Global mapping of H3K4me3 and H3K27me3 reveals chromatin state-based regulation of human monocyte-derived dendritic cells in different environments

Department of Immunology, Nankai University School of Medicine, Nankai University, Tianjin, China.
Genes and immunity (Impact Factor: 2.91). 01/2012; 13(4):311-20. DOI: 10.1038/gene.2011.87
Source: PubMed
ABSTRACT
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 histone modifications can regulate the differentiation of human monocyte-derived DCs (moDCs) into either activated or tolerized DCs. The inhibition or silencing of methyltransferases or methylation-associated factors affects the expression of multiple genes. Genome mapping of transforming growth factor (TGF-β)- or lipopolysaccharide (LPS)-associated H3K4 trimethylation (H3K4me3) and H3K27 trimethylation (H3K27me3) demonstrated the presence of histone modification of gene expression in human TGF-β- or LPS-conditioned moDCs. Although the upregulated or downregulated genes were not always associated with H3K4me3 and/or H3K27me3 modifications in TGF-β-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-β-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.

Full-text

Available from: Yuanhang Liu
ORIGINAL ARTICLE
Global mapping of H3K4me3 and H3K27me3 reveals chromatin
state-based regulation of human monocyte-derived dendritic cells
in different environments
Y Huang
1
,SMin
1
,YLui
1
, J Sun
1
,XSu
1
, Y Liu
1
, Y Zhang
1
, D Han
2
, Y Che
1
, C Zhao
3
,BMa
4
and R Yang
1,5
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 histone modifications
can regulate the differentiation of human monocyte-derived DCs (moDCs) into either activated or tolerized DCs. The inhibition
or silencing of methyltransferases or methylation-associated factors affects the expression of multiple genes. Genome mapping
of transforming growth factor (TGF-b)- or lipopolysaccharide (LPS)-associated H3K4 trimethylation (H3K4me3) and H3K27
trimethylation (H3K27me3) demonstrated the presence of histone modification of gene 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; epigenetics; H3K4me3; H3K27me3
INTRODUCTION
Dendritic cells (DCs) are antigen (Ag)-presenting cells, with a
unique capacity to stimulate naive T cells and initiate primary
immune responses. However, DCs also have critical roles in the
induction of central and peripheral immunological tolerance and
the regulation of the types of T-cell immune responses.
1,2
The
diverse functions of DCs in immune regulation not only depend
on the heterogeneity of DC subsets but also on their functional
plasticity.
1,2
DCs express a variety of pathogen recognition
receptors, such as Toll-like receptors. The detection of damage-
or pathogen-associated molecular patterns, such as lipopolysac-
charides (LPSs), by tissue-resident DCs initiates DC maturation and
migration to the regional lymph nodes.
3,4
Upon exposure to
transforming growth factor (TGF)-b, DCs become tolerogenic and
can thereby suppress T-cell alloreactivity
5
and induce Th2 or
T regulatory responses.
3,4
Studies have shown that the expression
of genes coding costimulatory molecules, cytokines, chemokines
and their receptors is remarkably different in the conditioned DCs;
those DCs activated by LPS express higher levels of the
costimulatory molecules, such as CD80 and CD86, and the
Ag-presenting molecule major histocompatibility complex (MHC)
II
3,4
than those conditioned with TGF-b.
6
Previous reports
6--8
and microarray data from the NCBI-GEO
database (LPS-treated DC gene expression from the GSE10316
record and TGF-b-treated DC gene expression from the GDS2940
record) have shown that both LPS and TGF-b induce the
expression of hundreds of genes in human monocyte-derived
DCs (moDCs). The responses induced by treatment with LPS or
TGF-b most likely employ different regulatory requirements that
reflect their functions. As the expression of different classes of
genes can be induced by the same receptor, differential regulation
occurs via a gene-specific mechanism. Although multiple mechan-
isms may be involved in the regulation of gene expression in
different kinds of cells,
9
epigenetic modification has served as an
important mechanism in regulating the cellular differentiation.
10
Indeed, a genome-wide promoter of histone modifications has
been identified in human moDCs.
11
In LPS-conditioned macro-
phages, gene-specific regulation occurs at the level of chromatin
and includes nucleosome remodeling and covalent histone
modifications.
12
Histone deacetylase (HDAC) inhibition can also
affect the phenotype and function of human moDCs.
13
Global
mapping of H4K4me3 and H3K27 trimethylation (H3K27me3) has
revealed the specificity and plasticity of lineage fate determination
in differentiating CD4
þ
T cells
10,14
and in memory CD8
þ
T cells.
14
Thus, the covalent modifications of histone N-terminal tails, such
as methylation, can act to regulate chromatin states.
15
moDCs develop from peripheral blood monocytes under the
influence of granulocyte-macrophage colony-stimulating factor
(GM-CSF) alone or GM-CSF with interleukin-4 (IL-4),
16,17
and are a
subset of DCs that are involved in inflammatory processes and
infection clearance.
3,18,19
These moDCs could become either
active or tolerogenic after exposed to different stimuli. Here, we
have used chromatin immunoprecipitation (Chip) and Chip-
sequencing (Chip-seq) to characterize the modification of H3K4
trimethylation (H3K4me3) and H3K27me3 in LPS- and TGF-b-
conditioned moDCs. We have found that during the transition of
Received 19 August 2011; revised 24 October 2011; accepted 28 November 2011; published online 26 January 2012
1
Department of Immunology, Nankai University School of Medicine, Nankai University, Tianjin, China;
2
Research and Cooperation Division, BGI-Shenzhen, Shenzhen, China;
3
Center for Tissue Engineering, Chinese Academy of Medical Science, Peking Union Medical College, Beijing, PR China;
4
College of Life Science, Tianjin, China and
5
Key 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: ryang@nankai.edu.cn
Genes and Immunity (2012) 13, 311 -- 320
&
2012 Macmillan Publishers Limited All rights reserved 1466-4879/12
www.nature.com/gene
Page 1
immature moDCs into activated moDCs (LPS conditioning) and
tolerized moDCs (TGF-b conditioning), changes in the modifica-
tion of H3K4me3 or H3K27me3 as well as alteration in these
epigenetic marks have a role in the resulting up- or down-
regulation of genes in these treated cells.
RESULTS
Histone methylation is involved in the regulation of the gene
expression in human moDCs
Histone methyltransferases have a critical role in epigenetic
modification during the cellular differentiation.
20
To investigate the
effect of methylation on the expression of TGF-b-andLPS-associated
genes, we first employed the methyltransferase inhibitor adenosine
dialdehyde (an adenosine analog and S-adenosylmethionine-depen-
dent methyltransferase inhibitor) to observe the effect on the gene
expression in human moDCs. As shown in Supplementary Figure S1,
adenosine dialdehyde clearly promoted the expression of the surface
molecules CD14, CD11C, CD80, CD86 and MHC-DR, the expression of
the cytokines and chemokines IL-1A, IL-12A, interferon alpha 6
(IFNA6), tumor necrosis factor (ligand) superfamily, member 10
(TNFSF10), chemokine (C-C motif) ligand (CCL)3, CCL4, chemokine (C-
C motif) receptor (CCR)7 and chemokine (C-X-C motif) ligand (CXCL)16,
and also the expression of the transcription factors signal transducer
and activator of transcription (STAT)3, STAT4, nuclear factor (NF)-kB1
and NF-kB2 in human moDCs. Euchromatic histone lysine N-
methyltransferase 2 (EHMT2) is a H3K9- and H3K27-specific histone
methyltransferase
21
that is remarkably downregulated in human
moDCs after exposure to LPS (LPS-treated DC gene expression,
GSE10316 record and Supplementary Figure S2), and has the
potential to affect the expression of multiple genes. Indeed, the
silencing of EHMT2 with demonstrated transfection (Figure 1a) was
found to promote the expression of surface molecules (CD14, CD11c,
CD80, CD86 and MHC-DR), cytokines (IL-1A, IL-12A, IFNA and
TNFSF10), chemokines (CCL3, CCL4, CCR7 and CXCL16) and
transcription factors (STAT3, STAT4, NF-kB1 and NF-kB2; Figure 1b).
Importantly, silencing EHMT2 also affected the Ag-presenting function
of moDCs (Supplementary Figure S3). Others genes such as enhancer
of zeste homol og 1 (EZH1), chromobox homolog 5 (CBX5)andSET
domain-containing 6 (SETD6), which are associated with the methyla-
tion of histones,
22 --24
were also involved in the regulation of gene
expression in moDCs. The silencing of SETD6 was found to
downregulate the expression of IL-1A and upregulate that of STAT3,
whereas the expression of NF-kB1 was not remarkably altered
(Figures 1c and d). The silencing of EZH1 was found to downregulate
IL-1A expression and upregulate both STAT3 and NF-kB 1 expression,
but the silencing of CBX5 downregulated both IL-1A and NF-kB1
expression and upregulated STAT3 expression (Figures 1c and d).
Taken together, these results suggest that histone methylation may
be involved in the gene regulation of human moDCs.
Genome-wide maps of H3K4me3 and H3K27me3 modification in
TGF-b- and LPS-conditioned DCs
To reveal the epigenetic features present during the process of
differentiation of moDCs in response to different environments,
we generated global maps of H3K4me3 and H3K27me3 modifica-
tion using the Chip-seq approach. The LPS-conditioned moDCs
had typical morphology of DC; they expressed higher levels of
costimulatory molecules, such as CD11C, CD40, CD80 and CD86,
and Ag-presenting molecules, MHC class I and class II (Supple-
mentary Figure S4). Consistent with previous reports,
6
TGF-b-
conditioned DCs exhibited an immature phenotype; they
expressed lower levels of CD40, CD80, CD86, MHC I, MHC II and
CD11C than did LPS-conditioned moDCs (Supplementary Figure S4).
Similar to unconditioned moDCs, the Ag-presenting function of
these human LPS- or TGF-b-conditioned moDCs could also be
altered after silencing EHMT2, suggesting that histone methylation
may be also involved in the gene regulation of human LPS- or TGF-
b-conditioned moDCs (Supplementary Figure S3). Using specific
antibodies, we next immunoprecipitated H3K4me3-or K3K27me3-
associated genomic DNAs from TGF-b- or LPS-conditioned moDCs
and control moDCs. The H3K4me3 and H3K27me3 peak (island,
Chip-seq-enriched region) distribution near the transcription start
site (
±
5 kb) of each annotated RefSeq (2012) 13, 311--320. gene
was analyzed. H3K4me3 islands were enriched in the region closest
to the transcription start site (from þ 2.5 kb to 100 bp; Figure 2a),
whereas H3K27me3 peaks were enriched in the region upstream of
the transcription start site sites (from þ 2.5 to þ 5 kb; Figure 2a).
To examine the overall H3K4me3 and H3K27me3 distribution,
we divided the human genome into four types of regions, which
included the proximal promoter (2 kb upstream and downstream
of the transcription start site), the exons, introns and intergenic
regions. These regions were based on the annotations of ‘known
genes’ in the UCSC genome browser (http://genome.UCSC.edu).
H3K4me3 islands were found in B20% of the proximal promoter
regions of TGF-b-treated moDCs (23%) and LPS-treated moDCs
(21%), whereas H3K27me3 islands were only found in 3% of the
proximal promoter regions of both TGF-b-treated moDCs (3%) and
LPS-treated moDCs (3%). The examination of H3K4me3 and
K3K27me3 islands within gene bodies revealed that B1% of these
was enriched for H3K4me3 or K3K27me3 islands (Figure 2b). Most
of H3K4me3 and H3K27me3 modifications were located in the
introns and the intergenic regions (Figure 2b). Lineage- specific
H3K4me3 and H3K27me3 marks in LPS- or TGF-b-conditioned DCs
were remarkably different than those in control moDCs. The
number of lineage-specific H3K4me3 islands was significantly
greater in TGF-b-conditioned moDCs than in LPS-conditioned
moDCs, which indicated that the active genes in TGF-b-condi-
tioned moDCs were modified by H3K4me3. However, the number
of lineage-specific H3K27me3 islands was similar between LPS- and
TGF-b-conditioned moDCs (Figure 2c). During the differentiation of
the immature DCs into LPS-activated DCs, the proportion of genes
with H3K4me3 marks alone decreased from 13 to 6.5% (Figure 2c).
We also performed gene ontology (GO) analyses for the
H3K4me3- and H3K27me3-marked genes. On the basis of the
genes marked by H3K4me3 and/or H3K27me3 (Supplementary
Table S1), it was clear that these epigenetic modifications decorate
many genes with functions related to differentiation, development
and transcriptional regulation (Supplementary Figure S5 and
Supplementary Table S2). As many as 30 --60% of the genes in
both the TGF-b-conditioned DCs and LPS-conditioned DCs were
modified by H3K4me3, but less genes were modified by
H3K27me3 (Supplementary Figure S5 and Supplementary Table S2).
Global analysis of H3K4me3 and H3K27me3 modification in
TGF-b- and LPS-conditioned 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
next co-analyzed the modification of H3K4me3 and H3K27me3 on
the upregulated, downregulated and unaltered genes, which are
originated from the microarray data of TGF-b-conditioned moDCs
(NCBI-GEO Database: TGF-b-treated DC gene expression, GDS2940
record), and the data are shown in Supplementary Table S3. An
analysis of the more highly expressed genes in TGF-b-conditioned
DCs revealed that almost half (50%) have the H3K4me3
modification on their promoter, whereas only 10% have the
H3K27me3 mark (Figure 3a). There was a highly significant
correlation between the H3K4me3 marks and the high expression
level of genes related to TGF-b activation, such as that of NFKB2,
HSP90AB1, IL-32, TGFBR1, PLCB3, PDIA3, NFKB1, PRKACA, ACVR1B,
MED24 and IL-1RAP (Figure 3b, only H3K4me3 marks of NFKB2,
HSP90AB1 and IL-32 are shown) were seen in TGF-b-conditioned
DCs. These genes are involved in the PPARa/RXRa-activated
pathway that may limit inflammatory responses. Conversely, the
Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells
Y Huang et al
312
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Page 2
presence of H3K27me3 marks in TGF-b-conditioned DCs was
correlated with lower levels of immune gene expression, such as
that of RYR3, HDAC9, DCC, PLCB4 and PRKCH were related to
H3K27me3 marks (Figure 3c, only H3K27me3 marks of RYR3 are
shown). However, 40% of the upregulated genes, 50% of the
downregulated genes and 20% of the unaltered genes were not
modified by H3K4me3 or H3K37me3 (Figure 3a).
We also analyzed the modification of H3K4me3 and H3K27me3
on the upregulated, downregulated and unaltered genes, which
are originated from the microarray data of LPS-conditioned DCs
(NCBI-GEO Database, LPS-treated DC gene expression, GSE10316
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
downregulated genes were modified by H3K27me3 in LPS-
conditioned DCs (Figure 4a). Similar to TGF-b-conditioned DCs,
many genes were not modified by either H3K4me3 or H3K27me3.
The JAK/STAT family of transcription factors is critical for DC
function. We found that signaling molecules in this signal pathway
were modified with H3K4me3 (Figure 4b, only H3K4me3 marks of
STAT3, JAK3 and STAT5A are shown). Interestingly, the upregula-
tion of genes such as PTPN1, STAT3, JAK3, STAT5B, STAT4, STAT5A
and JAK1 in the JAK/STAT signal pathway was not significantly
related to increased H3K4me3 modification in these LPS-
conditioned DCs (Figure 4b), which implied the involvement of
other mechanisms in the regulation of the expression of these
genes. Downregulated genes such as VAV3, PAK1, PRKCA and GNB5,
which are associated with phagocytosis and pinocytosis, were
indeed modified by the increased H3K27me3 in LPS-conditioned
DCs (Figure 4c, only H3K27me3 marks of VAV3 are shown).
Additionally, in the LPS-conditioned DCs, STAT3, STAT5B and STAT5A
genes were marked in their promoter regions with H3K4me3, but
these genes did not have substantial H3K27me3 signals, indicating
these active genes are mainly modified by H3K4me3.
Characterization of H3K4me3 and H3K27me3 modification and
the expression of the surface molecules, cytokines, chemokines
and their receptors
CD14 and CD83 are the critical markers of the process of the DC
differentiation. Surface molecule CD14, which is highly expressed
by monocytes, is downregulated during the differentiation after
exposure to LPS but is regulated after exposure to TGF-b
(Figure 5a). Consistent with other reporters,
11
the promoter of
the CD14 genes had multiple H3K4me3 marks with high CD14
expression (Figure 5a), where these marks were lost after exposure
ab
cd
1.2
8
6
4
2
0
8
6
4
2
0
8
6
4
2
0
CD14 CD11c CD80 CD86 MHC-DR
R.ER.E
R.E R.E
Counts
IL1A
IL12A
IFNA6
TNFSP10
GAPDH
CCL3
CCL4
CCR7
CXCL16
GAPDH
STAT3
STAT4
NFKB1
NFKB2
GAPDH
0.9
0.6
0.3
0
1.2
0.9
0.6
0.3
0
1.2
6
6
4
2
0
4
2
0
IL1A STAT3 NFKB1
0.9
0.6
0.3
0
EHMT2
Actin
Untr.
Untr.
SETD6
SETD6siRNA
EZH1
EZH1siRNA
CBX5
CBX5siRNA
SETD6siRNA
EZH1siRNA
CBX5siRNA
Oligo
Ctr.
Oligo.ctr
Oligo.ctr
Oligo.ctr
SETD6siRNA
EZH1siRNA
CBX5siRNA
Oligo.ctr
Oligo ctr.
Oligo ctr.
EHMT2
siRNA
siRNA
EHMT2 siRNA
EHMT2 siRNA
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.
Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells
Y Huang et al
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Page 3
to LPS, which is in agreement with transcriptional downregulation
of this surface molecule in LPS-conditioned DCs. However, TGF-b-
conditioned DCs maintained these H3K4me3 marks (Figure 5a) at
the CD14 promoter. These suggest that H3K4me3 modification
has an important role in regulating the expression of CD14. The
expression of CD83, a mature marker that is increased following
DC maturation, was lower in TGF-b-conditioned DCs than LPS-
conditioned DCs. However, the promoter region of CD83 did not
show decreased H3K4me3 marks in TGF-b-conditioned moDCs as
compared with untreated or LPS-conditioned moDCs. Interest-
ingly, there was remarkably increased H3K27me3 modification
near the CD83 locus in TGF-b-conditioned DCs (Figure 5a), which
implied that the modification of H3K27me3 marks has a role in
regulating the expression of CD83.
Consistent with previously published data,
11
genes active in
human moDCs often exhibit H3K4me3 modification. As shown in
Figures 5 and 6, the genes for surface molecules CD14, CD83,
CD86 and HLA-DRB1 (only H3K4me3 and H3K27me3 marks of
CD40 and CD86 are shown) as well as those for the cytokines/
chemokines and their receptors, such as IL-1A, IL-12A, TNFSF10,
CCL3, CCL4, IFNGR2 and CCR7, were modified by H3K4me3 in their
promoter regions. In addition, inconsistent with previous data,
11
we found that the CD40 gene in moDCs was also remarkably
modified by H3K4me3 in its promoter region. The genes for the
cytokines IL-2, IL-5, IL-7, IL-15 and IL-16, which are only expressed
at low levels by DCs, showed very minor H3K4me3 modification
but showed strong K3H27me3 modification (Supplementary
Figure S7). Thus, our results confirm that H3K4me3 modification
is related to active genes, and that H3K27me3 marks are
associated with repressive modification.
LPS as a potent inducer of DC maturation can promote the
surface expression of costimulatory and Ag-presenting molecules,
whereas TGF-b attenuates many of these processes.
25
Compared
with unconditioned DCs, TGF-b significantly downregulated the
expression of the costimulatory molecules CD80, CD86 and CD40
and Ag-presenting molecules (Figure 5), whereas the expression of
these molecules was higher in LPS-conditioned DCs. Importantly,
the genes for these costimulatory molecules were modified with
H3K4me3 and H3K27me3 upon exposure to LPS or TGF-b
(Figure 5). TGF-b and LPS treatment not only affects the
ab
c
H3K4me3
H3K27me3
60
50
40
30
20
10
0
30
Sample ID
Ctr.moDC
H3K4me3
H3K27me3
LPS moDC
To ta l
Proximal
promoter
Exons
Introns
Intergenic
region
5825 (65%)
1038 (12%)
427 (17%)
1353 (12%)
1249 (77%)
1542 (77%)
1569 (78%)
1556 (61%)
6916 (63%)
306 (18%)
390 (18%)
388(18%)
8927
1909 (21%)
155 (2%)
33 (1%)
172 (2%)
4 (1%)
6 (1%)
5 (1%)
549 (21%)
2479 (23%)
61 (4%)
69 (3%)
60 (3%)
2565
10920
1620
2027
2022
25
20
15
10
5
0
3.5e-09
3e-09
2.5e-09
2e-09
1.5e-09
1e-09
1e-09
5e-10
9e-10
8e-10
7e-10
6e-10
5e-10
4e-10
3e-10
2e-10
1e-10
LPS moDC
LPS
TGF-β moDC
TGF β
Ctr. moDC
Ctr
LPS
TGF β
TGF-β moDC
TGF-β moDC
Ctr.moDC
LPS moDC
Ctr
up5k
up2.5k
Tag density
Lineage specific H3K4me3
islands (%)
Lineage specific H3K27me3
islands (%)
TSS
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
TES
down2.5k
down5k
0
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.
Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells
Y Huang et al
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Page 4
expression of costimulatory and Ag-presenting molecules but also
alters the expression of cytokines, chemokines and their
receptors.
6,26
As shown in Figure 6, exposure to TGF-b and LPS
affected the expression of IL-1A, IL-12A, TNFSF10, CCL3, CCL4,
IFNGR2 and CCR7. In a similar manner as the genes for
costimulatory molecules, the loci of the genes for the cytokines/
chemokines and their receptors, including IL-1A, IL-12 A and
IFNGR2, which were downregulated following TGF-b treatment,
were modified by increased H3K27me3. The genes that were
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
both increased H3K4me3 and H3K27me3, their expressions were
not remarkably altered (Figure 6).
Interestingly, certain upregulated costimulatory molecules,
cytokines/chemokines and their receptors, such as CD40, CD80,
CD86 and HLA-DR, were not modified by increased H3K4me3
in LPS-conditioned DCs (Figure 5). However, the position of
H3K4me3 and/or H3K27me3 marks was altered in both upregu-
lated and downregulated genes from LPS-conditioned moDCs,
such as those for CD83, IL-1A, CCL3, CCL4 and IFNGR2 (Figures 5
and 6). This suggests that exposure to LPS induces both the
upregulation and downregulation of costimulatory molecules,
Ag-presenting molecules, cytokines/chemokines and their recep-
tors that may be partially dependent on the alteration of
H3K4me3 and/or H3K27me3 position.
Transcription factors are modified by H3K4me3 and H3K27me3 in
both LPS- and TGF-b-conditioned DCs
Transcription factors have a critical role in orchestrating the
differentiation and function of DCs. In particular, the Rel/NF-kB
family of transcription factors, which includes RelA, c-Rel, RelB,
NF-kB1 (p50 and its precursor p105) and NF-kB2 (p52 and its
a
c
b
80
H3K27me3
70
60
50
40
30
20
10
0
1.5
1.2
0.9
0.6
R.E
0.3
0
H3K4me3 H3K4me3+H3K27me3
No H3K4me3+H3K27me3
Percentage of
modification patterns
Upreg. Downreg. Unaltered
1
0
Ctr.
Ctr.
IL32
TFG β
Ctr.
TFG β
TFG β
NFKB2
chr10:104,138,582-104,157,906
chr6:44,307,800-44,330,960
chr16:3,047,040-3,067,943
chr15:31,124,956-32,001,106
HSP90AB1
NFKB 2
TGFBR 1
PLCB 3
PDIA 3
NFKB 1
PRKACA
ACVR1B
MED24
IL1RAP
GAPDH
IL 32
HSP90AB1
3
R.E
4
5
6
7
Ctr.
TGF-beta
2
Ctr.
RYR3
RYR3
HDAC9
DCC
PLCB4
PRKCH
PDIA3
GAPDH
TGF-beta
Ctr.
TFG β
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--PCR are one representative of three different healthy donors.
Gene struc tures 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.
Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells
Y Huang et al
315
Genes and Immunity (2012) 311 -- 320& 2012 Macmillan Publishers Limited
Page 5
precursor p100), has a central role in the immune system and
determines the expression of multiple cytokine and innate
immune responses.
27
We finally investigated histone modifica-
tions on genes associated with transcriptional processes. The
expressed genes encoding transcription factors NFKB1,
NFKB2, NFATC1, STAT3, STAT4, STAT5A and STAT5B had
H3K4me3 modifications at their promoters, and a few of these
had H3K27me3 modification (Figures 3 and 4 and Supplementary
Figure S6). TGF-b-induced expression of the NFKB2, STAT5A
and STAT5B genes in conditioned moDCs was also modified by
H3K4me3 marks (Figure 3 and Supplementary Figure S6).
Conversely, TGF-b downregulated the expression of the
NF-AC1 gene in conditioned moDCs, which was clearly modified
with increased H3K27me3 marks (Supplementary Figure S6).
However, similar to other genes in LPS-conditioned
moDCs, upregulated transcription factors such as NFKB1, NFKB2,
NFATC1, STAT3, STAT4, STAT5A and STAT5B were not modified
with increased H3K4me3 marks (Figure 4 and Supplementary
Figure S6).
DISCUSSION
In this study, we have shown that human moDCs can be regulated
by epigenetic histone modifications during their differentiation
into activated DCs or tolerized DCs. Meanwhile, we have mapped
the TGF-b- and LPS-associated H3K4me3 and H3K27me3
marks across the genome. We have demonstrated that the
upregulation of certain costimulatory molecules, cytokines/
chemokines as well as their receptors is related to the increased
H3K4me3 marks, whereas the downregulation of other genes is
associated with H3K27me3 modifications in TGF-b- and LPS-
conditioned DCs. However, the expression of the LPS- or TGF-b
induced genes is not always dependent on the increased level
of H3K4me3, and the expression of the downregulated genes is
not always modified by H3K27me3. Particularly, the expression
of costimulatory molecules, cytokines and chemokines and
their receptors after exposure to LPS is often independent
of the modifications of the H3K4me3 marks, but it is often
associated with the alteration of the H3K4me3 and/or H3K27me3
marks.
a
80
70
60
50
40
30
20
10
0
Upreg.
H3K4me3 H3K4me3+H3K27me3
No H3K4me3+H3K27me3H3K27me3
Downreg.
Unaltered
Percentage of
modification patterns
c
VAV3
PAK1
PRKCA
GNB5
GAPDH
1.2
0.9
0.6
0.3
0
R.E
Ctr.
LPS
STAT3
Ctr.
LPS
chr17:37,708,289-37,820,493
Ctr.
LPS
JAK3
chr19:17,780,320-17,836,112
STAT5A
Ctr.
LPS
chr17:37,676,017-37,734,557
VAV3
Ctr.
LPS
chr1:107,805,929-
108,508,443
b
6
Ctr.
LPS
5
4
3
2
R.E
1
0
STAT3
STAT5A
STAT5B
STAT4
PTPN1
JAK3
JAK1
GAPDH
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.
Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells
Y Huang et al
316
Genes and Immunity (2012) 311 -- 320 & 2012 Macmillan Publishers Limited
Page 6
DCs are extremely versatile Ag-presenting cells involved in the
initiation of both innate and adaptive immunity and also in the
maintenance of self-tolerance. These diverse and almost contra-
dictory functions are dependent on the plasticity of these cells to
allow them to undergo a complete genetic reprogramming in
response to different stimuli.
1,2
Our results showed that methyl-
transferase inhibitors not only affect the expression of certain
critical genes, but more importantly also alter DC functions,
suggesting that epigenetic modifications are involved in the
plasticity of human moDCs in different environments. Indeed,
upregulation of certain costimulatory molecules, cytokines/
chemokines as well as their receptors is related to the increased
H3K4me3 marks, and the downregulation of other genes is
associated with H3K27me3 modifications in the TGF-b- and LPS-
conditioned DCs.
The basic structural unit of eukaryotic chromatin is the
nucleosome, which consists of 146 bp of DNA wrapped around
an octamer of four core histones (H2A, H2B, H3 and H4). Histone
modifications within promoter regions have an important function
in the regulation of gene expression. The majority of modifications
occur at the N-terminal ends of the core histones. Studies from
several model systems have determined that the methylation of
lysines 4, 36 and 79 of histone H3 are typically associated with
active gene expression,
28 -- 30
whereas repressive genes are
associated with H3K27me3 modification. Indeed, many active
genes have increased H3K4me3 marks, especially on gene
promoters, whereas some repressive genes are modified by
H3K27me3 marks in TGF-b- or LPS-conditioned moDCs. This is in
agreement with other studies using embryonic stem cells
31,32
and
moDCs,
11
or CD4
þ
and CD8
þ
T cells,
10,14,33
which have reported
that H3K4me3 marks are the most characteristic modification in
promoter regions. In addition, H3K4me3 and H3K27me3 marks are
usually enriched at the active or inactive chromatin regions,
respectively.
34,35
Our data have also shown that both H3K4me3
and H3K27me3 marks were often colocalized to certain genomic
regions.
The decreased K3K4me3 marks and the increased expression of
genes with or without the H3K27me3 mark in the LPS-conditioned
DCs suggest that ex vivo differentiation in the presence of Toll-like
receptor ligands induces a global shift toward less active
chromatin in the presence of Toll-like receptor ligands. Although
our results have shown that the expression of some costimulatory
molecules, cytokines and chemokines and their receptors is
associated with increased H3K4me3 marks in TGF-b-conditioned
moDCs, the upregulation of some of these molecules was not
associated with increased H3K4me3 marks in LPS-conditioned
moDCs. Others have also found that transcriptionally active genes
can have reduced H3K4me3 marks during DC differentiation.
11
Some genes in human moDCs have been shown to lose their
H3K4me3 mark independent of their gene expression,
11
which
suggests that both the H3K4m3 and H3K27me3 marks are
unstable and are prone to either a loss of differentiation state or
a
b
CD14
CD40
CD80
CD86
GAPDH
Ctr.
LPS
TGF-β
Ctr.
LPS
TGF-β
HLA-DR
CD40
chr20:44,168,304-44,204,392 chr3:120,701,091-120,785,910
chr5:139,983,503-139,998,799 chr6:14,185,420-14,271,696
CD80
– LPS
– TGF β
– Ctr.
– LPS
– TGF β
– Ctr.
5
4
2
3
1
0
5
4
2
3
1
0
2
1
0
5
6
7
8
4
2
3
1
0
5
6
4
2
3
1
0
Isotypic ctr.
LPS
Ctr.
TGF-β
CD14
CD83
Counts
CD83
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 ml
1
) or TGF-b (10 ng ml
1
) 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--PCR are 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.
Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells
Y Huang et al
317
Genes and Immunity (2012) 311 -- 320& 2012 Macmillan Publishers Limited
Page 7
a gain of other permissive mark such as H3K9me3.
36,37
Addition-
ally, an alteration of the H3K4me3 and/or H3K27me3 positions is
often found in the upregulated and downregulated genes from
LPS-conditioned moDCs. H3K4me3 at certain chromatin loci may
also prevent aberrant gene expression or modulate transcriptional
response.
38
However, additional genome-wide localization and
functional studies will provide important new insights into the role
of other epigenetic marks, chromatin-modifying enzymes and
chromatin remodelers.
MATERIALS AND METHODS
Preparation and isolation of human moDCs
Immature DCs were prepared from the buffy coats of healthy donor
samples obtained from the blood bank of Tianjin, according to our
previously described method.
39
Briefly, peripheral blood mononuclear cells
were separated by Ficoll-Paque gradient centrifugation, and CD14
þ
cells
were purified using antibody-coated microbeads and magnetic separation.
Selected CD14
þ
cells were cultured for 5 days at a concentration of
2 10
6
ml
1
in the presence of GM-CSF (500 U ml
1
) and IL-4 (500 U ml
1
;
R&D Systems, Minneapolis, MN, USA) for the generation of immature
moDCs. Immature moDCs were matured by exposure to LPS (100 ng
ml
1
ml
1
, InvivoGen, San Diego, CA, USA) or tolerized by exposure to TGF-
b (10 ng ml
1
R&D Systems) for 48 h, unless otherwise stated.
Flow cytometric analyses
For surface staining, cells were collected in ice-cold phosphate-buffered
saline and incubated with the indicated phycoerythrin- or fluorescein
isothiocyanate-labeled antibodies. For each analysis, isotype-matched
control monoclonal antibodies were used as negative controls. The
phenotypes of human moDCs were analyzed using phycoerythrin- or
fluorescein isothiocyanate-labeled anti-CD14 (61D3), anti-CD83 (HB15e),
anti-CD11c (3.9), anti-CD86 (IT2.2), anti-CD80 (2D10.4), anti-CD40 (HB14)
and anti-CD11b (ICRF44), which were purchased from Pharmingen (San
Diego, CA, USA).
Transfection
For small interfering RNAs (siRNAs) and negative control oligonucleotides,
cells were transfected with the indicated oligos (100 nM) using Entranster-R
(Engreen Biosystem, Beijing, China) according to the manufacturer’s
instruction. The transfection rate of the human moDCs was as high as
85%. For validation of the siRNA(s), human moDCs were cultured in six-well
plate. The following day cells were transfected in fetal calf serum-free
medium. One day post transfection, the cells were treated with TRIzol
(Invitrogen, San Diego, CA, USA, 15596-026) for RNA extraction.
RNA isolation and quantitative real-time PCR
Total RNA was extracted using the TRIzol reagent (Invitrogen, 15596-026),
and reverse transcription was carried out with Superscript III (Invitrogen)
abcd
CCL3
CCL4
CCR7
– Ctr.
– LPS
– TGF β
– Ctr.
– LPS
– TGF β
– Ctr.
– LPS
– TGF β
chr17:31,431,539-31,449,795
chr21:31,447,621-31,464,837
chr17:35,941,314-35,997,482
IL1A
IL12A
IFNGR2
TNFSF10
– Ctr.
– Ctr.
– LPS
– TGF β
– LPS
– TGF β
– Ctr.
– LPS
– TGF β
– Ctr.
– LPS
– TGF β
chr2: 113,239,928-113,267,476
chr3:161,175,691-161,210,131
chr21:33,672,834-33,755,934
chr3:173,693,695-173,736,426
R.E
CCL3
CCL4
CCR7
GAPDH
1
0
8
10
2
4
6
0
8
2
4
6
0
0
2
2
4
6
IL1A
1
0
2
3
4
1
0
2
3
4
5
6
IL12A
IFNGR2
TNFSF10
R.E
GAPDH
1
1
0
0
0
2
2
2
3
4
4
6
Ctr.
LPS
TGF-β
Ctr.
LPS
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 cytokine receptor 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 chemokine receptor 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.
Epigenetic modification in TGF-b- and LPS-conditioned dendritic cells
Y Huang et al
318
Genes and Immunity (2012) 311 -- 320 & 2012 Macmillan Publishers Limited
Page 8
according to the manufacturer’s protocol. To measure gene expression,
quantitative real-time PCR was performed using the Quantitect SYBR PCR
kit with a specific set of primers according to the manufacture’s
recommendations (Qiagen, Valencia, CA, USA). Amplification of glycer-
aldehyde-3-phosphate dehydrogenase mRNA for gene was performed for
each experimental sample as an endogenous control for gene expression.
Fold changes were calculated using the DDC
t
method according to the
manufacturer’s protocol (Applied Biosystems, Foster City, CA, USA). All
reactions were run in triplicate, and the primer sequences are listed in
Supplementary Table S5.
siRNA experiments
siRNAs for SETD6, CBX5, EZH1, EHMT2 and control oligos were purchased
from Guangzhou Ribobio, Guangzhou, China, and were used to transfect
cells with Entranster-R according to the manufacturer’s recommendations
(Engreen Biosystem). EHMT2 siRNA of the sequence 5-CATGAC
TGCGTGCTGTTATTC-3
40
was synthesized by Guangzhou Ribobio. CBX5,
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
(EZH1) and 5-CGCCAATCTAGAATACTCT-3 (SETD6). The nonsilencing oligo
control is an irrelevant siRNA with random nucleotides (5-ACTATCTAAGTT
ACTACCC-3). To determine the effect of EHMT2, SETD6, EZH1 and CBX5
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
listed in Supplementary Table S5.
Chip, Chip-seq and bioinformatics
Chip and Chip-seq, followed by high-throughput sequencing were
performed by the Research and Cooperation Division of BGI-Shenzhen,
according to previously described methods.
41
In brief, 3 10
7
immature
moDCs (control), TGF-b-conditioned DCs or LPS-conditioned DCs were
digested with micrococcal nuclease (2 U) for 10 min in a 37 1C water bath
to generate native chromatin template consisting primarily of mono-
nucleosomes. The native chromatin templates were then incubated with
anti-histone H3K4me3 (Kit cat. no. GAH-8208, SABiosciences, Frederick, MD,
USA), H3K27me3 (Kit cat. no., GAH-9205, SABiosciences) or rabbit control IgG
(SABiosciences). Antibody-bound DNA fragments were extracted, and the
DNA fragments were modified to construct a sequencing library after PCR
amplification according to the manufacturer’s protocol (Illumina/Solexa,
San Diego, CA, USA). The specificity of the immunoprecipitation was
confirmed by analyzing known genes by quantitative real-time PCR,
including glyceraldehyde-3-phosphate dehydrogenase for transcriptionally
active euchromatin, human MYOD1 for transcriptionally inactive euchro-
matin and human SAT2 for heterochromatin. Primers used were from
SABiosciences. DNA fragments of B200 bp (mononucleosomal
DNA þ adaptor) were selectively recovered from 2% agarose gels for
further cluster generation and sequencing with the Solexa/Illumina 1G
Genome Analyzer.
The image processing and base calling were performed using the
Illumina pipeline. The adapter sequences were detected using the Perl
program (http://www.perl.com/pub/2006/08/03/sequence-diagrams.html).
Any reads with greater than 10% Ns (indicating ambiguous residues) or
over 50% bases with a quality score less than 20 were trimmed. The
remaining sequences were then aligned to the hg18 human reference
genome using SOAP2.21 (http://soap.genomics.org.cn/soapaligner.html),
and alignments with p2 errors were retained. The locations of repetitive
sequences in the hg18 genome (RepeatMasker) were obtained using the
UCSC (the University of California, Santa Cruz) Genome Brower (http://
hgdownload.cse.ucsc.edu/goldenPath/hg18/database/). The overlap of the
Chip-seq reads with these repeats was obtained by intersecting the
coordinates of the RepeatMasker data with the coordinates of read
alignments. The UCSC annotation data were used for general reference
(http://hgdownload.cse.ucsc.edu/goldenPath/hg18/database/). To obtain a
set of gene regions, the overlap between reads and each gene region is
used, and the reads number for each region is counted. For the peak
calling process, the intensities of control rabbit immunoglobulin G (IgG),
H3K4me3 and H3K27me3 were first normalized to the random genomic
background and then further normalized by subtracting control IgG
intensities from the corresponding modification intensities. The enriched
regions were identified using the model-based analysis of Chip-seq, a P-
value cutoff of 1 10
5
and a high-confidence fold enrichment. This
method was based on the Poisson distribution. The advantage of this
model was that it could capture both the mean and the variance of the
distribution. Background biases were controlled by themselves. Subse-
quently, the peak calling results were estimated according to the peak
number and the peak length distribution. The peak distributions of gene
regions were analyzed as read distributions. Finally, peak-related genes
were defined by the positional relationship of the islands and genes.
Several figures show the read distribution of a gene region of the gene of
interest. Difference analysis on peak-related genes was used to identify
genes sharing partial sequences that could provide information on the
regulatory strength for each gene.
GO analysis
GO analysis was performed using the online GO tool (http://go.princeton.
edu/). The complete set of all RefSeq genes was used as a background.
Complete GO analysis results are provided (Supplementary Table S2).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
This research was supported by the NSFC grants 91029736, 30771967 and 30872315;
Ministry of Science and Technology grants (863 program, 2008AA02Z129); the
National Key Basic Research and Development Program of China (973 Program ,
2007CB914803); and the National Key Scientific Program (2011CB964902).
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Supplementary Information accompanies the paper on Genes and Immunity website (http://www.nature.com/gene)
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    • "The expression of miRNAs in moDCs was also modulated by methyltransferase inhibitor AdOx, histone-lysine N-methyltransferases EZH1 and EHMT2, and chromobox homolog 5 (CBX5) (data not shown). In addition, by silencing MLL, RBBP5, EZH1, CBX5 and EHMT2, cytokine production and expression of costimulatory molecules was suppressed (Fig. S1 and [27]). All of these data suggest that H3K4me3 and H3K27me3 modifications are involved in miRNAs regulation in moDCs. "
    [Show abstract] [Hide abstract] ABSTRACT: Epigenetic modification plays a critical role in regulating gene expression. To understand how epigenetic modification alters miRNA expression in monocyte-derived dendritic cells (moDCs) in different environments, we analyzed the connections between H3K4me3 and H3K27me3 modification and the expression of miRNAs in LPS- and TGF-β-conditioned moDCs. In moDCs, H3K4me3 modification was strongly associated with the expression of activating miRNAs, whereas H3K27me3 was related to repressive miRNAs. The regulation of miRNA expression by H3K4me3 and H3K27me3 was further confirmed by silencing or inhibiting methyltransferases or methylation-associated factors in LPS- and TGF-β-conditioned moDCs. siRNAs targeting H3K4me3-associated mixed lineage leukemia (MLL) and retinoblastoma binding protein 5 (RBBP5) reduced H3K4me3 enrichment and downregulated miRNA expression; conversely, silencing H3K27me3-associated enhancer of zeste homolog 2 (EZH2) and embryonic ectoderm development (EED) genes upregulated the DC-associated miRNAs. However, LPS-mediated miRNAs were often associated with H3K4me3 redistribution from the transcription start site (TSS) to the miRNA-coding region. Silencing LPS-associated NF-κB p65 and CBP/p300 not only inhibited H3K4m3 redistribution but also reduced miRNA expression. LPS-upregulated RBBP4 and RBBP7, which are involved in chromatin remodeling, also affected the redistribution of H3K4me3 and reduced the expression of miRNAs. In LPS- and TGF-β-conditioned moDCs, miRNAs may be modulated not only by H3K4m3 and H3K27me3 modification but also by redistribution of H3K4me3 around the transcriptional start site of miRNAs. Thus, H3K4me3 and H3K27me3 epigenetic modification may play an important role in regulating DC differentiation and function in the presence of tumor or inflammatory environments.
    Full-text · Article · Apr 2014 · PLoS ONE
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    • "We found that, in both organs and at both ages, the histone mark H3K4me3 was associated with high levels of gene expression, and H3K27me3 was associated with low levels of expression. These associations are similar to associations identified in other biological systems, including human embryonic stem cells differentiation [22]–[24], erythroid cells development [8], thyroid hormone (T3)-dependent metamorphosis [25], and dendritic cells activation [26]. Our findings suggest that this general concept – that H3K4me3 is associated with active transcription and H3K27me3 is associated with gene silencing – applies also to postnatal tissues in vivo. "
    [Show abstract] [Hide abstract] ABSTRACT: During early postnatal life, extensive changes in gene expression occur concomitantly in multiple major organs, indicating the existence of a common core developmental genetic program. This program includes hundreds of growth-promoting genes that are downregulated with age in liver, kidney, lung, and heart, and there is evidence that this component of the program drives the widespread decline in cell proliferation that occurs in juvenile life, as organs approach adult sizes. To investigate epigenetic changes that might orchestrate this program, we performed chromatin immunoprecipitation-promoter tiling array to assess temporal changes in histone H3K4 and H3K27 trimethylation (me3) at promoter regions throughout the genome in kidney and lung, comparing 1- to 4-wk-old mice. We found extensive genome-wide shifts in H3K4me3 and H3K27me3 occurring with age in both kidney and lung. The number of genes with concordant changes in the two organs was far greater than expected by chance. Temporal changes in H3K4me3 showed a strong, positive association with changes in gene expression, assessed by microarray, whereas changes in H3K27me3 showed a negative association. Gene ontology analysis indicated that shifts in specific histone methylation marks were associated with specific developmental functions. Of particular interest, genes with decreases in H3K4me3 with age in both organs were strongly implicated in cell cycle and cell proliferation functions. Taken together, the findings suggest that the common core developmental program of gene expression which occurs in multiple organs during juvenile life is associated with a common core developmental program of histone methylation. In particular, declining H3K4me3 is strongly associated with gene downregulation and occurs in the promoter regions of many growth-regulating genes, suggesting that this change in histone methylation may contribute to the component of the genetic program that drives juvenile body growth deceleration.
    Full-text · Article · Jan 2014 · PLoS ONE
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    [Show abstract] [Hide abstract] ABSTRACT: At mucosal surfaces, phagocytes such as macrophages coexist with microbial communities; highly controlled regulation of these interactions is essential for immune homeostasis. Pattern-recognition receptors (PRRs) are critical in recognizing and responding to microbial products, and they are subject to negative regulation through various mechanisms, including downregulation of PRR-activating components or induction of inhibitors. Insights into these regulatory mechanisms have been gained through human genetic disease-association studies, in vivo mouse studies utilizing disease models or targeted gene perturbations, and in vitro and ex vivo human cellular studies examining phagocytic cell functions. Although mouse models provide an important approach to study macrophage regulation, human and mouse macrophages exhibit differences, which must be considered when extrapolating mouse findings to human physiology. This review discusses inhibitory regulation of PRR-induced macrophage functions and the consequences of dysregulation of these functions and highlights mechanisms that have a role in intestinal macrophages and in human macrophage studies.Mucosal Immunology advance online publication 23 January 2013; doi:10.1038/mi.2012.139.
    Preview · Article · Jan 2013 · Mucosal Immunology
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