1 spatial interplay between polycomb and trithorax complexes

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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762

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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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 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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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%!

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16% !co-occupied!

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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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1 spatial interplay between polycomb and trithorax complexes

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