1 spatial interplay between polycomb and trithorax complexes
TRANSCRIPT
1
Spatial interplay between Polycomb and Trithorax complexes controls 1
transcriptional activity in T lymphocytes. 2
3
Atsushi Onoderaa, Damon J. Tumesa, Yukiko Watanabea, Kiyoshi Hiraharab, 4
Atsushi Kanedac, Fumihiro Sugiyamad, Yutaka Suzukie and Toshinori 5
Nakayamaa,f. 6
7
aDepartment of Immunology, bDepartment of Advanced Allegology of the Airway, 8
cDepartment of Molecular Oncology, Graduate School of Medicine, Chiba 9
University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, dLaboratory Animal 10
Resource Center, University of Tsukuba, Tsukuba, Ibaraki 305-8575, 11
eLaboratory of Functional Genomics, Department of Medical Genome Sciences, 12
Graduate School of Frontier Sciences, University of Tokyo, 5-1-5, Kashiwanoha, 13
Kashiwa, Chiba, 277-8562, and f AMED -CREST, AMED, 1-8-1 Inohana, 14
Chuo-ku, Chiba 260-8670, Japan. 15
Running title: Ezh2/Menin co-occupancy and lymphocyte development 16
Key words: Menin; Ezh2; T cells; ES cells; transcriptional regulation; transcription start 17
site 18
Correspondence: Dr. Toshinori Nakayama, Department of Immunology, 19
Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 20
260-8670 Japan. Phone number: +81-43-226-2200, FAX number: 21
+81-43-227-1498, e-mail address: [email protected] 22
MCB Accepted Manuscript Posted Online 31 August 2015Mol. Cell. Biol. doi:10.1128/MCB.00677-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Abstract 24
Trithorax group (TrxG) and Polycomb group (PcG) proteins are two 25
mutually antagonistic chromatin modifying complexes, however, how they 26
together mediate transcriptional counterregulation remains unknown. 27
Genome-wide analysis revealed that binding of Ezh2 and Menin, central 28
members of the PcG and TrxG complexes, respectively, were reciprocally 29
correlated. Moreover, we identified a developmental change in the positioning 30
of Ezh2 and Menin in differentiated T lymphocytes compared to embryonic stem 31
cells. Ezh2-binding upstream and Menin-binding downstream of the 32
transcription start site (TSS) was frequently found at genes with higher 33
transcriptional levels, and Ezh2-binding downstream and Menin-binding 34
upstream was found at genes with lower expression in T lymphocytes. 35
Interestingly, of the Ezh2 and Menin co-occupied genes, those exhibiting 36
occupancy at the same position displayed greatly enhanced sensitivity to loss of 37
Ezh2. Finally, we also found that different combinations of Ezh2 and Menin 38
occupancy were associated with expression of specific functional gene groups 39
important for T cell development. Therefore, spatial cooperative gene 40
regulation by the PcG and TrxG complexes may represent a novel mechanism 41
regulating the transcriptional identity of differentiated cells. 42
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Introduction 44
Trithorax group (TrxG) and Polycomb group (PcG) complexes exert 45
opposing effects on the maintenance of transcriptional status and play a critical 46
role in the expression of developmentally regulated transcription factors through 47
methylation at histone H3-K4 (H3K4me3; a permissive mark) and H3-K27 48
(H3K27me3; a repressive mark), respectively (1-5). In embryonic stem (ES) 49
cells, a set of genes encoding developmental regulators was found to be 50
controlled by the PcG complex (4, 6) and the TrxG complex was found to be 51
essential for self-renewal and reprogramming (7, 8). TrxG and PcG complexes 52
have also been recognized as crucial factors regulating the terminal 53
differentiation of some cell types, such as epidermal cells (9), germ cells (10), 54
muscle cells (11) and T lymphocytes (3, 12-14), and mutations in these proteins 55
are often associated with tumorigenic potential (15). 56
PcG proteins are subdivided into two major repressive complexes: 57
polycomb repressive complex 1 (PRC1) and PRC2. The PcG protein Enhancer 58
of Zeste Homolog 2 (Ezh2) is a histone methyltransferase specific for H3K27 59
and is essential for repression of target gene transcription (16). In CD4+ T cells, 60
Ezh2 has been shown to directly bind and facilitate correct expression the Gata3 61
gene during differentiation into effector cells (12, 14). In addition, Ezh2 has 62
recently been found to play an essential role in regulating the germinal center 63
(GC) response by facilitating normal activation-induced cytidine deaminase 64
(AID) function and preventing terminal differentiation of GC B cells (17, 18). 65
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Compared to PcG proteins, TrxG proteins show more diversity 66
regarding the different molecules that they form complexes with. Menin is 67
found in TrxG complexes containing MLL1 or MLL2, which are responsible for 68
H3K4me3 (2, 19). The protein Menin is encoded by the MEN1 gene, which is 69
mutated in patients with multiple endocrine neoplasia type 1 (MEN1) syndrome 70
(20, 21). Menin can act as a tumour suppressor and is required for binding of 71
the complex to DNA (2). Menin also plays a crucial role in immune system 72
since it is shown to be important for Th2 cell function both in mice and humans 73
(14, 22). Although a considerable number of studies have been carried out on 74
the nature of PcG proteins or TrxG proteins individually, it has not been well 75
defined how transcriptional counterregulation is organized by the TrxG and PcG 76
complexes. With the exception of recent pioneering work demonstrating 77
dynamic transformations of histone modifications during T cell development (23), 78
how the global signature of TrxG and PcG co-occupied genes is changed during 79
developmental processes remains unclear. 80
Here we address these unresolved but important biological questions 81
by assessment of spatial interaction of chromatin regulators on a genome-wide 82
scale in ES cells and mature B and T lymphocytes using chromatin 83
immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-Seq) 84
(24). This study reveals a new cooperative regulation by the PcG/TrxG 85
complex that controls the transcriptional identity of differentiated cells. 86
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Materials and Methods 88
Mice 89
C57BL/6 mice were purchased from CLEA (Tokyo, Japan). Mice with loxp 90
sites flanking the SET domain of Ezh2 were generated as previously described 91
(25), and backcrossed with C57BL/6 mice for 10 generations. These mice were 92
then bred with mice expressing transgenic constructs for Cre recombinase under 93
the control of the CD4 promoter, allowing for conditional knock out (KO) of Ezh2 94
function in CD4+ T cells (CD4-Cre). CD4-Cre mice were purchased from Taconic. 95
All mice used in this study were maintained under specific pathogen-free 96
conditions and ranged from 6-8 weeks of age. All experimental protocols using 97
mice were approved by the Chiba University animal committee. All animal care 98
was performed in accordance with the guidelines of Chiba University. 99
100
Antibodies 101
The antibodies used for the ChIP assay were anti-Bmi1 (Santa cruz: sc-10745), 102
anti-Ezh2 (diagenode: pAb-039-050), anti-Menin (bethyl: A300-105A), and 103
anti-Ser5-P RNA polymerase II (Abcam: ab5408). 104
105
Isolation of B220+ B cells and CD4+ T cells from mouse spleen 106
B220+ B and CD4+ T cells were purified using magnetic beads and an 107
AutoMACS sorter (Miltenyi Biotec) that yielded purity of >98%. 108
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Generation Th2 cells and trichostatin A (TSA) treatment 110
Th2 cells were generated as previously described(13). In brief, splenic CD4+ T 111
cells were stimulated with 3 μg/ml of immobilized anti–TCR-β mAb plus 1 μg/ml 112
anti-CD28 mAb under Th2-culture conditions for 5 days in vitro. Th2 113
conditions; 15 ng/ml IL-2, 10 ng/ml IL-4. These cells were used as Th2 cells. 114
For TSA treatment, splenic CD4 cells were cultured under Th2 conditions and 10 115
nM TSA (Sigma, St Louis, MO) was added in the culture on day 2 and after 116
another 3-days of culture, CD4 T cells were collected for analysis. 117
118
ES cell culture 119
B6N-22 ES cells (26) were established and characterized previously. ES cells 120
were cultivated on mitomycin C (MMC) -treated mouse embryonic fibroblast 121
(MEF) in DMEM containing 0.1 mM 2-mercaptoethanol, 1000 units/ml leukemia 122
inhibitory factor (LIF), nonessential amino acids (NEAA), sodium pyruvate and 123
20% fetal bovine serum (FBS). 124
125
Chromatin immunoprecipitation (ChIP) assay 126
ChIP experiments for Bmi1, Ezh2, Menin, RNAPII and control antibody were 127
carried out using dynabeads (Invitrogen). In brief, 1 × 107 ES, B and CD4+ T 128
cells were fixed with 1% paraformaldehyde at 37 °C for 10 minutes. Cells were 129
sedimented, washed, lysed with SDS lysis buffer (50 mM Tris-HCl, 1% SDS, 10 130
mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, and 1 μg/ml 131
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leupeptin). The lysates were sonicated to reduce DNA lengths to between 150 132
and 300 bp. The soluble fraction was diluted in ChIP dilution buffer, and 133
incubated with antibody conjugated with dynabeads protein A and G overnight at 134
4°C. Then immune complexes were captured by magnet and washed with low 135
salt, high salt, LiCl, as well as TE wash buffer. Enriched chromatin fragments 136
were eluted with elution buffer (0.1 M NaHCO3 containing 1% SDS). The 137
eluted material was incubated at 65 °C for 6 hours to reverse the formaldehyde 138
cross-links, and treated with RNase A (10 ug/ml) and Proteinase K (40 ug/ml). 139
DNA was extracted with a QIAquick PCR purification kit (QIAGEN). Total input 140
DNA (cellular DNA without immunoprecipitation) was purified in parallel. 141
142
ChIP-Seq and Illumina sequencing 143
Antibody-specific immunoprecipitates and total input DNA samples were 144
prepared using a ChIP-Seq Sample Prep kit (Illumina). Adaptor-ligated DNA of 145
170 - 250 bp was recovered by size-fractionation on an acrylamide gel. This 146
DNA was then amplified by 18 cycles of PCR and one nanogram used for 36 147
cycles of sequencing reaction on an Illumina Genome Analyser IIx. Read 148
sequences (36 bp) were then aligned to the mm9 mouse reference genome 149
(University of California, Santa Cruz (UCSC) July 2007) using Eland software 150
(Illumina). Only sequences with two or less mismatches were considered for 151
alignment. 152
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ChIP-Seq data analysis 154
Each aligned read sequence was extended to 120 bp in order to efficiently 155
detect duplicate reads aligned to identical locations. These 120 bp tags were 156
used for further analysis (Bed file). For visualization of binding, data was 157
converted to BedGraph file format using a 500 bp sliding window with step size 158
100 bp and uploaded to the IGV genome browser 159
(http://www.broadinstitute.org/igv/). 160
161
ChIP-Seq peak calling 162
The numbers of tags at each base were calculated and normalized to total tag 163
number for both antibody-precipitated and total input DNA (cellular DNA without 164
immunoprecipitation). Binding peaks were defined as a 10-fold increase in 165
normalized tag count at all bases at any successive 121 bp window, compared to 166
the normalized tag count obtained from input DNA samples at the same position. 167
A cutoff of 10 ChIP tags at each base was used to exclude peaks with very low 168
ChIP tag and low Input DNA tag counts (27-29). 169
170
Definition of promoter regions and target genes 171
We defined 21035 mouse promoters, corresponding to each RefSeq gene, as 172
the sequences between -5 kb and +3 kb of the annotated transcription start site 173
(TSS), using the mouse mm9 genome build from the RefSeq gene database 174
http://hgdownload.cse.ucsc.edu/goldenPath/mm9/database/. We first defined 175
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target promoters that contained at least one peak in the promoter region. We 176
also used fold enrichment of ChIP tag counts compared to input DNA to 177
calculate the binding level. A 2-fold increase in ChIP tag count compared to 178
input DNA in the -5kb to +3kb region was empirically shown to indicate 179
enrichment of binding. In addition, we excluded promoter regions with very low 180
normalized ChIP tag counts (<4 ppm) (see Fig. S7 in the supplemental material). 181
Thus, promoter regions showing 2-fold increased ChIP tag counts compared to 182
input DNA and containing more than 4 normalized ChIP tag counts were defined 183
as binding targets. We therefore took into account both defined peaks of binding 184
for Bmi1, Ezh2, Menin and RNAPII, and also extended areas of increased 185
binding (2-fold increase around TSS). Genes that passed either of these two 186
criteria were selected for further analysis (i.e. target genes). 187
188
Compiled tag density profiles 189
We compiled tag density profiles by dividing the promoter region into 100 bp 190
bins and counted tag base numbers in each bin. Tag base counts were 191
normalized by total tag counts for representation. 192
193
Definition of UD index 194
At each gene promoter, UD index was calculated as follows: 195
UD index = (Tag count in the 3 kb region downstream of the TSS) / (Tag count in 196
the 8 kb region across the TSS) 197
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For comparison purposes, we also used UD indices normalized by input DNA 198
values or those calculated in the region -5 kb to +5 kb relative to the TSS of each 199
gene. 200
201
Model-based Analysis of ChIP-seq (MACS) 202
For comparison purposes, MACS 1.4.2 software (p-value for peak calling set at 203
0.0001) was also used (30). We selected genes that contained at least one 204
peak in the region between -5 kb and +3 kb of the annotated TSS. Total peak 205
length of each protein (Ezh2 and Menin) was calculated by the summation of 206
length of all peaks found in the -5 kb to +3 kb regions of these selected genes. 207
Ezh2 and Menin co-occupied genes were ranked based on total peak length with 208
the ranking determined by the shorter peak of Ezh2 or Menin. Correlation 209
coefficient between Ezh2 UD indices and Menin UD indices was calculated 210
focusing on the top 50, 100, 150, 200, 250, 300, 350 and 400 co-occupied genes 211
rank-ordered as described above (Fig. 3D). 212
213
Microarray data collection and analysis 214
Total cellular RNA was extracted with TRIzol reagent (Invitrogen) according to 215
the manufacturer's instructions. RNA was labeled using a 3’ IVT Express kit 216
(Affymetrix) and hybridized to GeneChip Mouse Genome 430 2.0 arrays 217
(Affymetrix) according to the manufacturer's protocols. Expression values were 218
determined with Affymetrix GeneChip Command Console Software (AGCC) and 219
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Console Software (Expression Console). Upregulation or downregulation of 220
Refseq gene mRNA level was defined if at least one Affymetrix-GeneChip probe 221
corresponding to the RefSeq gene showed upregulation or downregulation 222
(4-fold), respectively. The maximum Affymetrix-GeneChip probe data 223
corresponding to each RefSeq gene were used for scatter plots in Fig. 4A and 224
B; Fig. 6B, C and E. 225
226
RNA-seq 227
Total cellular RNA was extracted with TRIzol reagent (Invitrogen). For cDNA 228
library construction, we used TruSeq RNA Sample Prep Kit v2 (Illumina) 229
according to the manufacturer's protocol. Sequencing the library fragments 230
was performed on the HiSeq 1500 System. For data analysis, read sequences 231
(50 bp) were aligned to the mm10 mouse reference genome (University of 232
California, Santa Cruz (UCSC) December 2011) using Bowtie (version 0.12.8) 233
and TopHat (version 1.3.2). Fragments per kilobase of exon per million 234
mapped reads (FPKM) for each gene were calculated using Cufflinks (version 235
2.0.2). Genes with absolute FPKM >1 (mean from duplicate samples) were 236
defined as expressed genes (Fig. 2C-E). Differentially regulated genes were 237
selected with following criteria (1) absolute FPKM >1 in at least 1 condition (Th2 238
and TSA-treated Th2), and (2) expression change > 2 or < -2 (Fig. 7). 239
240
Gene ontology analysis 241
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Gene ontology (GO) functional annotation for Ezh2, Bmi1 and Menin target 242
genes was performed using the DAVID analysis tool 243
(http://david.abcc.ncifcrf.gov/home.jsp). 244
245
Accession number 246
The ChIP-Seq data sets of Bmi1, Ezh2, Menin and RNAPII, and the microarray 247
data for the ES, B and T cells are available in the Gene Expression Omnibus 248
(GEO) database (http://www.ncbi.nlm.nih.gov/geo) under accession number 249
GSExxxxx. The ChIP-Seq data sets for Ezh2 and Menin, and the microarray 250
data for WT Th2 and Ezh2 KO Th2 cells are available in the GEO database 251
under accession number GSE51079 and GSE50729. For comparison 252
purposes, Ezh2Ref (GSE23943) Dpy30 (GSE26136) and Histone modification 253
(GSE23943) datasets obtained from the GEO database. 254
255
Statistics 256
Welch's s t-test was used to compare Ezh2 binding levels at the expressed 257
genes with those at the non-expressed genes. Pearson correlation coefficient 258
was used to measure the correlation between two samples and p-values were 259
calculated by test for no correlation. Fisher's exact test was used to analyze 260
2x2 contingency tables. 261
262
Results 263
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Genome-wide comparison of Ezh2 and Menin binding between ES cells 264
and B and T lymphocytes 265
We first assessed the genome-wide binding pattern of Ezh2, a central 266
member of PRC2 and Menin, a critical component of the TrxG complex, using 267
ES cells and B220+ B and CD4+ T lymphocytes by chromatin 268
immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-Seq) 269
(24) (see Table S1 in the supplemental material). To identify target genes, we 270
first called peaks by an established method whose validity is verified by using 271
Poisson probabilities (27-29). We identified 8148 and 2352 peaks for Ezh2 and 272
Menin, respectively, in T cells. However, manual inspection of peaks using the 273
IGV browser revealed a considerable number of genes that exhibited strong 274
ChIP signals were classified as “peak-less” genes (e.g. Cd69, Cd28, Stat3, 275
Nfkb1, Tox, etc. for Menin). Based on these observations and the fact that this 276
peak calling algorism is optimized for “sharp peaks”, a simple fold enrichment 277
value (ChIP / Input DNA) was taken into consideration (31, 32), and target genes 278
were defined as described in the Method section. We focused on the region –5 279
kb to +3 kb relative to the TSS of each gene (23, 33, 34). In addition, our 280
previous analysis of the Gata3 gene showed that this region contained both 281
Ezh2 and Menin peaks (14). As shown in Fig. 1A, this analysis defined a clear 282
reciprocal pattern of binding between Ezh2 and Menin in both ES cells and B 283
and T lymphocytes. In addition, a considerable number of genes (731) were 284
co-occupied by both Ezh2 and Menin in ES cells and co-occupancy was less 285
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frequent (101 in B, 118 in T cells) in lymphocytes (Fig. 1B). In B and T 286
lymphocytes, Ezh2 and Menin co-occupancy was preserved at only a few 287
percent (~4%) of the genes that were co-occupied in ES cells (see left-most bars 288
in B cell and T cell panels in Fig. 1C). Of the co-occupied genes in ES cells, 289
approximately 40 and 20% of these genes showed Ezh2 or Menin 290
mono-occupancy in lymphocytes, respectively, and approximately half of the 291
co-occupied genes lost both Ezh2 and Menin in lymphocytes (45% in B, 40% in 292
T cells). Mono-occupied genes in ES cells tended to either lose Ezh2 or Menin 293
binding or preserve their original binding characteristics in lymphocytes (the 294
second and third bars in B cell and T cell panels in Fig. 1C). Only about 10 % 295
of genes that were not bound by either Ezh2 or Menin in ES cells acquired either 296
Ezh2 or Menin binding in lymphocytes. These genome-wide analyses revealed 297
that: (1) A reciprocal binding pattern of Ezh2 and Menin is observed. (2) 298
Co-occupancy is observed far more frequently in ES cells and tends to 299
disappear during development into lymphocytes. (3) The exchange from Ezh2 300
single-occupancy to Menin single-occupancy rarely occurred, and vice versa. 301
Similar results were obtained in the analysis of Bmi1, a central member of PRC1 302
(see Fig. S1A to C in the supplemental material). 303
304
Conserved signatures of PcG occupancy between ES cells and B and T 305
lymphocytes 306
A considerable percentage of the Ezh2 target genes were shared by ES 307
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cells and lymphocytes (61% in B cells and 42% in T cells) (Fig. 2A). In addition, 308
the intensity of Ezh2 binding appeared to be relatively higher for the genes that 309
showed Ezh2 binding in ES cells and also in B and T cells (see Fig. S2A and B in 310
the supplemental material). Gene ontology analyses in lymphocytes revealed 311
that the Ezh2 targets contained a marked enrichment of genes encoding 312
developmental proteins, including members of the Hox family (Fig. 2B). As 313
expected, we found similar groups of genes enriched for targeting by Ezh2 in ES 314
cells, in agreement with previously reported findings (6, 35), and comparable 315
results were obtained for the PRC1 protein Bmi1 (see Fig. S2C and D in the 316
supplemental material). We next compared Ezh2 binding levels in ES cells and 317
lymphocytes at the polycomb targeted transcription factor (TF) genes (6) (Fig. 318
2C; see Table S2 in the supplemental material) with all known cytokine and 319
cytokine receptor genes, whose expression is important for normal lymphocyte 320
effector function (Fig. 2D; also see Table S2). In each cell type analyzed, TF 321
genes showed much stronger Ezh2 binding as compared to cytokine and 322
cytokine receptor genes (see Fig. 2C and D; mean values=12.309 vs. 1.467 in 323
ES cells, 4.552 vs. 1.128 in B cells, and 6.175 vs. 1.356 in T cells). In ES cells, 324
35% of the TF genes showed mRNA expression (1 or greater FPKM value). 325
Ezh2 binding levels were lower at these expressed genes than the 326
non-expressed genes (Fig. 2E). In B and T lymphocytes, comparatively less 327
TF genes (13% and 15% for B and T cells, respectively) showed mRNA 328
expression, and at these loci Ezh2 binding levels were lower than the 329
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non-expressed genes, indicating that Ezh2-mediated repression was involved in 330
these TF gene mRNA expression. In contrast, a considerable number of 331
cytokine and cytokine receptor genes exhibited mRNA expression (36%, 32% 332
and 39% for ES, B and T cells, respectively). Ezh2 binding levels were low for 333
almost all cytokine and cytokine receptor genes, indicating that Ezh2 was not 334
involved in the direct repression of these gene (Fig. 2E). Again, similar results 335
were obtained for Bmi1 (see Fig. S2G and H in the supplemental material). 336
These results indicate that the PcG complex favors genes encoding transcription 337
factors, and that this bias is conserved between ES cells and lymphocytes. In 338
contrast, no functional bias was observed in the type of genes that Menin targets 339
(see Fig. S2E-H in the supplemental material). 340
341
ChIP-seq binding profiles of Ezh2 and/or Menin in ES cells and B and T 342
lymphocytes 343
To investigate the nature of Ezh2 binding in more detail, we performed 344
assessment of the localization of Ezh2 binding at its target genes by generating 345
tag density profiles relative the annotated TSS of each Ezh2 target gene in ES 346
cells and B and T lymphocytes (Fig. 3A upper) (32, 36). Ezh2 bound broadly 347
across the gene promoters with peaks either side of the TSS and an apparent 348
exclusion of Ezh2 binding from the TSS itself. The clear definition of Ezh2 349
binding on either side of the TSS prompted us to measure the proportion of tags 350
upstream and downstream of the TSS, termed an “UD index”, in which the 351
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proportion of tag counts downstream of TSS is displayed numerically (see 352
Methods). Heatmap displays (37) based on the UD index revealed large 353
variation in the positioning of Ezh2 binding relative to the TSS (Fig. 3A lower). 354
Some Ezh2 target genes showed strong binding at regions downstream of the 355
TSS whereas others displayed strong binding only at regions upstream of the 356
TSS. Menin also showed similar large variation in positioning relative to the 357
TSS (see Fig. S3A in the supplemental material). Next, we compared UD 358
indices for co-occupied genes for at all combinations of Bmi1, Ezh2, Menin and 359
also the RNA polymerase II complex (RNAPII) that composes the TrxG complex. 360
The correlation coefficients for each pair of molecules in ES, B and T cells are 361
represented in the correlation matrix shown in Fig. 3B (36). This allowed us to 362
assess similarities in the positioning of these regulators of chromatin and gene 363
expression in ES, B and T cells. The position of binding relative to the TSS was 364
highly conserved between ES cells and B and T lymphocytes for any one 365
particular molecule (Fig. 3B). Moreover, the positioning of the PcG 366
components; Bmi1 (PRC1) and Ezh2 (PRC2) were also highly conserved, 367
indicating that these two PcG proteins possess similar positioning patterns in 368
these three cell types (see upper left part in Fig. 3B). Likewise the two 369
molecules associated with gene activation, Menin and RNAPII, also displayed 370
comparatively strong conservation (see lower right in Fig. 3B). 371
372
A developmental change in the positioning of Ezh2 and Menin between ES 373
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and T cells 374
Interestingly, when we compared the correlation coefficients of Ezh2 375
and Menin between ES cells and T cells, we found that the positioning patterns 376
of Ezh2 and Menin were relatively similar in ES cells (0.351) but different in T 377
cells (-0.263) (see yellow outlined boxes in Fig. 3B). To visualize this finding 378
more precisely, we used scatter plots comparing Ezh2 UD indices versus Menin 379
UD indices at the co-occupied genes (731, 101 and 118 genes in ES, B and T 380
cells, respectively; Fig. 3C). In ES cells, UD indices of Ezh2 and Menin were 381
positively correlated (R = 0.351, p-value < 2.2E-16), i.e. frequently found in a 382
similar position relative to the annotated TSS (i.e. 88.9% genes showed 383
subtraction values of -2.5 to +2.5; left panel in Fig. 3C), whereas they were 384
negatively correlated (R = -0.263, p-value = 0.004), i.e. frequently found in 385
discrete positions either side of the annotated TSS in T cells (20.3 + 11.9 = 386
32.2%; right panel in Fig. 3C). The redistribution of Ezh2 and Menin relative to 387
the TSS of co-occupied genes in B cells was not as obvious at that observed for 388
T cells (R = 0.056; middle panel in Fig. 3C). We also confirmed these results 389
by using another approach. We performed Model-based Analysis of ChIP-seq 390
(MACS) and determined 1062, 139 and 390 co-occupied genes in ES, B and T 391
cells, respectively (30). Next, we rank-ordered the co-occupied genes by 392
MACS peak length to examine rank-dependent changes in correlation coefficient 393
between Ezh2 UD indices and Menin UD indices (38). In ES cells, positive 394
correlation between Ezh2 UD indices and Menin UD indices was observed in a 395
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rank-independent manner (Fig. 3D). In T cells, the negative correlation 396
become more evident when focusing on the top 50, 100, 150 and 200 397
co-occupied genes. When we used UD indices normalized by input DNA 398
values or those calculated in the region -5 kb to +5 kb relative to the TSS of each 399
gene, similar results were obtained (see Fig. S3B in the supplemental material). 400
Furthermore, these results were reproducible in another independent 401
experiment (see Fig. S4 in the supplemental material). We also analyzed Ezh2 402
(GSE23943) and Dpy30 (GSE26136) ChIP-seq datasets downloaded from GEO 403
and found that UD indices of Ezh2 and Dpy30, a component of TrxG complex 404
were positively correlated at 731 co-occupied genes in ES cells (R = 0.423, 405
p-value < 2.2E-16). A clear negative correlation between Ezh2 and Menin 406
positioning (UD indices) was also observed in Th2 cells, a functional CD4+ T 407
helper cell subset (R = -0.409, p-value = 1.1E-10; Fig. 3E). Next, we compared 408
expression levels of each co-occupied gene in wild-type and Ezh2-deficient Th2 409
cells, and genes with increased mRNA levels in Ezh2-deficient Th2 cells are 410
marked in the UD index scatter plots (Fig. 3E). Most of the Ezh2 and Menin 411
co-occupied genes (16 out of 17 genes) that were upregulated in Ezh2-deficient 412
cells were located in the central sector (red dots in Fig. 3E left panel, Fig. 3E 413
right panel). These results indicate that co-occupied genes in which Ezh2 and 414
Menin bound at the same position in Th2 cells were highly sensitive to loss of 415
Ezh2. 416
417
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Connection between gene expression and the position of binding of Ezh2 418
and Menin relative to the TSS 419
Next, in order to identify a functional link between mRNA levels and 420
Ezh2 and Menin positioning, we counter plotted absolute gene expression levels 421
against the subtraction of Menin UD index from Ezh2 UD index in ES and T cells 422
(see a scheme in Fig. S5A in the supplemental material). In ES cells, a weak 423
inverse correlation was observed (R = -0.244, p-value = 4.1E-11; Fig. 4A), 424
indicating that the binding position of Ezh2 and Menin had little effect on mRNA 425
levels in ES cells. A list of co-occupied genes in ES cells (see Table S3 in the 426
supplemental material) and actual binding patterns of Ezh2 and Menin at some 427
examples (Kcnc2, Fam184b, Gli2 and Tox) are shown in Fig. 5A to D. These 428
genes had varied levels of expression and similar binding positioning for Ezh2 429
and Menin, typical of the co-occupied genes in ES cells (Fig. 4A). In contrast, 430
in T cells, we found that mRNA levels displayed a strong negative correlation 431
with the subtraction values of Menin UD index from Ezh2 UD index (R = -0.490, 432
p-value = 2.7E-8 in Fig. 4B). This was also true in B and Th2 cells (see Fig. 433
S5B in the supplemental material). A strong negative correlation between 434
mRNA levels and the subtraction values of Menin UD index from Ezh2 UD index 435
in T cells was confirmed in the co-occupied genes identified by MACS (Fig. 5C 436
and D). These results indicate that the genes where Ezh2-binding tended to be 437
upstream and Menin-binding tended to be downstream of the TSS (see Fig. S5A, 438
left) had higher mRNA levels, and the genes with Ezh2-binding at the 439
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downstream region and Menin-binding at the upstream region in relation to the 440
TSS had lower mRNA levels (see Fig. S5A, right). Two typical examples of 441
co-occupied genes in T cells (Gata3 and Rab30) are shown in Fig. 5E and F. 442
These results indicate that positioning of Ezh2 and Menin at co-occupied genes 443
appear to control both sensitivity to the presence of Ezh2 and the overall 444
transcriptional state in T cells. 445
446
Changes in the binding states of Ezh2 and Menin during T cell 447
development, and association with T cell function 448
Finally, we analyzed the relationship between Ezh2 and Menin binding 449
states (co-occupancy or mono-occupancy) and up- or down-regulation of mRNA 450
levels between T lymphocytes and ES cells (CIRCOS visualization, Fig. 6A; see 451
Fig. S6A and Table S4 in the supplemental material). This analysis allowed 452
visualization of several important facets of Ezh2 and Menin function in 453
lymphocytes. In agreement with the role of Ezh2 as a positive regulator of 454
genetic repression, the majority of genes bound only by Ezh2 in T cells showed 455
lower levels of mRNA in T cells than ES cells (blue links, Fig. 6A; see third row in 456
Fig. S6A). In addition, these downregulated genes displayed a strong 457
functional bias for genes associated with embryonic morphogenesis and 458
development (see Fig. S6B in the supplemental material) that was largely 459
independent of the Ezh2/Menin binding states in ES cells. In T cells, mRNA 460
levels of genes bound only by Menin in ES cells and only by Ezh2 in T cells was 461
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most frequently decreased (170/387) (Fig. 6A left, “B” arrow and Fig. 6B upper). 462
The Ezh2 UD index in T cells was positively correlated with the Menin UD index 463
in ES cells (R = 0.341, p-value = 5.3E-6), indicating that relative to the TSS, 464
Ezh2 binding in T cells was frequently found at a similar position as Menin in ES 465
cells (Fig. 6B lower). 466
The majority of genes bound only by Menin in T cells showed higher 467
mRNA levels in T cells compared to ES cells, (pink links, Fig. 6A; see second 468
row in Fig. S6A). Most of the genes bound only by Ezh2 in ES cells and only by 469
Menin in T cells were up-regulated (40/52) (Fig. 6A “C” arrow and Fig. 6C 470
upper). The Menin UD index in T cells was positively correlated with the Ezh2 471
UD index in ES cells (R = 0.313, p-value = 0.049), suggesting that the position of 472
Menin binding in T cells was similar to the Ezh2 binding position in ES cells 473
relative to the TSS (Fig. 6C lower). 474
Interestingly, in contrast to the case for Ezh2 binding in T cells, the 475
functional biases found for Menin bound genes in T cells were highly dependent 476
on the Ezh2/Menin binding states in ES cells (Fig. 6D; see Fig. S6C in the 477
supplemental material). Genes that were bound by neither Ezh2 nor Menin in 478
ES cells contained a large number of genes broadly involved in regulation of 479
immune responses (p-value = 5.9E-17), whereas genes with Menin 480
mono-occupancy (Menin only) in both ES and T cells displayed a functional bias 481
for genes encoding chromatin regulators (p-value = 3.7E-11). 482
Co-occupancy-derived genes were enriched for transcription factors (p-value = 483
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3.6E-7) that included a set of genes essential for T cell development including 484
Bcl11b, Bach2, Ikzf1, Ikzf2, Satb1 and Tox. Additionally, although the overall 485
number was relatively small, Ezh2 mono-occupancy-derived genes showed 486
strong enrichment for genes associated with intracellular signaling (p-value = 487
4.3E-3). 488
Finally, we analyzed the genes that were co-occupied by Ezh2 and 489
Menin in T cells (first row in Fig. S6A). Of the genes co-occupied in T cells and 490
Menin mono-occupied in ES cells, 9 were upregulated and 10 were 491
downregulated (see Fig. S6A in the supplemental material). Genes 492
co-occupied in both ES and T cells were more often upregulated (11/26) than 493
downregulated (1/26) in T cells compared to ES cells (see Fig. S6A in the 494
supplemental material). However, in these groups no significant tendency was 495
found regarding the positioning of Ezh2 or Menin relative to the TSS. In 496
contrast, of the 11 genes co-occupied in T cells, and Ezh2 mono-occupied in ES 497
cells, 9 were expressed more strongly in T cells (Fig. 6A right, “E” arrow and Fig. 498
6E upper), moreover, all of these upregulated genes displayed a lower Ezh2 UD 499
index in T cells than ES cells, indicating that Ezh2 binding position had shifted 500
upstream relative to the TSS during development from ES cells into T cells (Fig. 501
6E lower). This group also includes essential T cell-related transcriptional 502
regulators such as Gata3, Fli1, Nfatc1, Gfi1 and Bcl11a. The binding patterns 503
of Ezh2 and Menin at the genes indicated above are shown in Fig. 5E and F 504
(also see Fig. S6D to K). 505
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Genome-wide comparison between ES cells and T cells argued that 506
physiological changes in the binding states of Ezh2 and Menin during T cell 507
development from ES cells were functionally associated with changes in 508
transcriptional states. To explore the biological relevance of Ezh2/Menin 509
co-occupancy in a given cell type, we next examine whether experimental 510
alternation of occupancy of Ezh2 may alter transcriptional at the co-occupied 511
genes. We used Ttrichostatin A (TSA) to alter Ezh2 binding because TSA 512
treatment was reported to reduce PcG protein binding levels at several gene loci 513
(14, 39). TSA treatment up-regulated 44 of 230 co-occupied genes in Th2 cells. 514
80% of these up-regulated genes showed loss of Ezh2 binding, indicating that 515
disruption of Ezh2/Menin co-occupancy by TSA relives Ezh2-dependent gene 516
silencing (Fig. 7). Thus, Ezh2/Menin co-occupancy is fundamental for 517
maintaining the transcriptional states at target genes. 518
Our analysis of co- and mono-occupancy characteristics of members of 519
the PcG and TrxG chromatin regulator complexes identified differences in the 520
functional biases of differentially regulated genes, depending on the 521
combinations of Ezh2 and Menin binding in ES cells and T cells. We propose 522
that the positioning of Ezh2 and Menin may control both sensitivity to the 523
presence of chromatin regulators and also the overall transcriptional state. 524
525
Discussion 526
We have here characterized the binding position of Ezh2 and Menin at 527
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all annotated genes in ES cells and B and T lymphocytes. Our data defines a 528
clear reciprocal pattern of binding between Ezh2 and Menin in these three cell 529
types. We also demonstrate a dynamic developmental change in the 530
positioning of Ezh2 and Menin in differentiated T lymphocytes compared to ES 531
cells at their co-occupied genes. Interestingly, different combinations of mono- 532
or co-occupancy of Ezh2 and Menin during development into T lymphocytes 533
appear to regulate expression of different functional groupings of genes in T 534
cells. 535
Our data indicate that the biological consequences of Ezh2-Menin 536
co-occupancy may be different between multipotent ES cells and differentiated T 537
cells. In ES cells, Ezh2-Menin co-occupancy is generally found at poised 538
genes (40, 41). PcG proteins repress expression of these genes so that they 539
are not activated without specific developmental cues (6) whereas TrxG proteins 540
are required for immediate activation of these poised genes after receiving 541
signals for differentiation (8). The present study identifies several previously 542
unappreciated characteristics of these poised genes. In ES cells the 543
positioning of Ezh2 and Menin were relatively similar, and deficiency of PcG 544
proteins in these cells often cause de-repression of poised genes, including 545
transcription factors important for normal tissue development (6). In contrast, in 546
T cells, Ezh2-Menin co-occupied genes exhibited variations in both the 547
positioning of Ezh2 and Menin and their transcriptional states. Among them, 548
genes where Ezh2 and Menin bound at the same position had similar 549
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characteristics as those in ES cells, i.e. sensitivity to loss of Ezh2. However, 550
co-occupied genes where Ezh2 and Menin were found at discrete positions on 551
either side of TSS had defined characteristics in differentiated lymphocytes. 552
Genes with Ezh2-binding upstream and Menin-binding downstream of the TSS 553
showed higher mRNA levels (active co-occupied genes), and genes with 554
Ezh2-binding downstream and Menin-binding upstream showed lower 555
expression (silent co-occupied genes) in T lymphocytes. In active co-occupied 556
genes, Menin is likely acting as a positive regulator of transcription, however, the 557
role of Ezh2 at these active co-occupied genes is currently unknown. We 558
postulate that Ezh2 may serve as a regulator of expression at co-occupied 559
genes where expression is essential while requiring tight control. A typical 560
example of an active co-occupied gene is Gata3. Consistent with our previous 561
report (14), the Gata3 gene exhibits Ezh2-binding upstream and Menin-binding 562
downstream of the TSS. As basal levels of Gata3 expression are required for 563
CD4+ T cell development and survival (42), Menin binding to the Gata3 gene 564
may allow positive regulation of its transcription while Ezh2 enables restriction of 565
Gata3 expression. In differentiated CD4 T lymphocytes Ezh2-deficiency results 566
in enhanced expression of Gata3 and hyper-production of Th2 cytokines (12). 567
Thus, Ezh2-Menin co-occupancy at the Gata3 gene likely regulates both 568
adequate expression for T cell development while maintaining multi-potency of 569
CD4+ T cells for differentiation into effector helper T cell subsets. 570
Our analysis also defined novel functional biases regarding Ezh2 and 571
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Menin-mediated gene regulation. Menin was found at immune response genes 572
that were active in T cells. In contrast, Ezh2 was not detected at genes 573
encoding cytokines, cytokine receptors or other immune-related molecules that 574
are silent in ES and B cells. For example, the Cd4, Cd28 and Cd247 (encoding 575
CD3 zeta chain) genes were highly expressed and showed Menin 576
mono-occupancy in T cells. However, these genes showed low level 577
expression and low level binding of Ezh2 in ES and B cells. Instead, Ezh2 578
binding was detected at many genes encoding transcription factors. These 579
results indicate that Ezh2 indirectly regulates several genes including 580
immune-related genes via controlling expression of the upstream transcription 581
factors. In T cells, Ezh2 binding was also found at many transcription factor 582
genes that are important for non-immune systems, and while Ezh2 appears 583
indispensable for repression of these transcription factor genes during 584
development it was largely dispensable in differentiated cells. Our data 585
indicates that in differentiated cells, the majority of Ezh2 target genes are 586
insensitive to Ezh2-deficiency in the absence of specific activating signals. 587
In summary, we have identified a novel mechanism of gene regulation 588
that is dependent on the spatial interplay between members of the PcG and 589
TrxG complexes. We propose that the positioning of these chromatin 590
regulators is an important determinant of their function at co-occupied genes 591
during cellular development. We expect our data set to serve as a resource for 592
the study of epigenetic regulatory mechanisms in ES cells and B and T 593
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lymphocytes. Further analysis of Ezh2 and/or Menin target genes identified in 594
this study will provide important insight for understanding lymphocyte 595
development and immune responses of B and T lymphocytes. 596
597
598
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Acknowledgments 599
The authors are grateful to Drs. John J. O’Shea, Yuka Kanno, Golnaz Vahedi 600
and for their helpful comments and constructive criticisms in the preparation of 601
the manuscript. We thank Drs. Atsushi Iwama and Satoru Miyagi for their 602
excellent experimental suggestions. This work was supported by the Global 603
COE Program (Global Center for Education and Research in Immune System 604
Regulation and Treatment), and by grants from the Ministry of Education, 605
Culture, Sports, Science and Technology (MEXT Japan) (Grants-in-Aid: for 606
Scientific Research (S) #26221305, (C) #24592083, #15K08522, Young 607
Scientists (B) #23790523, and #25860351, and for Scientific Research on 608
Innovative Areas “Genome Science” #221S0002), the Ministry of Health, Labour 609
and Welfare, The Uehara Memorial Foundation, Princes Takamatsu Cancer 610
Research Fund, and Takeda Science Foundation. D.J.T. was supported by a 611
Japanese Society for the Promotion of Science postdoctoral fellowship 612
(#2109747). 613
614
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763
Figure legends 764
Figure 1. Genome-wide comparison of Ezh2-Menin co-occupancy 765
between ES cells and lymphocytes. 766
(A) Comparison of Ezh2 and Menin binding in ES cells (left), B cells (middle), 767
and T cells (right). Of all target genes shown in (B), genes with more than 768
2-fold enrichment (ChIP / Input DNA) in Ezh2 and/or Menin binding were used 769
for the depiction. (B) Bar graph indicates frequency of Ezh2 and Menin 770
co-occupancy and mono-occupancy. (C) Co-occupied, mono-occupied and 771
unbound gene groups in ES cells are compared for relative percentage of Ezh2 772
and Menin occupancy in B cells (left) and T cells (right). Ezh2 and Menin 773
binding states at the ES cell stage are shown above the bars. For panel A to C, 774
Ezh2 mono-occupancy, Menin mono-occupancy and Ezh2-Menin co-occupancy 775
are indicated as light blue, orange and red, respectively. 776
Figure 2. Conserved signatures of PcG occupancy between ES cells and 777
lymphocytes. 778
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(A) Venn diagram shows numbers of cell type-specific and non-cell type-specific 779
Ezh2 target genes. (B) Gene Ontology categories over-represented in 780
Ezh2-positive gene sets in ES, B, and T cells. (C and D) mRNA levels is plotted 781
against Ezh2 binding levels at 205 genes encoding transcription factors that are 782
identified as PcG quadruple positive genes in ES cells(6) (C, yellow), or at 211 783
cytokine and cytokine receptor genes (D, brown). (E) Means of Ezh2 binding 784
levels at the expressed and non-expressed genes are shown. Error bars 785
indicate Standard Error of Mean (SEM). p-values were calculated by Welch 786
two sample t-test (*p<0.05). 787
788
Figure 3. ChIP-seq binding profiles reveal a novel feature of 789
co-occupancy with Ezh2 and Menin. 790
(A) Compiled tag density profiles (upper) and heatmap representation of binding 791
profiles (lower) across the TSS -5 kb and +3 kb flanking regions with 100-bp 792
resolution for Ezh2 are shown. The heatmap is rank-ordered from genes with 793
the highest UD indices to the lowest UD indices. (B) Correlation matrix shows 794
Pearson correlations of UD indices between indicated data sets. (Bright pink), 795
Positive correlation (0.75< R); (pink), Positive correlation (0.25 < R < 0.75); 796
(white), no correlation (-0.25 < R < 0.25); (light blue), negative correlation (R < 797
-0.25). (C) Comparison of UD indices of Ezh2 to those of Menin at co-occupied 798
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genes. Scatter plots compare Ezh2 UD indices (x axis) against Menin UD 799
indices (y axis) in ES (left panel), B (middle panel) and T cells (right panel). 800
Sectors are demarcated at subtraction of Menin UD index from Ezh2 UD index 801
of ±0.25. (D) Comparison of UD indices of Ezh2 and Menin at co-occupied 802
genes defined by MACS peak calling. The co-occupied genes were 803
rank-ordered by MACS peak length and rank-dependent changes in correlation 804
coefficient between Ezh2 UD indices and Menin UD indices were examined (see 805
also Methods). X axis indicates number of analyzed genes (e.g. “x=100” 806
means that top 100 co-occupied genes are used for calculating correlation 807
coefficient indicated in y axis). p-values were calculated by test for no 808
correlation (*p<0.05). (E) Scatter plots compare Ezh2 UD indices (x axis) 809
against Menin UD indices (y axis) in Th2 cells (left). Red dots indicate genes 810
with increased expression (4-fold) in Ezh2-deficient Th2 cells, and blue dots 811
indicate genes with decreased expression (4-fold) in Ezh2-deficient Th2 cells 812
compared to wild type Th2 cells. Sectors are demarcated at subtraction of 813
Menin UD index from Ezh2 UD index of ±0.25. Ratios of the number of genes 814
in the central sector to that in the peripheral sectors (right). 815
Figure 4. Comparison of mRNA levels with positions of Ezh2/Menin 816
binding. 817
(A and B) DNA microarray signal intensity in ES (A) and T (B) cells is plotted 818
against subtraction of Menin UD index from Ezh2 UD index. The maximum 819
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Affymetrix-GeneChip probe data corresponding to each RefSeq gene were used 820
for scatter plots. All dots corresponding to the co-occupied genes shown in Fig. 821
4A to F; Fig. S6J and K are highlighted. (C and D) Comparison of mRNA 822
levels with positions of Ezh2/Menin binding at co-occupied genes defined by 823
MACS peak calling. The co-occupied genes were rank-ordered by MACS peak 824
length and rank-dependent changes in correlation coefficient between mRNA 825
levels (after Log10 transformed) and subtraction of Menin UD index from Ezh2 826
UD index were examined. p-values were calculated by test for no correlation 827
(*p<0.05). 828
Figure 5. Ezh2 and Menin binding profiles at genes showing examples of 829
co-occupancy in ES or T cells. 830
(A to F) Binding of Ezh2, Dpy30 and Menin, and modifications of histone 831
H3K27me3 and H3K4me3 at representative loci in ES (pink) and T cells (green). 832
ChIP-Seq profiles are shown across six loci (chromosome 10: 833
111,650,000-111,750,000 (C); chromosome 5: 46,000,000-46,050,000 (D); 834
chromosome 1: 120,900,000-121,000,000 (E); chromosome 4: 835
6,873,211-6,950,000 (F); chromosome 2: 9,750,000-9,850,000 (G); 836
chromosome 7: 99,850,000-99,950,000 (H)). Ezh2Ref (GSE23943) Dpy30 837
(GSE26136) and Histone modification (GSE23943) datasets obtained from the 838
GEO database. For visualization of binding, datasets from GSE23943 839
underwent the same data processing as datasets of the present study, described 840
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in the Method section. Datasets from GSE26136 were used without data 841
processing. The Gata3 gene showed low UD index for Ezh2 and high UD index 842
for Menin in T cells (Ezh2: 0.109, Menin: 0.501), and was highly transcribed (G) 843
(also see Fig. 4B). The binding region of Ezh2 and Menin around the Gata3 844
TSS region are not overlapped, consistent with our previous findings (14). The 845
Rab30 gene was expressed at low levels, and Ezh2 bound mainly downstream 846
of the TSS with Menin binding mainly upstream of the TSS (H) (also see Fig. 847
4B). 848
Figure 6. Changes in the binding states of Ezh2 and Menin during T cell 849
development from ES cells. 850
(A) Circos visualization of comparison of Ezh2 and Menin binding states 851
between ES and T cells (43). Colors of the outer arch indicate Ezh2/Menin 852
binding states in ES (left) and T cells (right). Colors of the inner arch on the 853
right side indicate the original binding states of Ezh2 and Menin in ES cells. 854
Links of genes up-regulated or down-regulated during T cell development are 855
indicated by pink or blue, respectively. A green rectangle indicates the region of 856
enlarged view shown in the right panel. (B and C) Comparison of transcription 857
levels (upper), and their binding positioning (lower) between ES and T cells. 858
Genes showing Menin mono-occupancy in ES cells and Ezh2 mono-occupancy 859
in T cells were analyzed and genes down-regulated in T cells compared to ES 860
cells were used for the assessment of Ezh2 and Menin binding (B). Genes 861
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showing Ezh2 mono-occupancy in ES cells and Menin mono-occupancy in T 862
cells were analyzed and genes up-regulated in T cells compared to ES cells 863
were used for the assessment of Ezh2 and Menin binding (C). (D) Percentage 864
of co-occupancy (red), Ezh2 mono-occupancy (light blue), Menin 865
mono-occupancy (orange) or null-occupancy (gray) -derived Menin 866
mono-occupied genes in T cells for the category shown on the left side of each 867
bar. (E) Genes showing Ezh2 mono-occupancy in ES cells and Ezh2 and 868
Menin co-occupancy in T cells were analyzed and genes up-regulated in T cells 869
compared to ES cells were used for the assessment of Ezh2 and Menin binding. 870
All dots corresponding to the genes shown in Fig. 4G; Fig. S5N to U are 871
highlighted (B, C and E). 872
Figure 7. Disruption of Ezh2/Menin co-occupancy by Trichostatin A (TSA). 873
(A)TSA treatment up-regulated 44 of 230 co-occupied genes in Th2 cells. Pie 874
chart illustrating frequency of Ezh2 and Menin co-occupancy and 875
mono-occupancy in these 44 genes. (B and C) Binding of Ezh2 and Menin at 876
representative loci in Th2 (red) and TSA-treated Th2 cells (orange). ChIP-Seq 877
profiles are shown across two loci (chromosome 4: 3,875,000-3,845,000 (B); 878
chromosome 2: 127,940,000-127,960,000 (C)). 879
880
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Ezh2 binding (ChIP/Input) Ezh2 binding (ChIP/Input) Ezh2 binding (ChIP/Input)
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Figure 1. Genome-wide comparison of Ezh2-Menin co-occupancy between ES cells and lymphocytes. !(A) Comparison of Ezh2 and Menin binding in ES cells (left), B cells (middle), and T cells (right). Of all target genes shown in (B), genes with more than 2-fold enrichment (ChIP / Input DNA) in Ezh2 and/or Menin binding were used for the depiction. (B) Bar graph indicates frequency of Ezh2 and Menin co-occupancy and mono-occupancy. (C) Co-occupied, mono-occupied and unbound gene groups in ES cells are compared for relative percentage of Ezh2 and Menin occupancy in B cells (left) and T cells (right). Ezh2 and Menin binding states at the ES cell stage are shown above the bars. For panel A to C, Ezh2 mono-occupancy, Menin mono-occupancy and Ezh2-Menin co-occupancy are indicated as light blue, orange and red, respectively.!
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ES Developmental protein!Homeobox!DNA-binding!Transcription regulation
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3.5!5.5!2.5!2.2
3.20E-104!1.10E-92!1.50E-75!1.90E-58
B Homeobox!DNA-binding!Developmental protein!Transcription regulation
11.0 %!24.9 %!18.2 %!23.1 %
7.9!3.3!4.2!2.8
1.20E-92!1.20E-90!1.80E-85!1.50E-65
T Developmental protein!Homeobox!DNA-binding!Transcription regulation
12.7 %! 6.1 %!16.5 %!16.1 %
3.0!4.4!2.2!2.0
2.40E-87!2.10E-72!5.40E-68!2.30E-49
A B
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Ezh2 binding in ES (ChIP/Input)
Ezh2 binding in B (ChIP/Input)
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* *
*
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Figure 2. Conserved signatures of PcG occupancy between ES cells and lymphocytes. (A) Venn diagram shows numbers of cell type-specific and non-cell type-specific Ezh2 target genes. (B) Gene Ontology categories over-represented in Ezh2-positive gene sets in ES, B, and T cells. (C and D) mRNA expression is plotted against Ezh2 binding levels at 205 genes encoding transcription factors that are identified as PcG quadruple positive genes in ES cells(6) (C, yellow), or at 211 cytokine and cytokine receptor genes (D, brown). (E) Means of Ezh2 binding levels at the expressed and non-expressed genes are shown. Error bars indicate Standard Error of Mean (SEM). p-values were calculated by Welch two sample t-test (*p<0.05). !
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Figure 3. ChIP-seq binding profiles reveal a novel feature of co-occupancy with Ezh2 and Menin. !(A) Compiled tag density profiles (upper) and heatmap representation of binding profiles (lower) across the TSS -5 kb and +3 kb flanking regions with 100-bp resolution for Ezh2 are shown. The heatmap is rank-ordered from genes with the highest UD indices to the lowest UD indices. (B) Correlation matrix shows Pearson correlations of UD indices between indicated data sets. (Bright pink), Positive correlation (0.75< R); (pink), Positive correlation (0.25 < R < 0.75); (white), no correlation (-0.25 < R < 0.25); (light blue), negative correlation (R < -0.25). (C) Comparison of UD indices of Ezh2 to those of Menin at co-occupied genes. Scatter plots compare Ezh2 UD indices (x axis) against Menin UD indices (y axis) in ES (left panel), B (middle panel) and T cells (right panel). Sectors are demarcated at subtraction of Menin UD index from Ezh2 UD index of ±0.25. (D) Comparison of UD indices of Ezh2 and Menin at co-occupied genes defined by MACS peak calling. The co-occupied genes were rank-ordered by MACS peak length and rank-dependent changes in correlation coefficient between Ezh2 UD indices and Menin UD indices were examined (see also Methods). X axis indicates number of analyzed genes (e.g. “x=100” means that top 100 co-occupied genes are used for calculating correlation coefficient indicated in y axis). p-values were calculated by test for no correlation (*p<0.05). (E) Scatter plots compare Ezh2 UD indices (x axis) against Menin UD indices (y axis) in Th2 cells (left). Red dots indicate genes with increased expression (4-fold) in Ezh2-deficient Th2 cells, and blue dots indicate genes with decreased expression (4-fold) in Ezh2-deficient Th2 cells compared to wild type Th2 cells. Sectors are demarcated at subtraction of Menin UD index from Ezh2 UD index of ±0.25. Ratios of the number of genes in the central sector to that in the peripheral sectors (right). !
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Figure 4. Comparison of mRNA expression with positions of Ezh2/Menin binding. (A and B) DNA microarray signal intensity in ES (A) and T (B) cells is plotted against subtraction of Menin UD index from Ezh2 UD index. The maximum Affymetrix-GeneChip probe data corresponding to each RefSeq gene were used for scatter plots. All dots corresponding to the co-occupied genes shown in Fig. 4A to F; Fig. S6J and K are highlighted. (C and D) Comparison of mRNA expression with positions of Ezh2/Menin binding at co-occupied genes defined by MACS peak calling. The co-occupied genes were rank-ordered by MACS peak length and rank-dependent changes in correlation coefficient between mRNA expression levels (after Log10 transformed) and subtraction of Menin UD index from Ezh2 UD index were examined. p-values were calculated by test for no correlation (*p<0.05).
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Figure 5. Ezh2 and Menin binding profiles at genes showing examples of co-occupancy in ES or T cells. (A to F) Binding of Ezh2, Dpy30 and Menin, and modifications of histone H3K27me3 and H3K4me3 at representative loci in ES (pink) and T cells (green). ChIP-Seq profiles are shown across six loci (chromosome 10: 111,650,000-111,750,000 (C); chromosome 5: 46,000,000-46,050,000 (D); chromosome 1: 120,900,000-121,000,000 (E); chromosome 4: 6,873,211-6,950,000 (F); chromosome 2: 9,750,000-9,850,000 (G); chromosome 7: 99,850,000-99,950,000 (H)). Ezh2Ref (GSE23943) Dpy30 (GSE26136) and Histone modification (GSE23943) datasets obtained from the GEO database. For visualization of binding, datasets from GSE23943 underwent the same data processing as datasets of the present study, described in the Method section. Datasets from GSE26136 were used without data processing. The Gata3 gene showed low UD index for Ezh2 and high UD index for Menin in T cells (Ezh2: 0.109, Menin: 0.501), and was highly transcribed (G) (also see Fig. 4B). The binding region of Ezh2 and Menin around the Gata3 TSS region are not overlapped, consistent with our previous findings (25). The Rab30 gene was expressed at low levels, and Ezh2 bound mainly downstream of the TSS with Menin binding mainly upstream of the TSS (H) (also see Fig. 4B).
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0.8
1.0
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
01
23
45
01
23
45
01
23
45
Log
10(D
NA
Mic
roar
ray
sign
al in
tens
ity)
B C
Ezh2 UD index in T Ezh2 UD index in ES Ezh2 UD index in ES&T
Men
in U
D in
dex
in E
S
Men
in U
D in
dex
in T
E
Bcl11a
Alcam
Tm6sf1
Aim1
Nfatc1
Tgfbr1
Gata3
Fli1
Gfi1
R = 0.341 R = 0.313
Log
10(D
NA
Mic
roar
ray
sign
al in
tens
ity)
Log
10(D
NA
Mic
roar
ray
sign
al in
tens
ity)
up down
Menin Ezh2 ES T ES T ES T
ES T
ES T Cxxc5
Ikzf3
Pag1
Igf2bp1 Rbpms2
Nab1
up down up down
down up up
Fig6 A ES cell T cell
B C
E
Co-occupied
Menin only
Ezh2 only None
down-regulated in T cell up-regulated in T cell
Ezh2 Menin Ezh2 Co-occupied
ES
T
TOTAL
transcription
immune response
chromosome organization
intracellular signaling cascade
D
Co-occupied (ES)
Menin only (ES)
Ezh2 only (ES)
None (ES)
%
870
114
43
42
56
Menin only (T)
Menin only (T)
Menin only (T)
Menin only (T)
ES T
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Figure 6. Changes in the binding states of Ezh2 and Menin during T cell development from ES cells. (A) Circos visualization of comparison of Ezh2 and Menin binding states between ES and T cells (16). Colors of the outer arch indicate Ezh2/Menin binding states in ES (left) and T cells (right). Colors of the inner arch on the right side indicate the original binding states of Ezh2 and Menin in ES cells. Links of genes up-regulated or down-regulated during T cell development are indicated by pink or blue, respectively. A green rectangle indicates the region of enlarged view shown in the right panel. (B and C) Comparison of transcription levels (upper), and their binding positioning (lower) between ES and T cells. Genes showing Menin mono-occupancy in ES cells and Ezh2 mono-occupancy in T cells were analyzed and genes down-regulated in T cells compared to ES cells were used for the assessment of Ezh2 and Menin binding (B). Genes showing Ezh2 mono-occupancy in ES cells and Menin mono-occupancy in T cells were analyzed and genes up-regulated in T cells compared to ES cells were used for the assessment of Ezh2 and Menin binding (C). (D) Percentage of co-occupancy (red), Ezh2 mono-occupancy (light blue), Menin mono-occupancy (orange) or null-occupancy (gray) -derived Menin mono-occupied genes in T cells for the category shown on the left side of each bar. (E) Genes showing Ezh2 mono-occupancy in ES cells and Ezh2 and Menin co-occupancy in T cells were analyzed and genes up-regulated in T cells compared to ES cells were used for the assessment of Ezh2 and Menin binding. All dots corresponding to the genes shown in Fig. 4G; Fig. S5N to U are highlighted (B, C and E).
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44 genes were upregulated by TSA treatment! 80%!
Menin only!
16% !co-occupied!
Ezh2 only!
B
Th2
TSA
-trea
ted
Th2
Ezh2
Men
in
Ezh2
Men
in
10
10
0
0
0
0
7
7
30kb
0
0
0
0
7
7
6
6
20kb
C
Bcl2l11 Chchd7 Plag1
A
Figure 7. Disruption of Ezh2/Menin co-occupancy by Trichostatin A (TSA) accompanied with gene de-repression.!(A) TSA treatment up-regulated 44 of 230 co-occupied genes in Th2 cells. Pie chart illustrating frequency of Ezh2 and Menin co-occupancy and mono-occupancy in these 44 genes. (B and C) Binding of Ezh2 and Menin at representative loci in Th2 (red) and TSA-treated Th2 cells (orange). ChIP-Seq profiles are shown across two loci (chromosome 4: 3,875,000-3,845,000 (B); chromosome 2: 127,940,000-127,960,000 (C)).
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