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and Immunity (2012) 13, 311 --320 & 2012 Macmillan Publishers Limited All rights reserved 1466-4879/12 www.nature.com/gene

ORIGINAL ARTICLE Global mapping of H3K4me3 and reveals state-based regulation of human monocyte-derived dendritic cells in different environments

Y Huang1,SMin1,YLui1, J Sun1,XSu1, Y Liu1, Y Zhang1, D Han2, Y Che1, C Zhao3,BMa4 and R Yang1,5

Depending on the environment, dendritic cells (DCs) may become active or tolerogenic, but little is known about whether heritable epigenetic modifications are involved in these processes. Here, we have found that epigenetic modifications can regulate the differentiation of human monocyte-derived DCs (moDCs) into either activated or tolerized DCs. The inhibition or silencing of methyltransferases or -associated factors affects the expression of multiple genes. Genome mapping of transforming (TGF-b)- or lipopolysaccharide (LPS)-associated H3K4 trimethylation (H3K4me3) and H3K27 trimethylation (H3K27me3) demonstrated the presence of histone modification of expression in human TGF-b-or LPS-conditioned moDCs. Although the upregulated or downregulated genes were not always associated with H3K4me3 and/or H3K27me3 modifications in TGF-b-conditioned (tolerized) or LPS-conditioned (activated) moDCs, some of these genes may be regulated by the increased and/or decreased H3K4me3 or H3K27me3 levels or by the alteration of these epigenetic marks, especially in TGF-b-conditioned moDCs. Thus, our results suggested that the differentiation and function of moDCs in tumor and inflammation environments are associated with the modification of the H3K4me3 and K3K27me3 epigenetic marks.

Genes and Immunity (2012) 13, 311--320; doi:10.1038/gene.2011.87; published online 26 January 2012 Keywords: dendritic cells; ; H3K4me3; H3K27me3

INTRODUCTION TGF-b most likely employ different regulatory requirements that Dendritic cells (DCs) are antigen (Ag)-presenting cells, with a reflect their functions. As the expression of different classes of unique capacity to stimulate naive T cells and initiate primary genes can be induced by the same receptor, differential regulation immune responses. However, DCs also have critical roles in the occurs via a gene-specific mechanism. Although multiple mechan- induction of central and peripheral immunological tolerance and isms may be involved in the regulation of gene expression in the regulation of the types of T-cell immune responses.1,2 The different kinds of cells,9 epigenetic modification has served as an diverse functions of DCs in immune regulation not only depend important mechanism in regulating the .10 on the heterogeneity of DC subsets but also on their functional Indeed, a genome-wide of histone modifications has plasticity.1,2 DCs express a variety of pathogen recognition been identified in human moDCs.11 In LPS-conditioned macro- receptors, such as Toll-like receptors. The detection of damage- phages, gene-specific regulation occurs at the level of chromatin or pathogen-associated molecular patterns, such as lipopolysac- and includes remodeling and covalent histone charides (LPSs), by tissue-resident DCs initiates DC maturation and modifications.12 Histone deacetylase (HDAC) inhibition can also migration to the regional lymph nodes.3,4 Upon exposure to affect the phenotype and function of human moDCs.13 Global transforming growth factor (TGF)-b, DCs become tolerogenic and mapping of H4K4me3 and H3K27 trimethylation (H3K27me3) has can thereby suppress T-cell alloreactivity5 and induce Th2 or revealed the specificity and plasticity of lineage fate determination T regulatory responses.3,4 Studies have shown that the expression in differentiating CD4 þ T cells10,14 and in memory CD8 þ T cells.14 of genes coding costimulatory molecules, , Thus, the covalent modifications of histone N-terminal tails, such and their receptors is remarkably different in the conditioned DCs; as methylation, can act to regulate chromatin states.15 those DCs activated by LPS express higher levels of the moDCs develop from peripheral blood monocytes under the costimulatory molecules, such as CD80 and CD86, and the influence of granulocyte-macrophage colony-stimulating factor Ag-presenting molecule major histocompatibility complex (MHC) (GM-CSF) alone or GM-CSF with -4 (IL-4),16,17 and are a II3,4 than those conditioned with TGF-b.6 subset of DCs that are involved in inflammatory processes and Previous reports6--8 and microarray data from the NCBI-GEO infection clearance.3,18,19 These moDCs could become either database (LPS-treated DC gene expression from the GSE10316 active or tolerogenic after exposed to different stimuli. Here, we record and TGF-b-treated DC gene expression from the GDS2940 have used chromatin (Chip) and Chip- record) have shown that both LPS and TGF-b induce the sequencing (Chip-seq) to characterize the modification of H3K4 expression of hundreds of genes in human monocyte-derived trimethylation (H3K4me3) and H3K27me3 in LPS- and TGF-b- DCs (moDCs). The responses induced by treatment with LPS or conditioned moDCs. We have found that during the transition of

1Department of Immunology, Nankai University School of Medicine, Nankai University, Tianjin, China; 2Research and Cooperation Division, BGI-Shenzhen, Shenzhen, China; 3Center for Tissue Engineering, Chinese Academy of Medical Science, Peking Union Medical College, Beijing, PR China; 4College of Life Science, Tianjin, China and 5Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin, China. Correspondence: Dr R Yang or Dr B Ma, Department of Immunology, Nankai University School of Medicine, Nankai University, Tianjin 300071, China. E-mail: [email protected] Received 19 August 2011; revised 24 October 2011; accepted 28 November 2011; published online 26 January 2012 Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells Y Huang et al 312 immature moDCs into activated moDCs (LPS conditioning) and may be also involved in the gene regulation of human LPS- or TGF- tolerized moDCs (TGF-b conditioning), changes in the modifica- b-conditioned moDCs (Supplementary Figure S3). Using specific tion of H3K4me3 or H3K27me3 as well as alteration in these antibodies, we next immunoprecipitated H3K4me3-or K3K27me3- epigenetic marks have a role in the resulting up- or down- associated genomic from TGF-b- or LPS-conditioned moDCs regulation of genes in these treated cells. and control moDCs. The H3K4me3 and H3K27me3 peak (island, Chip-seq-enriched region) distribution near the start site (±5 kb) of each annotated RefSeq (2012) 13, 311--320. gene RESULTS was analyzed. H3K4me3 islands were enriched in the region closest is involved in the regulation of the gene to the transcription start site (from þ 2.5 kb to À100 bp; Figure 2a), expression in human moDCs whereas H3K27me3 peaks were enriched in the region upstream of Histone methyltransferases have a critical role in epigenetic the transcription start site sites (from þ 2.5 to þ 5 kb; Figure 2a). modification during the cellular differentiation.20 To investigate the To examine the overall H3K4me3 and H3K27me3 distribution, effect of methylation on the expression of TGF-b-andLPS-associated we divided the into four types of regions, which genes, we first employed the methyltransferase inhibitor adenosine included the proximal promoter (2 kb upstream and downstream dialdehyde (an adenosine analog and S-adenosylmethionine-depen- of the transcription start site), the exons, introns and intergenic dent methyltransferase inhibitor) to observe the effect on the gene regions. These regions were based on the annotations of ‘known expression in human moDCs. As shown in Supplementary Figure S1, genes’ in the UCSC genome browser (http://genome.UCSC.edu). B adenosine dialdehyde clearly promoted the expression of the surface H3K4me3 islands were found in 20% of the proximal promoter molecules CD14, CD11C, CD80, CD86 and MHC-DR, the expression of regions of TGF-b-treated moDCs (23%) and LPS-treated moDCs the cytokines and chemokines IL-1A, IL-12A, alpha 6 (21%), whereas H3K27me3 islands were only found in 3% of the (IFNA6), (ligand) superfamily, member 10 proximal promoter regions of both TGF-b-treated moDCs (3%) and (TNFSF10), (C-C motif) ligand (CCL)3, CCL4, chemokine (C- LPS-treated moDCs (3%). The examination of H3K4me3 and B C motif) receptor (CCR)7 and chemokine (C-X-C motif) ligand (CXCL)16, K3K27me3 islands within gene bodies revealed that 1% of these and also the expression of the transcription factors signal transducer was enriched for H3K4me3 or K3K27me3 islands (Figure 2b). Most and activator of transcription (STAT)3, STAT4, nuclear factor (NF)-kB1 of H3K4me3 and H3K27me3 modifications were located in the and NF-kB2 in human moDCs. Euchromatic histone N- introns and the intergenic regions (Figure 2b). Lineage- specific methyltransferase 2 (EHMT2) is a H3K9- and H3K27-specific histone H3K4me3 and H3K27me3 marks in LPS- or TGF-b-conditioned DCs methyltransferase21 that is remarkably downregulated in human were remarkably different than those in control moDCs. The moDCs after exposure to LPS (LPS-treated DC gene expression, number of lineage-specific H3K4me3 islands was significantly GSE10316 record and Supplementary Figure S2), and has the greater in TGF-b-conditioned moDCs than in LPS-conditioned potential to affect the expression of multiple genes. Indeed, the moDCs, which indicated that the active genes in TGF-b-condi- silencing of EHMT2 with demonstrated transfection (Figure 1a) was tioned moDCs were modified by H3K4me3. However, the number found to promote the expression of surface molecules (CD14, CD11c, of lineage-specific H3K27me3 islands was similar between LPS- and CD80, CD86 and MHC-DR), cytokines (IL-1A, IL-12A, IFNA and TGF-b-conditioned moDCs (Figure 2c). During the differentiation of TNFSF10), chemokines (CCL3, CCL4, CCR7 and CXCL16) and the immature DCs into LPS-activated DCs, the proportion of genes transcription factors (STAT3, STAT4, NF-kB1 and NF-kB2; Figure 1b). with H3K4me3 marks alone decreased from 13 to 6.5% (Figure 2c). Importantly, silencing EHMT2 also affected the Ag-presenting function We also performed (GO) analyses for the of moDCs (Supplementary Figure S3). Others genes such as H3K4me3- and H3K27me3-marked genes. On the basis of the of zeste homolog 1 (EZH1), chromobox homolog 5 (CBX5)andSET genes marked by H3K4me3 and/or H3K27me3 (Supplementary domain-containing 6 (SETD6), which are associated with the methyla- Table S1), it was clear that these epigenetic modifications decorate tion of ,22 --24 were also involved in the regulation of gene many genes with functions related to differentiation, development expression in moDCs. The silencing of SETD6 was found to and transcriptional regulation (Supplementary Figure S5 and downregulate the expression of IL-1A and upregulate that of STAT3, Supplementary Table S2). As many as 30--60% of the genes in whereas the expression of NF-kB1 was not remarkably altered both the TGF-b-conditioned DCs and LPS-conditioned DCs were (Figures 1c and d). The silencing of EZH1 was found to downregulate modified by H3K4me3, but less genes were modified by IL-1A expression and upregulate both STAT3 and NF-kB1 expression, H3K27me3 (Supplementary Figure S5 and Supplementary Table S2). but the silencing of CBX5 downregulated both IL-1A and NF-kB1 expression and upregulated STAT3 expression (Figures 1c and d). Global analysis of H3K4me3 and H3K27me3 modification in Taken together, these results suggest that histone methylation may TGF-b- and LPS-conditioned human moDCs be involved in the gene regulation of human moDCs. In addition to the microarray data on TGF-b-conditioned DCs and the global analysis of the Chip-seq in TGF-b-conditioned DCs, we Genome-wide maps of H3K4me3 and H3K27me3 modification in next co-analyzed the modification of H3K4me3 and H3K27me3 on TGF-b- and LPS-conditioned DCs the upregulated, downregulated and unaltered genes, which are To reveal the epigenetic features present during the process of originated from the microarray data of TGF-b-conditioned moDCs differentiation of moDCs in response to different environments, (NCBI-GEO Database: TGF-b-treated DC gene expression, GDS2940 we generated global maps of H3K4me3 and H3K27me3 modifica- record), and the data are shown in Supplementary Table S3. An tion using the Chip-seq approach. The LPS-conditioned moDCs analysis of the more highly expressed genes in TGF-b-conditioned had typical morphology of DC; they expressed higher levels of DCs revealed that almost half (50%) have the H3K4me3 costimulatory molecules, such as CD11C, CD40, CD80 and CD86, modification on their promoter, whereas only 10% have the and Ag-presenting molecules, MHC class I and class II (Supple- H3K27me3 mark (Figure 3a). There was a highly significant mentary Figure S4). Consistent with previous reports,6 TGF-b- correlation between the H3K4me3 marks and the high expression conditioned DCs exhibited an immature phenotype; they level of genes related to TGF-b activation, such as that of NFKB2, expressed lower levels of CD40, CD80, CD86, MHC I, MHC II and HSP90AB1, IL-32, TGFBR1, PLCB3, PDIA3, NFKB1, PRKACA, ACVR1B, CD11C than did LPS-conditioned moDCs (Supplementary Figure S4). MED24 and IL-1RAP (Figure 3b, only H3K4me3 marks of NFKB2, Similar to unconditioned moDCs, the Ag-presenting function of HSP90AB1 and IL-32 are shown) were seen in TGF-b-conditioned these human LPS- or TGF-b-conditioned moDCs could also be DCs. These genes are involved in the PPARa/RXRa-activated altered after silencing EHMT2, suggesting that histone methylation pathway that may limit inflammatory responses. Conversely, the

Genes and Immunity (2012) 311 --320 & 2012 Macmillan Publishers Limited Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells Y Huang et al 313 abCD14 CD11c CD80 CD86 MHC-DR Oligo ctr. 1.2 EHMT2 siRNA

0.9 Counts

0.6 R.E 8 8 0.3 8 Oligo ctr. 6 6 6 EHMT2 siRNA 0 4 4 4 EHMT2 2 2 2 Actin Untr. Oligo EHMT2 0 0 0 Ctr. siRNA IL1A CCL3 CCL4 IL12A CCR7 IFNA6 STAT3 STAT4 NFKB1 NFKB2 GAPDH GAPDH GAPDH CXCL16 TNFSP10

cd 1.2 1.2 IL1A6 STAT36 NFKB1 0.9 0.9 4 4 0.6 R.E R.E

R.E 0.6 2 2 0.3 0.3 0 0 0

0 SETD6 EZH1 CBX5 Oligo.ctr Oligo.ctr Oligo.ctr Untr. Oligo.ctr siRNA EZH1siRNA EZH1siRNA EZH1siRNA CBX5siRNA CBX5siRNA CBX5siRNA SETD6siRNA SETD6siRNA SETD6siRNA Figure 1. Histone methylation is involved in the regulation of the gene expression in human moDCs. (a) Validation of EHMT2 siRNA. Human moDCs were transfected with EHMT2 siRNA or an oligo control, and the transcriptional levels of EHMT2 were detected by quantitative real-time PCR (qRT--PCR).EHMT2 expression was analyzed with whole-cell extracts using anti-EHMT2 polyclonal antibody (SAB2100657, Sigma, St Louis, MO, USA) and anti-b-actin antibody (SAB3500350, Sigma) by western blot according to the previous method.42,43 (b) The histone methyltransferase EHMT2 affected the expression of gene expression. The expressions of CD14, CD11C, CD80, CD86 and MHC-DR were analyzed using FAScan (FASCAN International Inc., Baltimore, MD, USA), and the transcriptional levels of EHMT2, IL-1A (IL1A), IL-12A (IL12a), IFNa6 (IFNA6), TNFSP10, CCL3, CCL4, CCR7, CXCL16, STAT3, STAT4, NF-kB1, NF-kB2 and homing gene GAPDH were detected by qRT--PCR after transfection for 24 h according to the protocol described in the Materials and methods. (c, d) Histone methylation-associated factors affected the gene expression in human moDCs. Human moDCs were transfected with SETD6 siRNA (SETD6siRNA), EZH1 siRNA (EZH1siRNA) and CBX5 siRNAs (CBX5siRNAs) or an oligo control (oligo. ctr.), and their validation was demonstrated by qRT--PCR(c). The expressions of IL1A, STAT3 and NF-kB1 were analyzed by qRT--PCR (d). siRNA indicates SETD6siRNA, EZH1siRNA or CBX5siRNA. RE, relative expression; Untr., untransfected cells. presence of H3K27me3 marks in TGF-b-conditioned DCs was related to increased H3K4me3 modification in these LPS- correlated with lower levels of immune gene expression, such as conditioned DCs (Figure 4b), which implied the involvement of that of RYR3, HDAC9, DCC, PLCB4 and PRKCH were related to other mechanisms in the regulation of the expression of these H3K27me3 marks (Figure 3c, only H3K27me3 marks of RYR3 are genes. Downregulated genes such as VAV3, PAK1, PRKCA and GNB5, shown). However, 40% of the upregulated genes, 50% of the which are associated with phagocytosis and pinocytosis, were downregulated genes and 20% of the unaltered genes were not indeed modified by the increased H3K27me3 in LPS-conditioned modified by H3K4me3 or H3K37me3 (Figure 3a). DCs (Figure 4c, only H3K27me3 marks of VAV3 are shown). We also analyzed the modification of H3K4me3 and H3K27me3 Additionally, in the LPS-conditioned DCs, STAT3, STAT5B and STAT5A on the upregulated, downregulated and unaltered genes, which genes were marked in their promoter regions with H3K4me3, but are originated from the microarray data of LPS-conditioned DCs these genes did not have substantial H3K27me3 signals, indicating (NCBI-GEO Database, LPS-treated DC gene expression, GSE10316 these active genes are mainly modified by H3K4me3. record), and the data are shown in Supplementary Table S4. As shown in Figure 4, for LPS-conditioned DCs, 20% of the upregulated genes were modified by H3K4me3, and 8% of the Characterization of H3K4me3 and H3K27me3 modification and downregulated genes were modified by H3K27me3 in LPS- the expression of the surface molecules, cytokines, chemokines conditioned DCs (Figure 4a). Similar to TGF-b-conditioned DCs, and their receptors many genes were not modified by either H3K4me3 or H3K27me3. CD14 and CD83 are the critical markers of the process of the DC The JAK/STAT family of transcription factors is critical for DC differentiation. Surface molecule CD14, which is highly expressed function. We found that signaling molecules in this signal pathway by monocytes, is downregulated during the differentiation after were modified with H3K4me3 (Figure 4b, only H3K4me3 marks of exposure to LPS but is regulated after exposure to TGF-b STAT3, JAK3 and STAT5A are shown). Interestingly, the upregula- (Figure 5a). Consistent with other reporters,11 the promoter of tion of genes such as PTPN1, STAT3, JAK3, STAT5B, STAT4, STAT5A the CD14 genes had multiple H3K4me3 marks with high CD14 and JAK1 in the JAK/STAT signal pathway was not significantly expression (Figure 5a), where these marks were lost after exposure

& 2012 Macmillan Publishers Limited Genes and Immunity (2012) 311 --320 Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells Y Huang et al 314 abH3K4me3 3.5e-09

3e-09 Proximal Intergenic Sample ID Total promoter Exons Introns region 2.5e-09 Ctr.moDC 8927 1909 (21%) 155 (2%) 5825 (65%) 1038 (12%) 2e-09 H3K4me3 LPS moDC 2565 549 (21%) 33 (1%) 1556 (61%) 427 (17%)

1.5e-09 TGF-β moDC 10920 2479 (23%) 172 (2%) 6916 (63%) 1353 (12%)

Ctr.moDC 1620 61 (4%) 4 (1%) 306 (18%) 1249 (77%) 1e-09 H3K27me3 LPS moDC 2027 69 (3%) 6 (1%) 390 (18%) 1542 (77%)

5e-10 TGF-β moDC 2022 60 (3%) 5 (1%) 388(18%) 1569 (78%) 0 H3K27me3 1e-09 c

Tag density Tag 9e-10 30 60 8e-10 25 50 7e-10 20 40

6e-10 15 30 islands (%) 5e-10 10 20 islands (%)

4e-10 5 10 Lineage specific H3K4me3

3e-10 0 Lineage specific H3K27me3 0 Ctr LPS TGF β Ctr LPS TGF β 2e-10

1e-10 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 TSS TES up5k up2.5k down5k

LPS moDC down2.5k TGF-β moDC Ctr. moDC Figure 2. Genome-wide maps of H3K4me3 and H3K27me3 modification in the TGF-b- and LPS-conditioned DCs. (a) The distribution of H3K4me3 and H3K27me3 peaks in relation to the transcription start site (TSS) in each sample. The regions from 5.0 kb upstream to 5.0 kb downstream corresponding to the TSS were analyzed using bioinformatics according to the description in the Materials and methods. (b) The number of islands within genomic regions for each sample is shown, and the percentages are listed in the parenthesis. The human genome was divided into the following four types of regions: the proximal promoter (5 kb upstream and downstream of the TSS), exon, intron and intergenic regions. The numbers of peaks for each sample were assayed according to the description in the Materials and methods. (c) Lineage-specific H3K4me3 and H3K27me3 peaks are shown. According to the analysis data from each sample, the common peak is the shared peak between Ctr. moDCs, LPS moDCs and TGF-b moDCs (two peaks’ overlap 450% of the small peak’s length). The specific peak is the peak from the control moDCs (Ctr.), LPS-conditioned moDCs (LPS) or TGF-b-conditioned moDCs (TGF-b). Ctr. moDCs, control immature moDCs; LPS moDCs, LPS-conditioned moDCs; TGF-b moDCs, TGF-b-conditioned moDCs.

to LPS, which is in agreement with transcriptional downregulation CCL3, CCL4, IFNGR2 and CCR7, were modified by H3K4me3 in their of this surface molecule in LPS-conditioned DCs. However, TGF-b- promoter regions. In addition, inconsistent with previous data,11 conditioned DCs maintained these H3K4me3 marks (Figure 5a) at we found that the CD40 gene in moDCs was also remarkably the CD14 promoter. These suggest that H3K4me3 modification modified by H3K4me3 in its promoter region. The genes for the has an important role in regulating the expression of CD14. The cytokines IL-2, IL-5, IL-7, IL-15 and IL-16, which are only expressed expression of CD83, a mature marker that is increased following at low levels by DCs, showed very minor H3K4me3 modification DC maturation, was lower in TGF-b-conditioned DCs than LPS- but showed strong K3H27me3 modification (Supplementary conditioned DCs. However, the promoter region of CD83 did not Figure S7). Thus, our results confirm that H3K4me3 modification show decreased H3K4me3 marks in TGF-b-conditioned moDCs as is related to active genes, and that H3K27me3 marks are compared with untreated or LPS-conditioned moDCs. Interest- associated with repressive modification. ingly, there was remarkably increased H3K27me3 modification LPS as a potent inducer of DC maturation can promote the near the CD83 in TGF-b-conditioned DCs (Figure 5a), which surface expression of costimulatory and Ag-presenting molecules, implied that the modification of H3K27me3 marks has a role in whereas TGF-b attenuates many of these processes.25 Compared regulating the expression of CD83. with unconditioned DCs, TGF-b significantly downregulated the Consistent with previously published data,11 genes active in expression of the costimulatory molecules CD80, CD86 and CD40 human moDCs often exhibit H3K4me3 modification. As shown in and Ag-presenting molecules (Figure 5), whereas the expression of Figures 5 and 6, the genes for surface molecules CD14, CD83, these molecules was higher in LPS-conditioned DCs. Importantly, CD86 and HLA-DRB1 (only H3K4me3 and H3K27me3 marks of the genes for these costimulatory molecules were modified with CD40 and CD86 are shown) as well as those for the cytokines/ H3K4me3 and H3K27me3 upon exposure to LPS or TGF-b chemokines and their receptors, such as IL-1A, IL-12A, TNFSF10, (Figure 5). TGF-b and LPS treatment not only affects the

Genes and Immunity (2012) 311 --320 & 2012 Macmillan Publishers Limited Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells Y Huang et al 315 a b 7 Ctr. 80 6 70 5 TGF-beta 60 4 50 40 R.E 3 30 2 20

Percentage of Percentage 1 10

modification patterns 0 0 Upreg. Downreg. Unaltered

H3K4me3 H3K4me3+H3K27me3 IL 32 PDIA 3 MED24 PLCB 3 IL1RAP NFKB 2 NFKB 1 GAPDH ACVR1B PRKACA H3K27me3 No H3K4me3+H3K27me3 TGFBR 1 HSP90AB1

c 1.5 NFKB2 1.2 Ctr. TGF-beta 0.9

R.E 0.6 Ctr. 0.3 TFG β 0 chr10:104,138,582-104,157,906 HSP90AB1 DCC RYR3 PDIA3 HDAC9 PLCB4 PRKCH GAPDH

RYR3 Ctr.

Ctr. TFG β chr6:44,307,800-44,330,960

TFG β IL32

chr15:31,124,956-32,001,106 Ctr.

TFG β chr16:3,047,040-3,067,943 Figure 3. Global analysis of H3K4me3 and H3K27me3 modification in TGF-b-conditioned human moDCs. (a) Percentages of H3K4me3 and H3K27me3 modification in upregulated (more than twofold transcriptional change), downregulated (less than twofold transcriptional change) and unaltered genes (no significant change) in TGF-b-conditioned human moDCs. The upregulated, downregulated and unaltered genes on the gene-chip microarray of TGF-b-conditioned moDCs (NCBI-GEO Database: TGF-b-treated DC gene expression, GDS2940 record) were co-analyzed with the data from the Chip-seq (Supplementary Table S1), and the data are shown in Supplementary Table S3. (b) The confirmation of upregulated genes from the gene-chip microarray of TGF-b-conditioned moDCs and the modification by H3K4me3 marks. (c) The confirmation of downregulated genes from the gene-chip microarray of TGF-b-conditioned moDCs and the modification by H3K27me3 marks. TGF-b-conditioned human moDCs were prepared from immature human moDCs after exposure to TGF-b. Up- and downregulated genes NFKB2, HSP90AB1, CD32, TGFBR1, PLCB3, PDIA3, NFKB1, PRKACA, ACVR1B, MED24, IL-1RAP, RYR3, HDAC9, DCC, PLCB4, PRKCH and PDIA3 from the TGF-b conditioned moDC gene-chip microarray and control homing gene GAPDH after exposure to TGF-b were detected by quantitative real-time PCR (qRT--PCR) using the primer sets listed in Supplementary Table S5 and according to the protocols described in the Materials and Methods. RE represents relative expression. The data from qRT--PCRare one representative of three different healthy donors. Gene structures of NFKB2, HSP90AB1, IL-32 and RYR3 were downloaded from the UCSC genome browser. The peaks labeled in blue represent H3K4me3 modification, whereas the peaks labeled in red represent H3K27me3 modification. The arrow represents the direction of gene transcription. Ctr., human immature moDCs; TGF-b, TGF-b-conditioned DCs. expression of costimulatory and Ag-presenting molecules but also in LPS-conditioned DCs (Figure 5). However, the position of alters the expression of cytokines, chemokines and their H3K4me3 and/or H3K27me3 marks was altered in both upregu- receptors.6,26 As shown in Figure 6, exposure to TGF-b and LPS lated and downregulated genes from LPS-conditioned moDCs, affected the expression of IL-1A, IL-12A, TNFSF10, CCL3, CCL4, such as those for CD83, IL-1A, CCL3, CCL4 and IFNGR2 (Figures 5 IFNGR2 and CCR7. In a similar manner as the genes for and 6). This suggests that exposure to LPS induces both the costimulatory molecules, the loci of the genes for the cytokines/ upregulation and downregulation of costimulatory molecules, chemokines and their receptors, including IL-1A, IL-12 A and Ag-presenting molecules, cytokines/chemokines and their recep- IFNGR2, which were downregulated following TGF-b treatment, tors that may be partially dependent on the alteration of were modified by increased H3K27me3. The genes that were H3K4me3 and/or H3K27me3 position. upregulated following TGF-b treatment, such as IL-32, were modified by increased K3K4me3 (Figure 3). However, although genes such as TNFSF10, CCL3, CCL4 and CCR7 were modified by Transcription factors are modified by H3K4me3 and H3K27me3 in both increased H3K4me3 and H3K27me3, their expressions were both LPS- and TGF-b-conditioned DCs not remarkably altered (Figure 6). Transcription factors have a critical role in orchestrating the Interestingly, certain upregulated costimulatory molecules, differentiation and function of DCs. In particular, the Rel/NF-kB cytokines/chemokines and their receptors, such as CD40, CD80, family of transcription factors, which includes RelA, c-Rel, RelB, CD86 and HLA-DR, were not modified by increased H3K4me3 NF-kB1 (p50 and its precursor p105) and NF-kB2 (p52 and its

& 2012 Macmillan Publishers Limited Genes and Immunity (2012) 311 --320 Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells Y Huang et al 316

a b 6 Ctr. 80 5 LPS 70 60 4 50 3 R.E 40 2 30 20 1

Percentage of 10 0 0 modification patterns Upreg. Downreg. Unaltered JAK3 JAK1 STAT3 STAT4 PTPN1

H3K4me3 H3K4me3+H3K27me3 GAPDH STAT5A STAT5B STAT3 H3K27me3 No H3K4me3+H3K27me3 c 1.2 Ctr. LPS Ctr. 0.9 LPS 0.6 chr17:37,708,289-37,820,493 R.E

0.3 JAK3 0

VAV3 PAK1 GNB5 PRKCA GAPDH Ctr. VAV3 LPS chr19:17,780,320-17,836,112

Ctr. STAT5A

LPS

Ctr.

chr1:107,805,929- LPS 108,508,443 chr17:37,676,017-37,734,557 Figure 4. Global analysis of H3K4me3 and H3K27me3 modification in LPS-conditioned human moDCs. (a) Percentages of H3K4me3 and H3K27me3 modification in upregulated (more than twofold transcriptional change), downregulated (less than twofold transcriptional change) and unaltered genes (no significant change) in LPS-conditioned human moDCs. The upregulated, downregulated and unaltered genes on the gene chip from LPS-conditioned DCs (NCBI-GEO Database, LPS-treated DC gene expression, GSE10316 record) were co-analyzed using the data from the Chip-seq (Supplementary Table S1), and the data are shown in Supplementary Table S4. (b) The confirmation of upregulated genes from the gene-chip microarray of LPS-conditioned moDCs and the modification by H3K4me3. (c) The confirmation of the downregulated genes from the gene-chip microarray of LPS-conditioned moDCs and the modification by H3K27me3. Up- and downregulated genes STAT3, JAK3, STAT5A, PTPN1, STAT5B, STAT4, JAK1, VAV3, PAK1, PRKCA,and GNB5 from the LPS-conditioned moDC gene-chip microarray and control homing gene GAPDH after exposure to LPS were detected by quantitative real-time PCR (qRT--PCR) according to the protocol described in the Materials and methods. The data from qRT--PCR are one representative of three different healthy donors. RE represents relative expression. The gene structures of STAT3, JAK3, STAT5A and VAV3 were downloaded from UCSC genome browser. The peaks labeled in blue represent H3K4me3 modification, whereas the peaks labeled in red represent H3K27me3 modification. The arrow represents the direction of gene transcription.

precursor p100), has a central role in the immune system and DISCUSSION determines the expression of multiple and innate In this study, we have shown that human moDCs can be regulated immune responses.27 We finally investigated histone modifica- by epigenetic histone modifications during their differentiation tions on genes associated with transcriptional processes. The into activated DCs or tolerized DCs. Meanwhile, we have mapped expressed genes encoding transcription factors NFKB1, the TGF-b- and LPS-associated H3K4me3 and H3K27me3 NFKB2, NFATC1, STAT3, STAT4, STAT5A and STAT5B had marks across the genome. We have demonstrated that the H3K4me3 modifications at their promoters, and a few of these upregulation of certain costimulatory molecules, cytokines/ had H3K27me3 modification (Figures 3 and 4 and Supplementary chemokines as well as their receptors is related to the increased Figure S6). TGF-b-induced expression of the NFKB2, STAT5A H3K4me3 marks, whereas the downregulation of other genes is and STAT5B genes in conditioned moDCs was also modified by associated with H3K27me3 modifications in TGF-b- and LPS- H3K4me3 marks (Figure 3 and Supplementary Figure S6). conditioned DCs. However, the expression of the LPS- or TGF-b Conversely, TGF-b downregulated the expression of the induced genes is not always dependent on the increased level NF-AC1 gene in conditioned moDCs, which was clearly modified of H3K4me3, and the expression of the downregulated genes is with increased H3K27me3 marks (Supplementary Figure S6). not always modified by H3K27me3. Particularly, the expression However, similar to other genes in LPS-conditioned of costimulatory molecules, cytokines and chemokines and moDCs, upregulated transcription factors such as NFKB1, NFKB2, their receptors after exposure to LPS is often independent NFATC1, STAT3, STAT4, STAT5A and STAT5B were not modified of the modifications of the H3K4me3 marks, but it is often with increased H3K4me3 marks (Figure 4 and Supplementary associated with the alteration of the H3K4me3 and/or H3K27me3 Figure S6). marks.

Genes and Immunity (2012) 311 --320 & 2012 Macmillan Publishers Limited Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells Y Huang et al 317 a

CD14 CD83 CD14 CD83

– Ctr. Counts

– LPS

Isotypic ctr. LPS – TGF β Ctr. TGF-β

CD40 chr5:139,983,503-139,998,799 chr6:14,185,420-14,271,696 b 5 4 3 HLA-DR 2 6 1 5 CD40 CD80 0 4 5 CD80 3 4 2 3 1 2 0 – Ctr. 1 0 2 GAPDH

CD86 – LPS 8 7 1 6 5 4 – TGF β 3 2 0 1 β 0 Ctr. LPS chr20:44,168,304-44,204,392 chr3:120,701,091-120,785,910 TGF- β Ctr. LPS TGF- Figure 5. Characterization of H3K4me3 and H3K27me3 modification in LPS- and TGF-b-conditioned moDCs. (a) The expression and H3K4me3 and H3K27me3 modifications of the CD14 and CD83 genes from control immature moDCs (Ctr.), LPS-conditioned moDCs (LPS) and TGF-b- conditioned DCs (TGF-b). Immature human moDCs were exposed to LPS (100 ng mlÀ1) or TGF-b (10 ng mlÀ1) for 48 h, and the expressions of the surface molecules CD14 and CD83 were analyzed using FAScan after staining by anti-CD14 and CD83 antibodies. (b) The expression and H3K4me3 and H3K27me3 modifications of surface molecules in immature moDCs (Ctr.), LPS-conditioned DCs (LPS) and TGF-b-conditioned DCs (TGF-b). The expressions of costimulatory molecules CD40, CD80, CD86 and Ag-presenting molecule HLA-DR, as well as control homing gene GAPDH by immature moDCs (Ctr.), LPS-conditioned DCs (LPS) and TGF-b-conditioned DCs (TGF-b) were analyzed using quantitative real-time PCR (qRT--PCR) according to the protocol described in the Materials and methods. RE represents relative expression. The data from qRT--PCRare one representative of three different healthy donors. The gene structures of CD14, CD83, CD40 and CD86 were downloaded from UCSC genome browser. The islands labeled in blue represent H3K4me3 modification, whereas the islands labeled in red represent H3K27me3 modification. The arrow represents the direction of gene transcription.

DCs are extremely versatile Ag-presenting cells involved in the promoters, whereas some repressive genes are modified by initiation of both innate and adaptive immunity and also in the H3K27me3 marks in TGF-b- or LPS-conditioned moDCs. This is in maintenance of self-tolerance. These diverse and almost contra- agreement with other studies using embryonic stem cells31,32 and dictory functions are dependent on the plasticity of these cells to moDCs,11 or CD4 þ and CD8 þ T cells,10,14,33 which have reported allow them to undergo a complete genetic reprogramming in that H3K4me3 marks are the most characteristic modification in response to different stimuli.1,2 Our results showed that methyl- promoter regions. In addition, H3K4me3 and H3K27me3 marks are transferase inhibitors not only affect the expression of certain usually enriched at the active or inactive chromatin regions, critical genes, but more importantly also alter DC functions, respectively.34,35 Our data have also shown that both H3K4me3 suggesting that epigenetic modifications are involved in the and H3K27me3 marks were often colocalized to certain genomic plasticity of human moDCs in different environments. Indeed, regions. upregulation of certain costimulatory molecules, cytokines/ The decreased K3K4me3 marks and the increased expression of chemokines as well as their receptors is related to the increased genes with or without the H3K27me3 mark in the LPS-conditioned H3K4me3 marks, and the downregulation of other genes is DCs suggest that ex vivo differentiation in the presence of Toll-like associated with H3K27me3 modifications in the TGF-b- and LPS- receptor ligands induces a global shift toward less active conditioned DCs. chromatin in the presence of Toll-like receptor ligands. Although The basic structural unit of eukaryotic chromatin is the our results have shown that the expression of some costimulatory nucleosome, which consists of 146 bp of DNA wrapped around molecules, cytokines and chemokines and their receptors is an octamer of four core histones (H2A, H2B, H3 and H4). Histone associated with increased H3K4me3 marks in TGF-b-conditioned modifications within promoter regions have an important function moDCs, the upregulation of some of these molecules was not in the regulation of gene expression. The majority of modifications associated with increased H3K4me3 marks in LPS-conditioned occur at the N-terminal ends of the core histones. Studies from moDCs. Others have also found that transcriptionally active genes several model systems have determined that the methylation of can have reduced H3K4me3 marks during DC differentiation.11 4, 36 and 79 of are typically associated with Some genes in human moDCs have been shown to lose their active gene expression,28 --30 whereas repressive genes are H3K4me3 mark independent of their gene expression,11 which associated with H3K27me3 modification. Indeed, many active suggests that both the H3K4m3 and H3K27me3 marks are genes have increased H3K4me3 marks, especially on gene unstable and are prone to either a loss of differentiation state or

& 2012 Macmillan Publishers Limited Genes and Immunity (2012) 311 --320 Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells Y Huang et al 318 abcd 4 IL1A IL1A 3 CCL3 CCL3 10 2 – Ctr. – Ctr. 8 1 6 – LPS 0 – LPS 4 β – TGF 2 6 IL12A β chr2: 113,239,928-113,267,476 5 – TGF 0 4 IL12A 3 chr17:31,431,539-31,449,795 6 CCL4 2 1 4 – Ctr. 0 CCL4 – LPS 4 IFNGR2 2 – TGF β 3 0 2 – Ctr. R.E chr3:161,175,691-161,210,131 R.E 8 CCR7 1 – LPS IFNGR2 0 6 6 TNFSF10 – TGF β 4 – Ctr. 4 chr21:31,447,621-31,464,837 2 – LPS 0 2 – TGF β CCR7 2 GAPDH chr21:33,672,834-33,755,934 0 – Ctr. 2 GAPDH TNFSF10 – LPS 1 1 – Ctr. – TGF β – LPS 0 0 – TGF β chr17:35,941,314-35,997,482 Ctr. Ctr. chr3:173,693,695-173,736,426 LPS LPS TGF-β TGF-β Figure 6. Characterization of H3K4me3 and H3K27me3 modification of the genes for cytokines, chemokines and their receptors in LPS- or TGF-b-conditioned human moDCs. (a) H3K4me3 and H3K27me3 modifications of cytokine and loci (including the genomic region and the upstream and intergenic regions) in immature moDCs (Ctr.), LPS-conditioned-moDCs (LPS) and TGF-b-conditioned moDCs (TGF-b). (b) The expressions of cytokine and cytokine receptor in immature moDCs (Ctr.), LPS-conditioned moDCs (LPS) and TGF-b- conditioned moDCs (TGF-b). (c) H3K4me3 and H3K27me3 modifications of chemokine and loci (including the genomic region and the upstream and intergenic regions) in immature moDCs (Ctr.), LPS-conditioned moDCs (LPS) and TGF-b-conditioned moDCs (TGF-b). (d) The expressions of chemokine and chemokine receptor in immature moDCs (Ctr.), LPS-conditioned moDCs (LPS) and TGF-b- conditioned moDCs (TGF-b). The gene structure was downloaded from the UCSC genome browser. The islands labeled in blue represent H3K4me3 modification, whereas the islands labeled in red represent H3K27me3 modification. The expressions of cytokines, chemokines and their receptors were analyzed using quantitative real-time PCR (qRT--PCR) according to the protocol described in the Materials and methods. RE represents relative expression. The data from qRT--PCR are one representative of three different healthy donors. The arrow represents the direction of gene transcription.

a gain of other permissive mark such as .36,37 Addition- Flow cytometric analyses ally, an alteration of the H3K4me3 and/or H3K27me3 positions is For surface staining, cells were collected in ice-cold phosphate-buffered often found in the upregulated and downregulated genes from saline and incubated with the indicated phycoerythrin- or fluorescein LPS-conditioned moDCs. H3K4me3 at certain chromatin loci may isothiocyanate-labeled antibodies. For each analysis, isotype-matched also prevent aberrant gene expression or modulate transcriptional control monoclonal antibodies were used as negative controls. The 38 response. However, additional genome-wide localization and phenotypes of human moDCs were analyzed using phycoerythrin- or functional studies will provide important new insights into the role fluorescein isothiocyanate-labeled anti-CD14 (61D3), anti-CD83 (HB15e), of other epigenetic marks, chromatin-modifying enzymes and anti-CD11c (3.9), anti-CD86 (IT2.2), anti-CD80 (2D10.4), anti-CD40 (HB14) chromatin remodelers. and anti-CD11b (ICRF44), which were purchased from Pharmingen (San Diego, CA, USA).

MATERIALS AND METHODS Transfection Preparation and isolation of human moDCs For small interfering RNAs (siRNAs) and negative control oligonucleotides, Immature DCs were prepared from the buffy coats of healthy donor cells were transfected with the indicated oligos (100 nM) using Entranster-R samples obtained from the blood bank of Tianjin, according to our (Engreen Biosystem, Beijing, China) according to the manufacturer’s previously described method.39 Briefly, peripheral blood mononuclear cells instruction. The transfection rate of the human moDCs was as high as were separated by Ficoll-Paque gradient centrifugation, and CD14 þ cells 85%. For validation of the siRNA(s), human moDCs were cultured in six-well were purified using antibody-coated microbeads and magnetic separation. plate. The following day cells were transfected in fetal calf serum-free Selected CD14 þ cells were cultured for 5 days at a concentration of medium. One day post transfection, the cells were treated with TRIzol 2 Â 106 mlÀ1 in the presence of GM-CSF (500 U mlÀ1) and IL-4 (500 U mlÀ1; (Invitrogen, San Diego, CA, USA, 15596-026) for RNA extraction. R&D Systems, Minneapolis, MN, USA) for the generation of immature moDCs. Immature moDCs were matured by exposure to LPS (100 ng RNA isolation and quantitative real-time PCR mlÀ1mlÀ1, InvivoGen, San Diego, CA, USA) or tolerized by exposure to TGF- Total RNA was extracted using the TRIzol reagent (Invitrogen, 15596-026), b (10 ng mlÀ1 R&D Systems) for 48 h, unless otherwise stated. and reverse transcription was carried out with Superscript III (Invitrogen)

Genes and Immunity (2012) 311 --320 & 2012 Macmillan Publishers Limited Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells Y Huang et al 319 according to the manufacturer’s protocol. To measure gene expression, used, and the reads number for each region is counted. For the peak quantitative real-time PCR was performed using the Quantitect SYBR PCR calling process, the intensities of control rabbit immunoglobulin G (IgG), with a specific set of primers according to the manufacture’s H3K4me3 and H3K27me3 were first normalized to the random genomic recommendations (Qiagen, Valencia, CA, USA). Amplification of glycer- background and then further normalized by subtracting control IgG aldehyde-3-phosphate dehydrogenase mRNA for gene was performed for intensities from the corresponding modification intensities. The enriched each experimental sample as an endogenous control for gene expression. regions were identified using the model-based analysis of Chip-seq, a P- À5 Fold changes were calculated using the DDCt method according to the value cutoff of 1 Â 10 and a high-confidence fold enrichment. This manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). All method was based on the Poisson distribution. The advantage of this reactions were run in triplicate, and the primer sequences are listed in model was that it could capture both the mean and the variance of the Supplementary Table S5. distribution. Background biases were controlled by themselves. Subse- quently, the peak calling results were estimated according to the peak siRNA experiments number and the peak length distribution. The peak distributions of gene regions were analyzed as read distributions. Finally, peak-related genes siRNAs for SETD6, CBX5, EZH1, EHMT2 and control oligos were purchased were defined by the positional relationship of the islands and genes. from Guangzhou Ribobio, Guangzhou, China, and were used to transfect Several figures show the read distribution of a gene region of the gene of cells with Entranster-R according to the manufacturer’s recommendations interest. Difference analysis on peak-related genes was used to identify (Engreen Biosystem). EHMT2 siRNA of the sequence 5-CATGAC genes sharing partial sequences that could provide information on the TGCGTGCTGTTATTC-340 was synthesized by Guangzhou Ribobio. CBX5, regulatory strength for each gene. EZH1, JHDM1D and SETD6 siRNAs were designed and synthesized by Guangzhou Ribobio. The following sequences of these siRNAs were used: 5-TAAACCCAGGGAGAAGTCA-3 (CBX5); 5-GCCAACATATGTTAATGAG-3 GO analysis (EZH1) and 5-CGCCAATCTAGAATACTCT-3 (SETD6). The nonsilencing oligo GO analysis was performed using the online GO tool (http://go.princeton. control is an irrelevant siRNA with random nucleotides (5-ACTATCTAAGTT edu/). The complete set of all RefSeq genes was used as a background. ACTACCC-3). To determine the effect of EHMT2, SETD6, EZH1 and CBX5 Complete GO analysis results are provided (Supplementary Table S2). knockdown on the expression of genes, the cells were transfected using different siRNAs and the expression of surface markers were analyzed after 48 h. The transcriptional levels of the genes were detected with quantitative real-time PCR. The primers used in these experiments are CONFLICT OF INTEREST listed in Supplementary Table S5. The authors declare no conflict of interest.

Chip, Chip-seq and bioinformatics Chip and Chip-seq, followed by high-throughput sequencing were ACKNOWLEDGEMENTS performed by the Research and Cooperation Division of BGI-Shenzhen, This research was supported by the NSFC grants 91029736, 30771967 and 30872315; according to previously described methods.41 In brief, 3 Â 107 immature Ministry of Science and Technology grants (863 program, 2008AA02Z129); the moDCs (control), TGF-b-conditioned DCs or LPS-conditioned DCs were National Key Basic Research and Development Program of China (973 Program, digested with micrococcal nuclease (2 U) for 10 min in a 37 1C water bath 2007CB914803); and the National Key Scientific Program (2011CB964902). to generate native chromatin template consisting primarily of mono- . The native chromatin templates were then incubated with anti-histone H3K4me3 (Kit cat. no. 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Supplementary Information accompanies the paper on Genes and Immunity website (http://www.nature.com/gene)

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