0.0007761 0.0318675

caspase recruitment domain family, member 10 [Source:MGI Symbol;Acc:MGI:2146012]

ENSMUSG00000025044 Msr1 1254.8956 2.2690 -1.1820 0.3519 -3.3592 0.0007817 0.0319694

macrophage scavenger receptor 1 [Source:MGI Symbol;Acc:MGI:98257]

ENSMUSG00000003585 Sec14l2 593.8487 0.5935 0.7528 0.2252 3.3428 0.0008293 0.0337123

SEC14-like 2 (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1915065]

ENSMUSG00000031605 Klhl2 882.9198 1.4257 -0.5117 0.1531 -3.3423 0.0008308 0.0337123kelch-like 2, Mayven [Source:MGI Symbol;Acc:MGI:1924363]

144

ENSMUSG00000038412 Higd1a 1302.1943 1.9485 -0.9623 0.2882 -3.3394 0.0008395 0.0339366

HIG1 domain family, member 1A [Source:MGI Symbol;Acc:MGI:1930666]

ENSMUSG00000050856 Atp5k 4252.6581 2.1639 -1.1137 0.3336 -3.3379 0.0008441 0.0339888

ATP synthase, H+ transporting, mitochondrial F1F0 complex, subunit E [Source:MGI Symbol;Acc:MGI:106636]

ENSMUSG00000014226 Cacybp 4192.7988 1.4306 -0.5166 0.1548 -3.3368 0.0008476 0.0339972calcyclin binding protein [Source:MGI Symbol;Acc:MGI:1270839]

ENSMUSG00000027163 Commd9 803.8579 1.7190 -0.7815 0.2346 -3.3316 0.0008636 0.0343743

COMM domain containing 9 [Source:MGI Symbol;Acc:MGI:1923751]

ENSMUSG00000047721 Bola2 2510.9171 2.2413 -1.1643 0.3494 -3.3320 0.0008623 0.0343743

bolA-like 2 (E. coli) [Source:MGI Symbol;Acc:MGI:1913412]

ENSMUSG00000052698 Tln2 129.8258 2.9540 -1.5627 0.4696 -3.3279 0.0008752 0.0347043talin 2 [Source:MGI Symbol;Acc:MGI:1917799]

ENSMUSG00000034573 Ptpn13 103.9611 0.4586 1.1248 0.3384 3.3241 0.0008869 0.0350368

protein tyrosine phosphatase, non-receptor type 13 [Source:MGI Symbol;Acc:MGI:103293]

ENSMUSG00000037972 Snn 2283.2775 0.4184 1.2571 0.3785 3.3212 0.0008963 0.035272stannin [Source:MGI Symbol;Acc:MGI:1276549]

ENSMUSG00000002731 Prkra 772.4206 1.5942 -0.6728 0.2028 -3.3180 0.0009068 0.0355527

protein kinase, interferon inducible double stranded RNA dependent activator [Source:MGI Symbol;Acc:MGI:1344375]

ENSMUSG00000031722 Hp 35401.5797 1.8157 -0.8606 0.2600 -3.3104 0.0009317 0.0362567

haptoglobin [Source:MGI Symbol;Acc:MGI:96211]

145

ENSMUSG00000047222 Ear11 401.0168 1.6772 -0.7461 0.2254 -3.3105 0.0009314 0.0362567

ribonuclease, RNase A family, 2A (liver, eosinophil-derived neurotoxin) [Source:MGI Symbol;Acc:MGI:1890465]

ENSMUSG00000036138 Acaa1a 1963.1702 1.5690 -0.6498 0.1965 -3.3075 0.0009413 0.0363583

acetyl-Coenzyme A acyltransferase 1A [Source:MGI Symbol;Acc:MGI:2148491]

ENSMUSG00000038811 Gngt2 1121.0652 1.8164 -0.8611 0.2603 -3.3084 0.0009383 0.0363583

guanine nucleotide binding protein (G protein), gamma transducing activity polypeptide 2 [Source:MGI Symbol;Acc:MGI:893584]

ENSMUSG00000030872 Gga2 2187.4696 0.6130 0.7061 0.2136 3.3048 0.0009504 0.0365747

golgi associated, gamma adaptin ear containing, ARF binding protein 2 [Source:MGI Symbol;Acc:MGI:1921355]

ENSMUSG00000079111 Kdelr2 2659.0419 1.6548 -0.7266 0.2200 -3.3035 0.0009548 0.0366095

KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 2 [Source:MGI Symbol;Acc:MGI:1914163]

ENSMUSG00000026849 Tor1a 2339.9350 1.5491 -0.6314 0.1912 -3.3021 0.0009596 0.0366588

torsin family 1, member A (torsin A) [Source:MGI Symbol;Acc:MGI:1353568]

ENSMUSG00000029390 Tmed2 11227.6793 1.3690 -0.4531 0.1374 -3.2968 0.0009779 0.0369696

transmembrane emp24 domain trafficking protein 2 [Source:MGI Symbol;Acc:MGI:1929269]

ENSMUSG00000062169 Cnih4 1240.5464 1.6318 -0.7065 0.2143 -3.2964 0.0009795 0.0369696

cornichon homolog 4 (Drosophila) [Source:MGI Symbol;Acc:MGI:1925828]

146

ENSMUSG00000068735 Trp53i11 1070.4180 0.5388 0.8922 0.2707 3.2957 0.0009819 0.0369696

transformation related protein 53 inducible protein 11 [Source:MGI Symbol;Acc:MGI:2670995]

ENSMUSG00000073678 Pgap1 108.1731 0.5153 0.9566 0.2901 3.2975 0.0009755 0.0369696

post-GPI attachment to proteins 1 [Source:MGI Symbol;Acc:MGI:2443342]

ENSMUSG00000004552 Ctse 6825.4366 0.6874 0.5409 0.1655 3.2678 0.0010838 0.0376893

cathepsin E [Source:MGI Symbol;Acc:MGI:107361]

ENSMUSG00000005371 Fbxo11 1525.5255 0.6843 0.5473 0.1673 3.2706 0.0010731 0.0376893

F-box protein 11 [Source:MGI Symbol;Acc:MGI:2147134]

ENSMUSG00000015597 Zfp318 568.1947 0.5136 0.9614 0.2937 3.2735 0.0010621 0.0376893

zinc finger protein 318 [Source:MGI Symbol;Acc:MGI:1889348]

ENSMUSG00000015750 Aph1a 3709.8356 1.3667 -0.4507 0.1371 -3.2887 0.0010064 0.0376893

anterior pharynx defective 1a homolog (C. elegans) [Source:MGI Symbol;Acc:MGI:2385110]

ENSMUSG00000019188 H13 5961.0291 1.6466 -0.7195 0.2200 -3.2701 0.0010751 0.0376893

histocompatibility 13 [Source:MGI Symbol;Acc:MGI:95886]

ENSMUSG00000019876 Pkib 479.6772 1.4591 -0.5451 0.1662 -3.2791 0.0010413 0.0376893

protein kinase inhibitor beta, cAMP dependent, testis specific [Source:MGI Symbol;Acc:MGI:101937]

ENSMUSG00000020387 Phf15 575.6449 0.5815 0.7820 0.2381 3.2841 0.0010232 0.0376893jade family PHD finger 2 [Source:MGI Symbol;Acc:MGI:1924151]

ENSMUSG00000021061 Spnb1 2616.5980 0.6285 0.6700 0.2050 3.2689 0.0010798 0.0376893

spectrin beta, erythrocytic [Source:MGI Symbol;Acc:MGI:98387]

ENSMUSG00000027533 Fabp5 877.3174 1.8858 -0.9152 0.2797 -3.2719 0.0010681 0.0376893

fatty acid binding protein 5, epidermal [Source:MGI Symbol;Acc:MGI:101790]

147

ENSMUSG00000029649 Pomp 4660.8216 1.7727 -0.8259 0.2512 -3.2880 0.0010092 0.0376893

proteasome maturation protein [Source:MGI Symbol;Acc:MGI:1913787]

ENSMUSG00000031897 Psmb10 4240.8310 1.9738 -0.9810 0.3002 -3.2677 0.0010842 0.0376893

proteasome (prosome, macropain) subunit, beta type 10 [Source:MGI Symbol;Acc:MGI:1096380]

ENSMUSG00000034612 Chst11 647.6083 0.7026 0.5092 0.1558 3.2686 0.0010809 0.0376893

carbohydrate sulfotransferase 11 [Source:MGI Symbol;Acc:MGI:1927166]

ENSMUSG00000037999 Arap2 489.6614 0.4847 1.0449 0.3186 3.2801 0.0010377 0.0376893

ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2 [Source:MGI Symbol;Acc:MGI:2684416]

ENSMUSG00000038725 Pkhd1l1 205.7559 0.6439 0.6351 0.1935 3.2832 0.0010263 0.0376893

polycystic kidney and hepatic disease 1-like 1 [Source:MGI Symbol;Acc:MGI:2183153]

ENSMUSG00000042851 Zc3h6 133.8091 0.4832 1.0493 0.3196 3.2827 0.0010282 0.0376893

zinc finger CCCH type containing 6 [Source:MGI Symbol;Acc:MGI:1926001]

ENSMUSG00000043091 Tuba1c 4437.8076 1.7764 -0.8289 0.2531 -3.2756 0.0010545 0.0376893

tubulin, alpha 1C [Source:MGI Symbol;Acc:MGI:1095409]

ENSMUSG00000051355 Commd1 2014.2125 1.8156 -0.8604 0.2626 -3.2760 0.0010528 0.0376893

COMM domain containing 1 [Source:MGI Symbol;Acc:MGI:109474]

ENSMUSG00000053113 Socs3 902.6390 1.9293 -0.9481 0.2893 -3.2767 0.0010502 0.0376893

suppressor of cytokine signaling 3 [Source:MGI Symbol;Acc:MGI:1201791]

ENSMUSG00000056629 Fkbp2 2093.6492 2.0683 -1.0484 0.3205 -3.2716 0.0010696 0.0376893

FK506 binding protein 2 [Source:MGI Symbol;Acc:MGI:95542]

148

ENSMUSG00000057244 Gm6139 541.3756 2.2056 -1.1412 0.3490 -3.2696 0.0010769 0.0376893

predicted gene 6139 [Source:MGI Symbol;Acc:MGI:3643279]

ENSMUSG00000059796 Eif4a1 22883.0836 1.4578 -0.5438 0.1659 -3.2770 0.0010492 0.0376893

eukaryotic translation initiation factor 4A1 [Source:MGI Symbol;Acc:MGI:95303]

ENSMUSG00000075289 Carns1 254.6907 0.5573 0.8434 0.2576 3.2735 0.0010624 0.0376893carnosine synthase 1 [Source:MGI Symbol;Acc:MGI:2147595]

ENSMUSG00000078941 Taf9 716.9213 1.7878 -0.8382 0.2560 -3.2742 0.0010596 0.0376893

adenylate kinase 6 [Source:MGI Symbol;Acc:MGI:5510732]

ENSMUSG00000023055 Calcoco1 854.1152 0.6185 0.6931 0.2122 3.2665 0.001089 0.0377299

calcium binding and coiled coil domain 1 [Source:MGI Symbol;Acc:MGI:1914738]

ENSMUSG00000032221 Mns1 1330.5622 0.6947 0.5256 0.1611 3.2627 0.0011037 0.0381154

meiosis-specific nuclear structural protein 1 [Source:MGI Symbol;Acc:MGI:107933]

ENSMUSG00000057130 Txnl4a 643.2355 1.9234 -0.9437 0.2894 -3.2612 0.0011096 0.0381918

thioredoxin-like 4A [Source:MGI Symbol;Acc:MGI:1351613]

ENSMUSG00000017781 Pitpna 8601.8945 1.3975 -0.4828 0.1482 -3.2590 0.0011182 0.0383002

phosphatidylinositol transfer protein, alpha [Source:MGI Symbol;Acc:MGI:99887]

ENSMUSG00000045538 Ddx28 655.6697 1.6374 -0.7114 0.2183 -3.2585 0.0011201 0.0383002

DEAD (Asp-Glu-Ala-Asp) box polypeptide 28 [Source:MGI Symbol;Acc:MGI:1919236]

ENSMUSG00000023216 Epb4.2 330.7008 0.5457 0.8738 0.2683 3.2565 0.0011279 0.0384403

erythrocyte protein band 4.2 [Source:MGI Symbol;Acc:MGI:95402]

ENSMUSG00000005125 Ndrg1 1305.3827 1.5265 -0.6102 0.1877 -3.2518 0.0011469 0.0389603

N-myc downstream regulated gene 1 [Source:MGI Symbol;Acc:MGI:1341799]

149

ENSMUSG00000032301 Psma4 4240.8398 1.5401 -0.6230 0.1918 -3.2487 0.0011593 0.0391285


ENSMUSG00000047842 Diras2 127.0844 0.4672 1.0979 0.3379 3.2495 0.0011562 0.0391285

DIRAS family, GTP-binding RAS-like 2 [Source:MGI Symbol;Acc:MGI:1915453]

ENSMUSG00000029142 Mrpl33 5717.7833 2.1222 -1.0856 0.3343 -3.2475 0.0011641 0.039163 NA

ENSMUSG00000011752 Pgam1 8734.8355 1.7055 -0.7702 0.2374 -3.2441 0.001178 0.0392519

phosphoglycerate mutase 1 [Source:MGI Symbol;Acc:MGI:97552]

ENSMUSG00000038967 Pdk2 274.2446 0.6129 0.7063 0.2177 3.2445 0.0011767 0.0392519

pyruvate dehydrogenase kinase, isoenzyme 2 [Source:MGI Symbol;Acc:MGI:1343087]

ENSMUSG00000057605 Gm6807 201.5407 2.6253 -1.3925 0.4291 -3.2452 0.0011738 0.0392519


ENSMUSG00000062963 Ufc1 2451.1403 1.7452 -0.8034 0.2479 -3.2411 0.0011905 0.039541

ubiquitin-fold modifier conjugating enzyme 1 [Source:MGI Symbol;Acc:MGI:1913405]

ENSMUSG00000009687 Fxyd5 7933.1317 1.5645 -0.6457 0.1995 -3.2364 0.0012107 0.0400825

FXYD domain-containing ion transport regulator 5 [Source:MGI Symbol;Acc:MGI:1201785]

ENSMUSG00000009863 Sdhb 4370.4343 1.5537 -0.6357 0.1966 -3.2337 0.001222 0.0402629

succinate dehydrogenase complex, subunit B, iron sulfur (Ip) [Source:MGI Symbol;Acc:MGI:1914930]

ENSMUSG00000046959 Slc26a1 415.7931 0.5066 0.9810 0.3034 3.2333 0.0012238 0.0402629

solute carrier family 26 (sulfate transporter), member 1 [Source:MGI Symbol;Acc:MGI:2385894]

150

ENSMUSG00000026672 Optn 726.1729 0.7073 0.4997 0.1548 3.2283 0.0012451 0.0408339

optineurin [Source:MGI Symbol;Acc:MGI:1918898]

ENSMUSG00000067653 Ankrd23 101.8361 0.5302 0.9153 0.2836 3.2268 0.0012517 0.0409203

ankyrin repeat domain 23 [Source:MGI Symbol;Acc:MGI:1925571]

ENSMUSG00000021214 Akr1c18 120.0640 2.1952 -1.1343 0.3518 -3.2242 0.0012634 0.0411744

aldo-keto reductase family 1, member C18 [Source:MGI Symbol;Acc:MGI:2145420]

ENSMUSG00000034751 Mast4 184.7416 0.5164 0.9533 0.2959 3.2222 0.0012721 0.0413306

microtubule associated serine/threonine kinase family member 4 [Source:MGI Symbol;Acc:MGI:1918885]

ENSMUSG00000038510 Rpf2 1292.8160 1.5262 -0.6099 0.1897 -3.2159 0.0013002 0.0421113

ribosome production factor 2 homolog (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1914489]

ENSMUSG00000026547 Tagln2 13093.8658 1.5687 -0.6495 0.2021 -3.2132 0.0013125 0.0422485

transgelin 2 [Source:MGI Symbol;Acc:MGI:1312985]

ENSMUSG00000055943 2900064A13 2051.3176 1.4682 -0.5541 0.1724 -3.2140 0.001309 0.0422485

ER membrane protein complex subunit 7 [Source:MGI Symbol;Acc:MGI:1920274]

ENSMUSG00000018442 Derl2 1493.0133 1.5065 -0.5912 0.1843 -3.2076 0.0013383 0.0427749

Der1-like domain family, member 2 [Source:MGI Symbol;Acc:MGI:2151483]

ENSMUSG00000030268 Bcat1 162.5849 1.6370 -0.7111 0.2217 -3.2076 0.0013384 0.0427749

branched chain aminotransferase 1, cytosolic [Source:MGI Symbol;Acc:MGI:104861]

ENSMUSG00000040532 Abhd11 1144.8003 1.5384 -0.6214 0.1938 -3.2067 0.0013428 0.0427749

abhydrolase domain containing 11 [Source:MGI Symbol;Acc:MGI:1916008]

151

ENSMUSG00000041035 Dpcd 723.4666 2.0355 -1.0254 0.3198 -3.2061 0.0013453 0.0427749

deleted in primary ciliary dyskinesia [Source:MGI Symbol;Acc:MGI:1924407]

ENSMUSG00000026489 Adck3 417.6566 0.5994 0.7383 0.2306 3.2022 0.0013637 0.0430965

aarF domain containing kinase 3 [Source:MGI Symbol;Acc:MGI:1914676]

ENSMUSG00000032733 Snx33 230.0158 0.6147 0.7020 0.2192 3.2026 0.0013621 0.0430965

sorting nexin 33 [Source:MGI Symbol;Acc:MGI:2443239]

ENSMUSG00000006360 Crip1 8862.1661 2.1571 -1.1091 0.3469 -3.1976 0.001386 0.0434067

cysteine-rich protein 1 (intestinal) [Source:MGI Symbol;Acc:MGI:88501]

ENSMUSG00000025466 1810014F10 176.5644 2.0682 -1.0484 0.3277 -3.1989 0.0013796 0.0434067fucose mutarotase [Source:MGI Symbol;Acc:MGI:1916314]

ENSMUSG00000028478 Clta 8762.1226 1.4718 -0.5575 0.1744 -3.1976 0.0013859 0.0434067

clathrin, light polypeptide (Lca) [Source:MGI Symbol;Acc:MGI:894297]

ENSMUSG00000040276 Pacsin1 183.6015 0.5343 0.9042 0.2829 3.1965 0.0013911 0.043435

protein kinase C and casein kinase substrate in neurons 1 [Source:MGI Symbol;Acc:MGI:1345181]

ENSMUSG00000021520 Uqcrb 2722.6905 1.9565 -0.9682 0.3035 -3.1907 0.0014194 0.0441866

ubiquinol-cytochrome c reductase binding protein [Source:MGI Symbol;Acc:MGI:1914780]

ENSMUSG00000027776 Il12a 337.4807 0.5974 0.7433 0.2331 3.1889 0.0014284 0.0443356

interleukin 12a [Source:MGI Symbol;Acc:MGI:96539]

ENSMUSG00000016024 Lbp 1666.5307 1.6959 -0.7621 0.2391 -3.1876 0.0014346 0.0443486

lipopolysaccharide binding protein [Source:MGI Symbol;Acc:MGI:1098776]

ENSMUSG00000031266 Gla 427.4288 1.8301 -0.8719 0.2736 -3.1870 0.0014373 0.0443486

galactosidase, alpha [Source:MGI Symbol;Acc:MGI:1347344]

152

ENSMUSG00000006301 Tmbim1 556.6762 1.5096 -0.5941 0.1866 -3.1842 0.0014516 0.0443963

transmembrane BAX inhibitor motif containing 1 [Source:MGI Symbol;Acc:MGI:1916910]

ENSMUSG00000030760 Acer3 2051.2056 1.6736 -0.7429 0.2333 -3.1849 0.001448 0.0443963alkaline ceramidase 3 [Source:MGI Symbol;Acc:MGI:1913440]

ENSMUSG00000090166 Ear10 249.4202 2.6536 -1.4079 0.4422 -3.1842 0.0014516 0.0443963

eosinophil-associated, ribonuclease A family, member 10 [Source:MGI Symbol;Acc:MGI:1890464]

ENSMUSG00000028998 Tomm7 3846.4129 2.0291 -1.0208 0.3209 -3.1815 0.0014653 0.0446842

translocase of outer mitochondrial membrane 7 homolog (yeast) [Source:MGI Symbol;Acc:MGI:1913419]

ENSMUSG00000006517 Mvd 633.1381 1.5572 -0.6389 0.2011 -3.1763 0.0014916 0.0450223

mevalonate (diphospho) decarboxylase [Source:MGI Symbol;Acc:MGI:2179327]

ENSMUSG00000037601 Nme1 1985.6752 2.3344 -1.2231 0.3849 -3.1773 0.0014868 0.0450223

NME/NM23 nucleoside diphosphate kinase 1 [Source:MGI Symbol;Acc:MGI:97355]

ENSMUSG00000058715 Fcer1g 7715.7357 1.6051 -0.6826 0.2149 -3.1759 0.0014937 0.0450223

Fc receptor, IgE, high affinity I, gamma polypeptide [Source:MGI Symbol;Acc:MGI:95496]

ENSMUSG00000060992 Copz1 6388.0189 1.5701 -0.6508 0.2048 -3.1776 0.0014848 0.0450223

coatomer protein complex, subunit zeta 1 [Source:MGI Symbol;Acc:MGI:1929063]

ENSMUSG00000054676 1600014C10 3107.1754 1.5298 -0.6133 0.1932 -3.1740 0.0015038 0.0451962

RIKEN cDNA 1600014C10 gene [Source:MGI Symbol;Acc:MGI:1919494]

153

ENSMUSG00000071072 Ptges3 7646.3448 1.6023 -0.6802 0.2144 -3.1730 0.0015085 0.045207

prostaglandin E synthase 3 (cytosolic) [Source:MGI Symbol;Acc:MGI:1929282]

ENSMUSG00000032553 Srprb 954.6565 1.6099 -0.6869 0.2167 -3.1702 0.0015231 0.0453835

signal recognition particle receptor, B subunit [Source:MGI Symbol;Acc:MGI:102964]

ENSMUSG00000059040 Gm5506 3059.1572 1.5191 -0.6032 0.1902 -3.1710 0.0015192 0.0453835

enolase 1B, retrotransposed [Source:MGI Symbol;Acc:MGI:3648653]

ENSMUSG00000006567 Atp7b 694.3826 0.6345 0.6563 0.2072 3.1681 0.0015343 0.0455871

ATPase, Cu++ transporting, beta polypeptide [Source:MGI Symbol;Acc:MGI:103297]

ENSMUSG00000018770 Atp5g3 10374.4972 1.8674 -0.9011 0.2847 -3.1650 0.001551 0.0457036

ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C3 (subunit 9) [Source:MGI Symbol;Acc:MGI:2442035]

ENSMUSG00000020340 Cyfip2 5923.8385 0.6070 0.7203 0.2276 3.1651 0.00155 0.0457036

cytoplasmic FMR1 interacting protein 2 [Source:MGI Symbol;Acc:MGI:1924134]

ENSMUSG00000020490 Butr1 543.0699 0.6710 0.5755 0.1818 3.1649 0.0015514 0.0457036

butyrophilin-like 10 [Source:MGI Symbol;Acc:MGI:2182073]

ENSMUSG00000035649 Zcchc7 1153.4243 0.7199 0.4741 0.1499 3.1638 0.001557 0.0457419

zinc finger, CCHC domain containing 7 [Source:MGI Symbol;Acc:MGI:2442912]

ENSMUSG00000028496 Mllt3 784.0092 0.6952 0.5246 0.1659 3.1622 0.0015659 0.0458729

myeloid/lymphoid or mixed-lineage leukemia (trithorax homolog, Drosophila); translocated to, 3 [Source:MGI Symbol;Acc:MGI:1917372]

154

ENSMUSG00000026532 Spna1 1064.6117 0.5755 0.7971 0.2523 3.1596 0.0015798 0.0461507

spectrin alpha, erythrocytic 1 [Source:MGI Symbol;Acc:MGI:98385]

ENSMUSG00000039686 Zer1 1010.0528 0.6458 0.6309 0.2001 3.1534 0.0016136 0.0470062

zyg-11 related, cell cycle regulator [Source:MGI Symbol;Acc:MGI:2442511]

ENSMUSG00000021290 2010107E04 3773.4103 2.0159 -1.0114 0.3209 -3.1519 0.0016222 0.0470769

RIKEN cDNA 2010107E04 gene [Source:MGI Symbol;Acc:MGI:1917507]

ENSMUSG00000078193 Gm2000 635.4233 2.4832 -1.3122 0.4164 -3.1514 0.0016251 0.0470769


ENSMUSG00000028837 Psmb2 3494.7921 1.5621 -0.6435 0.2043 -3.1497 0.0016342 0.0472101


ENSMUSG00000019782 Rwdd1 1836.9629 1.5908 -0.6698 0.2130 -3.1450 0.001661 0.0475898

RWD domain containing 1 [Source:MGI Symbol;Acc:MGI:1913771]

ENSMUSG00000038970 Lmtk2 1198.8733 0.6678 0.5826 0.1852 3.1458 0.0016563 0.0475898

lemur tyrosine kinase 2 [Source:MGI Symbol;Acc:MGI:3036247]

ENSMUSG00000040447 Spns2 440.6433 0.5882 0.7656 0.2434 3.1456 0.0016575 0.0475898

spinster homolog 2 [Source:MGI Symbol;Acc:MGI:2384936]

ENSMUSG00000030104 Edem1 6564.9499 1.4586 -0.5446 0.1732 -3.1440 0.0016664 0.0476126

ER degradation enhancer, mannosidase alpha-like 1 [Source:MGI Symbol;Acc:MGI:2180139]

ENSMUSG00000024163 Mapk8ip3 1201.4242 0.7093 0.4955 0.1577 3.1415 0.0016809 0.0478977

mitogen-activated protein kinase 8 interacting protein 3 [Source:MGI Symbol;Acc:MGI:1353598]

155

ENSMUSG00000032330 Cox7a2 4313.0154 1.9108 -0.9342 0.2978 -3.1367 0.0017088 0.0484273

cytochrome c oxidase subunit VIIa 2 [Source:MGI Symbol;Acc:MGI:1316715]

ENSMUSG00000040964 Arhgef10l 1139.6885 1.8845 -0.9142 0.2914 -3.1374 0.0017046 0.0484273

Rho guanine nucleotide exchange factor (GEF) 10-like [Source:MGI Symbol;Acc:MGI:1920004]

ENSMUSG00000032123 Dpagt1 1093.0633 1.4865 -0.5719 0.1827 -3.1309 0.0017426 0.0492505

dolichyl-phosphate (UDP-N-acetylglucosamine) acetylglucosaminephosphotransferase 1 (GlcNAc-1-P transferase) [Source:MGI Symbol;Acc:MGI:1196396]

ENSMUSG00000013160 Atp6v0d1 4264.1555 1.5526 -0.6347 0.2030 -3.1270 0.0017658 0.0495307

ATPase, H+ transporting, lysosomal V0 subunit D1 [Source:MGI Symbol;Acc:MGI:1201778]

ENSMUSG00000029066 Mrpl20 2457.9474 1.7935 -0.8428 0.2695 -3.1269 0.0017667 0.0495307


ENSMUSG00000050379 40791 2014.0474 0.6061 0.7223 0.2309 3.1280 0.0017598 0.0495307

septin 6 [Source:MGI Symbol;Acc:MGI:1888939]

ENSMUSG00000024121 Atp6v0c 10107.6062 1.7197 -0.7821 0.2502 -3.1255 0.0017752 0.0496351

ATPase, H+ transporting, lysosomal V0 subunit C [Source:MGI Symbol;Acc:MGI:88116]

ENSMUSG00000026750 Psmb7 5137.2642 1.6619 -0.7329 0.2346 -3.1236 0.0017865 0.0498177


156

ENSMUSG00000060591 Ifitm2 15103.0691 1.4517 -0.5378 0.1723 -3.1207 0.0018045 0.0501834

interferon induced transmembrane protein 2 [Source:MGI Symbol;Acc:MGI:1933382]

ENSMUSG00000026433 Rab7l1 929.7305 1.5634 -0.6447 0.2067 -3.1197 0.0018106 0.0502199

RAB29, member RAS oncogene family [Source:MGI Symbol;Acc:MGI:2385107]

ENSMUSG00000078572 1810043H0 621.5653 1.9937 -0.9955 0.3192 -3.1183 0.0018191 0.0503208

RIKEN cDNA 1810043H04 gene [Source:MGI Symbol;Acc:MGI:1913676]

ENSMUSG00000062127 Cttnbp2nl 127.4342 2.6429 -1.4021 0.4505 -3.1124 0.001856 0.0512069CTTNBP2 N-terminal like [Source:MGI Symbol;Acc:MGI:1933137]

ENSMUSG00000000563 Atp5f1 10310.5744 1.5642 -0.6454 0.2076 -3.1092 0.0018761 0.0512773

ATP synthase, H+ transporting, mitochondrial F0 complex, subunit B1 [Source:MGI Symbol;Acc:MGI:1100495]

ENSMUSG00000016933 Plcg1 588.5833 0.5923 0.7556 0.2429 3.1101 0.0018699 0.0512773

phospholipase C, gamma 1 [Source:MGI Symbol;Acc:MGI:97615]

ENSMUSG00000022193 Psmb5 2904.5231 1.9604 -0.9711 0.3123 -3.1092 0.0018757 0.0512773


ENSMUSG00000026187 Xrcc5 507.4842 0.6613 0.5966 0.1919 3.1088 0.0018782 0.0512773

X-ray repair complementing defective repair in Chinese hamster cells 5 [Source:MGI Symbol;Acc:MGI:104517]

ENSMUSG00000029462 Vps29 1510.9531 1.3649 -0.4488 0.1444 -3.1074 0.0018872 0.0513888

vacuolar protein sorting 29 (S. pombe) [Source:MGI Symbol;Acc:MGI:1928344]

157

ENSMUSG00000024424 Ttc39c 364.8239 1.7848 -0.8358 0.2691 -3.1062 0.0018948 0.0514609

tetratricopeptide repeat domain 39C [Source:MGI Symbol;Acc:MGI:1919997]

ENSMUSG00000039081 Zfp503 103.2163 1.7761 -0.8287 0.2669 -3.1051 0.0019024 0.0515341


ENSMUSG00000031851 Ntpcr 765.7581 1.4554 -0.5414 0.1744 -3.1036 0.0019119 0.0516566

nucleoside-triphosphatase, cancer-related [Source:MGI Symbol;Acc:MGI:1913816]

ENSMUSG00000028334 Nans 3018.7745 1.4188 -0.5047 0.1628 -3.1009 0.0019295 0.0518636

N-acetylneuraminic acid synthase (sialic acid synthase) [Source:MGI Symbol;Acc:MGI:2149820]

ENSMUSG00000053617 Sh3pxd2a 285.8627 0.5488 0.8658 0.2792 3.1010 0.0019289 0.0518636SH3 and PX domains 2A [Source:MGI Symbol;Acc:MGI:1298393]

ENSMUSG00000030225 Dera 1736.1144 1.3582 -0.4417 0.1425 -3.0988 0.0019428 0.0520865

2-deoxyribose-5-phosphate aldolase homolog (C. elegans) [Source:MGI Symbol;Acc:MGI:1913762]

ENSMUSG00000050914 Ankrd37 155.2917 1.9710 -0.9789 0.3162 -3.0957 0.0019635 0.0525055

ankyrin repeat domain 37 [Source:MGI Symbol;Acc:MGI:3603344]

ENSMUSG00000016319 Slc25a5 18801.4806 1.4229 -0.5089 0.1645 -3.0941 0.0019744 0.0526625

solute carrier family 25 (mitochondrial carrier, adenine nucleotide translocator), member 5 [Source:MGI Symbol;Acc:MGI:1353496]

ENSMUSG00000000028 Cdc45 2352.2479 1.4302 -0.5162 0.1670 -3.0903 0.0019994 0.0527221

cell division cycle 45 [Source:MGI Symbol;Acc:MGI:1338073]

158

ENSMUSG00000022453 Naga 1965.3742 1.4702 -0.5561 0.1799 -3.0908 0.0019962 0.0527221

N-acetyl galactosaminidase, alpha [Source:MGI Symbol;Acc:MGI:1261422]

ENSMUSG00000034932 Mrpl54 1521.5204 1.9497 -0.9633 0.3117 -3.0899 0.0020019 0.0527221


ENSMUSG00000050373 Snx21 155.9071 1.6000 -0.6781 0.2193 -3.0915 0.0019916 0.0527221

sorting nexin family member 21 [Source:MGI Symbol;Acc:MGI:1917729]

ENSMUSG00000052419 2610001J05 1226.1825 1.4712 -0.5570 0.1801 -3.0923 0.0019864 0.0527221

RIKEN cDNA 2610001J05 gene [Source:MGI Symbol;Acc:MGI:1913770]

ENSMUSG00000009621 Vav2 1003.2984 0.5648 0.8243 0.2669 3.0882 0.0020136 0.0527642

vav 2 oncogene [Source:MGI Symbol;Acc:MGI:102718]

ENSMUSG00000038670 Mybpc2 109.5246 0.4350 1.2009 0.3888 3.0888 0.0020098 0.0527642

myosin binding protein C, fast-type [Source:MGI Symbol;Acc:MGI:1336170]

ENSMUSG00000024959 Bad 1073.8899 1.7598 -0.8154 0.2642 -3.0860 0.002029 0.0530332

BCL2-associated agonist of cell death [Source:MGI Symbol;Acc:MGI:1096330]

ENSMUSG00000027175 Tcp11l1 109.6288 1.7591 -0.8148 0.2642 -3.0836 0.0020452 0.0531904

t-complex 11 like 1 [Source:MGI Symbol;Acc:MGI:2444263]

ENSMUSG00000031545 Agpat6 2131.1616 1.3516 -0.4347 0.1410 -3.0837 0.0020444 0.0531904

1-acylglycerol-3-phosphate O-acyltransferase 6 (lysophosphatidic acid acyltransferase, zeta) [Source:MGI Symbol;Acc:MGI:2142716]

ENSMUSG00000059336 Slc14a1 1409.1294 0.6938 0.5274 0.1711 3.0819 0.0020567 0.0533558

solute carrier family 14 (urea transporter), member 1 [Source:MGI Symbol;Acc:MGI:1351654]

159

ENSMUSG00000079018 Ly6c1 764.1658 1.5157 -0.6000 0.1948 -3.0802 0.0020687 0.0535356

lymphocyte antigen 6 complex, locus C1 [Source:MGI Symbol;Acc:MGI:96882]

ENSMUSG00000001847 Rac1 12027.0857 1.3750 -0.4595 0.1492 -3.0791 0.0020763 0.053598

RAS-related C3 botulinum substrate 1 [Source:MGI Symbol;Acc:MGI:97845]

ENSMUSG00000023944 Hsp90ab1 54763.6234 1.3110 -0.3906 0.1269 -3.0772 0.0020893 0.0537394

heat shock protein 90 alpha (cytosolic), class B member 1 [Source:MGI Symbol;Acc:MGI:96247]

ENSMUSG00000069744 Psmb3 4989.3089 1.8318 -0.8733 0.2838 -3.0768 0.0020921 0.0537394


ENSMUSG00000035585 Tsen34 1032.9682 1.8040 -0.8512 0.2768 -3.0749 0.002106 0.0539636

tRNA splicing endonuclease 34 homolog (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1913328]

ENSMUSG00000015568 Lpl 626.4462 0.5556 0.8478 0.2760 3.0717 0.0021286 0.0544094

lipoprotein lipase [Source:MGI Symbol;Acc:MGI:96820]

ENSMUSG00000022353 Mtss1 1598.6345 0.5538 0.8525 0.2777 3.0695 0.0021441 0.0546723

metastasis suppressor 1 [Source:MGI Symbol;Acc:MGI:2384818]

ENSMUSG00000024208 2900010M2 1736.3413 1.9970 -0.9978 0.3252 -3.0687 0.0021501 0.0546917

ubiquinol-cytochrome c reductase complex assembly factor 2 [Source:MGI Symbol;Acc:MGI:1914517]

ENSMUSG00000026203 Dnajb2 160.9200 0.5723 0.8053 0.2628 3.0636 0.002187 0.0549046

DnaJ (Hsp40) homolog, subfamily B, member 2 [Source:MGI Symbol;Acc:MGI:1928739]

160

ENSMUSG00000035459 Stab2 95.9393 0.5356 0.9008 0.2940 3.0636 0.0021871 0.0549046

stabilin 2 [Source:MGI Symbol;Acc:MGI:2178743]

ENSMUSG00000036053 Fmnl2 587.2168 0.6923 0.5304 0.1732 3.0632 0.0021901 0.0549046

formin-like 2 [Source:MGI Symbol;Acc:MGI:1918659]

ENSMUSG00000037922 Bank1 983.7946 0.4375 1.1927 0.3891 3.0654 0.0021735 0.0549046

B cell scaffold protein with ankyrin repeats 1 [Source:MGI Symbol;Acc:MGI:2442120]

ENSMUSG00000040669 Phc1 383.5313 0.6547 0.6112 0.1995 3.0641 0.0021829 0.0549046

polyhomeotic-like 1 (Drosophila) [Source:MGI Symbol;Acc:MGI:103248]

ENSMUSG00000049751 Rpl36al 10404.8345 1.9720 -0.9797 0.3196 -3.0648 0.0021779 0.0549046

ribosomal protein L36A-like [Source:MGI Symbol;Acc:MGI:1913733]

ENSMUSG00000015127 Unkl 557.3719 0.7112 0.4916 0.1606 3.0612 0.0022048 0.055142

unkempt-like (Drosophila) [Source:MGI Symbol;Acc:MGI:1921404]

ENSMUSG00000023243 Kcnk5 219.4451 0.6342 0.6569 0.2147 3.0597 0.0022159 0.0552872

potassium channel, subfamily K, member 5 [Source:MGI Symbol;Acc:MGI:1336175]

ENSMUSG00000047547 Cltb 773.0069 1.6637 -0.7344 0.2401 -3.0586 0.0022234 0.0553421

clathrin, light polypeptide (Lcb) [Source:MGI Symbol;Acc:MGI:1921575]

ENSMUSG00000020635 Fkbp1b 222.4819 1.7523 -0.8092 0.2650 -3.0542 0.0022566 0.055978

FK506 binding protein 1b [Source:MGI Symbol;Acc:MGI:1336205]

ENSMUSG00000063556 Gm10132 278.4592 2.3272 -1.2186 0.3990 -3.0538 0.0022597 0.055978predicted gene 10132 [Source:MGI Symbol;Acc:MGI:3704449]

ENSMUSG00000025218 Poll 553.8798 1.5108 -0.5953 0.1951 -3.0514 0.0022777 0.0561734

polymerase (DNA directed), lambda [Source:MGI Symbol;Acc:MGI:1889000]

161

ENSMUSG00000027282 Mtch2 3024.7380 1.5136 -0.5979 0.1960 -3.0513 0.0022784 0.0561734

mitochondrial carrier homolog 2 (C. elegans) [Source:MGI Symbol;Acc:MGI:1929260]

ENSMUSG00000032216 Nedd4 3580.2298 0.5867 0.7692 0.2523 3.0489 0.0022965 0.0564337

neural precursor cell expressed, developmentally down-regulated 4 [Source:MGI Symbol;Acc:MGI:97297]

ENSMUSG00000048232 Fbxo10 647.2600 0.6641 0.5905 0.1937 3.0485 0.0022998 0.0564337

F-box protein 10 [Source:MGI Symbol;Acc:MGI:2686937]

ENSMUSG00000005779 Psmb4 8241.4917 1.8507 -0.8881 0.2917 -3.0445 0.0023309 0.0570631


ENSMUSG00000005732 Ranbp1 6607.4594 1.7492 -0.8067 0.2653 -3.0404 0.0023625 0.0575669RAN binding protein 1 [Source:MGI Symbol;Acc:MGI:96269]

ENSMUSG00000006095 Tbcb 1949.5438 1.7513 -0.8084 0.2658 -3.0410 0.0023579 0.0575669

tubulin folding cofactor B [Source:MGI Symbol;Acc:MGI:1913661]

ENSMUSG00000044229 Fam55d 294.8115 0.6732 0.5709 0.1878 3.0395 0.0023699 0.0576132

neurexophilin and PC-esterase domain family, member 4 [Source:MGI Symbol;Acc:MGI:1924792]

ENSMUSG00000028861 Mrps15 1866.2065 1.7048 -0.7696 0.2534 -3.0375 0.0023856 0.0578024

mitochondrial ribosomal protein S15 [Source:MGI Symbol;Acc:MGI:1913657]

ENSMUSG00000052949 Rnf157 418.9532 0.5652 0.8231 0.2710 3.0371 0.0023888 0.0578024

ring finger protein 157 [Source:MGI Symbol;Acc:MGI:2442484]

ENSMUSG00000038020 Rapgefl1 349.7939 0.5159 0.9548 0.3149 3.0322 0.0024276 0.0586056

Rap guanine nucleotide exchange factor (GEF)-like 1 [Source:MGI Symbol;Acc:MGI:3611446]

162

ENSMUSG00000022440 C1qtnf6 348.4578 1.8868 -0.9159 0.3025 -3.0282 0.0024599 0.0586342

C1q and tumor necrosis factor related protein 6 [Source:MGI Symbol;Acc:MGI:1919959]

ENSMUSG00000022684 Bfar 1337.5731 1.3646 -0.4484 0.1481 -3.0279 0.0024626 0.0586342

bifunctional apoptosis regulator [Source:MGI Symbol;Acc:MGI:1914368]

ENSMUSG00000026576 Atp1b1 2514.7619 0.4846 1.0451 0.3452 3.0273 0.0024673 0.0586342

ATPase, Na+/K+ transporting, beta 1 polypeptide [Source:MGI Symbol;Acc:MGI:88108]

ENSMUSG00000030245 Golt1b 1235.9534 1.4324 -0.5184 0.1711 -3.0293 0.0024515 0.0586342

golgi transport 1 homolog B (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1914214]

ENSMUSG00000030337 Vamp1 266.3441 0.5920 0.7562 0.2499 3.0256 0.0024811 0.0586342vesicle-associated membrane protein 1 [Source:MGI Symbol;Acc:MGI:1313276]

ENSMUSG00000030560 Ctsc 9524.6543 2.4066 -1.2670 0.4187 -3.0259 0.002479 0.0586342

cathepsin C [Source:MGI Symbol;Acc:MGI:109553]

ENSMUSG00000030842 2400001E08 3694.8908 1.5626 -0.6439 0.2125 -3.0302 0.002444 0.0586342


ENSMUSG00000038312 Edem2 2944.1285 1.4088 -0.4945 0.1634 -3.0253 0.0024836 0.0586342

ER degradation enhancer, mannosidase alpha-like 2 [Source:MGI Symbol;Acc:MGI:1915540]

ENSMUSG00000043424 Gm9781 627.8109 1.5305 -0.6140 0.2028 -3.0282 0.0024604 0.0586342

eukaryotic translation initiation factor 3, subunit J2 [Source:MGI Symbol;Acc:MGI:3704486]

163

ENSMUSG00000055681 Cope 4586.2845 1.5509 -0.6331 0.2093 -3.0252 0.002485 0.0586342

coatomer protein complex, subunit epsilon [Source:MGI Symbol;Acc:MGI:1891702]

ENSMUSG00000020386 Sar1b 1409.6230 1.3744 -0.4588 0.1518 -3.0230 0.0025028 0.0586781

SAR1 gene homolog B (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1913647]

ENSMUSG00000020834 Dhrs13 320.9175 1.9007 -0.9266 0.3065 -3.0234 0.0024999 0.0586781

dehydrogenase/reductase (SDR family) member 13 [Source:MGI Symbol;Acc:MGI:1917701]

ENSMUSG00000041642 Kif21b 2484.6094 0.6373 0.6500 0.2150 3.0229 0.0025038 0.0586781

kinesin family member 21B [Source:MGI Symbol;Acc:MGI:109234]

ENSMUSG00000022820 Ndufb4 1582.1843 1.9086 -0.9325 0.3086 -3.0219 0.002512 0.0587333

NADH dehydrogenase (ubiquinone) 1 beta subcomplex 4 [Source:MGI Symbol;Acc:MGI:1915444]

ENSMUSG00000028035 Dnajb4 510.1506 0.6945 0.5260 0.1741 3.0212 0.0025174 0.0587333

DnaJ (Hsp40) homolog, subfamily B, member 4 [Source:MGI Symbol;Acc:MGI:1914285]

ENSMUSG00000024065 Ehd3 373.9050 0.5609 0.8342 0.2764 3.0178 0.0025463 0.0592751

EH-domain containing 3 [Source:MGI Symbol;Acc:MGI:1928900]

ENSMUSG00000018858 Ict1 1081.4353 1.7617 -0.8170 0.2712 -3.0125 0.0025912 0.060096

immature colon carcinoma transcript 1 [Source:MGI Symbol;Acc:MGI:1915822]

ENSMUSG00000028398 3110001D0 1616.7606 1.8745 -0.9065 0.3010 -3.0123 0.0025931 0.060096


164

ENSMUSG00000021037 Ahsa1 4869.6113 1.4125 -0.4983 0.1655 -3.0102 0.0026108 0.0602394

AHA1, activator of heat shock protein ATPase 1 [Source:MGI Symbol;Acc:MGI:2387603]

ENSMUSG00000027610 Gss 525.3037 1.7878 -0.8382 0.2784 -3.0108 0.0026059 0.0602394

glutathione synthetase [Source:MGI Symbol;Acc:MGI:95852]

ENSMUSG00000014551 Mrps25 1066.5253 1.6351 -0.7094 0.2361 -3.0048 0.0026578 0.0609189


ENSMUSG00000027673 Ndufb5 2758.1808 1.8529 -0.8898 0.2960 -3.0057 0.0026501 0.0609189

NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 5 [Source:MGI Symbol;Acc:MGI:1913296]

ENSMUSG00000036526 Card11 1058.2846 0.4673 1.0976 0.3653 3.0050 0.0026559 0.0609189


ENSMUSG00000008333 Snrpb2 2246.9720 1.4776 -0.5633 0.1876 -3.0030 0.002673 0.0609621

U2 small nuclear ribonucleoprotein B [Source:MGI Symbol;Acc:MGI:104805]

ENSMUSG00000016534 Lamp2 7725.2939 1.7022 -0.7674 0.2558 -2.9999 0.0027006 0.0609621

lysosomal-associated membrane protein 2 [Source:MGI Symbol;Acc:MGI:96748]

ENSMUSG00000029759 Pon3 641.9170 1.6451 -0.7182 0.2392 -3.0021 0.0026811 0.0609621

paraoxonase 3 [Source:MGI Symbol;Acc:MGI:106686]

ENSMUSG00000034203 Chchd4 1054.7236 1.7013 -0.7667 0.2554 -3.0014 0.0026872 0.0609621

coiled-coil-helix-coiled-coil-helix domain containing 4 [Source:MGI Symbol;Acc:MGI:1919420]

ENSMUSG00000037606 Osbpl5 224.5730 0.5464 0.8719 0.2905 3.0012 0.0026888 0.0609621

oxysterol binding protein-like 5 [Source:MGI Symbol;Acc:MGI:1930265]

165

ENSMUSG00000045071 E130308A19 308.8580 0.6710 0.5755 0.1918 3.0003 0.0026969 0.0609621

RIKEN cDNA E130308A19 gene [Source:MGI Symbol;Acc:MGI:2442164]

ENSMUSG00000057789 Bak1 2302.6645 1.5666 -0.6476 0.2158 -3.0006 0.0026947 0.0609621

BCL2-antagonist/killer 1 [Source:MGI Symbol;Acc:MGI:1097161]

ENSMUSG00000036751 Cox6b1 7949.0928 1.9512 -0.9644 0.3218 -2.9972 0.0027247 0.0613603

cytochrome c oxidase, subunit VIb polypeptide 1 [Source:MGI Symbol;Acc:MGI:107460]

ENSMUSG00000078485 Plekhn1 118.6749 0.6184 0.6935 0.2314 2.9966 0.00273 0.0613603

pleckstrin homology domain containing, family N member 1 [Source:MGI Symbol;Acc:MGI:2387630]

ENSMUSG00000021113 Snapc1 232.1131 1.5175 -0.6017 0.2009 -2.9950 0.0027447 0.0614907

small nuclear RNA activating complex, polypeptide 1 [Source:MGI Symbol;Acc:MGI:1922877]

ENSMUSG00000050965 Prkca 369.5964 0.6356 0.6537 0.2183 2.9947 0.0027476 0.0614907

protein kinase C, alpha [Source:MGI Symbol;Acc:MGI:97595]

ENSMUSG00000045404 Kcnk13 115.8797 2.4984 -1.3210 0.4413 -2.9934 0.0027586 0.0616044

potassium channel, subfamily K, member 13 [Source:MGI Symbol;Acc:MGI:2384976]

ENSMUSG00000020180 Snrpd3 4172.5806 1.6765 -0.7454 0.2491 -2.9924 0.0027682 0.0616869

small nuclear ribonucleoprotein D3 [Source:MGI Symbol;Acc:MGI:1914582]

ENSMUSG00000071528 Usmg5 2343.4172 1.9423 -0.9578 0.3203 -2.9902 0.0027884 0.0618099

upregulated during skeletal muscle growth 5 [Source:MGI Symbol;Acc:MGI:1891435]

ENSMUSG00000072324 Gm8420 246.9287 1.9461 -0.9606 0.3213 -2.9898 0.0027915 0.0618099


166

ENSMUSG00000078484 Klhl17 528.7390 0.7158 0.4824 0.1613 2.9900 0.0027897 0.0618099

kelch-like 17 [Source:MGI Symbol;Acc:MGI:2678948]

ENSMUSG00000033739 Fkbpl 216.0742 1.5877 -0.6669 0.2232 -2.9878 0.0028103 0.0619309

FK506 binding protein-like [Source:MGI Symbol;Acc:MGI:1932127]

ENSMUSG00000044080 S100a1 1031.4601 1.8568 -0.8928 0.2989 -2.9873 0.0028147 0.0619309


ENSMUSG00000063952 Brpf3 844.6299 0.7122 0.4897 0.1639 2.9873 0.0028148 0.0619309

bromodomain and PHD finger containing, 3 [Source:MGI Symbol;Acc:MGI:2146836]

ENSMUSG00000020019 Ntn4 114.8244 0.6165 0.6979 0.2339 2.9833 0.0028515 0.0623867netrin 4 [Source:MGI Symbol;Acc:MGI:1888978]

ENSMUSG00000021832 Psmc6 3595.7234 1.3433 -0.4258 0.1427 -2.9831 0.0028534 0.0623867

proteasome (prosome, macropain) 26S subunit, ATPase, 6 [Source:MGI Symbol;Acc:MGI:1914339]

ENSMUSG00000026796 Fam129b 1329.8966 2.1054 -1.0741 0.3600 -2.9838 0.0028472 0.0623867

family with sequence similarity 129, member B [Source:MGI Symbol;Acc:MGI:2442910]

ENSMUSG00000001056 Nhp2 3614.0623 1.9224 -0.9429 0.3167 -2.9772 0.0029093 0.0627378

NHP2 ribonucleoprotein [Source:MGI Symbol;Acc:MGI:1098547]

ENSMUSG00000024675 Ms4a4c 3146.1491 2.0321 -1.0230 0.3433 -2.9797 0.0028856 0.0627378

membrane-spanning 4-domains, subfamily A, member 4C [Source:MGI Symbol;Acc:MGI:1927656]

ENSMUSG00000025357 Dgka 1195.5857 0.4879 1.0354 0.3479 2.9763 0.0029176 0.0627378diacylglycerol kinase, alpha [Source:MGI Symbol;Acc:MGI:102952]

167

ENSMUSG00000026928 Card9 2511.7100 1.4980 -0.5830 0.1958 -2.9781 0.0029003 0.0627378


ENSMUSG00000030335 Mrpl51 2929.2075 1.7972 -0.8457 0.2840 -2.9785 0.0028966 0.0627378


ENSMUSG00000038175 Mylip 966.4871 0.5878 0.7666 0.2573 2.9796 0.0028861 0.0627378

myosin regulatory light chain interacting protein [Source:MGI Symbol;Acc:MGI:2388271]

ENSMUSG00000038418 Egr1 1325.1945 0.7180 0.4779 0.1606 2.9764 0.0029163 0.0627378

early growth response 1 [Source:MGI Symbol;Acc:MGI:95295]

ENSMUSG00000041926 Rnpep 1797.6073 1.8104 -0.8563 0.2875 -2.9784 0.0028976 0.0627378

arginyl aminopeptidase (aminopeptidase B) [Source:MGI Symbol;Acc:MGI:2384902]

ENSMUSG00000044068 Zrsr1 309.4604 0.6463 0.6296 0.2117 2.9749 0.0029312 0.062901

zinc finger (CCCH type), RNA binding motif and serine/arginine rich 1 [Source:MGI Symbol;Acc:MGI:98885]

ENSMUSG00000004451 Ralb 2307.4448 1.3800 -0.4647 0.1564 -2.9710 0.0029683 0.0635649

v-ral simian leukemia viral oncogene homolog B (ras related) [Source:MGI Symbol;Acc:MGI:1927244]

ENSMUSG00000013593 Ndufs2 5684.9777 1.5602 -0.6417 0.2161 -2.9699 0.0029786 0.0636264

NADH dehydrogenase (ubiquinone) Fe-S protein 2 [Source:MGI Symbol;Acc:MGI:2385112]

168

ENSMUSG00000020085 Aifm2 329.3904 1.6197 -0.6957 0.2344 -2.9682 0.0029955 0.0636264

apoptosis-inducing factor, mitochondrion-associated 2 [Source:MGI Symbol;Acc:MGI:1918611]

ENSMUSG00000028132 Tmem56 1172.9337 0.6324 0.6612 0.2227 2.9687 0.0029903 0.0636264


ENSMUSG00000029632 Ndufa4 6162.3222 1.9014 -0.9271 0.3123 -2.9690 0.0029874 0.0636264

NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4 [Source:MGI Symbol;Acc:MGI:107686]

ENSMUSG00000001467 Cyp51 728.7373 1.4098 -0.4955 0.1670 -2.9674 0.0030036 0.063667

cytochrome P450, family 51 [Source:MGI Symbol;Acc:MGI:106040]

ENSMUSG00000029650 Slc46a3 522.5531 0.6585 0.6028 0.2032 2.9667 0.0030105 0.0636712

solute carrier family 46, member 3 [Source:MGI Symbol;Acc:MGI:1918956]

ENSMUSG00000036986 Pml 2521.4624 0.6017 0.7330 0.2472 2.9655 0.0030216 0.0636712

promyelocytic leukemia [Source:MGI Symbol;Acc:MGI:104662]

ENSMUSG00000037152 Ndufc1 1357.6570 1.9161 -0.9381 0.3164 -2.9655 0.0030221 0.0636712

NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 1 [Source:MGI Symbol;Acc:MGI:1913627]

ENSMUSG00000042476 Abcb4 1651.6114 0.6097 0.7137 0.2408 2.9642 0.0030349 0.0638119

ATP-binding cassette, sub-family B (MDR/TAP), member 4 [Source:MGI Symbol;Acc:MGI:97569]

169

ENSMUSG00000031458 2410022L05 193.6231 1.9328 -0.9507 0.3208 -2.9635 0.0030413 0.0638177

coordinator of PRMT5, differentiation stimulator [Source:MGI Symbol;Acc:MGI:1913673]

ENSMUSG00000043323 Fbrsl1 481.1882 0.6988 0.5170 0.1746 2.9615 0.0030613 0.0640744

fibrosin-like 1 [Source:MGI Symbol;Acc:MGI:1920907]

ENSMUSG00000072214 40790 1313.5719 0.5609 0.8342 0.2817 2.9611 0.0030658 0.0640744

septin 5 [Source:MGI Symbol;Acc:MGI:1195461]

ENSMUSG00000020219 Timm13 3877.9131 2.1332 -1.0930 0.3692 -2.9603 0.003073 0.064076

translocase of inner mitochondrial membrane 13 [Source:MGI Symbol;Acc:MGI:1353432]

ENSMUSG00000034343 Ube2f 1936.3020 1.3799 -0.4645 0.1569 -2.9598 0.0030782 0.064076

ubiquitin-conjugating enzyme E2F (putative) [Source:MGI Symbol;Acc:MGI:1915171]

ENSMUSG00000017778 Cox7c 5706.0053 2.0641 -1.0455 0.3534 -2.9588 0.0030887 0.0641677

cytochrome c oxidase subunit VIIc [Source:MGI Symbol;Acc:MGI:103226]

ENSMUSG00000001666 Ddt 176.5334 1.9596 -0.9705 0.3284 -2.9553 0.0031233 0.0647069

D-dopachrome tautomerase [Source:MGI Symbol;Acc:MGI:1298381]

ENSMUSG00000035595 1600002K03 401.0018 1.9769 -0.9832 0.3327 -2.9550 0.0031271 0.0647069

RIKEN cDNA 1600002K03 gene [Source:MGI Symbol;Acc:MGI:1917020]

ENSMUSG00000026399 Cd55 1679.9545 0.5297 0.9167 0.3104 2.9530 0.0031471 0.0649397


ENSMUSG00000063856 Gpx1 37168.8017 1.7129 -0.7764 0.2630 -2.9526 0.0031508 0.0649397

glutathione peroxidase 1 [Source:MGI Symbol;Acc:MGI:104887]

ENSMUSG00000043671 Dpy19l3 227.1154 0.6396 0.6447 0.2187 2.9487 0.0031913 0.0656454

dpy-19-like 3 (C. elegans) [Source:MGI Symbol;Acc:MGI:2443952]

170

ENSMUSG00000022956 Atp5o 5772.2209 1.7953 -0.8442 0.2865 -2.9466 0.0032129 0.0659601

ATP synthase, H+ transporting, mitochondrial F1 complex, O subunit [Source:MGI Symbol;Acc:MGI:106341]

ENSMUSG00000003882 Il7r 1128.8405 0.4986 1.0041 0.3410 2.9442 0.0032382 0.0661178

interleukin 7 receptor [Source:MGI Symbol;Acc:MGI:96562]

ENSMUSG00000006057 Atp5g1 4880.3719 1.7621 -0.8173 0.2776 -2.9440 0.0032396 0.0661178

ATP synthase, H+ transporting, mitochondrial F0 complex, subunit C1 (subunit 9) [Source:MGI Symbol;Acc:MGI:107653]

ENSMUSG00000026073 Il1r2 831.6501 2.0465 -1.0331 0.3508 -2.9452 0.0032272 0.0661178

interleukin 1 receptor, type II [Source:MGI Symbol;Acc:MGI:96546]

ENSMUSG00000027199 Gatm 3557.9059 1.9468 -0.9611 0.3268 -2.9409 0.0032729 0.0665366

glycine amidinotransferase (L-arginine:glycine amidinotransferase) [Source:MGI Symbol;Acc:MGI:1914342]

ENSMUSG00000028080 Lrba 629.6852 0.6411 0.6415 0.2181 2.9414 0.0032673 0.0665366

LPS-responsive beige-like anchor [Source:MGI Symbol;Acc:MGI:1933162]

ENSMUSG00000002102 Psmc3 5493.6290 1.4489 -0.5350 0.1820 -2.9387 0.0032964 0.0666851

proteasome (prosome, macropain) 26S subunit, ATPase 3 [Source:MGI Symbol;Acc:MGI:1098754]

ENSMUSG00000020056 Ccdc53 948.6881 1.6362 -0.7104 0.2418 -2.9378 0.0033058 0.0666851

coiled-coil domain containing 53 [Source:MGI Symbol;Acc:MGI:1914532]

ENSMUSG00000021607 Mrpl36 1552.4220 1.6544 -0.7263 0.2474 -2.9359 0.0033262 0.0666851


171

ENSMUSG00000021759 Ppap2a 150.7778 0.6186 0.6929 0.2359 2.9368 0.0033167 0.0666851

phosphatidic acid phosphatase type 2A [Source:MGI Symbol;Acc:MGI:108412]

ENSMUSG00000023828 Slc22a3 154.3309 0.5934 0.7529 0.2565 2.9351 0.0033342 0.0666851

solute carrier family 22 (organic cation transporter), member 3 [Source:MGI Symbol;Acc:MGI:1333817]

ENSMUSG00000030532 Hddc3 610.2557 1.6474 -0.7202 0.2452 -2.9366 0.003318 0.0666851

HD domain containing 3 [Source:MGI Symbol;Acc:MGI:1915945]

ENSMUSG00000031543 Ank1 1744.4817 0.5940 0.7515 0.2560 2.9352 0.0033338 0.0666851ankyrin 1, erythroid [Source:MGI Symbol;Acc:MGI:88024]

ENSMUSG00000032035 Ets1 4706.3799 0.5180 0.9489 0.3230 2.9377 0.0033062 0.0666851E26 avian leukemia oncogene 1, 5' domain [Source:MGI Symbol;Acc:MGI:95455]

ENSMUSG00000038690 Atp5j2 5127.3488 1.8644 -0.8987 0.3062 -2.9348 0.0033378 0.0666851

ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F2 [Source:MGI Symbol;Acc:MGI:1927558]

ENSMUSG00000024902 Mrpl11 1376.9903 1.8569 -0.8929 0.3044 -2.9331 0.003356 0.0669216


ENSMUSG00000029918 Mrps33 787.3293 1.4953 -0.5804 0.1980 -2.9317 0.0033713 0.0670978


ENSMUSG00000002308 Cd320 269.1783 1.7931 -0.8425 0.2875 -2.9305 0.0033838 0.0672179


ENSMUSG00000002477 Snrpd1 4349.0796 1.8371 -0.8774 0.2997 -2.9278 0.0034139 0.0675048

small nuclear ribonucleoprotein D1 [Source:MGI Symbol;Acc:MGI:98344]

172

ENSMUSG00000025781 Atp5c1 7954.5253 1.3701 -0.4543 0.1552 -2.9274 0.0034176 0.0675048

ATP synthase, H+ transporting, mitochondrial F1 complex, gamma polypeptide 1 [Source:MGI Symbol;Acc:MGI:1261437]

ENSMUSG00000076437 2700094K13 2461.2543 1.8093 -0.8554 0.2921 -2.9284 0.0034074 0.0675048


ENSMUSG00000024800 Rpp30 757.1214 1.4632 -0.5491 0.1877 -2.9255 0.0034393 0.0676764

ribonuclease P/MRP 30 subunit [Source:MGI Symbol;Acc:MGI:1859683]

ENSMUSG00000053398 Phgdh 4222.7257 1.5824 -0.6622 0.2263 -2.9257 0.0034368 0.0676764

3-phosphoglycerate dehydrogenase [Source:MGI Symbol;Acc:MGI:1355330]

ENSMUSG00000062981 Mrpl42 1646.3699 1.6111 -0.6880 0.2354 -2.9231 0.0034651 0.0680561


ENSMUSG00000033793 Atp6v1h 2016.1466 1.4944 -0.5796 0.1985 -2.9204 0.0034961 0.0682779

ATPase, H+ transporting, lysosomal V1 subunit H [Source:MGI Symbol;Acc:MGI:1914864]

ENSMUSG00000038965 Ube2l3 2992.5881 1.6058 -0.6833 0.2339 -2.9208 0.0034914 0.0682779

ubiquitin-conjugating enzyme E2L 3 [Source:MGI Symbol;Acc:MGI:109240]

ENSMUSG00000073400 Trim10 659.4642 0.6695 0.5789 0.1982 2.9215 0.0034837 0.0682779

tripartite motif-containing 10 [Source:MGI Symbol;Acc:MGI:1338757]

ENSMUSG00000015671 Psma2 5529.0678 1.5442 -0.6269 0.2150 -2.9158 0.0035473 0.0691047


173

ENSMUSG00000048351 2010305A19 331.5993 1.5907 -0.6697 0.2297 -2.9155 0.0035516 0.0691047

cytochrome c oxidase assembly factor 7 [Source:MGI Symbol;Acc:MGI:1917143]

ENSMUSG00000000318 Clec10a 633.2429 1.4869 -0.5723 0.1964 -2.9139 0.0035698 0.0693279

C-type lectin domain family 10, member A [Source:MGI Symbol;Acc:MGI:96975]

ENSMUSG00000038717 Atp5l 6986.2034 2.0442 -1.0316 0.3546 -2.9091 0.0036252 0.0702745

ATP synthase, H+ transporting, mitochondrial F0 complex, subunit G [Source:MGI Symbol;Acc:MGI:1351597]

ENSMUSG00000061981 Flot2 4498.1307 1.4247 -0.5107 0.1756 -2.9082 0.0036355 0.070343

flotillin 2 [Source:MGI Symbol;Acc:MGI:103309]

ENSMUSG00000026728 Vim 30980.1488 1.5306 -0.6141 0.2112 -2.9073 0.0036453 0.0704009

vimentin [Source:MGI Symbol;Acc:MGI:98932]

ENSMUSG00000041849 Card6 519.9112 0.5467 0.8713 0.2999 2.9055 0.003667 0.0706895


ENSMUSG00000024181 Mrpl28 3169.8320 1.9751 -0.9819 0.3383 -2.9024 0.0037036 0.0712638


ENSMUSG00000027712 Anxa5 4716.2464 1.3921 -0.4773 0.1646 -2.8994 0.0037387 0.0713766

annexin A5 [Source:MGI Symbol;Acc:MGI:106008]

ENSMUSG00000027822 Slc33a1 700.4138 1.3928 -0.4780 0.1649 -2.8979 0.0037572 0.0713766

solute carrier family 33 (acetyl-CoA transporter), member 1 [Source:MGI Symbol;Acc:MGI:1332247]

ENSMUSG00000030588 Yif1b 706.3646 1.4167 -0.5025 0.1734 -2.8978 0.0037574 0.0713766

Yip1 interacting factor homolog B (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1924504]

174

ENSMUSG00000032890 Rims3 102.6801 0.5357 0.9006 0.3107 2.8987 0.0037473 0.0713766

regulating synaptic membrane exocytosis 3 [Source:MGI Symbol;Acc:MGI:2443331]

ENSMUSG00000070056 Mfhas1 719.1263 0.6078 0.7184 0.2477 2.9001 0.00373 0.0713766

malignant fibrous histiocytoma amplified sequence 1 [Source:MGI Symbol;Acc:MGI:1098644]

ENSMUSG00000074677 Sirpb1c 1260.8468 1.9599 -0.9707 0.3348 -2.8997 0.0037349 0.0713766

signal-regulatory protein beta 1C [Source:MGI Symbol;Acc:MGI:3807521]

ENSMUSG00000089847 Fxc1 352.8341 2.0526 -1.0374 0.3577 -2.9001 0.0037302 0.0713766

translocase of inner mitochondrial membrane 10B [Source:MGI Symbol;Acc:MGI:1315196]

ENSMUSG00000031765 Mt1 3553.3098 2.1729 -1.1196 0.3866 -2.8965 0.0037736 0.0715545

metallothionein 1 [Source:MGI Symbol;Acc:MGI:97171]

ENSMUSG00000052397 Ezr 5861.3822 0.6782 0.5601 0.1935 2.8943 0.0037997 0.0719187ezrin [Source:MGI Symbol;Acc:MGI:98931]

ENSMUSG00000021285 Ppp1r13b 317.5626 0.4922 1.0228 0.3535 2.8934 0.0038109 0.0719995

protein phosphatase 1, regulatory (inhibitor) subunit 13B [Source:MGI Symbol;Acc:MGI:1336199]

ENSMUSG00000004110 Cacna1e 187.7615 0.4602 1.1198 0.3871 2.8927 0.0038193 0.0720274

calcium channel, voltage-dependent, R type, alpha 1E subunit [Source:MGI Symbol;Acc:MGI:106217]

ENSMUSG00000031875 Cmtm3 703.6299 1.4048 -0.4904 0.1696 -2.8907 0.003844 0.0723621

CKLF-like MARVEL transmembrane domain containing 3 [Source:MGI Symbol;Acc:MGI:2447162]

175

ENSMUSG00000024240 Epc1 1405.1638 0.7322 0.4497 0.1557 2.8887 0.0038682 0.0725598

enhancer of polycomb homolog 1 (Drosophila) [Source:MGI Symbol;Acc:MGI:1278322]

ENSMUSG00000026797 Stxbp1 240.1307 0.6577 0.6044 0.2092 2.8887 0.0038684 0.0725598

syntaxin binding protein 1 [Source:MGI Symbol;Acc:MGI:107363]

ENSMUSG00000030647 Ndufc2 2531.1562 1.8878 -0.9167 0.3175 -2.8871 0.003888 0.0726586

NADH dehydrogenase (ubiquinone) 1, subcomplex unknown, 2 [Source:MGI Symbol;Acc:MGI:1344370]

ENSMUSG00000038372 Gmds 463.2900 1.5738 -0.6542 0.2266 -2.8866 0.0038945 0.0726586

GDP-mannose 4, 6-dehydratase [Source:MGI Symbol;Acc:MGI:1891112]

ENSMUSG00000044627 Swi5 4089.7130 1.5356 -0.6188 0.2143 -2.8874 0.0038843 0.0726586SWI5 recombination repair homolog (yeast) [Source:MGI Symbol;Acc:MGI:1920181]

ENSMUSG00000059518 Znhit1 1632.2240 1.7932 -0.8425 0.2919 -2.8859 0.0039033 0.0726912

zinc finger, HIT domain containing 1 [Source:MGI Symbol;Acc:MGI:1917353]

ENSMUSG00000037204 9430023L20 2164.6843 1.8277 -0.8701 0.3015 -2.8853 0.0039105 0.0726954autophagy related 101 [Source:MGI Symbol;Acc:MGI:1915368]

ENSMUSG00000007987 Rabl5 419.3664 1.5200 -0.6041 0.2094 -2.8846 0.0039189 0.0727223

intraflagellar transport 22 [Source:MGI Symbol;Acc:MGI:1914536]

ENSMUSG00000052151 Ppap2c 230.0238 1.6011 -0.6791 0.2356 -2.8828 0.0039416 0.0730136

phosphatidic acid phosphatase type 2C [Source:MGI Symbol;Acc:MGI:1354945]

176

ENSMUSG00000006058 Snf8 3409.4530 1.8222 -0.8656 0.3004 -2.8818 0.0039543 0.07312

SNF8, ESCRT-II complex subunit, homolog (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1343161]

ENSMUSG00000073616 Myeov2 2670.4596 2.1183 -1.0829 0.3760 -2.8804 0.0039723 0.0733222

myeloma overexpressed 2 [Source:MGI Symbol;Acc:MGI:1914165]

ENSMUSG00000002602 Axl 1570.0408 0.6048 0.7254 0.2521 2.8774 0.0040097 0.0733639

AXL receptor tyrosine kinase [Source:MGI Symbol;Acc:MGI:1347244]

ENSMUSG00000029198 Grpel1 2320.1086 1.5745 -0.6549 0.2276 -2.8779 0.0040028 0.0733639

GrpE-like 1, mitochondrial [Source:MGI Symbol;Acc:MGI:1334417]

ENSMUSG00000029430 Ran 15183.4165 1.5252 -0.6090 0.2116 -2.8780 0.0040026 0.0733639

RAN, member RAS oncogene family [Source:MGI Symbol;Acc:MGI:1333112]

ENSMUSG00000041697 Cox6a1 8979.7484 2.0170 -1.0122 0.3517 -2.8778 0.0040047 0.0733639

cytochrome c oxidase subunit VIa polypeptide 1 [Source:MGI Symbol;Acc:MGI:103099]

ENSMUSG00000045948 Mrps12 1029.4812 1.8539 -0.8905 0.3093 -2.8796 0.0039823 0.0733639


ENSMUSG00000021807 2700060E02 3470.5409 1.5564 -0.6382 0.2219 -2.8760 0.004028 0.0734758

RIKEN cDNA 2700060E02 gene [Source:MGI Symbol;Acc:MGI:1915295]

ENSMUSG00000026833 Olfm1 1319.7388 2.0441 -1.0315 0.3587 -2.8758 0.0040299 0.0734758

olfactomedin 1 [Source:MGI Symbol;Acc:MGI:1860437]

ENSMUSG00000025487 Psmd13 4875.4893 1.7394 -0.7986 0.2782 -2.8703 0.0041002 0.0738533


177

ENSMUSG00000030298 Sec13 3070.8785 1.3999 -0.4853 0.1689 -2.8725 0.0040729 0.0738533

SEC13 homolog (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:99832]

ENSMUSG00000033735 Spr 738.2997 1.5819 -0.6617 0.2304 -2.8712 0.0040889 0.0738533

sepiapterin reductase [Source:MGI Symbol;Acc:MGI:103078]

ENSMUSG00000041939 Mvk 377.3561 1.5928 -0.6716 0.2339 -2.8707 0.0040956 0.0738533mevalonate kinase [Source:MGI Symbol;Acc:MGI:107624]

ENSMUSG00000042462 Dctpp1 1804.2254 1.9557 -0.9677 0.3369 -2.8720 0.004079 0.0738533

dCTP pyrophosphatase 1 [Source:MGI Symbol;Acc:MGI:1913672]

ENSMUSG00000062421 Arf2 1278.2807 1.3866 -0.4716 0.1641 -2.8733 0.0040621 0.0738533ADP-ribosylation factor 2 [Source:MGI Symbol;Acc:MGI:99595]

ENSMUSG00000066861 Oas1g 101.4582 2.5200 -1.3334 0.4641 -2.8729 0.0040677 0.0738533

2'-5' oligoadenylate synthetase 1G [Source:MGI Symbol;Acc:MGI:97429]

ENSMUSG00000040363 Bcor 1536.7577 0.6548 0.6109 0.2129 2.8695 0.0041109 0.0739186

BCL6 interacting corepressor [Source:MGI Symbol;Acc:MGI:1918708]

ENSMUSG00000053553 3110082I17 557.4079 1.6865 -0.7541 0.2629 -2.8685 0.004124 0.0740253

RIKEN cDNA 3110082I17 gene [Source:MGI Symbol;Acc:MGI:1920462]

ENSMUSG00000030000 Add2 1347.6077 0.5818 0.7815 0.2727 2.8660 0.0041576 0.0745002

adducin 2 (beta) [Source:MGI Symbol;Acc:MGI:87919]

ENSMUSG00000060063 Alox5ap 18687.9789 1.7238 -0.7856 0.2742 -2.8648 0.0041734 0.0746556

arachidonate 5-lipoxygenase activating protein [Source:MGI Symbol;Acc:MGI:107505]

ENSMUSG00000024533 Spire1 1439.3687 0.6702 0.5773 0.2017 2.8624 0.0042044 0.0749539

spire homolog 1 (Drosophila) [Source:MGI Symbol;Acc:MGI:1915416]

178

ENSMUSG00000028343 Erp44 2650.4135 1.3743 -0.4587 0.1602 -2.8625 0.0042028 0.0749539

endoplasmic reticulum protein 44 [Source:MGI Symbol;Acc:MGI:1923549]

ENSMUSG00000017400 Stac2 181.4205 0.4423 1.1770 0.4114 2.8607 0.0042275 0.0750772

SH3 and cysteine rich domain 2 [Source:MGI Symbol;Acc:MGI:2144518]

ENSMUSG00000025232 Hexa 5764.5590 1.4596 -0.5456 0.1907 -2.8603 0.0042329 0.0750772

hexosaminidase A [Source:MGI Symbol;Acc:MGI:96073]

ENSMUSG00000040521 Tsfm 1229.5078 1.8784 -0.9095 0.3179 -2.8609 0.0042239 0.0750772

Ts translation elongation factor, mitochondrial [Source:MGI Symbol;Acc:MGI:1913649]

ENSMUSG00000020022 Ndufa12 1797.2511 1.6471 -0.7199 0.2518 -2.8589 0.0042515 0.0752791


ENSMUSG00000032294 Pkm2 31899.7753 1.5151 -0.5994 0.2097 -2.8580 0.0042635 0.0753634pyruvate kinase, muscle [Source:MGI Symbol;Acc:MGI:97591]

ENSMUSG00000024099 Ndufv2 4629.0531 1.7745 -0.8274 0.2896 -2.8574 0.004271 0.0753671

NADH dehydrogenase (ubiquinone) flavoprotein 2 [Source:MGI Symbol;Acc:MGI:1920150]

ENSMUSG00000023089 Ndufa5 1754.2816 2.0526 -1.0375 0.3632 -2.8563 0.0042864 0.0754928


ENSMUSG00000063787 Chchd1 1719.8198 1.8697 -0.9028 0.3161 -2.8558 0.0042926 0.0754928

coiled-coil-helix-coiled-coil-helix domain containing 1 [Source:MGI Symbol;Acc:MGI:1913371]

179

ENSMUSG00000059278 Lsmd1 554.4201 1.6478 -0.7205 0.2525 -2.8540 0.0043177 0.0758075

N(alpha)-acetyltransferase 38, NatC auxiliary subunit [Source:MGI Symbol;Acc:MGI:1925554]

ENSMUSG00000021843 Ktn1 989.7170 0.6878 0.5398 0.1894 2.8510 0.0043588 0.0764005

kinectin 1 [Source:MGI Symbol;Acc:MGI:109153]

ENSMUSG00000052013 Btla 911.2475 0.5424 0.8824 0.3098 2.8488 0.0043878 0.0767799

B and T lymphocyte associated [Source:MGI Symbol;Acc:MGI:2658978]

ENSMUSG00000063931 Pepd 1606.1175 1.3454 -0.4280 0.1503 -2.8477 0.0044035 0.0769255

peptidase D [Source:MGI Symbol;Acc:MGI:97542]

ENSMUSG00000025950 Idh1 2745.3036 1.5200 -0.6041 0.2122 -2.8466 0.0044183 0.0769426

isocitrate dehydrogenase 1 (NADP+), soluble [Source:MGI Symbol;Acc:MGI:96413]

ENSMUSG00000029486 Mrpl1 853.6925 1.5096 -0.5942 0.2088 -2.8460 0.0044267 0.0769426


ENSMUSG00000074781 Ube2n 4340.2814 1.3928 -0.4780 0.1679 -2.8463 0.0044235 0.0769426

ubiquitin-conjugating enzyme E2N [Source:MGI Symbol;Acc:MGI:1934835]

ENSMUSG00000042111 Ccdc115 1997.6155 1.5984 -0.6766 0.2378 -2.8450 0.0044418 0.0770768


ENSMUSG00000038642 Ctss 11979.1606 1.6149 -0.6914 0.2431 -2.8444 0.0044495 0.0770821

cathepsin S [Source:MGI Symbol;Acc:MGI:107341]

ENSMUSG00000041775 Mapk1ip1 221.3770 0.6694 0.5791 0.2038 2.8419 0.0044844 0.0775589

mitogen-activated protein kinase 1 interacting protein 1 [Source:MGI Symbol;Acc:MGI:1916796]

180

ENSMUSG00000075266 Cenpw 695.5466 1.8257 -0.8684 0.3056 -2.8413 0.0044925 0.07757centromere protein W [Source:MGI Symbol;Acc:MGI:1913561]

ENSMUSG00000020802 Ube2o 871.3948 0.7137 0.4866 0.1713 2.8400 0.0045113 0.0777654

ubiquitin-conjugating enzyme E2O [Source:MGI Symbol;Acc:MGI:2444266]

ENSMUSG00000021458 2010111I01 602.6910 1.6457 -0.7187 0.2535 -2.8351 0.004581 0.078579


ENSMUSG00000030879 Mrpl17 1790.0246 1.8373 -0.8776 0.3095 -2.8358 0.0045714 0.078579


ENSMUSG00000049191 Rgag4 180.7972 0.5904 0.7601 0.2681 2.8351 0.0045811 0.078579

retrotransposon gag domain containing 4 [Source:MGI Symbol;Acc:MGI:3045324]

ENSMUSG00000060073 Psma3 5128.3766 1.4978 -0.5828 0.2056 -2.8344 0.0045907 0.0786151


ENSMUSG00000008036 Ap2s1 6361.4856 1.9406 -0.9565 0.3376 -2.8330 0.0046114 0.0788401

adaptor-related protein complex 2, sigma 1 subunit [Source:MGI Symbol;Acc:MGI:2141861]

ENSMUSG00000031609 Sap30 1671.0450 1.6156 -0.6921 0.2444 -2.8312 0.0046373 0.0788941

sin3 associated polypeptide [Source:MGI Symbol;Acc:MGI:1929129]

ENSMUSG00000058833 2810428I15 1585.7109 2.0139 -1.0100 0.3567 -2.8315 0.0046334 0.0788941


ENSMUSG00000073676 Hspe1 5534.1489 1.7982 -0.8466 0.2990 -2.8317 0.0046302 0.0788941

heat shock protein 1 (chaperonin 10) [Source:MGI Symbol;Acc:MGI:104680]

181

ENSMUSG00000059534 Uqcr10 3247.0320 1.8780 -0.9092 0.3212 -2.8306 0.0046457 0.0789093

ubiquinol-cytochrome c reductase, complex III subunit X [Source:MGI Symbol;Acc:MGI:1913402]

ENSMUSG00000007050 Lsm2 2127.6781 1.7717 -0.8251 0.2917 -2.8285 0.0046761 0.0791665

LSM2 homolog, U6 small nuclear RNA associated (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:90676]

ENSMUSG00000008193 Spib 5150.1474 0.4655 1.1031 0.3899 2.8289 0.004671 0.0791665Spi-B transcription factor (Spi-1/PU.1 related) [Source:MGI Symbol;Acc:MGI:892986]

ENSMUSG00000002804 Nudt14 383.1529 1.7334 -0.7936 0.2807 -2.8274 0.0046925 0.0793166

nudix (nucleoside diphosphate linked moiety X)-type motif 14 [Source:MGI Symbol;Acc:MGI:1913424]

ENSMUSG00000018377 Vezf1 1859.0426 0.7195 0.4750 0.1681 2.8247 0.0047325 0.0796477

vascular endothelial zinc finger 1 [Source:MGI Symbol;Acc:MGI:1313291]

ENSMUSG00000045438 Cox19 841.2900 1.6047 -0.6823 0.2416 -2.8245 0.004735 0.0796477

cytochrome c oxidase assembly protein 19 [Source:MGI Symbol;Acc:MGI:1915283]

ENSMUSG00000070780 Rbm47 375.1897 1.6056 -0.6831 0.2418 -2.8254 0.0047228 0.0796477

RNA binding motif protein 47 [Source:MGI Symbol;Acc:MGI:2384294]

ENSMUSG00000005465 Il27ra 451.9435 0.5523 0.8564 0.3034 2.8228 0.0047605 0.07969

interleukin 27 receptor, alpha [Source:MGI Symbol;Acc:MGI:1355318]

ENSMUSG00000038347 Tcte2 112.9956 1.8988 -0.9251 0.3277 -2.8232 0.0047546 0.07969

t-complex-associated testis expressed 2 [Source:MGI Symbol;Acc:MGI:98641]

182

ENSMUSG00000060288 Ppih 1919.3226 1.6892 -0.7563 0.2679 -2.8229 0.0047593 0.07969

peptidyl prolyl isomerase H [Source:MGI Symbol;Acc:MGI:106499]

ENSMUSG00000018750 Zbtb4 440.6009 0.5361 0.8994 0.3188 2.8212 0.0047841 0.0798857

zinc finger and BTB domain containing 4 [Source:MGI Symbol;Acc:MGI:1922830]

ENSMUSG00000035547 Capn5 380.9074 0.7022 0.5100 0.1808 2.8207 0.0047921 0.0798857

calpain 5 [Source:MGI Symbol;Acc:MGI:1100859]

ENSMUSG00000039745 Htatip2 467.7452 1.4558 -0.5418 0.1921 -2.8205 0.0047951 0.0798857

HIV-1 tat interactive protein 2, homolog (human) [Source:MGI Symbol;Acc:MGI:1859271]

ENSMUSG00000029651 Mtus2 136.8639 1.7351 -0.7950 0.2820 -2.8195 0.0048103 0.0800099

microtubule associated tumor suppressor candidate 2 [Source:MGI Symbol;Acc:MGI:1915388]

ENSMUSG00000045598 Zfp553 464.6361 0.6779 0.5609 0.1990 2.8183 0.0048273 0.0801654


ENSMUSG00000021497 Txndc15 1005.4323 1.4114 -0.4971 0.1764 -2.8175 0.0048395 0.0802411

thioredoxin domain containing 15 [Source:MGI Symbol;Acc:MGI:1916922]

ENSMUSG00000022604 Cep97 385.4267 0.6820 0.5522 0.1961 2.8162 0.0048591 0.0804378centrosomal protein 97 [Source:MGI Symbol;Acc:MGI:1921451]

ENSMUSG00000016756 Cmah 1636.4658 0.5682 0.8154 0.2898 2.8134 0.0049016 0.0805268

cytidine monophospho-N-acetylneuraminic acid hydroxylase [Source:MGI Symbol;Acc:MGI:103227]

ENSMUSG00000021018 Polr2h 1177.5312 1.8749 -0.9068 0.3223 -2.8133 0.0049031 0.0805268

polymerase (RNA) II (DNA directed) polypeptide H [Source:MGI Symbol;Acc:MGI:2384309]

183

ENSMUSG00000030029 Lrig1 125.0541 0.5513 0.8591 0.3052 2.8147 0.0048827 0.0805268

leucine-rich repeats and immunoglobulin-like domains 1 [Source:MGI Symbol;Acc:MGI:107935]

ENSMUSG00000032010 Usp2 178.5191 1.6679 -0.7380 0.2623 -2.8140 0.0048925 0.0805268

ubiquitin specific peptidase 2 [Source:MGI Symbol;Acc:MGI:1858178]

ENSMUSG00000050708 Ftl1 20391.4112 1.3954 -0.4806 0.1707 -2.8151 0.0048755 0.0805268

ferritin light chain 1 [Source:MGI Symbol;Acc:MGI:95589]

ENSMUSG00000073240 2410017P09 221.8104 1.8296 -0.8715 0.3099 -2.8126 0.0049144 0.080585 NA

ENSMUSG00000021606 Ndufs6 2374.2086 2.0303 -1.0217 0.3634 -2.8113 0.0049347 0.0807354


ENSMUSG00000035246 Pcyt1b 183.5652 0.6312 0.6639 0.2362 2.8110 0.004939 0.0807354

phosphate cytidylyltransferase 1, choline, beta isoform [Source:MGI Symbol;Acc:MGI:2147987]

ENSMUSG00000020277 Pfkl 2729.3588 1.5263 -0.6100 0.2173 -2.8075 0.0049928 0.0811176

phosphofructokinase, liver, B-type [Source:MGI Symbol;Acc:MGI:97547]

ENSMUSG00000031818 Cox4i1 15123.3060 1.8797 -0.9105 0.3243 -2.8079 0.0049867 0.0811176

cytochrome c oxidase subunit IV isoform 1 [Source:MGI Symbol;Acc:MGI:88473]

ENSMUSG00000037416 Dmxl1 990.8064 0.5613 0.8332 0.2968 2.8075 0.0049935 0.0811176

Dmx-like 1 [Source:MGI Symbol;Acc:MGI:2443926]

ENSMUSG00000071014 Ndufb6 921.6658 1.6895 -0.7566 0.2694 -2.8089 0.0049707 0.0811176

NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 6 [Source:MGI Symbol;Acc:MGI:2684983]

184

ENSMUSG00000020163 Uqcr11 3498.9096 1.9447 -0.9595 0.3421 -2.8052 0.0050292 0.0815704

ubiquinol-cytochrome c reductase, complex III subunit XI [Source:MGI Symbol;Acc:MGI:1913844]

ENSMUSG00000027784 Ppm1l 320.5986 0.6223 0.6843 0.2440 2.8045 0.0050392 0.0816055

protein phosphatase 1 (formerly 2C)-like [Source:MGI Symbol;Acc:MGI:2139740]

ENSMUSG00000078348 Sf3b5 3256.2805 1.9576 -0.9691 0.3458 -2.8022 0.005075 0.0820573

splicing factor 3b, subunit 5 [Source:MGI Symbol;Acc:MGI:1913375]

ENSMUSG00000031698 Mylk3 472.8282 0.6330 0.6598 0.2355 2.8016 0.0050855 0.0821005

myosin light chain kinase 3 [Source:MGI Symbol;Acc:MGI:2443063]

ENSMUSG00000024414 Mrpl27 1039.3076 1.7544 -0.8110 0.2897 -2.7990 0.0051257 0.0824939


ENSMUSG00000041774 Ydjc 373.6345 1.8651 -0.8993 0.3212 -2.7993 0.0051207 0.0824939

YdjC homolog (bacterial) [Source:MGI Symbol;Acc:MGI:1916351]

ENSMUSG00000091625 Lsm5 1781.6629 1.9522 -0.9651 0.3449 -2.7982 0.005139 0.0825798


ENSMUSG00000057572 Zbtb8os 988.8843 1.4937 -0.5789 0.2071 -2.7956 0.0051811 0.0828744

zinc finger and BTB domain containing 8 opposite strand [Source:MGI Symbol;Acc:MGI:1914356]

ENSMUSG00000058624 Gda 4506.2849 1.5575 -0.6392 0.2286 -2.7957 0.0051794 0.0828744guanine deaminase [Source:MGI Symbol;Acc:MGI:95678]

ENSMUSG00000071414 Gm6736 245.9936 1.8952 -0.9224 0.3299 -2.7959 0.0051749 0.0828744


185

ENSMUSG00000035891 Cerk 3086.2368 0.6450 0.6327 0.2264 2.7942 0.0052032 0.0829733

ceramide kinase [Source:MGI Symbol;Acc:MGI:2386052]

ENSMUSG00000050271 D8Ertd82e 138.0762 0.4712 1.0855 0.3884 2.7946 0.0051963 0.0829733

DNA segment, Chr 8, ERATO Doi 82, expressed [Source:MGI Symbol;Acc:MGI:1196223]

ENSMUSG00000044348 Mcart6 276.4277 0.6199 0.6900 0.2470 2.7933 0.0052178 0.0830788

solute carrier family 25, member 53 [Source:MGI Symbol;Acc:MGI:1914312]

ENSMUSG00000024516 Sec11c 6539.3439 1.6819 -0.7501 0.2687 -2.7917 0.0052431 0.0832297

SEC11 homolog C (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1913536]

ENSMUSG00000048277 Syngr2 5053.7084 1.5871 -0.6664 0.2387 -2.7917 0.0052433 0.0832297

synaptogyrin 2 [Source:MGI Symbol;Acc:MGI:1328324]

ENSMUSG00000002778 Kdelr1 3637.5329 1.3873 -0.4723 0.1693 -2.7896 0.0052778 0.0836508

KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 1 [Source:MGI Symbol;Acc:MGI:1915387]

ENSMUSG00000020657 Dnajc27 301.4454 0.6879 0.5398 0.1936 2.7881 0.0053021 0.0837864

DnaJ (Hsp40) homolog, subfamily C, member 27 [Source:MGI Symbol;Acc:MGI:2443036]

ENSMUSG00000040270 Bach2 1035.7237 0.4652 1.1041 0.3960 2.7881 0.0053024 0.0837864

BTB and CNC homology 2 [Source:MGI Symbol;Acc:MGI:894679]

ENSMUSG00000011832 Evi5l 215.4554 0.6571 0.6058 0.2174 2.7871 0.0053186 0.0839149

ecotropic viral integration site 5 like [Source:MGI Symbol;Acc:MGI:2442167]

ENSMUSG00000035385 Ccl2 104.4780 1.9125 -0.9355 0.3357 -2.7863 0.0053317 0.0839939

chemokine (C-C motif) ligand 2 [Source:MGI Symbol;Acc:MGI:98259]

186

ENSMUSG00000025130 P4hb 17791.0830 1.6800 -0.7484 0.2687 -2.7857 0.0053412 0.084017

prolyl 4-hydroxylase, beta polypeptide [Source:MGI Symbol;Acc:MGI:97464]

ENSMUSG00000023044 Csad 284.1244 0.7054 0.5036 0.1808 2.7848 0.0053553 0.0841117

cysteine sulfinic acid decarboxylase [Source:MGI Symbol;Acc:MGI:2180098]

ENSMUSG00000008845 Cd163 670.5249 0.6111 0.7105 0.2553 2.7832 0.0053831 0.0843736


ENSMUSG00000028648 Ndufs5 1493.1899 1.9705 -0.9785 0.3518 -2.7819 0.0054043 0.0843736


ENSMUSG00000059495 Arhgef12 496.1936 0.6455 0.6315 0.2269 2.7828 0.0053899 0.0843736

Rho guanine nucleotide exchange factor (GEF) 12 [Source:MGI Symbol;Acc:MGI:1916882]

ENSMUSG00000071451 Psmg4 900.6598 1.8594 -0.8948 0.3217 -2.7820 0.0054021 0.0843736

proteasome (prosome, macropain) assembly chaperone 4 [Source:MGI Symbol;Acc:MGI:1916916]

ENSMUSG00000027997 Casp6 786.7267 1.5056 -0.5903 0.2123 -2.7807 0.0054236 0.0845477

caspase 6 [Source:MGI Symbol;Acc:MGI:1312921]

ENSMUSG00000019494 Cops6 2683.2812 1.4326 -0.5187 0.1866 -2.7797 0.0054417 0.0847041

COP9 (constitutive photomorphogenic) homolog, subunit 6 (Arabidopsis thaliana) [Source:MGI Symbol;Acc:MGI:1349439]

ENSMUSG00000017716 Birc5 5694.6150 1.8345 -0.8754 0.3152 -2.7770 0.0054861 0.0847671

baculoviral IAP repeat-containing 5 [Source:MGI Symbol;Acc:MGI:1203517]

187

ENSMUSG00000032215 Rsl24d1 2398.1516 1.7781 -0.8304 0.2989 -2.7782 0.005466 0.0847671

ribosomal L24 domain containing 1 [Source:MGI Symbol;Acc:MGI:2681840]

ENSMUSG00000036372 1810006K21 2645.1502 1.8009 -0.8487 0.3056 -2.7773 0.0054817 0.0847671


ENSMUSG00000040229 Gpr34 107.9052 0.6002 0.7364 0.2650 2.7785 0.0054611 0.0847671

G protein-coupled receptor 34 [Source:MGI Symbol;Acc:MGI:1346334]

ENSMUSG00000042810 Krba1 137.4033 0.5946 0.7500 0.2701 2.7765 0.0054945 0.0847671

KRAB-A domain containing 1 [Source:MGI Symbol;Acc:MGI:1925077]

ENSMUSG00000078812 Eif5a 38838.9446 1.6575 -0.7290 0.2625 -2.7768 0.0054894 0.0847671

eukaryotic translation initiation factor 5A [Source:MGI Symbol;Acc:MGI:106248]

ENSMUSG00000017417 Plxdc1 587.8993 0.6365 0.6518 0.2349 2.7745 0.0055283 0.0849965

plexin domain containing 1 [Source:MGI Symbol;Acc:MGI:1919574]

ENSMUSG00000029313 Aff1 1885.7886 0.6915 0.5321 0.1918 2.7742 0.0055339 0.0849965

AF4/FMR2 family, member 1 [Source:MGI Symbol;Acc:MGI:1100819]

ENSMUSG00000074364 Ehd2 295.0861 0.5143 0.9594 0.3458 2.7743 0.0055319 0.0849965

EH-domain containing 2 [Source:MGI Symbol;Acc:MGI:2154274]

ENSMUSG00000018923 Med11 1003.4137 1.6253 -0.7007 0.2528 -2.7715 0.0055797 0.0852873

mediator complex subunit 11 [Source:MGI Symbol;Acc:MGI:1913422]

ENSMUSG00000034744 Nagk 463.6283 1.4312 -0.5172 0.1866 -2.7716 0.005579 0.0852873 N-acetylglucosamine kinase [Source:MGI Symbol;Acc:MGI:1860418]

ENSMUSG00000039183 Nubp2 2251.1011 1.4025 -0.4880 0.1761 -2.7712 0.0055855 0.0852873

nucleotide binding protein 2 [Source:MGI Symbol;Acc:MGI:1347072]

188

ENSMUSG00000048058 Ldlrad3 484.0996 0.6233 0.6820 0.2460 2.7719 0.0055733 0.0852873

low density lipoprotein receptor class A domain containing 3 [Source:MGI Symbol;Acc:MGI:2138856]

ENSMUSG00000071653 1810009A15 1511.9204 1.6034 -0.6812 0.2459 -2.7703 0.0056013 0.0854035

RIKEN cDNA 1810009A15 gene [Source:MGI Symbol;Acc:MGI:1913526]

ENSMUSG00000027346 Gpcpd1 2545.8251 0.6912 0.5329 0.1925 2.7688 0.0056256 0.0856333

glycerophosphocholine phosphodiesterase GDE1 homolog (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:104898]

ENSMUSG00000050608 Minos1 4561.9756 1.9366 -0.9535 0.3444 -2.7684 0.0056328 0.0856333

mitochondrial inner membrane organizing system 1 [Source:MGI Symbol;Acc:MGI:1913628]

ENSMUSG00000057363 Uxs1 1250.9886 1.4293 -0.5153 0.1862 -2.7673 0.0056529 0.0858138

UDP-glucuronate decarboxylase 1 [Source:MGI Symbol;Acc:MGI:1915133]

ENSMUSG00000032265 Fam46a 1214.0542 1.4049 -0.4905 0.1773 -2.7659 0.005676 0.086039


ENSMUSG00000020843 Timm22 935.7133 1.5110 -0.5955 0.2155 -2.7635 0.0057179 0.0861793


ENSMUSG00000029833 Trim24 689.3400 0.7192 0.4756 0.1722 2.7621 0.0057431 0.0861793


ENSMUSG00000031068 Glrx3 4003.9298 1.4796 -0.5652 0.2045 -2.7642 0.0057063 0.0861793

glutaredoxin 3 [Source:MGI Symbol;Acc:MGI:1353653]

189

ENSMUSG00000035215 Lsm7 1454.0935 1.9306 -0.9490 0.3435 -2.7629 0.0057294 0.0861793


ENSMUSG00000044678 Ly6k 253.1998 2.1008 -1.0709 0.3877 -2.7623 0.0057397 0.0861793

lymphocyte antigen 6 complex, locus K [Source:MGI Symbol;Acc:MGI:1923736]

ENSMUSG00000046985 Tapt1 3001.7867 0.7071 0.5000 0.1809 2.7638 0.0057125 0.0861793

transmembrane anterior posterior transformation 1 [Source:MGI Symbol;Acc:MGI:2683537]

ENSMUSG00000066357 Wdr6 923.6693 0.5490 0.8652 0.3131 2.7631 0.0057261 0.0861793WD repeat domain 6 [Source:MGI Symbol;Acc:MGI:1930140]

ENSMUSG00000013701 Timm23 3647.0924 1.5103 -0.5949 0.2156 -2.7589 0.0057999 0.0866574


ENSMUSG00000019797 1700021F05 557.4634 1.6868 -0.7543 0.2734 -2.7592 0.005795 0.0866574

RIKEN cDNA 1700021F05 gene [Source:MGI Symbol;Acc:MGI:1915101]

ENSMUSG00000055485 9830001H0 294.0957 0.7049 0.5046 0.1829 2.7591 0.0057963 0.0866574

suppressor of glucose, autophagy associated 1 [Source:MGI Symbol;Acc:MGI:2444575]

ENSMUSG00000018286 Psmb6 4459.4964 1.7497 -0.8071 0.2930 -2.7541 0.005885 0.0873745


ENSMUSG00000022544 Fam86 419.6248 1.4093 -0.4950 0.1798 -2.7538 0.0058905 0.0873745

eukaryotic elongation factor 2 lysine methyltransferase [Source:MGI Symbol;Acc:MGI:1917761]

190

ENSMUSG00000022912 Pros1 516.8842 2.2975 -1.2001 0.4358 -2.7534 0.0058981 0.0873745

protein S (alpha) [Source:MGI Symbol;Acc:MGI:1095733]

ENSMUSG00000031388 Naa10 1438.7816 1.9041 -0.9291 0.3372 -2.7552 0.0058647 0.0873745

N(alpha)-acetyltransferase 10, NatA catalytic subunit [Source:MGI Symbol;Acc:MGI:1915255]

ENSMUSG00000035674 Ndufa3 3147.6173 1.9970 -0.9978 0.3622 -2.7546 0.0058769 0.0873745


ENSMUSG00000055839 Tceb2 4073.3058 1.8349 -0.8757 0.3178 -2.7557 0.005857 0.0873745

transcription elongation factor B (SIII), polypeptide 2 [Source:MGI Symbol;Acc:MGI:1914923]

ENSMUSG00000001054 Rmnd5b 1418.5350 0.7353 0.4436 0.1614 2.7494 0.0059711 0.0875681

required for meiotic nuclear division 5 homolog B (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1913339]

ENSMUSG00000003062 Stard3nl 951.5025 1.4337 -0.5198 0.1890 -2.7500 0.0059592 0.0875681STARD3 N-terminal like [Source:MGI Symbol;Acc:MGI:1923455]

ENSMUSG00000006818 Sod2 2177.9234 1.5016 -0.5865 0.2138 -2.7428 0.0060926 0.0875681

superoxide dismutase 2, mitochondrial [Source:MGI Symbol;Acc:MGI:98352]

ENSMUSG00000009739 Pou6f1 201.2406 0.5602 0.8359 0.3051 2.7396 0.0061511 0.0875681POU domain, class 6, transcription factor 1 [Source:MGI Symbol;Acc:MGI:102935]

ENSMUSG00000013707 Tnfaip8l2 2319.7777 1.5502 -0.6324 0.2306 -2.7426 0.0060947 0.0875681

tumor necrosis factor, alpha-induced protein 8-like 2 [Source:MGI Symbol;Acc:MGI:1917019]

191

ENSMUSG00000019842 Traf3ip2 166.2609 0.4977 1.0065 0.3670 2.7424 0.0060994 0.0875681

TRAF3 interacting protein 2 [Source:MGI Symbol;Acc:MGI:2143599]

ENSMUSG00000020921 Tmem101 648.3899 1.5874 -0.6667 0.2434 -2.7394 0.0061547 0.0875681


ENSMUSG00000022913 Psmg1 1018.7669 1.6102 -0.6873 0.2508 -2.7402 0.00614 0.0875681

proteasome (prosome, macropain) assembly chaperone 1 [Source:MGI Symbol;Acc:MGI:1860263]

ENSMUSG00000024074 Crim1 195.0751 0.5356 0.9007 0.3286 2.7407 0.0061311 0.0875681

cysteine rich transmembrane BMP regulator 1 (chordin like) [Source:MGI Symbol;Acc:MGI:1354756]

ENSMUSG00000025132 Arhgdia 17078.7475 1.3658 -0.4498 0.1639 -2.7435 0.0060793 0.0875681

Rho GDP dissociation inhibitor (GDI) alpha [Source:MGI Symbol;Acc:MGI:2178103]

ENSMUSG00000025940 Tmem70 908.3948 1.3863 -0.4712 0.1715 -2.7481 0.0059947 0.0875681


ENSMUSG00000026939 Tmem141 333.9610 1.7018 -0.7670 0.2794 -2.7450 0.0060505 0.0875681


ENSMUSG00000030591 Psmd8 5648.7606 1.5457 -0.6282 0.2288 -2.7452 0.0060482 0.0875681


ENSMUSG00000032370 Lactb 1091.1323 1.3912 -0.4763 0.1732 -2.7492 0.0059736 0.0875681

lactamase, beta [Source:MGI Symbol;Acc:MGI:1933395]

192

ENSMUSG00000033295 Ptprf 137.7173 0.6288 0.6692 0.2433 2.7506 0.0059488 0.0875681

protein tyrosine phosphatase, receptor type, F [Source:MGI Symbol;Acc:MGI:102695]

ENSMUSG00000033916 Chmp2a 4431.5591 1.6396 -0.7133 0.2598 -2.7453 0.0060456 0.0875681

charged multivesicular body protein 2A [Source:MGI Symbol;Acc:MGI:1916203]

ENSMUSG00000036561 Ppp6r2 636.5888 0.6352 0.6546 0.2385 2.7453 0.0060462 0.0875681protein phosphatase 6, regulatory subunit 2 [Source:MGI Symbol;Acc:MGI:1918724]

ENSMUSG00000036661 Dennd3 1016.7740 0.6881 0.5394 0.1966 2.7437 0.0060755 0.0875681

DENN/MADD domain containing 3 [Source:MGI Symbol;Acc:MGI:2146009]

ENSMUSG00000041857 Oosp1 248.1039 1.7292 -0.7901 0.2878 -2.7448 0.006054 0.0875681

oocyte secreted protein 1 [Source:MGI Symbol;Acc:MGI:2149290]

ENSMUSG00000042312 S100a13 2129.6954 1.9219 -0.9426 0.3439 -2.7409 0.0061264 0.0875681


ENSMUSG00000045827 Serpinb9 226.3865 0.6687 0.5807 0.2112 2.7492 0.0059739 0.0875681

serine (or cysteine) peptidase inhibitor, clade B, member 9 [Source:MGI Symbol;Acc:MGI:106603]

ENSMUSG00000047880 Cxcr5 585.7710 0.4659 1.1018 0.4019 2.7414 0.0061185 0.0875681

chemokine (C-X-C motif) receptor 5 [Source:MGI Symbol;Acc:MGI:103567]

ENSMUSG00000050552 0910001L09 5798.2894 2.0039 -1.0028 0.3655 -2.7434 0.0060804 0.0875681


193

ENSMUSG00000054836 2610002I17 348.5018 1.6027 -0.6805 0.2479 -2.7455 0.0060423 0.0875681

elongator acetyltransferase complex subunit 6 [Source:MGI Symbol;Acc:MGI:1919349]

ENSMUSG00000057286 St6galnac2 594.6192 0.7071 0.5001 0.1822 2.7455 0.006042 0.0875681

ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 2 [Source:MGI Symbol;Acc:MGI:107553]

ENSMUSG00000070808 Gltscr1 397.8840 0.4972 1.0081 0.3677 2.7419 0.006109 0.0875681

glioma tumor suppressor candidate region gene 1 [Source:MGI Symbol;Acc:MGI:2154263]

ENSMUSG00000079419 Ms4a6c 7344.2287 1.8739 -0.9061 0.3295 -2.7499 0.0059607 0.0875681

membrane-spanning 4-domains, subfamily A, member 6C [Source:MGI Symbol;Acc:MGI:2385644]

ENSMUSG00000090841 Myl6 22504.2820 1.9819 -0.9869 0.3591 -2.7481 0.0059943 0.0875681

myosin, light polypeptide 6, alkali, smooth muscle and non-muscle [Source:MGI Symbol;Acc:MGI:109318]

ENSMUSG00000091537 Ccdc72 4842.9312 1.7347 -0.7947 0.2895 -2.7452 0.0060474 0.0875681

translational machinery associated 7 homolog (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1913417]

ENSMUSG00000027131 Tmem85 786.3495 1.4034 -0.4890 0.1786 -2.7371 0.0061977 0.087637

ER membrane protein complex subunit 4 [Source:MGI Symbol;Acc:MGI:1915282]

194

ENSMUSG00000038387 Rras 433.8609 1.3809 -0.4656 0.1701 -2.7371 0.0061985 0.087637Harvey rat sarcoma oncogene, subgroup R [Source:MGI Symbol;Acc:MGI:98179]

ENSMUSG00000038578 Susd1 1597.8402 0.6044 0.7264 0.2654 2.7369 0.0062016 0.087637

sushi domain containing 1 [Source:MGI Symbol;Acc:MGI:3651543]

ENSMUSG00000042745 Id1 675.4254 1.8614 -0.8964 0.3275 -2.7369 0.0062014 0.087637

inhibitor of DNA binding 1 [Source:MGI Symbol;Acc:MGI:96396]

ENSMUSG00000043923 Ccdc84 169.9503 0.6011 0.7343 0.2681 2.7387 0.0061681 0.087637


ENSMUSG00000038604 Fam65a 1357.3803 0.7044 0.5056 0.1847 2.7365 0.0062103 0.087641


ENSMUSG00000003308 Keap1 1633.4212 1.3407 -0.4230 0.1547 -2.7332 0.0062719 0.087669

kelch-like ECH-associated protein 1 [Source:MGI Symbol;Acc:MGI:1858732]

ENSMUSG00000006998 Psmd2 6933.9844 1.3227 -0.4035 0.1475 -2.7348 0.0062425 0.087669


ENSMUSG00000016252 Atp5e 7237.1118 2.0757 -1.0536 0.3853 -2.7348 0.0062425 0.087669

ATP synthase, H+ transporting, mitochondrial F1 complex, epsilon subunit [Source:MGI Symbol;Acc:MGI:1855697]

ENSMUSG00000019362 D8Ertd738e 3380.0758 1.6619 -0.7329 0.2682 -2.7328 0.0062795 0.087669

DNA segment, Chr 8, ERATO Doi 738, expressed [Source:MGI Symbol;Acc:MGI:1289231]

195

ENSMUSG00000019823 Mical1 1091.1105 0.5434 0.8798 0.3218 2.7339 0.0062595 0.087669

microtubule associated monooxygenase, calponin and LIM domain containing 1 [Source:MGI Symbol;Acc:MGI:2385847]

ENSMUSG00000029101 Rgs12 1068.8509 0.7541 0.4072 0.1489 2.7344 0.0062491 0.087669

regulator of G-protein signaling 12 [Source:MGI Symbol;Acc:MGI:1918979]

ENSMUSG00000031584 Gsr 19393.4095 1.3872 -0.4722 0.1726 -2.7352 0.0062345 0.087669

glutathione reductase [Source:MGI Symbol;Acc:MGI:95804]

ENSMUSG00000078695 Cisd3 277.4731 1.6437 -0.7170 0.2623 -2.7333 0.0062694 0.087669

CDGSH iron sulfur domain 3 [Source:MGI Symbol;Acc:MGI:101788]

ENSMUSG00000028645 Slc2a1 881.6114 1.7151 -0.7783 0.2850 -2.7310 0.0063138 0.0880302

solute carrier family 2 (facilitated glucose transporter), member 1 [Source:MGI Symbol;Acc:MGI:95755]

ENSMUSG00000029490 Mfsd7a 268.3130 1.6312 -0.7059 0.2587 -2.7291 0.0063508 0.0884276

major facilitator superfamily domain containing 7A [Source:MGI Symbol;Acc:MGI:2442629]

ENSMUSG00000090258 Churc1 299.0195 2.0358 -1.0256 0.3760 -2.7278 0.0063759 0.0886592

churchill domain containing 1 [Source:MGI Symbol;Acc:MGI:1923684]

ENSMUSG00000003072 Atp5d 5444.2366 1.7375 -0.7970 0.2924 -2.7259 0.0064128 0.0888655

ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit [Source:MGI Symbol;Acc:MGI:1913293]

ENSMUSG00000005397 Nid1 140.0989 0.4688 1.0929 0.4012 2.7239 0.0064509 0.0888655

nidogen 1 [Source:MGI Symbol;Acc:MGI:97342]

196

ENSMUSG00000025521 Tmem192 879.4412 1.5586 -0.6402 0.2349 -2.7260 0.0064099 0.0888655


ENSMUSG00000027459 Fam110a 764.1077 1.7358 -0.7956 0.2921 -2.7232 0.0064662 0.0888655


ENSMUSG00000027642 Rpn2 9177.8540 1.4531 -0.5391 0.1980 -2.7231 0.0064669 0.0888655

ribophorin II [Source:MGI Symbol;Acc:MGI:98085]

ENSMUSG00000028567 Txndc12 1293.2168 1.3274 -0.4086 0.1500 -2.7243 0.0064444 0.0888655

thioredoxin domain containing 12 (endoplasmic reticulum) [Source:MGI Symbol;Acc:MGI:1913323]

ENSMUSG00000030284 Creld1 260.8314 1.4072 -0.4928 0.1810 -2.7232 0.0064661 0.0888655

cysteine-rich with EGF-like domains 1 [Source:MGI Symbol;Acc:MGI:2152539]

ENSMUSG00000030577 Cd22 2633.1163 0.4473 1.1607 0.4262 2.7231 0.0064674 0.0888655


ENSMUSG00000058443 Rpl10-ps3 455.1804 1.9166 -0.9385 0.3447 -2.7231 0.0064671 0.0888655

ribosomal protein L10, pseudogene 3 [Source:MGI Symbol;Acc:MGI:3704336]

ENSMUSG00000028644 Ermap 3432.6282 0.6906 0.5341 0.1963 2.7212 0.0065039 0.0892486

erythroblast membrane-associated protein [Source:MGI Symbol;Acc:MGI:1349816]

ENSMUSG00000021270 Hsp90aa1 19350.1054 1.3134 -0.3933 0.1446 -2.7200 0.0065282 0.0893471

heat shock protein 90, alpha (cytosolic), class A member 1 [Source:MGI Symbol;Acc:MGI:96250]

197

ENSMUSG00000021773 Comtd1 133.7305 2.0652 -1.0463 0.3846 -2.7203 0.006523 0.0893471

catechol-O-methyltransferase domain containing 1 [Source:MGI Symbol;Acc:MGI:1916406]

ENSMUSG00000000171 Sdhd 5550.9660 1.5333 -0.6167 0.2269 -2.7184 0.0065604 0.089478

succinate dehydrogenase complex, subunit D, integral membrane protein [Source:MGI Symbol;Acc:MGI:1914175]

ENSMUSG00000034345 Gtf2h5 1765.0600 1.7660 -0.8205 0.3018 -2.7182 0.0065635 0.089478

general transcription factor IIH, polypeptide 5 [Source:MGI Symbol;Acc:MGI:107227]

ENSMUSG00000063605 Ccdc102a 218.8927 1.4759 -0.5616 0.2066 -2.7182 0.006563 0.089478

coiled-coil domain containing 102A [Source:MGI Symbol;Acc:MGI:2686927]

ENSMUSG00000008200 Fnbp4 2286.5069 0.7225 0.4690 0.1727 2.7163 0.0066017 0.0898311

formin binding protein 4 [Source:MGI Symbol;Acc:MGI:1860513]

ENSMUSG00000037313 Tacc3 4108.0819 1.3830 -0.4678 0.1722 -2.7160 0.0066066 0.0898311

transforming, acidic coiled-coil containing protein 3 [Source:MGI Symbol;Acc:MGI:1341163]

ENSMUSG00000005873 Reep5 6100.6677 1.6124 -0.6892 0.2539 -2.7142 0.0066441 0.0902226

receptor accessory protein 5 [Source:MGI Symbol;Acc:MGI:1270152]

ENSMUSG00000000823 Znf512b 421.5152 0.5597 0.8374 0.3088 2.7120 0.0066882 0.0907038

zinc finger protein 512B [Source:MGI Symbol;Acc:MGI:2685478]

ENSMUSG00000037138 Aff3 781.9268 0.5808 0.7838 0.2891 2.7108 0.0067127 0.0909173

AF4/FMR2 family, member 3 [Source:MGI Symbol;Acc:MGI:106927]

198

ENSMUSG00000000088 Cox5a 1955.3717 1.5200 -0.6041 0.2230 -2.7091 0.0067463 0.0911535

cytochrome c oxidase subunit Va [Source:MGI Symbol;Acc:MGI:88474]

ENSMUSG00000047557 Lxn 284.1283 1.4284 -0.5144 0.1899 -2.7091 0.0067476 0.0911535latexin [Source:MGI Symbol;Acc:MGI:107633]

ENSMUSG00000020328 Nudcd2 1910.2636 1.5246 -0.6084 0.2249 -2.7051 0.0068279 0.0921197

NudC domain containing 2 [Source:MGI Symbol;Acc:MGI:1277103]

ENSMUSG00000057278 Snrpg 6074.5298 1.8274 -0.8698 0.3219 -2.7024 0.0068844 0.092762

small nuclear ribonucleoprotein polypeptide G [Source:MGI Symbol;Acc:MGI:1915261]

ENSMUSG00000069729 Arid1b 1660.1219 0.6759 0.5650 0.2092 2.7008 0.0069174 0.0930856

AT rich interactive domain 1B (SWI-like) [Source:MGI Symbol;Acc:MGI:1926129]

ENSMUSG00000025499 Hras1 1682.2796 1.7367 -0.7963 0.2950 -2.6994 0.0069471 0.093365

Harvey rat sarcoma virus oncogene [Source:MGI Symbol;Acc:MGI:96224]

ENSMUSG00000014769 Psmb1 7740.6988 1.6877 -0.7550 0.2800 -2.6963 0.0070109 0.0940441


ENSMUSG00000052738 Suclg1 2619.4609 1.4000 -0.4854 0.1801 -2.6961 0.0070157 0.0940441

succinate-CoA ligase, GDP-forming, alpha subunit [Source:MGI Symbol;Acc:MGI:1927234]

ENSMUSG00000044617 Zbtb39 392.1457 0.7087 0.4968 0.1843 2.6955 0.0070293 0.0941058

zinc finger and BTB domain containing 39 [Source:MGI Symbol;Acc:MGI:2443316]

ENSMUSG00000004610 Etfb 5029.9270 1.8039 -0.8511 0.3165 -2.6890 0.0071675 0.0943805

electron transferring flavoprotein, beta polypeptide [Source:MGI Symbol;Acc:MGI:106098]

199

ENSMUSG00000019189 Rnf145 2035.6308 0.7456 0.4236 0.1574 2.6915 0.0071123 0.0943805

ring finger protein 145 [Source:MGI Symbol;Acc:MGI:1921565]

ENSMUSG00000019916 P4ha1 1005.7132 1.6351 -0.7093 0.2634 -2.6925 0.0070913 0.0943805

procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha 1 polypeptide [Source:MGI Symbol;Acc:MGI:97463]

ENSMUSG00000021024 Psma6 6041.3874 1.6306 -0.7054 0.2623 -2.6894 0.007159 0.0943805


ENSMUSG00000022378 Fam49b 13410.4071 1.3511 -0.4341 0.1612 -2.6922 0.0070983 0.0943805

family with sequence similarity 49, member B [Source:MGI Symbol;Acc:MGI:1923520]

ENSMUSG00000028121 Bcar3 444.0172 0.5418 0.8843 0.3283 2.6938 0.0070647 0.0943805

breast cancer anti-estrogen resistance 3 [Source:MGI Symbol;Acc:MGI:1352501]

ENSMUSG00000029016 Clcn6 371.2953 0.6503 0.6208 0.2305 2.6932 0.007076 0.0943805

chloride channel 6 [Source:MGI Symbol;Acc:MGI:1347049]

ENSMUSG00000036295 Lrrn3 113.4629 0.5633 0.8280 0.3078 2.6901 0.007142 0.0943805

leucine rich repeat protein 3, neuronal [Source:MGI Symbol;Acc:MGI:106036]

ENSMUSG00000042148 Cox10 562.5081 1.4111 -0.4968 0.1847 -2.6902 0.0071402 0.0943805

cytochrome c oxidase assembly protein 10 [Source:MGI Symbol;Acc:MGI:1917633]

200

ENSMUSG00000059734 Ndufs8 2732.5557 1.6360 -0.7102 0.2641 -2.6896 0.0071534 0.0943805


ENSMUSG00000067995 Gtf2f2 1280.4200 1.4385 -0.5246 0.1949 -2.6910 0.0071228 0.0943805

general transcription factor IIF, polypeptide 2 [Source:MGI Symbol;Acc:MGI:1915955]

ENSMUSG00000078713 Tomm5 2720.4651 1.8189 -0.8630 0.3209 -2.6891 0.0071637 0.0943805

translocase of outer mitochondrial membrane 5 homolog (yeast) [Source:MGI Symbol;Acc:MGI:1915762]

ENSMUSG00000090213 Tmem189 1565.0207 1.4481 -0.5342 0.1986 -2.6904 0.0071359 0.0943805


ENSMUSG00000002379 Ndufa11 2146.9835 1.8617 -0.8966 0.3338 -2.6861 0.0072294 0.0946554

NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 11 [Source:MGI Symbol;Acc:MGI:1917125]

ENSMUSG00000015013 Trappc2l 1288.5370 1.7595 -0.8151 0.3034 -2.6863 0.0072253 0.0946554

trafficking protein particle complex 2-like [Source:MGI Symbol;Acc:MGI:1916295]

ENSMUSG00000038085 4921517L17 995.5739 1.7421 -0.8008 0.2981 -2.6861 0.00723 0.0946554

cyclic nucleotide binding domain containing 2 [Source:MGI Symbol;Acc:MGI:1918123]

ENSMUSG00000041237 Pklr 769.4644 0.5347 0.9031 0.3362 2.6859 0.0072337 0.0946554

pyruvate kinase liver and red blood cell [Source:MGI Symbol;Acc:MGI:97604]

ENSMUSG00000078784 1810022K09 829.2042 1.7083 -0.7726 0.2876 -2.6867 0.0072161 0.0946554


201

ENSMUSG00000041298 Katnal1 244.8039 0.7195 0.4749 0.1768 2.6852 0.0072481 0.0947244katanin p60 subunit A-like 1 [Source:MGI Symbol;Acc:MGI:2387638]

ENSMUSG00000019158 Tmem160 1837.0761 2.0197 -1.0142 0.3780 -2.6830 0.0072973 0.0947734


ENSMUSG00000024966 Stip1 5386.2753 1.2795 -0.3556 0.1325 -2.6840 0.0072753 0.0947734

stress-induced phosphoprotein 1 [Source:MGI Symbol;Acc:MGI:109130]

ENSMUSG00000028639 Ybx1 22572.7453 1.3096 -0.3891 0.1450 -2.6836 0.0072824 0.0947734

Y box protein 1 [Source:MGI Symbol;Acc:MGI:99146]

ENSMUSG00000029048 Rer1 4174.0257 1.4185 -0.5044 0.1880 -2.6830 0.0072965 0.0947734

RER1 retention in endoplasmic reticulum 1 homolog (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1915080]

ENSMUSG00000070733 Fryl 1720.9189 0.6294 0.6679 0.2489 2.6834 0.0072883 0.0947734

furry homolog-like (Drosophila) [Source:MGI Symbol;Acc:MGI:1919563]

ENSMUSG00000026365 Cfh 980.3570 2.2681 -1.1815 0.4412 -2.6778 0.0074098 0.096004

complement component factor h [Source:MGI Symbol;Acc:MGI:88385]

ENSMUSG00000042541 Shfm1 9924.7171 1.8410 -0.8805 0.3288 -2.6778 0.0074104 0.096004

split hand/foot malformation (ectrodactyly) type 1 [Source:MGI Symbol;Acc:MGI:109238]

ENSMUSG00000006800 Sulf2 713.2787 0.6618 0.5954 0.2225 2.6767 0.0074356 0.0962111

sulfatase 2 [Source:MGI Symbol;Acc:MGI:1919293]

ENSMUSG00000014599 Csf1 228.1942 0.6766 0.5637 0.2106 2.6761 0.0074484 0.0962566

colony stimulating factor 1 (macrophage) [Source:MGI Symbol;Acc:MGI:1339753]

202

ENSMUSG00000024201 Kdm4b 686.1434 0.6561 0.6080 0.2275 2.6724 0.0075304 0.0966657

lysine (K)-specific demethylase 4B [Source:MGI Symbol;Acc:MGI:2442355]

ENSMUSG00000025085 Ablim1 1300.9345 0.4882 1.0345 0.3871 2.6722 0.0075356 0.0966657

actin-binding LIM protein 1 [Source:MGI Symbol;Acc:MGI:1194500]

ENSMUSG00000029815 2410003K15 471.1783 1.6916 -0.7584 0.2837 -2.6730 0.0075165 0.0966657

mitochondrial assembly of ribosomal large subunit 1 [Source:MGI Symbol;Acc:MGI:1922843]

ENSMUSG00000030512 Snrpa1 2518.6610 1.6003 -0.6784 0.2538 -2.6725 0.0075288 0.0966657

small nuclear ribonucleoprotein polypeptide A' [Source:MGI Symbol;Acc:MGI:1916231]

ENSMUSG00000045409 Trim39 770.7066 0.7306 0.4528 0.1694 2.6736 0.0075042 0.0966657


ENSMUSG00000050490 Gm8394 402.5961 1.6351 -0.7093 0.2653 -2.6742 0.0074917 0.0966657


ENSMUSG00000039105 Atp6v1g1 5798.0868 1.7276 -0.7888 0.2953 -2.6714 0.0075539 0.0967809

ATPase, H+ transporting, lysosomal V1 subunit G1 [Source:MGI Symbol;Acc:MGI:1913540]

ENSMUSG00000006763 Saal1 817.8003 1.4295 -0.5155 0.1932 -2.6681 0.0076278 0.0973679

serum amyloid A-like 1 [Source:MGI Symbol;Acc:MGI:1926185]

ENSMUSG00000017002 Slpi 9950.7686 1.7607 -0.8162 0.3060 -2.6669 0.0076557 0.0973679secretory leukocyte peptidase inhibitor [Source:MGI Symbol;Acc:MGI:109297]

ENSMUSG00000021190 Lgmn 1796.9929 1.5847 -0.6642 0.2489 -2.6685 0.0076189 0.0973679

legumain [Source:MGI Symbol;Acc:MGI:1330838]

203

ENSMUSG00000025780 Itih5 336.8173 0.7025 0.5095 0.1910 2.6670 0.0076521 0.0973679

inter-alpha (globulin) inhibitor H5 [Source:MGI Symbol;Acc:MGI:1925751]

ENSMUSG00000027274 Mkks 625.2818 1.5036 -0.5884 0.2205 -2.6683 0.0076244 0.0973679

McKusick-Kaufman syndrome [Source:MGI Symbol;Acc:MGI:1891836]

ENSMUSG00000030750 Nsmce1 2054.2601 1.7853 -0.8361 0.3135 -2.6674 0.0076438 0.0973679

non-SMC element 1 homolog (S. cerevisiae) [Source:MGI Symbol;Acc:MGI:1914961]

ENSMUSG00000001999 Blvra 1265.8287 1.4259 -0.5119 0.1920 -2.6662 0.0076721 0.0974575biliverdin reductase A [Source:MGI Symbol;Acc:MGI:88170]

ENSMUSG00000026004 1110028C15 461.8705 0.5257 0.9276 0.3480 2.6652 0.0076936 0.0976108

KAT8 regulatory NSL complex subunit 1-like [Source:MGI Symbol;Acc:MGI:1915941]

ENSMUSG00000021660 Btf3 17891.9355 1.7590 -0.8147 0.3060 -2.6626 0.0077532 0.0980959 basic transcription factor 3 [Source:MGI Symbol;Acc:MGI:1202875]

ENSMUSG00000028932 Psmc2 4060.4196 1.3591 -0.4427 0.1663 -2.6623 0.00776 0.0980959

proteasome (prosome, macropain) 26S subunit, ATPase 2 [Source:MGI Symbol;Acc:MGI:109555]

ENSMUSG00000064326 Siva1 2184.2797 1.8349 -0.8757 0.3289 -2.6625 0.0077563 0.0980959

SIVA1, apoptosis-inducing factor [Source:MGI Symbol;Acc:MGI:1353606]

ENSMUSG00000004207 Psap 70277.2797 1.4069 -0.4926 0.1851 -2.6607 0.0077974 0.0983871

prosaposin [Source:MGI Symbol;Acc:MGI:97783]

ENSMUSG00000030341 Tnfrsf1a 4515.0541 1.3699 -0.4541 0.1707 -2.6605 0.0078019 0.0983871

tumor necrosis factor receptor superfamily, member 1a [Source:MGI Symbol;Acc:MGI:1314884]

204

ENSMUSG00000024217 Snrpc 2909.5577 1.7843 -0.8354 0.3141 -2.6598 0.0078185 0.0984774

U1 small nuclear ribonucleoprotein C [Source:MGI Symbol;Acc:MGI:109489]

ENSMUSG00000038524 Fchsd1 170.3887 0.5752 0.7978 0.3000 2.6592 0.0078324 0.0985331

FCH and double SH3 domains 1 [Source:MGI Symbol;Acc:MGI:2441771]

ENSMUSG00000030706 Mrpl48 1212.6525 1.6044 -0.6820 0.2565 -2.6583 0.0078531 0.0986741


ENSMUSG00000040767 Snrnp25 1312.8770 1.8743 -0.9063 0.3411 -2.6575 0.0078728 0.0986841

small nuclear ribonucleoprotein 25 (U11/U12) [Source:MGI Symbol;Acc:MGI:1925622]

ENSMUSG00000042207 Kdm5b 897.7698 0.7522 0.4109 0.1546 2.6577 0.0078671 0.0986841

lysine (K)-specific demethylase 5B [Source:MGI Symbol;Acc:MGI:1922855]

ENSMUSG00000027018 Hat1 2956.7700 1.4161 -0.5019 0.1890 -2.6553 0.0079232 0.099197

histone aminotransferase 1 [Source:MGI Symbol;Acc:MGI:96013]

ENSMUSG00000039640 Mrpl12 1396.9537 1.6651 -0.7356 0.2771 -2.6545 0.0079427 0.0993221


ENSMUSG00000019179 Mdh2 9224.0393 1.3885 -0.4735 0.1785 -2.6535 0.0079665 0.0995003

malate dehydrogenase 2, NAD (mitochondrial) [Source:MGI Symbol;Acc:MGI:97050]

ENSMUSG00000044708 Kcnj10 122.0057 0.5242 0.9319 0.3513 2.6524 0.0079917 0.0995764potassium inwardly-rectifying channel, subfamily J, member 10 [Source:MGI Symbol;Acc:MGI:1194504]

ENSMUSG00000062585 Cnr2 1688.4257 0.6465 0.6294 0.2372 2.6528 0.0079827 0.0995764

cannabinoid receptor 2 (macrophage) [Source:MGI Symbol;Acc:MGI:104650]

205

ENSMUSG00000000295 Hddc2 808.4635 1.7523 -0.8092 0.3055 -2.6492 0.0080682 0.0996436

HD domain containing 2 [Source:MGI Symbol;Acc:MGI:1916942]

ENSMUSG00000011958 Bnip2 3972.4021 1.3038 -0.3827 0.1444 -2.6503 0.0080409 0.0996436

BCL2/adenovirus E1B interacting protein 2 [Source:MGI Symbol;Acc:MGI:109327]

ENSMUSG00000014846 Tppp3 376.6771 1.7324 -0.7928 0.2992 -2.6498 0.008053 0.0996436

tubulin polymerization-promoting protein family member 3 [Source:MGI Symbol;Acc:MGI:1915221]

ENSMUSG00000021699 Pde4d 326.7377 0.7116 0.4909 0.1853 2.6496 0.0080592 0.0996436

phosphodiesterase 4D, cAMP specific [Source:MGI Symbol;Acc:MGI:99555]

ENSMUSG00000022890 Atp5j 4334.3162 1.4288 -0.5148 0.1943 -2.6490 0.0080735 0.0996436

ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F [Source:MGI Symbol;Acc:MGI:107777]

ENSMUSG00000028447 Dctn3 1655.6161 1.6268 -0.7021 0.2649 -2.6506 0.0080347 0.0996436

dynactin 3 [Source:MGI Symbol;Acc:MGI:1859251]

ENSMUSG00000032023 4931429I11 190.4175 0.4529 1.1427 0.4310 2.6512 0.0080204 0.0996436


ENSMUSG00000038065 2410066E13 237.6916 0.5371 0.8967 0.3384 2.6497 0.0080574 0.0996436

maturin, neural progenitor differentiation regulator homolog (Xenopus) [Source:MGI Symbol;Acc:MGI:1915485]

ENSMUSG00000006179 Prss16 278.2710 1.5211 -0.6051 0.2286 -2.6473 0.0081128 0.0997912

protease, serine 16 (thymus) [Source:MGI Symbol;Acc:MGI:1859181]

206

ENSMUSG00000024747 Aldh1a7 134.4085 0.5485 0.8664 0.3273 2.6471 0.0081185 0.0997912

aldehyde dehydrogenase family 1, subfamily A7 [Source:MGI Symbol;Acc:MGI:1347050]

ENSMUSG00000040658 BC048355 815.3288 1.9007 -0.9265 0.3500 -2.6473 0.0081139 0.0997912

2'-deoxynucleoside 5'-phosphate N-hydrolase 1 [Source:MGI Symbol;Acc:MGI:3039376]

ENSMUSG00000045411 2410002F23 819.6987 0.6764 0.5640 0.2131 2.6469 0.0081238 0.0997912

RIKEN cDNA 2410002F23 gene [Source:MGI Symbol;Acc:MGI:1914226]

207

selective hdac6 inhibition in systemic lupus erythematosus miranda diane … · 2020-01-16 ·...

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