Epigenetic Control of T Helper Cell

f o c u s o n c D 4 + T- c e l l D i v e r s i T y
REVIEWS
Epigenetic control of Thelpercell
differentiation
Christopher B. Wilson, Emily Rowell and Masayuki Sekimata
Abstract | Naive CD4+ T cells give rise to T-helper-cell subsets with functions that are tailored
to their respective roles in host defence. The specification of T-helper-cell subsets is
controlled by networks of lineage-specifying transcription factors, which bind to regulatory
elements in genes that encode cytokines and other transcription factors. The nuclear
context in which these transcription factors act is affected by epigenetic processes, which
allow programmes of gene expression to be inherited by progeny cells that at the same time
retain the potential for change in response to altered environmental signals. In this Review,
we describe these epigenetic processes and discuss how they collaborate to govern the fate
and function of T helper cells.
Epigenetic process
A process that affects gene
expression without altering
the sequences of bases in the
DNA. Epigenetic changes are
potentially heritable in the
absence of the factors that
initially induced them, and
some propose that this term
be restricted to those that are
demonstrably heritable
(although the broader
definition is used here).
In mammals, epigenetic
processes that affect gene
transcription include
methylation of cytosines
in CpG dinucleotides, posttranslational histone
modifications and changes
to higher-order chromatin
structure.
Chromatin
DNA and the proteins with
which it is associated in the
nucleus.
Department of Immunology,
University of Washington,
Seattle, Washington 98195,
USA.
Correspondence to C.B.W.
e-mail:
cbwilson@u.washington.edu
doi:10.1038/nri2487
Published online
19 January 2009
CD4+ T helper (TH)-cell subsets were first described

by Mossman and Coffman1, who found that mouse
T-cell clones segregated into two subsets they named
TH1 and TH2 cells based on their mutually exclusive
production of interferon- (IFN) or interleukin-4
(IL-4), IL-5 and IL-13, respectively. The relevance of
this distinction was subsequently shown by Locksley
and colleagues2, who found that mice which mounted
a TH1-cell-dominant response resolved infection with
Leishmania major, whereas mice that mounted a TH2cell-dominant response did not. Work by many groups
has since established the importance of TH1 cells in host
defence to intracellular pathogens and of TH2 cells in the
protection against helminth infections. More recently,
cells that produce IL-17A were shown to represent a distinct TH-cell lineage the TH17-cell subset that in
addition to IL-17A produces IL-17F, IL-21, IL-22 and,
in humans, IL-26. TH17 cells contribute to host defence
against extracellular bacteria and fungi, particularly at
mucosal sites, although the full extent of their contribution to host defence is still being investigated3. In contrast
to these protective functions of TH cells, inappropriate
TH2-cell responses give rise to allergic diseases, whereas
autoimmune diseases result from inappropriate TH1- and
TH17-cell responses36.
For the development of distinct TH-cell lineages, the
instructions that are received by naive CD4+ T cells during
initial encounters with antigen-presenting cells (APCs)
must be converted into cell-intrinsic changes. These
changes are passed on to progeny cells through multiple
cell divisions and occur over time and in various environments where effector and memory T cells carry out their
functions. These instructions are converted by responding T cells into changes in the abundance, interactions
and locations of transcription factors, which in turn
lead to changes in gene expression. The resulting information could in principle be propagated from one T-cell
generation to the next solely through self-reinforcing
transcription factor networks. In practice, more precise control of gene expression is achieved through
epigenetic processes7, which facilitate heritable and stable programmes of gene expression, while preserving
the potential for these programmes to be modified in
response to environmental changes.
In this Review, we describe the epigenetic processes
that help to regulate TH1-, TH2- and TH17-cell lineage
fate and function by affecting gene transcription. In
recent years, technological advances have accelerated
the pace of discovery in this field. As a result, progressively more comprehensive and higher resolution
maps of gene regulatory elements and their cell-typespecific epigenetic marks, including DNA methylation, histone modifications and three-dimensional
chromatin structure, are being derived from stem
cells and other cell types, including T cells. These
findings and their contribution to T H-cell differentiation and function are also discussed in this Review.
Epigenetic processes that influence mRNA splicing,
stability and translation in the immune system, such as
microRNAs, have been reviewed recently810 and are
therefore not discussed here. Follicular TH cells11 and
other proposed TH-cell subsets that might represent
distinct lineages but are not yet firmly established are
also not discussed in this Review.
NATuRE REvIEWs | Immunology
voLuME 9 | FEbRuARy 2009 | 91

2009 Macmillan Publishers Limited. All rights reserved
revieWs
Notch
A transmembrane receptor
that is involved in the pathway
for direct cellcell signalling
through its association with a
transmembrane ligand of the
Delta or Jagged family on a
neighbouring cell. The large
intracellular domain of Notch
is cleaved and travels to the
nucleus to become a direct
co-activator of the transcription
factor recombinationsignal-binding protein for
immunoglobulin- J region
(RBPJ).
Nucleosome
The basic structural subunit
of chromatin, which consists of
~156 base pairs of DNA
wrapped around an octamer
of histones.
Regulating lineage choice

Transcription factor networks. Nuclear factor of activated T cells (NFAT) and other transcription factors that
are activated in naive CD4+ T cells in response to signals
through the T-cell receptor (TCR) and co-stimulatory
molecules induce the production of IL-2, which leads to
IL-2-induced activation of signal transducer and activator of transcription 5 (sTAT5) and entry into the cell
cycle. Thereafter, TH-cell-lineage choice is determined,
for the most part, by the cytokine milieu and the network
of transcription factors that are induced downstream of
cytokine signalling pathways (FIG. 1).
TH1-cell development is initiated by sTAT1, which
is activated in response to IFN and IL-27 that are produced by natural killer (NK) cells and APCs, respectively. Together with TCR-induced transcription factors,
sTAT1 induces the transcription factor T-bet, a key (if
not master) regulator of the TH1-cell lineage, which
in turn induces the production of IFN, the activation of the transcription factors H2.0-like homeobox
(HLX) and runt-related transcription factor 3 (RuNX3),
and opposes the inhibitory effects of GATA-binding
protein 3 (GATA3; see later) on TH1-cell differentiation1217. The expression of IL-12 receptor-2 (IL-12R2)
is also induced in this process; IL-12R2 pairs with
IL-12R1 to form the IL-12 receptor, thereby allowing
APC-derived IL-12 to activate sTAT4. sTAT4, T-bet,
HLX and RuNX3 then bind to and activate Ifng, which
reinforces TH1-cell commitment through the activation
of sTAT1 in a positive-feedback loop. Concomitantly,
T-bet and RuNX3 bind to and repress Il4 to inhibit
TH2-cell differentiation.
TH2-cell differentiation is initiated by the activation of
sTAT6 by IL-4, which, together with TCR-induced transcription factors, binds to and activates Gata3 (REF. 5).
Alternatively, Notch signalling can induce Gata3 in a
sTAT6-independent manner 18,19. GATA3 induces the
transcription factor MAF, which helps to activate Il4, and
together GATA3 and sTAT6 activate the transcription of
Il4, Il5 and Il13. TH2-lineage commitment is stabilized by
the autoactivation of GATA3, the autocrine and paracrine activation of sTAT6 by IL-4, and the sTAT6- and
GATA3-dependent antagonism of IFN expression and
TH1-cell differentiation5.
The transcription factors that are involved in TH1and TH2-cell differentiation are not required for TH17cell differentiation and can antagonize this process20,21.
Transforming growth factor- (TGF) inhibits TH1and TH2-cell differentiation, and promotes regulatory
T (TReg)-cell and TH17-cell lineage commitment by
inducing the expression of the transcription factors forkhead box P3 (FoXP3) and retinoic-acid-receptor-related
orphan receptor-t (RoRt; also known as RoRC),
which are required for TReg- and TH17-cell lineage commitment, respectively 2023. In the absence of IL-6, FoXP3
inhibits RoRt and therefore blocks TH17-cell development, whereas in the presence of IL-6, sTAT3 is activated, which inhibits the expression of FoXP3 and its
interactions with RoRt. This results in an increase in
the expression of RoRt (as well as of RoR, which has
an ancillary role in TH17-cell induction)24, and TH17-cell
differentiation is favoured. once TH17-cell differentiation has been initiated, the T cells produce IL-21, which
activates sTAT3 and induces the expression of IL-23R.
This allows APC-derived IL-23 to activate sTAT3, which
dampens IL-10 production, drives IL-22 production
and stabilizes TH17-cell differentiation and commitment 21,2528. The transcription factor aryl-hydrocarbon
receptor (AHR) also influences the differentiation of TReg
and TH17 cells29,30. Although initial studies suggested otherwise, the cytokine and transcription factor networks
involved in human and mouse TH17-cell differentiation
seem to be similar 21,31,32.
Epigenetic processes. The ability of transcription factors
to bind to DNA at regulatory regions on genes is affected
by their concentration, post-translational modifications
and subcellular localization, as well as by the state of the
chromatin and underlying DNA. The epigenetic context
in which transcription factors function is provided by
the position and compaction of nucleosomes, the interactions of nucleosomes with the DNA, post-translational
histone modifications and the methylation status of the
DNA6,3336. Therefore, unlike genetic information, epigenetic information is not encoded by changes in the
sequence of the DNA but by differential methylation of
the DNA and modifications of chromatin, which affect
whether, when and to what level specific genes are
expressed in a given cell. because the DNA sequence
remains unchanged, epigenetic modifications and the
information that they encode can be heritable but plastic
the potential to erase modifications and inscribe new
ones is retained.
In mammals, DNA can be methylated on cytosines
in CpG dinucleotides. At present, DNA methylation is
the only proven mechanism by which epigenetic information is faithfully propagated from one cell generation
to the next. Heritability is achieved through copying
of the pattern of methylated cytosines from parental
to progeny DNA strands by DNA methyltransferase 1
(DNMT1) 37. DNA methylation at gene promoters,
and possibly at distal regulatory elements, can directly
inhibit transcription38,39; by contrast, DNA methylation
within transcribed sequences seems to have little effect
on transcription, although demethylation within the
transcribed sequences of Il4 and Ifng correlates with high
expression levels of these cytokines in TH2 and TH1 cells,
respectively 13,40. Methylated DNA directly represses gene
expression by blocking the binding of some transcription
factors to the promoter and other regulatory elements,
thereby inhibiting the recruitment of RNA polymerase II,
and indirectly by providing docking sites for methyl-CpGbinding domain proteins (MbDs)41,42. Although plants
contain enzymes that actively demethylate DNA, such
enzymes have not been identified in mammals, in which
the only established mechanism for DNA demethylation
is passive that is, the result of a failure to copy methylation patterns from the parental strand onto the daughter
strand during DNA replication.
Nucleosome composition and histone modifications
are diverse and dynamic. variant forms of histones H2
and H3 and the linker histone H1 can be incorporated
92 | FEbRuARy 2009 | voLuME 9
www.nature.com/reviews/immunol
f o c u s o n c D 4 + T- c e l l D irveevri se iW
Ty
s
Inititation; proliferation
Differentiation; stabilization
Epigenetic remodelling
a TH1-cell development
IL-2
IL-2R
MHC
class II
TCR
Ifng
STAT5 GATA3
Naive
CD4+ T cell IFNGR
CD80/CD86
T-bet
Heritability
HLX
STAT1
Ifng
STAT1
T-bet
STAT1
IFN
RUNX3
IFN
STAT4
CD28
DC
IL-12
IL-12R
IL-27R
NK cell
IL-12
IL-27
b TH2-cell development
Naive
CD4+ T cell
IL-4
GATA3
IL-4R
MAF
STAT6
IL-4
IL-2
Il4
Il5
Il13
STAT6
Il4
STAT5
Heritability
GATA3
RBPJ
DC
Notch
signalling
c TH17-cell development
Naive
CD4+ T cell
IL-2
STAT5
Il21
Il17
STAT3
RORt
IL-21
IL-21R
RORt
Il17
Il21
Il22
Heritability
STAT3
IL-6R
DC
STAT3
TGFR
IL-6
TGF
IL-23R
Epithelial
cell
IL-23
Figure 1 | Cytokines and transcription factor networks regulate Thelpercell differentiation. Activation-induced
division of naive CD4+ T cells provides a context that allows their differentiation into one of several T helper (TH)-cell lineages.
a | TH1-cell differentiation is initiated by the activation of signal transducer and activator of transcription 1 (STAT1) by
Nature
Reviewsof
| Immunology
interferon- (IFN)- and/or interleukin-27 (IL-27), both of which upregulate T-bet. T-bet induces the
expression
H2.0-like
homeobox (HLX), and together they collaborate with transcription factors that are activated following T-cell receptor (TCR)
signalling to activate Ifng transcription and to antagonize GATA-binding protein 3 (GATA3). These events result in the
expression of IL-12 receptor (IL-12R), which binds IL-12 that is secreted by antigen-presenting cells, such as dendritic cells
(DCs), and thereby mediates the activation of STAT4. T-bet also induces the activation of runt-related transcription factor 3
(RUNX3), and along with STAT4, these transcription factors drive TH1-cell differentiation. IL-2-induced STAT5 activation has a
permissive role in the initial stages of TH1-cell differentiation. b | T-cell differentiation to the TH2-cell lineage involves the
induction of GATA3. GATA3 activation is mediated by STAT6 and IL-4, which is activated by STAT5 and STAT6 and/or
recombination-signal-binding protein for immunoglobulin- J region (RBPJ). This establishes a positive-feedback loop that
drives TH2-cell differentiation and the expression of IL-4, IL-5 and IL-13. IL-2-induced STAT5 activation has a permissive role in
the initial stages of TH2-cell differentiation. c | TH17-cell differentiation is initiated by the activation of STAT3, which induces
the expression of IL-21 and cooperates with transforming growth factor- (TGF) signalling to induce the expression of
retinoic-acid-receptor-related orphan receptor-t (RORt), IL-17 and IL-23R, and STAT3 activation is attenuated by
IL-2-induced STAT5. IL-21 and IL-23 drive the production of IL-17 and IL-22 and TH17-cell differentiation. These pathways
also induce epigenetic remodelling at genes that encode lineage-restricted transcription factors and cytokines to facilitate
heritable patterns of gene expression and lineage commitment. IFNGR, IFN receptor; NK, natural killer.

revieWs
Histone code
Post-translational
modifications of histone tails
that involve characteristic
clusters of modifications,
including acetylation,
phosphorylation,
ubiquitylation, methylation,
sumoylation and ADPribosylation that combine
to create an epigenetic
mechanism for the regulation
of gene expression.
Heterochromatin
Highly compacted chromatin
that is transcriptionally
inactive. Includes structural
regions of the chromosome
that lack genes (for example,
centromeres; known as
constitutive heterochromatin)
as well as genes that are
silenced in a given cell type
(known as facultative
heterochromatin).
into nucleosomes or removed, and histones can be

modified by the enzyme-catalysed addition or removal
of acetyl, methyl, phosphate, ubiquitin, sumoyl or ADPribose groups34. Acetylation alters histone charge, which
reduces the interactions between histones and DNA and
enhances nucleosome mobility 36. In addition, incorporation of histone H1 condenses chromatin43, and posttranslational modifications of histones create or remove
binding sites for regulatory proteins that facilitate or
impede transcription.
The diversity of potential modifications led to the
histone code and later nucleosome code hypothesis,
which posits that histone modifications combine to create a code that is recognized by specific regulatory proteins or complexes that are involved in transcription36.
Genome-wide studies identified many different combinations of histone modifications that could allow for
fine-tuning of transcription, but a few sets of core modifications seem to be sufficient to characterize genes and
regulatory elements as active and accessible, inactive but
poised, or silent44 (FIG. 2). specifically, the promoters and
enhancers of active or recently transcribed genes can be
characterized by the presence of histones H3 and H4 that

are acetylated at various residues, by H3 lysine 4 (H3K4)
that is modified with one (monomethylated H3K4), two
(dimethylated H3K4) or three (trimethylated H3K4)
methyl groups (BOX 1; TABlE 1) and by an alternative
form of histone H2A, known as H2A.Z. These histone
modifications are absent from silenced genes, whereas
dimethylated and trimethylated H3K27, or dimethylated
and trimethylated H3K9 are present. Dimethylated and
trimethylated H3K27 are typically found throughout the
condensed, facultative heterochromatin of silenced tissuespecific loci, and dimethylated and trimethylated H3K9
are found throughout constitutive heterochromatin or in
discrete sites within active genes, where they may inhibit
inappropriate transcription33. Promoters of genes that
are poised to be either activated or silenced generally
do not have any of these histone modifications or have a
bivalent modification pattern that is, they have both
permissive (H3K4 dimethylation and trimethylation)
and repressive (H3K27 trimethylation) histone modifications33,4446. locus control regions (LCRs) and distal
enhancers (BOX 1) at an inactive locus that are bivalent or
Compact heterochromatin
DNAseI hypersensitive site

Nucleosome
Euchromatin
Silent
H4
Nucleosome
Transcriptional
regulatory factors
Inactive but poised
Active and accessible
Methylation
H3K4 and
H3K27 me3
H3K27 me2
or me3
H3
DNA
Faculative heterochromatin
Bivalent
Acetylation
H3K9 Ac,
H3K14 Ac
and H3K18 Ac Promoter
H3K4 me3
or me2
H4K5 Ac and
H4K8 Ac
OR
H3K9 me2
or me3
H3K4 me1,
me2 or me3
H3K18 Ac
Constitutive heterochromatin
or focal silencing in euchromatin
Null
Enhancer
Figure 2 | Chromatin and chromatin modifications. a | DNA is compacted through its association with histone proteins
to form chromatin, the basic unit of which is the nucleosome. Nucleosomes consist of two copies of histones H2A, H2B, H3
Nature Reviews | Immunology
and H4. Each nucleosome is encircled by approximately ~156 base pairs of DNA and interconnected by linker DNA.
Wrapping of DNA around nucleosomes generates a 10 nm fibre that is typical of euchromatin, which can be further
compacted into a 30 nm fibre that is typical of heterochromatin. b | Binding of transcriptional regulatory and chromatin
remodelling proteins displaces nucleosomes, and the sites at which nucleosomes have been displaced can be detected as
DNaseI hypersensitive sites. c | Representative acetylation (Ac) and methylation modifications to the tails of histones H3
(red line) and H4 (blue line) at promoters and enhancers of genes that are silent, inactive but poised, or active and
accessible. For simplicity, modifications are shown on only one of the two histone tails but may be present alone or in
combination on one or both. H3K4 modified with one, two and/or three (me1, me2 and/or me3) methyl groups is
permissive. H3K9 and H3K27 modified with two and/or three (me2 and/or me3) methyl groups are repressive.
Ty
s
only have methylated H3K4 may be pioneer elements
that are amenable to subsequent activation or silencing, at
which time histone modifications and DNA methylation
at these elements are altered accordingly 41,42.
The epigenetic state of regulatory elements is also
altered when transcription factors and RNA polymerase II
that are bound recruit chromatin-remodelling complexes
that displace or alter the conformation of nucleosomes.
such regions are accessible to cutting by DNaseI, allowing the regulatory elements to be detected as DNaseI
hypersensitive sites. Therefore, functional regulatory
elements in a given cell can be experimentally identified
by approaches that detect DNaseI hypersensitive sites,
post-translational histone modifications and differential
DNA methylation35.
Locus control region

A DNA sequence that is
defined by its ability (in
transgenic assays) to permit
high-level, tissue-specific
expression of a linked
promoter at all integration
sites.
Epigenetics and TH-cell subsets

Cause or consequence. Transcription factors may
directly transactivate (induce) or repress gene expression
and may also affect transcription by recruiting proteins
that modify the epigenetic state of genes to which they
bind or by blocking the recruitment of these proteins.
Epigenetic modifications may persist in the absence of
the transcription factors that initially induced them,
but do such modifications merely mark past events and
report transcriptional competence or do they also contribute to differences in competence? In other words, in
addition to the transcription factor networks described
above, which initiate and help to sustain TH-cell subset
differentiation, do epigenetic modifications also help to
maintain these differentiated states?
Early biochemical evidence that the epigenetic state
of a gene has a causal role in transcriptional competence came from studies in which treatment with 5-azacytidine, an inhibitor of DNA methylation, resulted in the
production of IL-2 (REF. 47) and IFN (REF. 48) by T-cell
lines that could not previously produce these cytokines.
subsequently, studies showed that treatment of CD4+

T cells with inhibitors of histone deacetylases (HDACs)
enhanced the expression of both IFN and TH2-type
cytokines49,50. Genetic evidence supported these findings: conditional ablation of DNMT1 or MbD2 which
recruit HDACs and chromatin-remodelling complexes
to methylated DNA and induce a repressive chromatin
state led to increased expression of IFN and TH2-type
cytokines and an inability of TH1 or TH2 cells to silence
the expression of cytokine genes that are associated
with the opposing lineage5154. These studies also suggested
that DNMT1 and MbD2 mediate gene silencing mostly,
if not wholly, by directly affecting the loci that encode
IFN and TH2-type cytokines. Thus, DNA methylation,
MbDs and histone deacetylation dampen the expression
of both TH1- and TH2-type cytokines, and help to restrict
cytokine expression to the appropriate lineage.
A prediction of these findings is that lineage-specific
transcription factors regulate TH1- and TH2-cell fate
in part through epigenetic processes. Consistent with
this possibility, chromatin-remodelling complexes that
contain brahma-related gene 1 (bRG1; also known as
sMARCA4) displace nucleosomes and remodel chromatin at the Ifng promoter in mouse TH1 cells in a sTAT4dependent manner; these complexes are required for
high Ifng expression55. In addition, mice that are haploinsufficient for the H3K4 methyltransferase MLL have
a defect in the maintenance but not the induction of
Gata3, Il4, Il5 and Il13 expression, whereas TH1-cell differentiation is not affected56. Conversely, mice that lack
expression of MEL18, a polycomb repressor complex 1
(PRC1) protein that binds to trimethylated H3K27, have
impaired GATA3 expression and TH2-cell differentiation, although the cause of these defects is not known57.
These findings, together with a large body of correlative data, suggest that epigenetic mechanisms are key
determinants of TH-cell differentiation and function.
Chromatinremodelling
complex
Box 1 | Promoters, other regulatory elements and their interactions
An enzymatic complex that

carries out the remodelling
of DNAnucleosomal
architecture and determines
transcriptional activity. The
SWISNF (switching-defective
sucrose non-fermenting)
ATPases are an example of
complexes that remodel
chromatin.
Promoters are located immediately upstream of the point where transcription starts. Mammalian promoters are
typically several hundred base pairs in length and contain binding sites for transcription factors, which together with
the position and orientation of their binding sites helps to determine the cells and the conditions under which that
gene will be expressed, as well as the magnitude of its expression. Transcription factors that are bound to the DNA
create a platform to recruit the basal transcriptional machinery, which is common to all cells and consists of RNA
polymerases and their associated co-factors. Protein coding (and microRNA) genes recruit RNA polymerase IIcontaining complexes and associated co-factors that can displace or remodel nucleosomes, can phosphorylate RNA
polymerase II and can add or remove acetyl, methyl, phosphate, ubiquitin, sumoyl or ADP-ribose groups to histones
and transcription factors. The content and post-translational modifications of these RNA polymerase II-containing
complexes are dynamic and determine whether binding leads to transcript initiation and elongation.
Promoters are sufficient for proper gene regulation in prokaryotes, but do so in concert with other regulatory elements
to achieve proper gene regulation in mammalian cells. These regulatory elements may be located just upstream of the
promoter, within introns or up to hundreds of kilobases upstream or downstream of the gene (or genes) they regulate, or
even on other chromosomes. Enhancers augment transcription either actively or by promoting permissive epigenetic
modifications, whereas silencers repress gene expression by promoting repressive epigenetic modifications. Unlike the
function of promoters, which depends on their proximity to the transcription start site and their 5 to 3 orientation,
the function of enhancers and silencers is independent of their orientation and location. Insulators create boundaries
between genes or genetic loci, thereby allowing genes in these loci to be regulated independently of neighbouring
regulatory elements, gene loci and chromosomal domains or territories. Locus control regions typically contain both
enhancer and insulator activity and have been functionally defined by their ability to permit copy number-dependent
expression of transgenes. Matrix attachment regions are found at the base of chromatin loops, which they tether to
structures such as the nuclear matrix.
DNaseI hypersensitive site

A region of chromatin (usually
less than a few hundred base
pairs) that is ~100 times more
sensitive to digestion by
DNaseI than bulk chromatin
and corresponds to regions
in which nucleosomes are
depleted. Regulatory elements,
including enhancers, promoters
and insulators, which are
functional in the cells being
assayed, typically map to
these sites.

revieWs
Table 1 | Histone lysine modifications
modification Histone lysines
modified
Transferases that Deacetylases or

add modification* demethylases that
remove modification
Function of histone modification
Effect on
transcription
Acetylation
H3K9, H3K14 and H3K18
KAT2A (GCN5) and

KAT2B (PCAF)
HDAC1 and HDAC2

(in SIN3A, NURD and
CoREST complexes)
Binds or recruits bromodomaincontaining proteins (such as TAF1)
Permissive
H4K5, H4K8, H4K12

and H4K16
KAT5 (TIP60)
HDAC1 and HDAC2

(in SIN3A, NURD and
CoREST complexes)
Permissive
H2aK5, H2bK12, H2bK15, KAT3A (CBP) and

H3K14, H3K18, H4K5
KAT3B (p300)
and H4K8
HDAC1 and HDAC2

(in SIN3A, NURD and
CoREST complexes)
Permissive
H3K4 monomethylation
KMT7 (SET7 or
SET9)
KDM1 (LSD1) and

KDM5B (JARID1B)
Binds or recruits the WDR5

component of the H3K4
methyltransferase MLL complex
Permissive
H3K4 dimethylation
and trimethylation
KMT2AKMT2E
(MLL1MLL5)
KDM1 (LSD1) and

KDM5AKDM5D
(JARID1AJARID1D)
Binds or recruits chromodomain,

PHD- and Tudor-domain-containing
proteins (such as TFIID, CHD1, BPTF
and WDR5)
Permissive
Binds or recruits NURD complexes

and DNMT3aDNMT3l
Repressive
Repressive
Methylation
Unmodified H3K4
H3K27 dimethylation
and trimethylation
KMT6 (EZH2)
KDM6A and KDM6B

(UTX and JMJD3)
Binds or recruits PRC1 complex and

DNA methyltransferases
H3K9 dimethylation
and trimethylation
KMT1B (SUV39H)
and KMT1C (G9a)
KDM1 (LSD1) and

KDM4AKDM4D
(JMJD2AJMJD2D)
Binds or recruits CBX5 (HP1) and DNA Repressive

methyltransferases
See REFS 34,36,44,141143. *Alternative name is included in brackets. Also note that histone acetyltransferases are now referred to as KATs in recognition of
their broader substrate specificity. BPTF, bromodomain and PHD finger transcription factor; CBP, CREB-binding protein; CBX5, chromobox homologue 5; CHD1,
chromodomain-helicase-DNA-binding protein 1; CoREST, corepressor of REST; DNMT3a, DNA methyltransferase 3a; EZH2, enhancer of zeste homologue 2; GCN5,
general control non-depressible 5; HDAC, histone deacetylase; HP1, heterochromatin protein 1; JARID1, jumonji AT-rich interactive domain 1; JMJD, jumonjidomain-containing protein histone demethylase; KAT, lysine acetyltransferase; KDM, lysine demethylase; KMT, lysine methyltransferase; LSD1, lysine-specific
histone demethylase 1; NURD, nucleosome remodelling and histone deacetylation; PCAF, p300/CBP-associated factor; PHD, plant homeodomain; PRC1, polycomb
repressive complex 1; SUV39H, suppressor of variegation 39 homologue; TAF1, TFIID subunit 1; TFIID, transcription factor IID; TIP60, Tat interactive protein, 60 kDa;
UTX, ubiquitously transcribed X chromosome tetratricopeptide repeat; WDR5, WD-repeat-containing domain 5.
However, it is worth noting that whether the specific

epigenetic modifications described below are causally
related to differences in T-cell function or whether they
merely report these differences has for the most part not
been directly determined.
The TH2-cytokine locus and lineage commitment. Just
over a decade ago, alterations to the chromatin structure
of the Il4 and Il13 genes were shown to occur as naive
T cells differentiated into TH2 or TH1 cells58,59. These
findings stimulated interest in the contribution of epigenetic processes to TH-cell differentiation and in the TH2cytokine locus as a model system by which to address the
coordinated regulation of clustered, functionally related
genes. The accrual of additional information was greatly
accelerated by the availability of complete genomic
sequences for humans, mice and other species, and
by improvements in the techniques used for assessing
epigenetic modifications.
The mouse TH2-cytokine locus contains Il4, Il5, Il13
and the constitutively expressed Rad50 gene (FIG. 3), and
is flanked by Irf1 and Kif3A. The gene composition of
this locus, as well as the linear relationship of the genes
within the locus, is conserved in the genomes of mammals, suggesting that these relationships are functionally important. Consistent with this possibility, the genes
encoding TH2-type cytokines are regulated through their
promoters and also by several additional regulatory elements, the locations of which have been experimentally
determined by the detection of DNaseI hypersensitive
sites, histone modifications and differential DNA methylation, as well as through computational identification
of conserved non-coding sequences (CNs). various
approaches have been used to test the function of the
elements that have been identified by these methods and
their interactions with transcription factors35.
In the mouse TH2-cytokine locus, the transcription
of Il4 is enhanced by regulatory elements that map to
DNaseI hypersensitive site I (HsI) and HsII in the second
intron of Il4, to DNaseI hypersensitive site vA (HsvA) and
Hsv located 3 of Il4, to DNaseI hypersensitive site s1
(Hss1) and Hss2 located between Il13 and Il4, and to the
TH2-cytokine LCR, which encompasses Rad50 hypersensitive site 4 (RHs4), RHs5, RHs6 and RHs7 (FIG. 3;
see figure legend for the convention used to name hypersensitive sites in this locus and note that Hsv maps to
CNs2, and Hss1 and Hss2 map to CNs1)5. The expression of Il13 is augmented by regulatory elements at CNs1,
the TH2-cytokine LCR and Hs1, which maps to the
CG-rich element (CGRE) upstream of the Il13 promoter.
Many of these enhancers and each of the TH2-cytokine
promoters are direct targets of NFAT, other TCR-induced
transcription factors and TH2-cytokine-promoting
transcription factors. For example, sTAT6 binds to the
Ty
s
RHS2 RHS3
RHS6 RHS7
Hss3
HSIV
CTCF
T-bet
RUNX3
Hss3
HSIV
TH1 cell
CTCF
RHS2 RHS3
RHS6
?
Naive T cell
CTCF
RHS2 RHS3
pro
TH2 cell
TH2 LCR
pro
GATA3
CTCF
Il5
Hss3
HSVA
HSII
Hss2
HS2
HS1 HS3 Hss1 HSI HSIII HSIV HSV
RH
S4
RH
S5
RH
S6
RH
S7
RHS1
CTCF
enh
STAT6 GATA3
STAT6
Rad50
pro
GATA3
STAT6
enh
CTCF
CGRE Il13
enh
pro
STAT6 STAT5
MAF
Il4
CNS1
sil
enh
GATA3
STAT6
CNS2
140 kilobases
Permissive histone
modifications
Bivalent histone
modifications
Repressive histone
modifications
CNS
Figure 3 | The T helper 2 cytokine locus in mouse T cells. Naive CD4+ T cells have DNaseI hypersensitive sites at
Nature
| Immunology
hypersensitive site s3 (Hss3), HSIV, the 5end of the Rad50 gene at Rad50 hypersensitive site 2 (RHS2)
andReviews
RHS3, and
perhaps
at RHS6 in the locus control region (LCR) of the T helper 2 (TH2)-cytokine locus. In addition, the locus has low levels of
permissive histone modifications (light green blocks) at LCR and HSV and substantial levels at RHS2, whereas HSIV has a
bivalent modification pattern with both permissive dimethylated and/or trimethylated H3K4 and repressive trimethylated
H3K27 (yellow blocks) and low levels of repressive modifications at Hss3 (light blue blocks). In TH2 cells, DNaseI hypersensitive
sites and substantial levels of permissive histone modifications (such as acetylated H3, acetylated H4 and dimethylated and/
or trimethylated H3K4; dark green blocks) are acquired at the promoters and enhancers of Il4 (interleukin-4), Il13 and Il5, and
repressive trimethylated H3K27 (blue blocks) is absent throughout the locus. DNA methylation is progressively reduced at
these cytokine genes and their enhancers over time in TH2 cells (not shown). Conversely, in TH1 cells repressive trimethylated
H3K27 spreads to encompass Il4, Il13 and a region that extends from conserved non-coding sequence 1 (CNS1) to CNS2 (to
our knowledge, data for Rad50 and Il5 are not yet available). The function of specific elements, such as promoters (pro),
enhancers (enh), silencers (sil) and the LCR are indicated. The binding sites for the lineage-restricted transcription factors
MAF, CCCTC-binding factor (CTCF), GATA-binding protein 3 (GATA3), runt-related transcription factor 3 (RUNX3), signal
transducer and activator of transcription 5 (STAT5), STAT6 and T-bet are also shown. Gene locations, intergenic CNS (that is,
intergenic regions where there is 70% sequence conservation between humans and mice that extends 100 base pairs as
identified at the VISTA web site) and the size of the region depicted are shown. DNaseI hypersensitive sites in the
TH2-cytokine locus are referred by their commonly used names, in which sites in or near Il4 are indicated as HS followed by a
roman numeral (for example, HSIV), sites between Il4 and Il13 are indicated as Hss followed by a number (for example, Hss1)
sites in or upstream of Il13 are indicated as HS followed by a number (for example, HS1), and sites in or near Rad50 are
indicated as RHS followed by a number (for example, RHS6). T-cell subset-specific patterns of DNA methylation in this locus
are described in the text.
Il4 and Il13 promoters and to HsvA, as well as to RHs6

and RHs7; GATA3 binds to the Il5 and Il13 promoters,
to the first intron of Il4, HsvA and RHs7, and to the Il13
Hs1CGRE region; and recombination-signal-binding
protein for immunoglobulin- J region (RbPJ), which
is the DNA-binding component of the Notch pathway,
binds to Hsv60,61. Perhaps surprisingly, none of these
elements, including the TH2-cytokine LCR, appears to
affect the expression of Il5, to which GATA3 binds5.
Naive CD4+ T cells express low levels of GATA3 and
T-bet and produce small but detectable amounts of Il4,
Il5 and Il13 mRNA before cell division when activated by
TCR ligation62,63. In these cells, the TH2-cytokine locus
is characterized by a paucity of DNaseI hypersensitive
sites and histone modifications, although some are still
present (FIG. 3). specifically, DNaseI hypersensitive sites
are found in the promoter of the constitutively expressed
Rad50 gene, at HsIv (the Il4 silencer) and Hss3 (one

of three DNaseI hypersensitive sites that are located
between Il4 and Il13, which is of unknown function), as
well as at RHs6 in the TH2-cytokine LCR (although this
was observed in one study 64 but not another 65). HsIv
also has bivalent histone modifications (that is, both
permissive H3K4 dimethylation and H3 acetylation,
and repressive H3K27 trimethylation are detectable)66,67,
whereas Hss3 is marked solely by repressive trimethylated H3K27. The 3 Il4 enhancer, Il4 CNs2 and the TH2cytokine LCR are marked weakly by dimethylated H3K4
and/or H3 acetylation64,67,68.
The expression of TH2-type cytokines is probably
restrained in naive T cells by the high degree of CpG
methylation (~90%) at their promoters, CNs1, CNs2
and the TH2-cytokine LCR40,51,53,64,69. The Il4 promoter
is less methylated (~60%) than the other promoters

revieWs
and lacks CpG motifs in the 250 base pairs that are
proximal to the start site53, which might facilitate the
early low-level expression of IL-4. Therefore, in naive
CD4 + T cells, modest amounts of permissive and
repressive epigenetic marks are focally targeted to a
subset of the known regulatory elements in the TH2cytokine locus. This bivalent epigenetic state may help
poise this locus, permitting TCR-induced transcription
factors to bind and induce early, low-level expression
of TH2-type cytokines and providing the potential for this
locus to convert to a fully permissive state during TH2cell differentiation or to a silenced state during TH1- and
TH17-cell differentiation5,6.
Permissive histone modifications are acquired in the
TH2-cytokine locus in the first 2448 hours following
activation of naive CD4+ T cells under both TH1- and
TH2-cell-polarizing conditions through the actions of
NFAT and other transcription factors that are induced
by TCR signalling 70,71. These transcription factors also
induce the expression of IL-2, which activates sTAT5,
which in turn binds to and induces chromatin remodelling at intron 2 of Il4 to promote TH2-cell differentiation72. However, the continued presence and progressive
increase in these permissive histone modifications, and
the subsequent acquisition of TH2-specific DNaseI
hypersensitive sites and DNA demethylation at the
TH2-cytokine locus are unique to TH2 cells40,6468,70,7274.
The active locus of effector TH2 cells contains the
hypersensitive sites that are found in naive T cells as
well as new sites at the Il4, Il5 and Il13 promoters, at
each of the known enhancers and at the LCR, although
the cells must be restimulated for HsvA to be detected.
Permissive H3 and H4 acetylation and H3K4 dimethylation are acquired or become more prominent at these
elements, and repressive H3K27 trimethylation is
lost throughout the locus. These changes are evident
in the first week of mouse TH2-cell differentiation in
culture. by this time, demethylation of the DNaseI
hypersensitive sites has commenced but it progresses
slowly by a passive replication-dependent process.
by contrast, RHs7 undergoes a more rapid and potentially active demethylation40,51,53,64,69,75. With the exception
of the demethylation of RHs5 and RHs6, these epigenetic changes are not found in the TH2-cytokine locus
of TH1 cells; instead, the locus is modified with repressive
H3K27 trimethylation66.
GATA3 is necessary and apparently sufficient to
induce most, if not all, of these TH2-cell-specific epigenetic modifications. It may do so through the direct
or indirect recruitment of histone acetyltransferases
(HATs) and histone H3K4 methyltransferases5,56, the displacement of MbD2 and associated HDAC-containing
complexes51, the displacement of DNMT1 and inhibition of DNA methylation53,69, and the recruitment of
chromatin-remodelling complexes to the mouse TH2cytokine locus76. However, the molecular details of the
mechanism by which GATA3 induces epigenetic modifications remain to be fully elucidated. In physiological
situations, sTAT6 and/or Notch signalling are activated
before GATA3 and help to induce GATA3 expression
by binding to, transactivating and recruiting HATs and
other chromatin modifiers to one or both of its promoters18,74,77. once induced, GATA3 binds to its promoter,
sustaining its own expression through direct activation and recruitment of the H3K4 methyltransferase
MLL56. sTAT6 also facilitates TH2-cell differentiation
by binding to multiple sites on the TH2-cytokine locus.
In addition to the sTAT6-dependent pathway, TH2-cell
differentiation can be induced independently of sTAT6,
partly through the conversion of RbPJ that is bound to
the Il4 CNs2 to a co-activator of Il4 in a process that
depends on Notch signalling 61. Therefore, although
TH2-cell differentiation can be initiated either through
sTAT6-dependent or Notch-dependent pathways, it is
stabilized by the autoactivation of GATA3 and by the
GATA3-mediated epigenetic modification of Gata3 and
the TH2-cytokine locus.
IFN and TH1-lineage commitment. IFNG is not clustered with other co-expressed cytokine genes. In all vertebrates except rodents, the nearest upstream neighbours
of IFNG are IL22 and IL26, which are mainly expressed
by TH17 cells21,31,78, with the housekeeping gene MDM1
located further upstream; the nearest downstream gene
to IFNG is ~500 kilobases away. In mice and rats, complex structural rearrangements are evident >70 kilobases
upstream of Ifng, and Il26 is absent; a few remnants of
sequences from Il26 remain, which is consistent with this
gene having been lost in rodents as a result of the structural rearrangements79 (FIG. 4). Despite these differences,
cell-type-specific patterns of IFNG expression are similar in rodents and humans, suggesting that the essential
regulatory elements and their relationships are conserved
and are proximal to these structural changes. Consistent
with this possibility, multiple regulatory elements and
CNs have been identified in a region that extends
6070 kilobases upstream and downstream of the mouse
Ifng locus. These include enhancers at CNs-34, CNs-22,
CNs-6, CNs+1820 and CNs+29, as well as a putative
insulator (BOX 1) at CNs+46, although enhancer function
has been confirmed in vivo for only CNs-22 (REF. 80) and
a previously described enhancer in intron 1 (REF. 81).
Recent genome-wide analyses of DNaseI hypersensitive
sites and histone modifications in human CD4+ T cells
suggest that a similar set of regulatory elements is present
in the human IFNG locus44,46,82 (FIG. 4).
Activated naive CD4+ T cells produce low levels
of IFN, indicating that the Ifng locus is in a poised
state. DNA at the mouse Ifng promoter and at CNs-34,
CNs-22, CNs+29 and CNs+46 is demethylated in naive
T cells, and CNs-34 and CNs-22 exhibit low levels of
permissive H3K4 dimethylation and H4 acetylation38,79,80.
Conversely, moderate levels of repressive H3K27 trimethylation are present between Ifng and CNs+1820, from
CNs+29 to CNs+46 and adjacent to CNs-22. Therefore,
overall the Ifng locus has bivalent histone modifications,
which makes it poised for either expression or silencing. TH1-cell differentiation in vitro and in response to
infection in vivo leads to a marked increase in H3K4
dimethylation, H3 and/or H4 acetylation, the acquisition
of DNaseI hypersensitive sites at regulatory elements
within the Ifng locus and a complete loss of repressive
Ty
s
TH2 cell
GATA3
BLIMP1
GATA3
STAT6
Naive T cell
CTCF
CTCF
HSII
HSI HSIII
TH1 cell
CTCF
Mouse
CNS 70
Human
CNS
T-bet
T-bet T-bet T-bet
enh
34
54
-63
-31
enh
-22
STAT5 CTCF
T-bet
RUNX3
T-bet
STAT4
T-bet
T-bet
STAT4
enh
22
enh
6
pro
Ifng
enh
+1820
enh
+29
ins
+46
-18
enh
-4
enh
pro
+22
enh
+40
enh
+80
ins
CTCF
+66
+119
HSI HSIII
HSII
Permissive histone
modifications
Bivalent histone
modifications
Repressive histone
modifications
CNS
Figure 4 | The Ifng locus in mouse naive, T helper 1 and T helper 2 cells and human CD4+ T cells. Naive mouse
CD4+ T cells have DNaseI hypersensitive sites at conserved non-coding sequence -34 (CNS-34)
and near CNS+46, low
Nature Reviews | Immunology
levels of permissive histone modifications (such as acetylated H3, acetylated H4 and dimethylated and/or
trimethylated H3K4; light green blocks) at Ifng (interferon-) CNS-22 and CNS-34 and repressive trimethylated
H3K27 (light blue blocks) at the 3 end of the locus. DNA at CNS-34, CNS-22, the Ifng promoter (pro), CNS+29 and
CNS+46 is demethylated. In T helper 1 (TH1) cells, hypersensitive site I (HSI), HSII and HSIII, DNaseI hypersensitive
sites at several CNS enhancers sites, and high levels of permissive histone modifications (dark green blocks) are
acquired, whereas trimethylated H3K27 is lost. In addition, DNA demethylation occurs at Ifng CNS-54, CNS-6 and
CNS+18 (not shown). The opposite occurs in TH2 cells, in which high levels of repressive trimethylated H3K27 (dark
blue blocks) spreads throughout the locus. The elements that have permissive chromatin modifications in mouse
TH1 cells are DNaseI hypersensitive in total human CD4+ T cells82 and in TH1 cells (C.B.W. and M.S., unpublished
observations). DNaseI hypersensitive sites in human CNS-4 and CNS+80 of the IFNG locus were detected only in
TH1 cells, and for this reason are denoted by red arrows rather than black arrows (which denote sites that are
also found in total human CD4+ T cells). The function of specific elements, such as promoters, enhancers (enh) and
insulators (ins) are indicated, as are the binding sites for the lineage-restricted transcription factors CCCTC-binding
factor (CTCF), GATA-binding protein 3 (GATA3), runt-related transcription factor 3 (RUNX3), signal transducer and
activator of transcription 4 (STAT4), STAT5, STAT6 and T-bet. Gene locations and intergenic CNS (that is, intergenic
regions where there is 70% sequence conservation between humans and mice that extends 100 base pairs as
identified at the VISTA web site) are shown at the bottom. T-cell subset-specific patterns of DNA methylation at this
locus are described in the text.
H3K27 trimethylation throughout the locus59,79,80,8385.

However, repressive H3K9 dimethylation is induced and
retained at specific sites of the Ifng locus in TH1 cells86,
in which it may help to prevent aberrant transcriptional
initiation33. by contrast, when naive T cells differentiate into TH2 cells, permissive histone modifications are
lost, repressive H3K27 trimethylation across the locus
and CpG methylation are increased and NFAT loses the
ability to bind to the Ifng promoter 38,79,83,86.
similarly to the TH2-cytokine locus, permissive histone modifications are initially acquired at the Ifng locus
under both TH1- and TH2-cell-inducing conditions, but
TH1-cell-specific changes are evident by 17 hours following stimulation70. sTAT1, sTAT4 and sTAT5 all contribute to these modifications, although they are not essential
for TH1-cell differentiation in vivo14. sTAT1 probably promotes the transcription of Ifng through T-bet expression
and has not been shown to bind to the endogenous Ifng

gene. As with TH2-cell differentiation, sTAT5 directly
promotes the transcription of Ifng by binding to CNs-6,
the Ifng promoter and CNs+1820, thereby facilitating
histone acetylation, chromatin remodelling and T-bet
binding to the Ifng promoter 87. sTAT4 binds to the Ifng
promoter and many other elements, including CNs-22,
leading to the induction of permissive epigenetic modifications and the activation of gene expression. In addition,
sTAT4 recruits bRG1-containing chromatin-remodelling
complexes to the Ifng promoter, induces the acquisition
of HsI and HsII and promotes permissive histone modifications at this site17,55.
Although sTAT4 supports Ifng expression and TH1cell differentiation synergistically with T-bet 88, the
expression of which is enhanced by sTAT4 (REF. 89),
T-bet can induce T H1-cell differentiation and the

revieWs
TH2 cell
TH1 cell
Naive T cell
TH17 cell
RORt STAT3
CNS1
CNS2
Pkhd1
STAT3
CNS3
Il17a
pro
enh pro
CNS4 CNS5 CNS6
CNS7 CNS8
Mcm3
Il17f
pro
200 kilobases
Permissive histone modifications
CNS
Figure 5 | The Il17aIl17f locus in mouse naive, T helper 1, T helper 2 and T helper 17 cells. Naive mouse CD4+
Nature
Reviews 1
| Immunology
T cells have weak permissive histone H3 acetylation (light green blocks) at conserved non-coding
sequence
(CNS1),
CNS5, CNS7 and CNS8, which is reduced in T helper 1 (TH1) and TH2 cells. By contrast, TH17 cells exhibit higher levels of
H3 acetylation (dark green blocks) at these regions, at other CNS in this region and at the Il17a (interleukin-17a) and Il17f
promoters101. Signal transducer and activator of transcription 3 (STAT3) binds to the Il17a and Il17f promoters in TH17
cells20,21,102 and retinoic-acid-receptor-related orphan receptor-t (RORt) has been shown to bind to CNS2, at least
when it is overexpressed24. DNaseI hypersensitive sites have not to our knowledge been mapped in this locus in TH17
cells. Gene locations, intergenic CNS (intergenic regions where there is 70% sequence conservation between humans
and mice that extends 100 base pairs as identified at VISTA web site) numbered as reported in REF. 101 and the size
of the region depicted are shown at the bottom. enh, enhancer; Mcm3, minichromosome maintenance deficient 3;
Pkhd1, polycystic kidney and hepatic disease 1; pro, promoter.
production of IFN in the absence of sTAT4 when the

expression of T-bet is forced71,90. However, under physiological conditions, sTAT4 and IL-12 are required for
TH1-cell differentiation, as their absence substantially
reduces or abolishes this process9193. T-bet directly
transactivates Ifng transcription and has many additional effects. More specifically, it binds to the Ifng promoter and to many enhancers80,94,95 (FIG. 4), it induces the
expression of HLX and RuNX3, it binds with these transcription factors to the Ifng promoter and with RuNX3
to the Il4 silencer 12,13,96 and it inhibits GATA3 expression and function16,17. T-bet binds to the Ifng promoter
even when its DNA is repressively methylated97, where
it displaces HDAC-containing complexes and possibly
recruits HATs98. Moreover, the Weinmann laboratory 99
recently showed that T-bet can directly recruit jumonjidomain-containing protein histone demethylase 3
(JMJD3) to remove repressive H3K27 trimethylation
and the histone methyltransferases sET7 (also known
as sET9 and KMT7) to induce H3K4 dimethylation,
thereby creating a permissive chromatin state. Together,
these functions probably explain how forced expression of T-bet can induce the expression of IFN even in
committed TH2 cells100.
TH17-cytokine loci and lineage commitment. Much less
is known about the regulatory mechanisms and epigenetic processes that control TH17-cell differentiation.
IL-17A and IL-17F are typically co-expressed by TH17
cells, and the genes that encode them are co-localized
in mammals, suggesting that they may be coordinately
regulated by shared regulatory elements (similarly to the
TH2-type cytokines). In mice, eight candidate regulatory
elements have been described in the Il17 locus based on

sequence conservation101 (FIG. 5). In these eight elements,
as well as the Il17a and Il17f promoters, permissive H3
acetylation is induced or increases solely or to a greater
degree in naive CD4+ T cells that are cultured under
TH17-cell-inducing conditions than in those that are
cultured under TH1- or TH2-cell-inducing conditions101.
Consistent with its crucial role in TH17-cell differentiation, sTAT3 binds to and induces H3 acetylation at
the Il17a and Il17f promoters20,21,102. The TH17-lineagespecific transcription factors RoRt and RoR do not
seem to bind to these promoters, but can bind to CNs2,
which is a RoR-dependent enhancer that is located just
upstream of Il17a24. The transcription factors that bind
to the other CNs and their function, if any, are unknown.
The genes that encode other TH17-type cytokines (IL-21,
IL-22 and IL-26) are located on different chromosomes
and, curiously, are close to genes that are expressed by
TH1 cells but not by TH17 cells. Il21 is located next to Il2,
and Il22 and Il26 (or its remnants in rodents) are located
next to Ifng. The juxtaposition of TH1-type cytokine
genes to TH17-type cytokine genes suggests that they
may be regulated in part by competition for or alternative
use of regulatory elements. However, these possibilities,
and the epigenetic processes by which this might be
achieved, have not been investigated.
Structuring regulatory relationships

The location of regulatory elements within the genome is
linear and fixed, whereas the actual relationships of these
elements to each other and to their cognate genes are nonlinear, mobile and dynamic. The three-dimensional structure of chromosomes changes during cell differentiation,
Ty
s
such that loops of chromatin containing relevant genes
can be extended outside the chromatin territory. such
chromatin looping can bring distal regulatory elements
in proximity to one another and to the promoters of their
target genes, thereby facilitating gene expression103105.
Chromatin looping allows the Il4, Il5 and Il13 promoters to come into proximity with each other in many
cell types, which results in a basal conformation that is
not specific to T cells. However, studies in mice have
shown that the LCR is located close to these promoters only in T cells, apparently helping to poise this locus
for subsequent activation106. sTAT6, the TH2 LCR and
probably GATA3 are required to establish this poised
conformation during T-cell development, but once it has
been established, the conformation is maintained and
is similar in naive, TH1 and TH2 cells. TH2-cell-specific
changes to this conformation are induced in response to
cell activation in TH2-cell-inducing conditions. This triggers the expression of special AT-rich sequence binding
protein 1 (sATb1; an architectural factor), which binds
to CNs1, CNs2 and other sites across the locus and promotes the formation of additional loops and more intimate interactions between regulatory elements and the
Il4, Il5 and Il13 promoters107. In the absence of sATb1
these changes are lost and TH2-type cytokine expression
is compromised. sATb1-induced chromatin looping
extends to include the flanking Kif3a gene, suggesting
that the regulatory domain of the TH2-cytokine locus
includes this gene.
Genome-wide studies suggest that CCCTC-binding
factor (CTCF) may also be involved in the three-dimensional organization of the TH2-cytokine locus. CTCF is a
self-interacting, insulator protein that can mediate chromatin looping to proximal elements within a locus and
can insulate a locus from surrounding chromatin, nearby
genes and regulatory elements105,108. CTCF co-localizes
with cohesins, which contributes to the CTCF-dependent
insulator function and perhaps to CTCF-mediated chromatin looping 109. In mouse and human T cells, CTCF
and cohesins have been shown to strongly bind to sites
that flank the TH2-cytokine locus as well as to RHs2 and
Hss3 within the locus46,109.
similar mechanisms may be involved at the Ifng and
Il17 loci, which are flanked by sites where CTCF and
cohesins are bound46,109. CTCF binds to Ifng in TH1 cells,
but not TH2 cells109, and may help to induce a TH1-specific
locus architecture (M.s. and C.b.W., unpublished observations), including TH1-specific chromatin looping that
brings Ifng CNs+1820 close to the Ifng promoter 110.
Interchromomal interactions between the Ifng and
the TH2-cytokine locus in naive T cells, which are lost
after TH1- and TH2-cell differentiation, have also been
described110, but the molecular basis and functional
importance of these interactions remain unclear.
Heritability, plasticity and diversity

The TH1- and TH2-cell paradigm arose from studies
of long-term T-cell clones1. Commitment to a single
lineage was later shown to be acquired after 4 cycles
of cell division under T-cell-lineage inducing conditions in vitro 62,63. Indeed, after 4 cell divisions TH1 or
TH2 cells did not express lineage-inappropriate cytokines

or lose robust expression of lineage-appropriate
cytokines when polarizing conditions were removed
or switched. Commitment was attributed to heritable
alterations to the cytokine genes themselves and/or to
the lineage-specifying transcription factors GATA3 and
T-bet. Recent studies have identified the mechanisms
that are involved in lineage commitment.
Silencing and remembering. silencing of Ifng in TH2
cells occurs at many levels. GATA3 and sTAT6 bind to
the Ifng promoter, and this is associated with binding
of PRC1 and the H3K27 methyltransferase EZH2 to
the Ifng locus, as well as with increased acquisition of
repressive H3K27 trimethylation, during TH2-cell differentiation86. As a result, IFN expression is repressed,
although another report suggests that DNA binding by
EZH2 occurs in both TH1 and TH2 cells and may have
other effects111. GATA3 also interacts with T-bet following its tyrosine phosphorylation by IL-2-inducible
T-cell kinase (ITK) and can inhibit T-bet function16,98;
this may account for the contribution of ITK to TH2-cell
differentiation112. Whether the expression of Tbx21 (the
gene encoding T-bet) is directly inhibited by GATA3 is
not known. GATA3 also binds HDACs98, which it may
recruit to Ifng, and inhibits the expression of IL-12R2
and sTAT4 (REF. 17). TH2-cell differentiation38 and/or
loss of sTAT4 signalling 113 result in the recruitment of
DNMT3a and an associated increase in CpG methylation at the Ifng promoter, perhaps as an indirect result of
reduced T-bet-mediated H3K4 methylation and binding of DNMT3aDNMT3l complexes to unmodified
H3K4 (REF. 114). Finally, the transcriptional repressor
b-lymphocyte-induced maturation protein 1 (bLIMP1)
represses IFN expression in TH2 cells, in which it is
highly expressed, possibly by binding to CNs-22 in
the Ifng locus (FIG. 4) and in many sites near or in the
Tbx21 gene115.
In addition to silencing Ifng, GATA3 is essential for
promoting the expression of TH2-type cytokines and
consequently for inducing and, at least partly, maintaining the TH2-cell response68,116,117. Deletion of GATA3 in
TH2 cells that had been generated in vivo in response
to infection with Nippostrongylus brasiliensis or in vitro
by culturing naive CD4+ T cells for 45 weeks in TH2inducing conditions resulted in reduced numbers of
IL-5- and IL-13-producing cells (but not IL-4-producing
cells) and in a reduction in the production of IL-5 and
IL-13, as well as a ~50% reduction in the amount of
IL-4 produced per cell117. similarly, silencing of Ifng
was markedly impaired when GATA3 was deleted at
the start of TH2-cell differentiation. Interestingly, however, when IFN expression was evaluated after Gata3
had been deleted from established TH2 cells, silencing
of Ifng was found to be less impaired117. These findings
suggest that GATA3 is not essential for but does help
to maintain the permissive epigenetic state of the TH2cytokine locus and the repressive state of the Ifng locus.
In addition, the data indicate that GATA3 has a modest
role in transactivating Il4 and a more important role
in transactivating Il5 and Il13 in committed TH2 cells.

revieWs
Maintenance of this permissive epigenetic state and the
expression of Gata3, Il4, Il5 and Il13 depends on the
H3K4 methyltransferase MLL, which binds to Gata3
and the TH2-cytokine locus in memory TH2 cells, but
not in naive T cells56.
The expression of T-bet seems to be crucial for the
induction of TH1-cell differentiation, but is less important for maintaining the differentiation state13,118. A
dominant-negative form of T-bet inhibits the expression of IFN and abolishes DNaseI hypersensitive sites
from the Ifng locus in developing TH1 cells, but not in
long-term TH1-cell lines. The stability of TH1 clones
correlates with marked DNA demethylation within the
Ifng locus, which is acquired more slowly than permissive chromatin modifications and may contribute to or
be a marker of heritable commitment. similarly to Il5
and Il13 in TH2 cells, the maintenance of the expression of IL-12R2 and HLX requires T-bet, as their
expression was lost when T-bet function was blocked.
Thus, the full programme of TH1- and TH2-cell-specific
gene expression cannot be sustained without continued expression of T-bet and GATA3, respectively. And
although the expression of Ifng in TH1 cells and Il4
in T H2 cells can be partly sustained through heritable epigenetic modifications in the absence of these
lineage-specifying transcription factors, in physiological
contexts the expression of Ifng and Il4 is collaboratively sustained by these transcription factors and by
epigenetic processes.
T-bet has also been shown to interact with GATA3.
Indeed, T-bet can bind to and inhibit GATA3 (REF. 16),
but this ITK-dependent interaction is not required for
silencing the TH2-cytokine locus112. T-bet also silences
the expression of GATA3 in TH1 cells17, but how this is
mediated and whether silencing of GATA3 and the TH2cytokine locus is heritable and sustained in the absence
of T-bet has not to our knowledge been determined. In
addition, T-bet is thought to be required for the silencing
of Il4 in TH1 cells mainly, although not completely, by
cooperatively binding with RuNX3 to the Il4 silencer,
which is located 3 of Il4 at HsIv12,96. RuNX proteins
induce heritable silencing in other contexts119, but the
mechanism by which they do so at the Il4 silencer is not
clear. The Il4 silencer is crucial for silencing the expression of IL-4 in TH1 cells120, possibly through a switch that
allows constitutively bound EZH2 to generate repressive
trimethylated H3K27 across this locus67.
TH17-cell differentiation is strongly inhibited by
IL-4, IL-27 and IFN, attenuated by IL-2, more readily achieved in T-bet-deficient cells and repressed by
forced expression of T-bet 20,21,121. The inhibitory effects
of IL-2 and IL-27 on the differentiation of this lineage
depend on sTAT5 and sTAT1, respectively. sTAT1,
sTAT5 and sTAT6 bind to the Il17a promoter, where
they may compete with sTAT3 for binding 121. Inhibition
by sTAT5 may also be mediated by the induction of
FoXP3, which binds to and inhibits RoRt 22. Whether
any of these sTAT-dependent effects contribute directly
to the silencing of IL-17 expression and the mechanisms
by which TH17-cell differentiation is stably repressed are
not known.
During the induction of TH17 cells, the expression

of TH1- and TH2-type cytokines is repressed by TGF,
which inhibits T-bet and GATA3 expression 122,123.
TGF-induced sMAD (mothers against decapentaplegic homologue) proteins can bind to the promoters
and probably repress Tbx21 and Ifng in TH17 cells124.
However, there is currently no evidence suggesting that
these effects are heritable in the absence of TGF, or
that TH17-lineage commitment can be epigenetically
maintained in the absence of sTAT3, RoRt and RoR.
There is some evidence in support of the contrary,
which shows that T-bet, IFN, RoRt and IL-17 are
often co-expressed by cells in vivo and that T-bet may
help to induce the expression of IL-23R and be induced
by IL-23 in TH17 cells125.
Forgetting or disregarding. A limitation of the TH1, TH2,
TH17 paradigm and most of the studies cited above is
that the systems that were used in each study were often
designed for the purpose of showing what is possible
rather than seeking to recapitulate what actually occurs
in vivo. Although the relevance of these subsets to host
defence, autoimmunity and allergy is clear, mutually
exclusive, canonical phenotypes and irreversible commitment are often violated in vivo. CD4+ T cells that express
IFN plus IL-17 and/or IL-22 are commonly generated
in vivo78,126131. And although TH17 and TH1 cells typically express either CC-chemokine receptor 6 (CCR6) or
CXC-chemokine receptor 3 (CXCR3), respectively, cells
that produce both TH1- and TH17-type cytokines typically
express both receptors31,78,128,132. Memory CD4+ T cells
that produce IFN in conjunction with IL-10, produce
IL-17 and IL-10 or produce IFN in conjunction with
IL-4 have been described in mice and humans133139.
The in vivo induction of cells that co-express cytokines
of mixed lineages and the lack of irreversible silencing of
lineage-inappropriate cytokines in some TH cells probably reflect the more diverse and resource-limited nature
of life in vivo as opposed to the homogeneous environments that are constructed in vitro140. Heterogeneity in
the mix and abundance of cytokines, Notch ligands,
peptideMHC complexes and co-stimulatory molecules,
asymmetric cell division and variation in the number of
times cells have divided provide ample opportunities for
diversification and retention of plasticity or heritability.
At present, little is known about the mechanisms
by which this diversity is achieved and whether such
diversity is itself heritable, reflecting a permanent
dtente that is achieved through the loss of cross-lineage
counter-regulatory mechanisms. The complex network
of epigenetic processes and feed-forward networks that
are evident in canonical TH-cell subsets indicate that lineage fidelity could be tweaked at many points to achieve
diversity and maintain a desirable degree of plasticity.
Concluding remarks
There is now a compelling body of evidence showing that
the state of chromatin and DNA methylation at lineagerestricted cytokine and transcription factor genes, as
well as their regulatory elements in TH cells, both reflects
and affects their functions in transcription. There is also
Ty
s
increasing clarity regarding the specific epigenetic modifications that are associated with active and accessible,
inactive but poised, and silenced loci, but the extent to
which these modifications work in a combinatorial
manner to encode and finely tune transcription is not
yet completely clear.
A future challenge will be to determine with greater
clarity how specific combinations of epigenetic modifications are established by networks of lineage-specifying
transcription factors, and whether, when and how they
can later be removed or selectively modified to achieve
or alter TH-lineage specification. such analyses should
help to unravel the basis for the non-canonical patterns of cytokines that are expressed by TH cells in vivo,
and whether these patterns reflect residual plasticity of
cells within the main TH lineages, stable subsets within
these lineages or a snapshot in time of cells in the process of shifting from one lineage to another. New, highresolution and comprehensive approaches provide tools
to probe for these mechanisms. Initial insights from such
studies show that total human resting CD4+ T cells exhibit
molecular signatures of open chromatin at the Ifng locus
(FIG. 4) that resemble those found in mouse and human
TH1 cells82. This indicates that resting memory T cells
maintain epigenetic signatures of previous events while
Mosmann, T. R., Cherwinski, H., Bond, M. W., Giedlin,

M. A. & Coffman, R. L. Two types of murine helper
T cell clone. I. Definition according to profiles of
lymphokine activities and secreted proteins.
J. Immunol. 136, 23482357 (1986).
2.
Heinzel, F. P., Sadick, M. D., Holaday, B. J.,
Coffman, R. L. & Locksley, R. M. Reciprocal
expression of interferon gamma or interleukin 4
during the resolution or progression of murine
leishmaniasis. Evidence for expansion of distinct
helper T cell subsets. J. Exp. Med. 169, 5972
(1989).
3.
Ouyang, W., Kolls, J. K. & Zheng, Y. The biological
functions of T helper 17 cell effector cytokines in
inflammation. Immunity 28, 454467 (2008).
4.
Murphy, K. M. & Reiner, S. L. The lineage decisions
of helper T cells. Nature Rev. Immunol. 2, 933944
(2002).
5.
Ansel, K. M., Djuretic, I., Tanasa, B. & Rao, A.
Regulation of Th2 differentiation and Il4 locus
accessibility. Annu. Rev. Immunol. 24, 607656
(2006).
6.
Lee, G. R., Kim, S. T., Spilianakis, C. G., Fields, P. E. &
Flavell, R. A. T helper cell differentiation: regulation by
cis elements and epigenetics. Immunity 24, 369379
(2006).
7.
Ledford, H. Language: disputed definitions. Nature
455, 10231028 (2008).
8.
Merkenschlager, M. & Wilson, C. B. RNAi and
chromatin in T cell development and function.
Curr. Opin. Immunol. 20, 131138 (2008).
9.
Anderson, P. Posttranscriptional control of cytokine
production. Nature Immunol. 9, 353359 (2008).
10. Baltimore, D., Boldin, M. P., OConnell, R. M., Rao,
D. S. & Taganov, K. D. MicroRNAs: new regulators of
immune cell development and function. Nature
Immunol. 9, 839845 (2008).
11. Nurieva, R. I. et al. Generation of T follicular helper
cells is mediated by interleukin21 but independent
of T helper 1, 2, or 17 cell lineages. Immunity 29,
138149 (2008).
12. Djuretic, I. M. et al. Transcription factors Tbet and
Runx3 cooperate to activate Ifng and silence Il4 in
T helper type 1 cells. Nature Immunol. 8, 145153
(2007).
This study shows that Tbet and RUNX3 together
bind to the Ifng promoter to activate IFN
expression and to the Il4 silencer in the
TH2cytokine locus to silence IL4 expression and,
consequently, TH2cell differentiation.
1.
retaining plasticity 136. Whether these epigenetic marks

are subject to further modulation in new environments
or are derived solely from a subset of committed cells
within a larger uncommitted population that lacks these
marks is unclear. Development of ways to apply comprehensive, systems-based approaches to specific cell subsets
as they arise in vivo will be important for our understanding of their inherent diversity and of processes by which
this diversity is achieved.
Note added in proof

Two reports provide additional insights into TH-cell lineage commitment and plasticity. Lee et al.144 showed that
even after 4 weeks in polarizing culture conditions, TH17
cells produce IL-17 and IFN or IFN alone if they are
not continually exposed to TGF. Wei et al.145 mapped
permissive H3K4 trimethylation and repressive H3K27
trimethylation in the genome of several CD4+ T-cell
subsets and showed that the promoters of the genes
encoding T-bet and GATA3 have both of these chromatin markers in TH17 and TReg cells. They also provided
evidence suggesting that this bivalent epigenetic state
may allow these cells to activate parts of the TH1 and TH2
transcriptional programme in response to alterations in
the types of cytokine to which they are exposed.
13. Mullen, A. C. et al. Hlx is induced by and genetically

interacts with Tbet to promote heritable TH1 gene
induction. Nature Immunol. 3, 652658 (2002).
14. Schoenborn, J. R. & Wilson, C. B. Regulation of
interferon during innate and adaptive immune
responses. Adv. Immunol. 96, 41101 (2007).
15. Townsend, M. J. et al. Tbet regulates the terminal
maturation and homeostasis of NK and V14i
NKT cells. Immunity 20, 477494 (2004).
16. Hwang, E. S., Szabo, S. J., Schwartzberg, P. L. &
Glimcher, L. H. T helper cell fate specified by kinase
mediated interaction of Tbet with GATA3. Science
307, 430433 (2005).
17. Usui, T. et al. Tbet regulates Th1 responses through
essential effects on GATA3 function rather than on
IFNG gene acetylation and transcription. J. Exp. Med.
203, 755766 (2006).
18. Amsen, D. et al. Direct regulation of Gata3
expression determines the T helper differentiation
potential of Notch. Immunity 27, 8999 (2007).
19. van Panhuys, N. et al. In vivo studies fail to reveal a
role for IL4 or STAT6 signaling in Th2 lymphocyte
differentiation. Proc. Natl Acad. Sci. USA 105,
1242312428 (2008).
20. Ivanov, I. I., Zhou, L. & Littman, D. R. Transcriptional
regulation of Th17 cell differentiation. Semin.
Immunol. 19, 409417 (2007).
21. McGeachy, M. J. & Cua, D. J. Th17 cell differentiation:
the long and winding road. Immunity 28, 445453
(2008).
22. Zhou, L. et al. TGFinduced Foxp3 inhibits TH17 cell
differentiation by antagonizing RORt function. Nature
453, 236240 (2008).
23. Dong, C. TH17 cells in development: an updated
view of their molecular identity and genetic
programming. Nature Rev. Immunol. 8, 337348
(2008).
24. Yang, X. O. et al. T helper 17 lineage differentiation is
programmed by orphan nuclear receptors ROR and
ROR. Immunity 28, 2939 (2008).
25. Korn, T. et al. IL21 initiates an alternative pathway to
induce proinflammatory TH17 cells. Nature 448,
484487 (2007).
26. Nurieva, R. et al. Essential autocrine regulation by
IL21 in the generation of inflammatory T cells.
Nature 448, 480483 (2007).
27. Onoda, T. et al. Human CD4+ central and effector
memory T cells produce IL21: effect on cytokine
driven proliferation of CD4+ T cell subsets.
Int. Immunol. 19, 11911199 (2007).
28. Zhou, L. et al. IL6 programs TH17 cell differentiation

by promoting sequential engagement of the IL21 and
IL23 pathways. Nature Immunol. 8, 967974
(2007).
29. Quintana, F. J. et al. Control of Treg and TH17 cell
differentiation by the aryl hydrocarbon receptor.
Nature 453, 6571 (2008).
30. Veldhoen, M. et al. The aryl hydrocarbon receptor
links TH17cellmediated autoimmunity to
environmental toxins. Nature 453, 106109
(2008).
31. Manel, N., Unutmaz, D. & Littman, D. R. The
differentiation of human TH17 cells requires
transforming growth factor and induction of the
nuclear receptor RORt. Nature Immunol. 9,
641649 (2008).
32. Volpe, E. et al. A critical function for transforming
growth factor, interleukin 23 and proinflammatory
cytokines in driving and modulating human TH17
responses. Nature Immunol. 9, 650657 (2008).
33. Berger, S. L. The complex language of chromatin
regulation during transcription. Nature 447,
407412 (2007).
34. Kouzarides, T. Chromatin modifications and their
function. Cell 128, 693705 (2007).
35. Rowell, E., Merkenschlager, M. & Wilson, C. B. Long
range regulation of cytokine gene expression.
Curr. Opin. Immunol. 20, 272280 (2008).
36. Ruthenburg, A. J., Li, H., Patel, D. J. & Allis, C. D.
Multivalent engagement of chromatin modifications
by linked binding modules. Nature Rev. Mol. Cell Biol.
8, 983994 (2007).
37. Wilson, C. B., Makar, K. W., Shnyreva, M. &
Fitzpatrick, D. R. DNA methylation and the expanding
epigenetics of T cell lineage commitment. Semin.
Immunol. 17, 105119 (2005).
38. Jones, B. & Chen, J. Inhibition of IFN transcription
by sitespecific methylation during T helper cell
development. EMBO J. 25, 24432452 (2006).
39. Schubeler, D. et al. Genomic targeting of methylated
DNA: influence of methylation on transcription,
replication, chromatin structure, and histone
acetylation. Mol. Cell. Biol. 20, 91039112 (2000).
40. Lee, D. U., Agarwal, S. & Rao, A. Th2 lineage
commitment and efficient IL4 production involves
extended demethylation of the IL-4 gene. Immunity
16, 649660 (2002).
41. Meissner, A. et al. Genomescale DNA methylation
maps of pluripotent and differentiated cells. Nature
454, 766770 (2008).

revieWs
42. Mohn, F. et al. Lineagespecific polycomb targets and
de novo DNA methylation define restriction and
potential of neuronal progenitors. Mol. Cell 30,
755766 (2008).
43. Trojer, P. & Reinberg, D. Facultative heterochromatin:
is there a distinctive molecular signature? Mol. Cell
28, 113 (2007).
44. Wang, Z. et al. Combinatorial patterns of histone
acetylations and methylations in the human genome.
Nature Genet. 40, 897903 (2008).
45. Azuara, V. et al. Chromatin signatures of pluripotent
cell lines. Nature Cell Biol. 8, 532538 (2006).
46. Barski, A. et al. Highresolution profiling of histone
methylations in the human genome. Cell 129,
823837 (2007).
References 44 and 46 provide a database of 39
different histone modifications, through which
common sets of modifications that are typical of
active and inactive promoters and enhancers can
be deduced.
47. Ballas, Z. I. The use of 5azacytidine to establish
constitutive interleukin 2producing clones of the EL4
thymoma. J. Immunol. 133, 79 (1984).
48. Young, H. A. et al. Differentiation of the T helper
phenotypes by analysis of the methylation state of the
IFNgamma gene. J. Immunol. 153, 36033610 (1994).
49. Bird, J. J. et al. Helper T cell differentiation is controlled
by the cell cycle. Immunity 9, 229237 (1998).
50. Valapour, M. et al. Histone deacetylation inhibits IL4
gene expression in T cells. J. Allergy Clin. Immunol.
109, 238245 (2002).
51. Hutchins, A. S. et al. Gene silencing quantitatively
controls the function of a developmental trans
activator. Mol. Cell 10, 8191 (2002).
This study shows that the MBD2 is present at the
Il4 gene in TH1 cells and can be displaced by
enforced expression of GATA3. MBD2deficient mice
showed modestly increased expression of IFN and
TH2type cytokines in the appropriate Tcell lineages,
but TH2 cells from these mice expressed IFN and
TH1 cells expressed Th2type cytokines.
52. Lee, P. P. et al. A critical role for Dnmt1 and DNA
methylation in T cell development, function, and
survival. Immunity 15, 763774 (2001).
53. Makar, K. W. et al. Active recruitment of DNA
methyltransferases regulates interleukin 4 in
thymocytes and T cells. Nature Immunol. 4,
11831190 (2003).
54. Makar, K. W. & Wilson, C. B. DNA methylation is a
nonredundant repressor of the Th2 effector program.
J. Immunol. 173, 44024406 (2004).
References 53 and 54 show that the DNA
methyltransferase DNMT1 is rapidly excluded from
the Il4 gene and CNS2 in TH2 cells. Conditional
ablation of DNMT1 leads to modestly increased
expression of IFN and TH2type cytokines in the
appropriate Tcell lineages, expression of IFN in
TH2 cells and marked expression of TH2type
cytokines in TH1 and CD8+ T cells.
55. Zhang, F. & Boothby, M. T helper type 1specific Brg1
recruitment and remodeling of nucleosomes
positioned at the IFN promoter are Stat4
dependent. J. Exp. Med. 203, 14931505 (2006).
56. Yamashita, M. et al. Crucial role of MLL for the
maintenance of memory T helper type 2 cell
responses. Immunity 24, 611622 (2006).
This study shows that the histone H3K4
methlytransferase MLL is recruited to Gata3 and
Il4 in TH2 cells and is required to maintain but not
to establish GATA3 and IL4 expression and
TH2cell differentiation.
57. Kimura, M. et al. Regulation of Th2 cell differentiation
by mel-18, a mammalian polycomb group gene.
Immunity 15, 275287 (2001).
58. Takemoto, N. et al. Th2specific DNase I
hypersensitivity sites in the murine IL13 and IL4
intergenic region. Int. Immunol. 10, 19811985
(1998).
59. Agarwal, S. & Rao, A. Modulation of chromatin
structure regulates cytokine gene expression during
T cell differentiation. Immunity 9, 765775 (1998).
60. Amsen, D. et al. Instruction of distinct CD4 T helper
cell fates by different Notch ligands on antigen
presenting cells. Cell 117, 515526 (2004).
61. Tanaka, S. et al. The interleukin4 enhancer CNS2 is
regulated by Notch signals and controls initial
expression in NKT cells and memorytype CD4 T cells.
Immunity 24, 689701 (2006).
62. Grogan, J. L. et al. Early transcription and silencing of
cytokine genes underlie polarization of T helper cell
subsets. Immunity 14, 205215 (2001).
63. Mullen, A. C. et al. Cell cycle controlling the silencing

and functioning of mammalian activators. Curr. Biol.
11, 16951699 (2001).
64. Fields, P. E., Lee, G. R., Kim, S. T., Bartsevich, V. V. &
Flavell, R. A. Th2specific chromatin remodeling and
enhancer activity in the Th2 cytokine locus control
region. Immunity 21, 865876 (2004).
This report shows that the 3 end of Rad50
contains clusters of DNaseI hypersensitive sites,
hyperacetylated histones and demethylated DNA
in TH2 cells, and that these sites have enhancer
activity in vivo and together are sufficient to
function as an LCR.
65. Lee, D. U. & Rao, A. Molecular analysis of a locus
control region in the T helper 2 cytokine gene cluster:
a target for STAT6 but not GATA3. Proc. Natl Acad.
Sci. USA 101, 1601016015 (2004).
This report identifies DNaseI hypersensitive sites in
the TH2cytokine LCR, although there are some
differences regarding the specificity of the sites for
distinct CD4+ Tcell subsets between this study and
reference 64.
66. Koyanagi, M. et al. EZH2 and histone 3 trimethyl
lysine 27 associated with Il4 and Il13 gene silencing in
TH1 cells. J. Biol. Chem. 280, 3147031477 (2005).
67. Baguet, A. & Bix, M. Chromatin landscape dynamics of
the Il4Il13 locus during T helper 1 and 2 development.
Proc. Natl Acad. Sci. USA 101, 1141011415 (2004).
68. Yamashita, M. et al. Essential role of GATA3 for the
maintenance of type 2 helper T (Th2) cytokine
production and chromatin remodeling at the Th2
cytokine gene loci. J. Biol. Chem. 279, 2698326990
(2004).
69. Tykocinski, L. O. et al. A critical control element for
interleukin4 memory expression in T helper
lymphocytes. J. Biol. Chem. 280, 2817728185
(2005).
70. Avni, O. et al. TH cell differentiation is accompanied by
dynamic changes in histone acetylation of cytokine
genes. Nature Immunol. 3, 643651 (2002).
71. Fields, P. E., Kim, S. T. & Flavell, R. A. Cutting Edge:
changes in histone acetylation at the IL-4 and IFN-
loci accompany Th1/Th2 differentiation. J. Immunol.
169, 647650 (2002).
72. Zhu, J., CoteSierra, J., Guo, L. & Paul, W. E. Stat5
activation plays a critical role in Th2 differentiation.
Immunity 19, 739748 (2003).
73. Lee, H. J. et al. GATA3 induces T helper cell type 2
(Th2) cytokine expression and chromatin remodeling
in committed Th1 cells. J. Exp. Med. 192, 105115
(2000).
74. Ouyang, W. et al. Stat6independent GATA3
autoactivation directs IL4independent Th2
development and commitment. Immunity 12,
2737 (2000).
75. Kim, S. T., Fields, P. E. & Flavell, R. A. Demethylation
of a specific hypersensitive site in the Th2 locus control
region. Proc. Natl Acad. Sci. USA 104, 1705217057
(2007).
76. Wurster, A. L. & Pazin, M. J. BRG1mediated
chromatin remodeling regulates differentiation and
gene expression of T helper cells. Mol. Cell. Biol. 28,
72747285 (2008).
77. Asnagli, H., Afkarian, M. & Murphy, K. M. Cutting
Edge: identification of an alternative GATA3
promoter directing tissuespecific gene expression in
mouse and human. J. Immunol. 168, 42684271
(2002).
78. AcostaRodriguez, E. V. et al. Surface phenotype and
antigenic specificity of human interleukin 17producing
T helper memory cells. Nature Immunol. 8, 639646
(2007).
79. Schoenborn, J. R. et al. Comprehensive epigenetic
profiling identifies multiple distal regulatory elements
directing transcription of the gene encoding
interferon. Nature Immunol. 8, 732742 (2007).
This study maps DNaseI hypersensitive sites,
histone modifications and DNA methylation on
the mouse Ifng locus and shows the structural
divergence of part of this locus ~70 kilobases
upstream of Ifng in rodents compared with
humans and other mammals.
80. Hatton, R. D. et al. A distal conserved sequence
element controls Ifng gene expression by T cells and
NK cells. Immunity 25, 717729 (2006).
This report identifies several distal regulatory
elements upstream of Ifng based on sequence
conservation and the presence of favourable histone
modifications. It also shows that Tbet binds to
several of these sites, one of which (CNS22) is
crucial for expression of Ifng from a BAC transgene.
81. Soutto, M., Zhou, W. & Aune, T. M. Cutting Edge:

distal regulatory elements are required to achieve
selective expression of IFN in Th1/Tc1 effector cells.
J. Immunol. 169, 66646667 (2002).
82. Boyle, A. P. et al. Highresolution mapping and
characterization of open chromatin across the
genome. Cell 132, 311322 (2008).
This study provides a comprehensive, genomewide
map of DNaseI hypersensitive sites in resting
human CD4+ T cells and compares the locations of
these sites with the histone modifications that were
identified by reference 46.
83. Agarwal, S. & Rao, A. Longrange transcriptional
regulation of cytokine gene expression. Curr. Opin.
Immunol. 10, 345352 (1998).
84. Lee, D. U., Avni, O., Chen, L. & Rao, A. A distal
enhancer in the interferon (IFN) locus revealed by
genome sequence comparison. J. Biol. Chem. 279,
48024810 (2004).
85. Shnyreva, M. et al. Evolutionarily conserved sequence
elements that positively regulate IFN expression in
T cells. Proc. Natl Acad. Sci. USA 101, 1262212627
(2004).
86. Chang, S. & Aune, T. M. Dynamic changes in histone
methylation marks across the locus encoding
interferon during the differentiation of T helper
type 2 cells. Nature Immunol. 8, 723731 (2007).
87. Shi, M., Lin, T. H., Appell, K. C. & Berg, L. J.
Januskinase3dependent signals induce chromatin
remodeling at the Ifng locus during T helper 1 cell
differentiation. Immunity 28, 763773 (2008).
88. Thieu, V. T. et al. Signal transducer and activator of
transcription 4 is required for the transcription factor
Tbet to promote T helper 1 cellfate determination.
Immunity 29, 679690 (2008).
89. Yang, Y., Ochando, J. C., Bromberg, J. S. & Ding, Y.
Identification of a distant Tbet enhancer responsive to
IL12/Stat4 and IFN/Stat1 signals. Blood 110,
24942500 (2007).
90. Mullen, A. C. et al. Role of Tbet in commitment of TH1
cells before IL12dependent selection. Science 292,
19071910 (2001).
91. Way, S. S., HavenarDaughton, C., Kolumam, G. A.,
Orgun, N. N. & MuraliKrishna, K. IL12 and typeI
IFN synergize for IFN production by CD4 T cells,
whereas neither are required for IFN production by
CD8 T cells after Listeria monocytogenes infection.
J. Immunol. 178, 44984505 (2007).
92. Cai, G., Radzanowski, T., Villegas, E. N., Kastelein, R.
& Hunter, C. A. Identification of STAT4dependent
and independent mechanisms of resistance to
Toxoplasma gondii. J. Immunol. 165, 26192627
(2000).
93. Kaplan, M. H., Sun, Y. L., Hoey, T. & Grusby, M. J.
Impaired IL12 responses and enhanced development
of Th2 cells in Stat4deficient mice. Nature 382,
174177 (1996).
94. Chang, S. & Aune, T. M. Histone hyperacetylated
domains across the Ifng gene region in natural killer
cells and T cells. Proc. Natl Acad. Sci. USA 102,
1709517100 (2005).
95. Beima, K. M. et al. Tbet binding to newly identified
target gene promoters is cell typeindependent but
results in variable contextdependent functional
effects. J. Biol. Chem. 281, 1199212000 (2006).
96. Naoe, Y. et al. Repression of interleukin4 in T helper
type 1 cells by Runx/Cbf binding to the Il4 silencer.
J. Exp. Med. 204, 17491755 (2007).
97. Tong, Y., Aune, T. & Boothby, M. Tbet antagonizes
mSin3a recruitment and transactivates a fully
methylated IFN promoter via a conserved Tbox
halfsite. Proc. Natl Acad. Sci. USA 102, 20342039
(2005).
98. Chen, G. Y., Osada, H., SantamariaBabi, L. F. &
Kannagi, R. Interaction of GATA3/Tbet transcription
factors regulates expression of sialyl Lewis X homing
receptors on Th1/Th2 lymphocytes. Proc. Natl Acad.
Sci. USA 103, 1689416899 (2006).
99. Miller, S. A., Huang, A. C., Miazgowicz, M. M.,
Brassil, M. M. & Weinmann, A. S. Coordinated, but
physically separable interaction with H3K27
demethylase and H3K4methyltransferase activities
are required for Tbox proteinmediated activation of
developmental gene expression. Genes Dev. 22,
29802993 (2008).
This study shows that Tbet interacts with an
H3K27 demethylase and can recruit it to target
genes to erase this repressive histone modification,
and at the same time can recruit an H3K4
methyltransferase to add this permissive histone
modification.
Ty
s
100. Szabo, S. J. et al. A novel transcription factor, Tbet,
directs Th1 lineage commitment. Cell 100, 655669
(2000).
101. Akimzhanov, A. M., Yang, X. O. & Dong, C. Chromatin
remodeling of interleukin17 (IL17)IL17F cytokine
gene locus during inflammatory helper T cell
differentiation. J. Biol. Chem. 282, 59695972
(2007).
102. Wei, L., Laurence, A., Elias, K. M. & OShea J., J.
IL21 is produced by TH17 cells and drives IL17
production in a STAT3dependent manner. J. Biol.
Chem. 282, 3460534610 (2007).
103. Decker, J. Gene regulation in the third dimension.
Science 319, 17931794 (2008).
104. Misteli, T. Beyond the sequence: cellular organization
of genome function. Cell 128, 787800 (2007).
105. Williams, A. & Flavell, R. A. The role of CTCF in
regulating nuclear organization. J. Exp. Med. 205,
747750 (2008).
106. Spilianakis, C. G. & Flavell, R. A. Longrange
intrachromosomal interactions in the T helper type 2
cytokine locus. Nature Immunol. 5, 10171027
(2004).
This report shows for the first time the existence
of longrange intrachromosomal interactions in
the TH2cytokine locus and the importance of
STAT6 and GATA3 in the establishment of these
interactions.
107. Cai, S., Lee, C. C. & KohwiShigematsu, T. SATB1
packages densely looped, transcriptionally active
chromatin for coordinated expression of cytokine
genes. Nature Genet. 38, 12781288 (2006).
This study identifies many SATB1 binding sites in
the TH2cytokine locus in TH2 cells, and shows that
activationinduced, SATB1dependent chromatin
looping of the TH2cytokine locus is important for
TH2type cytokine expression.
108. Filippova, G. N. Genetics and epigenetics of the
multifunctional protein CTCF. Curr. Top. Dev. Biol. 80,
337360 (2008).
109. Parelho, V. et al. Cohesins functionally associate with
CTCF on mammalian chromosome arms. Cell 132,
422433 (2008).
This study shows that cohesins colocalize
throughout the genome with CTCF and contribute
to the insulator function of CTCF in model systems.
Cohesin and CTCF binding sites are found at the
boundary and within the Ifng gene and do not to
interfere with its transcription.
110. Spilianakis, C. G., Lalioti, M. D., Town, T., Lee, G. R. &
Flavell, R. A. Interchromosomal associations between
alternatively expressed loci. Nature 435, 637645
(2005).
This report describes longrange interchromosomal
interactions between the TH2cytokine and Ifng loci
in naive T cells and suggests that these interactions
may poise these loci for expression during TH2 or
TH1cell differentiation.
111. Jacob, E., HodDvorai, R., SchifZuck, S. & Avni, O.
Unconventional association of the polycomb group
proteins with cytokine genes in differentiated
T helper cells. J. Biol. Chem. 283, 1347113481
(2008).
112. Miller, A. T., Wilcox, H. M., Lai, Z. & Berg, L. J.
Signaling through Itk promotes T helper 2
differentiation via negative regulation of Tbet.
Immunity 21, 6780 (2004).
113. Yu, Q., Thieu, V. T. & Kaplan, M. H. Stat4 limits DNA
methyltransferase recruitment and DNA methylation
of the IL18R gene during Th1 differentiation.
EMBO J. 26, 20522060 (2007).
114. Ooi, S. K. et al. DNMT3L connects unmethylated

lysine 4 of histone H3 to de novo methylation of DNA.
Nature 448, 714717 (2007).
115. Cimmino, L. et al. Blimp1 attenuates Th1
differentiation by repression of Ifng, Tbx21, and Bcl6
gene expression. J. Immunol. 181, 23382347 (2008).
116. Pai, S. Y., Truitt, M. L. & Ho, I. C. GATA3 deficiency
abrogates the development and maintenance of
T helper type 2 cells. Proc. Natl Acad. Sci. USA 101,
19931998 (2004).
117. Zhu, J. et al. Conditional deletion of Gata3 shows its
essential function in TH1TH2 responses. Nature
Immunol. 5, 11571165 (2004).
This report shows that GATA3 is essential for
the initiations of TH2cell differentiation and
contributes to but is not essential for its
maintenance.
118. Martins, G. A., Hutchins, A. S. & Reiner, S. L.
Transcriptional activators of helper T cell fate are
required for establishment but not maintenance of
signature cytokine expression. J. Immunol. 175,
59815985 (2005).
119. Taniuchi, I. et al. Differential requirements for Runx
proteins in CD4 repression and epigenetic silencing
during T lymphocyte development. Cell 111, 621633
(2002).
120. Ansel, K. M. et al. Deletion of a conserved Il4 silencer
impairs T helper type 1mediated immunity. Nature
Immunol. 5, 12511259 (2004).
121. Laurence, A. et al. Interleukin2 signaling via STAT5
constrains T helper 17 cell generation. Immunity 26,
371381 (2007).
122. Gorelik, L., Constant, S. & Flavell, R. A. Mechanism
of transforming growth factor induced inhibition of
T helper type 1 differentiation. J. Exp. Med. 195,
14991505 (2002).
123. Gorelik, L., Fields, P. E. & Flavell, R. A. Cutting Edge:
TGF inhibits Th type 2 development through
inhibition of GATA3 expression. J. Immunol. 165,
47734777 (2000).
124. Yu, J. et al. Pro and antiinflammatory cytokine
signaling: reciprocal antagonism regulates interferon
production by human natural killer cells. Immunity 24,
575590 (2006).
125. Chen, Y. et al. AntiIL23 therapy inhibits multiple
inflammatory pathways and ameliorates autoimmune
encephalomyelitis. J. Clin. Invest. 116, 13171326
(2006).
126. Laurence, A. & OShea, J. J. TH17 differentiation:
of mice and men. Nature Immunol. 8, 903905 (2007).
127. Wilson, N. J. et al. Development, cytokine profile and
function of human interleukin 17producing helper
T cells. Nature Immunol. 8, 950957 (2007).
128. Annunziato, F. et al. Phenotypic and functional
features of human Th17 cells. J. Exp. Med. 204,
18491861 (2007).
129. Zheng, Y. et al. Interleukin22, a TH17 cytokine,
mediates IL23induced dermal inflammation and
acanthosis. Nature 445, 648651 (2007).
130. Harrington, L. E. et al. Interleukin 17producing CD4+
effector T cells develop via a lineage distinct from the
T helper type 1 and 2 lineages. Nature Immunol. 6,
11231132 (2005).
131. Ivanov, I. et al. The orphan nuclear receptor
RORt directs the differentiation program of
proinflammatory IL17+ T helper cells. Cell 126,
11211133 (2006).
132. Singh, S. P., Zhang, H. H., Foley, J. F., Hedrick, M. N. &
Farber, J. M. Human T cells that are able to produce
IL17 express the chemokine receptor CCR6.
J. Immunol. 180, 214221 (2008).
133. McGeachy, M. J. et al. TGF and IL6 drive the

production of IL17 and IL10 by T cells and restrain
TH17 cellmediated pathology. Nature Immunol. 8,
13901397 (2007).
134. OGarra, A. & Vieira, P. TH1 cells control themselves by
producing interleukin10. Nature Rev. Immunol. 7,
425428 (2007).
135. Krawczyk, C. M., Shen, H. & Pearce, E. J. Functional
plasticity in memory T helper cell responses.
J. Immunol. 178, 40804088 (2007).
136. Messi, M. et al. Memory and flexibility of cytokine
gene expression as separable properties of human TH1
and TH2 lymphocytes. Nature Immunol. 4, 7886
(2003).
137. Sundrud, M. S. et al. Genetic reprogramming of
primary human T cells reveals functional plasticity in
Th cell differentiation. J. Immunol. 171, 35423549
(2003).
138. Adeeku, E., Gudapati, P., MendezFernandez, Y.,
Van Kaer, L. & Boothby, M. Flexibility accompanies
commitment of memory CD4 lymphocytes derived
from IL4 locusactivated precursors. Proc. Natl Acad.
Sci. USA 105, 93079312 (2008).
139. Lohning, M. et al. Longlived virusreactive memory
T cells generated from purified cytokinesecreting
T helper type 1 and type 2 effectors. J. Exp. Med.
205, 5361 (2008).
140. Reiner, S. L., Sallusto, F. & Lanzavecchia, A. Division of
labor with a workforce of one: challenges in specifying
effector and memory T cell fate. Science 317,
622625 (2007).
141. Allis, C. D. et al. New nomenclature for chromatin
modifying enzymes. Cell 131, 633636 (2007).
142. Ruthenburg, A. J., Allis, C. D. & Wysocka, J.
Methylation of lysine 4 on histone H3: intricacy of
writing and reading a single epigenetic mark. Mol. Cell
25, 1530 (2007).
143. Yang, X. J. & Seto, E. The Rpd3/Hda1 family of lysine
deacetylases: from bacteria and yeast to mice and
men. Nature Rev. Mol. Cell Biol. 9, 206218 (2008).
144. Lee, Y. et al. Late developmental plasticity in the
helper 17 lineage. Immunity 31 Dec 2008
(doi:10.1016/j.immuni.2008.11.005)
145. Wei, G. et al. Global mapping of histone H3 K4 and
K27 trimethylation reveals specificity and plasticity in
lineage fate determination of differentiating CD4+
T cells. Immunity 15 Jan 2008 (doi:10.1016/j.
immuni.2008.12.009)
Acknowledgements
We apologize to the colleagues whose work was not discussed

owing to space limitations. Work from the authors laborato
ries was supported by the National Institutes of Health,
USA (grant numbers R01AI071272, R01HD18184,
N01AI40069 and T32AI07411).
DATABASES
entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
AHR | GATA3 | HLX | IFN | IL-4 | IL-5 | IL-13 | IL-17A | IL-17F |
Rad50 | RBPJ | RUNX3 | RORt | STAT4 | STAT5 | STAT6 | Tbx21 |
TGF
FURTHER INFORMATION
christopher B. Wilsons homepage: http://depts.
washington.edu/immunweb/faculty/profiles/wilson.html
The visTA web site: http://pipeline.lbl.gov/cgi-bin/
gateway2
All lInks ArE ACTIvE In THE onlInE pDF


Epigenetic Control of T Helper Cell

Diunggah oleh

Informasi Dokumen

Judul Asli

Hak Cipta

Format Tersedia

Bagikan dokumen Ini

Bagikan atau Tanam Dokumen

Opsi Berbagi

Apakah menurut Anda dokumen ini bermanfaat?

Apakah konten ini tidak pantas?

Hak Cipta:

Format Tersedia

Epigenetic Control of T Helper Cell

Diunggah oleh

Hak Cipta:

Format Tersedia

f o c u s o n c D 4 + T- c e l l D i v e r s i T y

CD4+ T helper (TH)-cell subsets were first described

NATuRE REvIEWs | Immunology

voLuME 9 | FEbRuARy 2009 | 91

Regulating lineage choice

92 | FEbRuARy 2009 | voLuME 9

voLuME 9 | FEbRuARy 2009 | 93

into nucleosomes or removed, and histones can be

characterized by the presence of histones H3 and H4 that

DNAseI hypersensitive site

Inactive but poised

Active and accessible

Locus control region

Epigenetics and TH-cell subsets

subsequently, studies showed that treatment of CD4+

Box 1 | Promoters, other regulatory elements and their interactions

An enzymatic complex that

DNaseI hypersensitive site

NATuRE REvIEWs | Immunology

voLuME 9 | FEbRuARy 2009 | 95

Transferases that Deacetylases or

Function of histone modification

H3K9, H3K14 and H3K18

KAT2A (GCN5) and

HDAC1 and HDAC2

Binds or recruits bromodomaincontaining proteins (such as TAF1)

H4K5, H4K8, H4K12

HDAC1 and HDAC2

Binds or recruits bromodomaincontaining proteins (such as TAF1)

H2aK5, H2bK12, H2bK15, KAT3A (CBP) and

HDAC1 and HDAC2

Binds or recruits bromodomaincontaining proteins (such as TAF1)

KDM1 (LSD1) and

Binds or recruits the WDR5

KDM1 (LSD1) and

Binds or recruits chromodomain,

Binds or recruits NURD complexes

KDM6A and KDM6B

Binds or recruits PRC1 complex and

KDM1 (LSD1) and

Binds or recruits CBX5 (HP1) and DNA Repressive

However, it is worth noting that whether the specific

96 | FEbRuARy 2009 | voLuME 9

DNaseI hypersensitive site

Il4 and Il13 promoters and to HsvA, as well as to RHs6

Rad50 gene, at HsIv (the Il4 silencer) and Hss3 (one

NATuRE REvIEWs | Immunology

voLuME 9 | FEbRuARy 2009 | 97

98 | FEbRuARy 2009 | voLuME 9

T-bet T-bet T-bet

DNaseI hypersensitive site

H3K27 trimethylation throughout the locus59,79,80,8385.

and has not been shown to bind to the endogenous Ifng

NATuRE REvIEWs | Immunology

voLuME 9 | FEbRuARy 2009 | 99

CNS4 CNS5 CNS6

production of IFN in the absence of sTAT4 when the

elements have been described in the Il17 locus based on

Structuring regulatory relationships

100 | FEbRuARy 2009 | voLuME 9

Heritability, plasticity and diversity

TH2 cells did not express lineage-inappropriate cytokines

NATuRE REvIEWs | Immunology