QUICK TIPS (--THIS SECTION DOES NOT PRINT--)

This PowerPoint template requires basic PowerPoint (version 2007 or newer) skills. Below is a list of commonly asked questions specific to this template. If you are using an older version of PowerPoint some template features may not work properly.

Using the template

Verifying the quality of your graphics Go to the VIEW menu and click on ZOOM to set your preferred magnification. This template is at 100% the size of the final poster. All text and graphics will be printed at 100% their size. To see what your poster will look like when printed, set the zoom to 100% and evaluate the quality of all your graphics before you submit your poster for printing. Using the placeholders To add text to this template click inside a placeholder and type in or paste your text. To move a placeholder, click on it once (to select it), place your cursor on its frame and your cursor will change to this symbol: Then, click once and drag it to its new location where you can resize it as needed. Additional placeholders can be found on the left side of this template. Modifying the layout This template has four different column layouts. Right-click your mouse on the background and click on “Layout” to see the layout options. The columns in the provided layouts are fixed and cannot be moved but advanced users can modify any layout by going to VIEW and then SLIDE MASTER. Importing text and graphics from external sources TEXT: Paste or type your text into a pre-existing placeholder or drag in a new placeholder from the left side of the template. Move it anywhere as needed. PHOTOS: Drag in a picture placeholder, size it first, click in it and insert a photo from the menu. TABLES: You can copy and paste a table from an external document onto this poster template. To adjust the way the text fits within the cells of a table that has been pasted, right-click on the table, click FORMAT SHAPE then click on TEXT BOX and change the INTERNAL MARGIN values to 0.25 Modifying the color scheme To change the color scheme of this template go to the “Design” menu and click on “Colors”. You can choose from the provide color combinations or you can create your own.

QUICK DESIGN GUIDE (--THIS SECTION DOES NOT PRINT--)

This PowerPoint 2007 template produces a 36”x48” professional poster. It will save you valuable time placing titles, subtitles, text, and graphics. Use it to create your presentation. Then send it to PosterPresentations.com for premium quality, same day affordable printing. We provide a series of online tutorials that will guide you through the poster design process and answer your poster production questions. View our online tutorials at: http://bit.ly/Poster_creation_help (copy and paste the link into your web browser). For assistance and to order your printed poster call PosterPresentations.com at 1.866.649.3004

Object Placeholders

Use the placeholders provided below to add new elements to your poster: Drag a placeholder onto the poster area, size it, and click it to edit. Section Header placeholder Move this preformatted section header placeholder to the poster area to add another section header. Use section headers to separate topics or concepts within your presentation. Text placeholder Move this preformatted text placeholder to the poster to add a new body of text. Picture placeholder Move this graphic placeholder onto your poster, size it first, and then click it to add a picture to the poster.

RESEARCH POSTER PRESENTATION DESIGN © 2012

www.PosterPresentations.com

©  2012  PosterPresenta.ons.com          2117  Fourth  Street  ,  Unit  C          Berkeley  CA  94710          posterpresenter@gmail.com  

Student discounts are available on our Facebook page. Go to PosterPresentations.com and click on the FB icon.

Embryonic stem cells (ESCs) possess the capacity to differentiate into multiple cell types and to proliferate indefinitely. Locus-specific modifications of histones participate in the epigenetic regulation of genes involved in cell growth, differentiation and cell fate. Histone H3 lysine 18 acetylation (H3K18ac) plays a major role in cell cycle as it is abundant in growth-arrested cells and lost in cancer3,4,5. Furthermore, in growth-arrested cells, depletion of H3K18ac is sufficient to trigger re-entry into the cell cycle1,2. The H3K18ac-modification is produced by at least four lysine acetyltransferases including p300, CBP, PCAF and GCN5. Genetic studies targeting individual lysine acetyltransferases in mouse indicate redundancy between these enzymes, e.g. p300 and CBP and possible roles in early development, e.g. Gcn5 in mesoderm formation. These studies and others suggest that through H3K18ac, these enzymes may play a major role in the epigenetic control of cell identity. In this study, we found that H3K18ac is highly enriched at stem cell determining loci in ESC and investigate the developmental consequence of H3K18ac dependency on ESC identity and pluripotency.

RESULTS

RESULTS (continued) RESULTS (continued)

REFERENCES

ACKNOWLEDGEMENTS This work was supported by generous grants from the California Institute for Regenerative Medicine RN2-00908, and institutional funds to B.D.Y..

Division of Dermatology, Department of Medicine, Stem Cell Program, and Institute for Genomic Medicine, University of California, San Diego, La Jolla, CA 92093, USA.

Shantanu Kumar, Craig Ricker and Benjamin D. Yu

Functional role of histone H3 lysine 18 acetylation in the maintenance of pluripotency in embryonic stem cells

CONCLUSION We demonstrated that upon global reduction of H3K18ac by E1A oncoprotein, mESCs lose characteristics associated with pluripotency and fail to activate enhancers that are associated with stem cell identity. Lineage markers of neuroectoderm and endoderm were also suppressed by E1A suggesting a functional role of H3K18ac in lineage specifications. Loss of pluripotency by E1A requires binding to H3K18 acetyltransferases, P300/CBP, but does not require pRB- and P400-family interaction.

Fig. 3. Loss of pluripotency in mESCs expressing E1A (A), Bright field image of mES cells infected with control Ad and Ad E1A (B), Alkaline phosphatase (AP) staining of mESCs infected with control and Ad E1A (C), Immunofluorescence for Oct3/4 and E1a expression in MESCs 72h post transduction. Oct3/4, red; E1a, green (D), RT-qPCR measuring mRNA levels of pluripotency markers in MESCs expressing E1A relative to control. Data are represented as mean ± SD

Fig. 5. Change in expression of differentiation markers in mESCs cells expressing E1A (A), RT-qPCR measuring mRNA levels of differentiation markers in MESCs expressing E1a relative to control at 24, 48 and 72 hr post transduction. Data are represented as mean ± SD

C E1A

C E1A

E1A Protein Levels

Cel

l cou

nts

0.9 1.2 0.6 0.9 1.0 0.4 1.3 1.2 0.7

H3K18ac

Total H3

24h 48h 72h

Ratio of H3K18ac / Total H3

Ad Control Ad E1A

Loss of pluripotency in mESCs expressing E1A

Figure 1. H3K18ac occupancy in hESCs and derived germ layers. (A) ChIP-seq occupancy for H3K18ac over POU5F1, SOX2, KLF4 and NANOG (B) Distribution of ChIP-seq signal near TSS comparing global analysis of ESC Specific (Top 2% H1 ESC vs. H1 EB) and differentiation specific genes (Bottom 2%). (C) Box-plot for differences in H3K18ac ChIP-seq density in hESCs and derived germ layers comparing ESC genes. We found decreasing enrichment of the ESC related genes in progressively more differentiated cell types. P-values (<2.2e-16), calculated using two-tailed t-test. ChIP-seq data based on Xie et al. (2013)

Figure 2. Depletion of H3K18ac by E1A oncoprotein in mESCs. (A)  Immunofluorescence for E1a expression in mES cells 24h post transduction. (B), Flow cytometry analysis for E1a expression. (C), Western blot analysis for H3K18ac expression at various time point post Ad control, Ad R2G(Ad mutant does not bind p300/CBP) and Ad E1A transduction (D), CHIP-qRT PCR for H3K18ac occupancy on pluripotency genes promoters.

Depletion of H3K18ac by Adenovirus E1A oncoprotein

H3K18Ac occupancy in hESCs and derived germ layers

Loss of enhancer activity activity in mESCs expressing E1A

Figure 4. Loss of enhancer reporter in mESCs expressing E1A (A), mESCs expressing EOS-GFP reporter and loss of GFP expression after differentiation (B), Fluorescence and Bright field image of mESCs-EOS reporter cell line transduced with control and Ad E1a 72h post transduction.

0

1

2

3

4

5

6

7

Pax6 Notch BraT PECAM1 Gata4 Foxa2 Gata6

Control-24h

Control-48

Control-72h

E1a-24h

E1a-48h

E1A-72h

Effect of E1A on lineage differentiation

1.  Horwitz, G.A. et al. Adenovirus small e1a alters global patterns of histone modification. Science 321, 1084-1085 (2008).

2.  Ferrari, R. et al. Epigenetic reprogramming by adenovirus e1a. Science 321, 1086-1088 (2008).

3.  Barber, M.F. et al. SIRT7 links H3K18 deacetylation to maintenance of oncogenic transformation. Nature 487, 114-118 (2012).

4.  Seligson, D.B. et al. Global histone modification patterns predict risk of prostate cancer recurrence. Nature 435, 1262-1266 (2005).

5.  Pasqualucci L. et al. Inactivating mutations of acetyltransferases genes in B-cell lymphoma. Nature 471, 189-95 (2011)

6.  Xie W.et al. Epigenomic analysis of multilineage differentiation of human embryonic stem cells. Cell 153, 1134-48 (2013)

INTRODUCTION

EOS-GFP -LIF (5D) -LIF +RA (4D)

Control E1A

0

1

2

3

4

5

6

Bra Gsc Evx1 Eomes fgf-8 cdx2

Fold

cha

nge

Fold

cha

nge

CR4

RESULTS (continued)

H1 ESC

H1-derived mesendoderm

(ME)

H1-derived neural (NE)

H1-derived mesoderm

(MES)

*

*

B

C

A

A B

C D

A

B

Fig. 6. Loss of pluripotency requires E1A to bind p300/CBP (A) Schematics of E1A and their mutants. (B) Flow cytometer analysis of E1A and their mutants expression in mESCs (C) phase contrasts image of mESCs expressing E1A and their mutants, 72h post transduction.

Loss of pluripotency requires E1A to bind p300/CBP

A

A B

C

Top 2% Bot 2% All

T Gsc Evx1 Eomes Fgf8 Cdx2