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CD4+T helper (TH)-cell subsets were first described
by Mossman and Coffman1, who found that mouseT-cell clones segregated into two subsets they namedT
H1 and T
H2 cells based on their mutually exclusive
production of interferon- (IFN) or interleukin-4(IL-4), IL-5and IL-13, respectively. The relevance ofthis distinction was subsequently shown by Locksleyand colleagues2, who found that mice which mounteda T
H1-cell-dominant response resolved infection with
Leishmania major, whereas mice that mounted a TH2-
cell-dominant response did not. Work by many groupshas since established the importance of T
H1 cells in host
defence to intracellular pathogens and of TH2 cells in the
protection against helminth infections. More recently,cells that produce IL-17Awere shown to represent a dis-tinct T
H-cell lineage the T
H17-cell subset that in
addition to IL-17A produces IL-17F, IL-21, IL-22 and,in humans, IL-26. T
H17 cells contribute to host defence
against extracellular bacteria and fungi, particularly at
mucosal sites, although the full extent of their contribu-tion to host defence is still being investigated3. In contrastto these protective functions of T
Hcells, 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 T
H-cell lineages, the
instructions that are received by naive CD4+T cells duringinitial encounters with antigen-presenting cells (APCs)must be converted into cell-intrinsic changes. Thesechanges are passed on to progeny cells through multiplecell divisions and occur over time and in various environ-ments where effector and memory T cells carry out their
functions. These instructions are converted by respond-ing T cells into changes in the abundance, interactionsand locations of transcription factors, which in turnlead to changes in gene expression. The resulting infor-mation could in principle be propagated from one T-cellgeneration to the next solely through self-reinforcingtranscription factor networks. In practice, more pre-cise control of gene expression is achieved throughepigenetic processes7, which facilitate heritable and sta-ble programmes of gene expression, while preservingthe potential for these programmes to be modified inresponse to environmental changes.
In this Review, we describe the epigenetic processesthat help to regulate T
H1-, T
H2- and T
H17-cell lineage
fate and function by affecting gene transcription. Inrecent years, technological advances have acceleratedthe pace of discovery in this field. As a result, pro-gressively more comprehensive and higher resolutionmaps of gene regulatory elements and their cell-type-
specific epigenetic marks, including DNA methyla-tion, histone modifications and three-dimensionalchromatin structure, are being derived from stemcells and other cell types, including T cells. Thesefindings and their contribution to T
H-cell differen-
tiation and function are also discussed in this Review.Epigenetic processes that influence mRNA splicing,stability and translation in the immune system, such asmicroRNAs, have been reviewed recently810and aretherefore not discussed here. Follicular T
Hcells11and
other proposed TH
-cell subsets that might representdistinct lineages but are not yet firmly established arealso not discussed in this Review.
Department of Immunology,
University of Washington,
Seattle, Washington 98195,
USA.
Correspondence to C.B.W.
e-mail:
doi:10.1038/nri2487
Published online
19 January 2009
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, post-
translational histone
modifications and changes
to higher-order chromatin
structure.
ChromatinDNA and the proteins with
which it is associated in the
nucleus.
Epigenetic control of T-helper-celldifferentiationChristopher 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, whichallow 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.
REVIEWS
NATURE REVIEWS |IMMUNOLOGY VOLUME 9 | FEBRUARY 2009 |91
F OC US ON C D 4 +T-CELL DI VERSITY
2009 Macmillan Publishers Limited. All rights reserved
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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 largeintracellular domain of Notch
is cleaved and travels to the
nucleus to become a direct
co-activator of the transcription
factor recombination-
signal-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 acti-vated T cells (NFAT) and other transcription factors thatare activated in naive CD4+T cells in response to signalsthrough the T-cell receptor (TCR) and co-stimulatorymolecules induce the production of IL-2, which leads toIL-2-induced activation of signal transducer and activa-tor of transcription 5 (STAT5) and entry into the cellcycle. Thereafter, T
H-cell-lineage choice is determined,
for the most part, by the cytokine milieu and the networkof transcription factors that are induced downstream ofcytokine signalling pathways (FIG. 1).
TH1-cell development is initiated by STAT1, which
is activated in response to IFN and IL-27 that are pro-duced by natural killer (NK) cells and APCs, respec-tively. Together with TCR-induced transcription factors,STAT1 induces the transcription factor T-bet, a key (ifnot master) regulator of the T
H1-cell lineage, which
in turn induces the production of IFN, the activa-tion of the transcription factors H2.0-like homeobox(HLX) and runt-related transcription factor 3 (RUNX3),
and opposes the inhibitory effects of GATA-bindingprotein 3 (GATA3; see later) on T
H1-cell differentia-
tion1217. The expression of IL-12 receptor-2 (IL-12R2)is also induced in this process; IL-12R2 pairs withIL-12R1 to form the IL-12 receptor, thereby allowingAPC-derived IL-12 to activate STAT4. STAT4, T-bet,HLX and RUNX3 then bind to and activate Ifng, whichreinforces T
H1-cell commitment through the activation
of STAT1 in a positive-feedback loop. Concomitantly,T-bet and RUNX3 bind to and repress Il4 to inhibitT
H2-cell differentiation.T
H2-cell differentiation is initiated by the activation of
STAT6 by IL-4, which, together with TCR-induced tran-scription factors, binds to and activates Gata3(REF. 5).Alternatively, Notch signalling can induce Gata3in aSTAT6-independent manner18,19. GATA3 induces thetranscription factor MAF, which helps to activate Il4, andtogether GATA3 and STAT6 activate the transcription ofIl4, Il5and Il13. T
H2-lineage commitment is stabilized by
the autoactivation of GATA3, the autocrine and para-crine activation of STAT6 by IL-4, and the STAT6- andGATA3-dependent antagonism of IFN expression andT
H1-cell differentiation5.The transcription factors that are involved in T
H1-
and TH2-cell differentiation are not required for T
H17-
cell differentiation and can antagonize this process20,21.Transforming growth factor- (TGF)inhibits T
H1-
and TH2-cell differentiation, and promotes regulatoryT (T
Reg)-cell and T
H17-cell lineage commitment by
inducing the expression of the transcription factors fork-head box P3 (FOXP3) and retinoic-acid-receptor-relatedorphan receptor-t (RORt; also known as RORC),which are required for T
Reg- and T
H17-cell lineage com-
mitment, respectively2023. In the absence of IL-6, FOXP3inhibits RORt and therefore blocks T
H17-cell develop-
ment, whereas in the presence of IL-6, STAT3 is acti-vated, which inhibits the expression of FOXP3 and itsinteractions with RORt. This results in an increase inthe expression of RORt (as well as of ROR, which hasan ancillary role in T
H17-cell induction)24, and T
H17-cell
differentiation is favoured. Once TH17-cell differentia-
tion has been initiated, the T cells produce IL-21, whichactivates STAT3 and induces the expression of IL-23R.This allows APC-derived IL-23 to activate STAT3, whichdampens IL-10 production, drives IL-22 productionand stabilizes T
H17-cell differentiation and commit-
ment21,2528. The transcription factor aryl-hydrocarbonreceptor (AHR) also influences the differentiation of T
Reg
and TH17 cells29,30. Although initial studies suggested oth-
erwise, the cytokine and transcription factor networksinvolved in human and mouse T
H17-cell differentiation
seem to be similar21,31,32.
Epigenetic processes. The ability of transcription factorsto bind to DNA at regulatory regions on genesis affectedby their concentration, post-translational modificationsand subcellular localization, as well as by the state of thechromatin and underlying DNA. The epigenetic contextin which transcription factors function is provided bythe position and compaction of nucleosomes, the interac-tions of nucleosomes with the DNA, post-translational
histone modifications and the methylation status of theDNA6,3336. Therefore, unlike genetic information, epi-genetic information is not encoded by changes in thesequence of the DNA but by differential methylation ofthe DNA and modifications of chromatin, which affectwhether, when and to what level specific genes areexpressed in a given cell. Because the DNA sequenceremains unchanged, epigenetic modifications and theinformation that they encode can be heritable but plastic the potential to erase modifications and inscribe newones is retained.
In mammals, DNA can be methylated on cytosinesin CpG dinucleotides. At present, DNA methylation isthe only proven mechanism by which epigenetic infor-mation is faithfully propagated from one cell generationto the next. Heritability is achieved through copyingof the pattern of methylated cytosines from parentalto progeny DNA strands by DNA methyltransferase 1(DNMT1)37. DNA methylation at gene promoters,and possibly at distal regulatory elements, can directlyinhibit transcription38,39; by contrast, DNA methylationwithin transcribed sequences seems to have little effecton transcription, although demethylation within thetranscribed sequences of Il4 andIfng correlates with highexpression levels of these cytokines in T
H2 and T
H1 cells,
respectively13,40. Methylated DNA directly represses geneexpression 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-CpG-binding domain proteins (MBDs)41,42. Although plantscontain enzymes that actively demethylate DNA, suchenzymes have not been identified in mammals, in whichthe only established mechanism for DNA demethylationis passive that is, the result of a failure to copy methy-lation patterns from the parental strand onto the daughterstrand during DNA replication.
Nucleosomecomposition and histone modificationsare diverse and dynamic. Variant forms of histones H2and H3 and the linker histone H1 can be incorporated
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|
NaiveCD4+T cell
a TH1-cell development
b TH2-cell development
Inititat ion; proliferat ion Differentiat ion; stabilizat ion Epigenetic remodelling
CD28
CD80/CD86MHCclass II
TCR
DC
DC
IL-2IL-2R
IFN
IFNGR
IFN
NK cellIL-12
STAT5
STAT1
STAT4
RUNX3
T-betT-bet
HLXSTAT1
STAT1
Heritability
Ifng
Ifng
IL-27
IL-27RIL-12R
IL-12
IL-23
NaiveCD4+T cell
IL-4IL-4R
IL-4
IL-2
STAT6
GATA3
GATA3
GATA3
MAF
STAT5 STAT6
RBPJ
Heritability
Il4
Notchsignalling
Il4
Il5
Il13
c TH17-cell development
DC
NaiveCD4+T cell
IL-21
IL-2
STAT5
STAT3
STAT3
STAT3
RORt
RORtHeritability
Il21
Il17
Il17
Il21
Il22
TGFR
TGF
IL-23R
IL-21R
IL-6
IL-6R
Epithelialcell
Figure 1 |Cytokines and transcription factor networks regulate T-helper-cell 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
interferon-(IFN)- and/or interleukin-27 (IL-27), both of which upregulate T-bet. T-bet induces the expression of H2.0-likehomeobox (HLX), and together they collaborate with transcription factors that are activated following T-cell receptor (TCR)
signalling to activate Ifngtranscription and to antagonize GATA-binding protein 3 (GATA3). These events result in theexpression 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 T
H2-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 thatdrives T
H2-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| T
H17-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 ofretinoic-acid-receptor-related orphan receptor-t(RORt), IL-17 and IL-23R, and STAT3 activation is attenuated byIL-2-induced STAT5. IL-21 and IL-23 drive the production of IL-17 and IL-22 and T
H17-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, IFNreceptor; NK, natural killer.
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|
Euchromatin
Compact heterochromatin
Nucleosome
a b
c
MethylationH4
Nucleosome
H3K27 me2or me3
H3K9 me2or me3
H3K18 Ac
H3K4 me1,me2 or me3
OR
H3K4 me3or me2
Silent Inactive but poised Active and accessible
Faculative heterochromatin Bivalent
NullConstitutive heterochromatinor focal silencing in euchromatin
DNA
Promoter
Enhancer
H3K4 andH3K27 me3
H3K9 Ac,H3K14 Acand H3K18 Ac
DNAseI hypersensitive site
Acetylation
Transcriptionalregulatory factors
H3H4K5 Ac andH4K8 Ac
Histone code
Post-translational
modifications of histone tails
that involve characteristic
clusters of modifications,
including acetylation,
phosphorylation,
ubiquitylation, methylation,
sumoylation and ADP-
ribosylation 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 bemodified by the enzyme-catalysed addition or removalof acetyl, methyl, phosphate, ubiquitin, sumoyl or ADP-ribose groups34. Acetylation alters histone charge, whichreduces the interactions between histones and DNA andenhances nucleosome mobility36. In addition, incorpo-ration of histone H1 condenses chromatin43, and post-translational modifications of histonescreate or removebinding sites for regulatory proteins that facilitate orimpede transcription.
The diversity of potential modifications led to thehistone code and later nucleosome code hypothesis,which posits that histone modifications combine to cre-ate a code that is recognized by specific regulatory pro-teins or complexes that are involved in transcription36.Genome-wide studies identified many different com-binations of histone modifications that could allow forfine-tuning of transcription, but a few sets of core modi-fications seem to be sufficient to characterize genes andregulatory elements as active and accessible, inactive butpoised, 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 thatare 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 alternativeform of histone H2A, known as H2A.Z. These histonemodifications are absent from silenced genes, whereasdimethylated and trimethylated H3K27, or dimethylatedand trimethylated H3K9 are present. Dimethylated andtrimethylated H3K27 are typically found throughout thecondensed, facultative heterochromatinof silenced tissue-specific loci, and dimethylated and trimethylated H3K9are found throughout constitutive heterochromatin or indiscrete sites within active genes, where they may inhibitinappropriate transcription33. Promoters of genes thatare poised to be either activated or silenced generallydo not have any of these histone modifications or have abivalent modification pattern that is, they have bothpermissive (H3K4 dimethylation and trimethylation)and repressive (H3K27 trimethylation) histone modi-fications33,4446. Locus control regions (LCRs) and distal
enhancers (BOX 1)at an inactive locus that are bivalent or
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
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.
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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.
Chromatin-remodelling
complex
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.
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.
only have methylated H3K4 may be pioneer elementsthat are amenable to subsequent activation or silencing, atwhich time histone modifications and DNA methylationat these elements are altered accordingly41,42.
The epigenetic state of regulatory elements is alsoaltered when transcription factors and RNA polymerase IIthat are bound recruit chromatin-remodelling complexesthat displace or alter the conformation of nucleosomes.Such regions are accessible to cutting by DNaseI, allow-ing the regulatory elements to be detected as DNaseIhypersensitive sites. Therefore, functional regulatoryelements in a given cell can be experimentally identifiedby approaches that detect DNaseI hypersensitive sites,post-translational histone modifications and differentialDNA methylation35.
Epigenetics and TH-cell subsets
Cause or consequence. Transcription factors maydirectly transactivate (induce) or repress gene expressionand may also affect transcription by recruiting proteinsthat 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 ofthe transcription factors that initially induced them,but do such modifications merely mark past events andreport transcriptional competence or do they also con-tribute to differences in competence? In other words, inaddition to the transcription factor networks describedabove, which initiate and help to sustain T
H-cell subset
differentiation, do epigenetic modifications also help tomaintain these differentiated states?
Early biochemical evidence that the epigenetic stateof a gene has a causal role in transcriptional compe-tence came from studies in which treatment with 5-aza-cytidine, an inhibitor of DNA methylation, resulted in theproduction of IL-2 (REF. 47) and IFN (REF. 48) by T-celllines 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 T
H2-type
cytokines49,50. Genetic evidence supported these find-ings: conditional ablation of DNMT1 or MBD2 whichrecruit HDACs and chromatin-remodelling complexesto methylated DNA and induce a repressive chromatinstate led to increased expression of IFN and T
H2-type
cytokines and an inability of TH1 or T
H2 cells to silence
the expression of cytokine genes that are associatedwith the opposing lineage5154. These studies also suggestedthat DNMT1 and MBD2 mediate gene silencing mostly,if not wholly, by directly affecting the loci that encodeIFN and T
H2-type cytokines. Thus, DNA methylation,
MBDs and histone deacetylation dampen the expressionof both T
H1- and T
H2-type cytokines, and help to restrict
cytokine expression to the appropriate lineage.A prediction of these findings is that lineage-specific
transcription factors regulate TH
1- and TH2-cell fate
in part through epigenetic processes. Consistent withthis possibility, chromatin-remodelling complexes that
contain Brahma-related gene 1 (BRG1; also known asSMARCA4) displace nucleosomes and remodel chroma-tin at the Ifng promoter in mouse T
H1 cells in a STAT4-
dependent manner; these complexes are required forhigh Ifng expression55. In addition, mice that are haplo-insufficient for the H3K4 methyltransferase MLL havea defect in the maintenance but not the induction ofGata3, Il4, Il5 andIl13expression, whereas T
H1-cell dif-
ferentiation is not affected56. Conversely, mice that lackexpression of MEL18, a polycomb repressor complex 1(PRC1) protein that binds to trimethylated H3K27, haveimpaired GATA3 expression and T
H2-cell differentia-
tion, although the cause of these defects is not known57.These findings, together with a large body of correla-tive data, suggest that epigenetic mechanisms are keydeterminants of T
H-cell differentiation and function.
Box 1 |Promoters, other regulatory elements and their interactions
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 II-
containing 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-containingcomplexes 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 3orientation,
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.
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However, it is worth noting that whether the specificepigenetic modifications described below are causallyrelated to differences in T-cell function or whether theymerely report these differences has for the most part notbeen directly determined.
The TH2-cytokine locus and lineage commitment. Just
over a decade ago, alterations to the chromatin structureof the Il4and Il13genes were shown to occur as naiveT cells differentiated into T
H2 or T
H1 cells58,59. These
findings stimulated interest in the contribution of epige-netic processes to T
H-cell differentiation and in the T
H2-
cytokine locus as a model system by which to address thecoordinated regulation of clustered, functionally related
genes. The accrual of additional information was greatlyaccelerated by the availability of complete genomicsequences for humans, mice and other species, andby improvements in the techniques used for assessingepigenetic modifications.
The mouse TH2-cytokine locus containsIl4, Il5,Il13
and the constitutively expressed Rad50gene (FIG. 3), andis flanked by Irf1 and Kif3A. The gene composition ofthis locus, as well as the linear relationship of the geneswithin the locus, is conserved in the genomes of mam-mals, suggesting that these relationships are function-ally important. Consistent with this possibility, the genesencoding T
H2-type cytokines are regulated through their
promoters and also by several additional regulatory ele-ments, the locations of which have been experimentallydetermined by the detection of DNaseI hypersensitivesites, histone modifications and differential DNA methy-lation, as well as through computational identificationof conserved non-coding sequences (CNS). Variousapproaches have been used to test the function of theelements that have been identified by these methods andtheir interactions with transcription factors35.
In the mouse TH2-cytokine locus,the transcription
of Il4is enhanced by regulatory elements that map toDNaseI hypersensitive siteI (HSI) and HSII in the secondintron of Il4, to DNaseI hypersensitive site V
A(HSV
A) and
HSV located 3of Il4, to DNaseI hypersensitive site s1
(Hss1) and Hss2 located between Il13and Il4,and to theT
H2-cytokine LCR, which encompasses Rad50hyper-
sensitive site 4 (RHS4), RHS5, RHS6 and RHS7 (FIG. 3;see figure legend for the convention used to name hyper-sensitive sites in this locus and note that HSV maps toCNS2, and Hss1 and Hss2 map to CNS1)5. The expres-sion of Il13is augmented by regulatory elements at CNS1,the T
H2-cytokine LCR and HS1, which maps to the
CG-rich element (CGRE) upstream of the Il13promoter.Many of these enhancers and each of the T
H2-cytokine
promoters are direct targets of NFAT, other TCR-inducedtranscription factors and T
H2-cytokine-promoting
transcription factors. For example, STAT6 binds to the
Table 1 | Histone lysine modifications
Modification Histone lysinesmodified
Transferases thatadd modification*
Deacetylases ordemethylases thatremove modification
Function of histone modification Effect ontranscription
Acetylation H3K9, H3K14 and H3K18 KAT2A (GCN5) andKAT2B (PCAF)
HDAC1 and HDAC2(in SIN3A, NURD andCoREST complexes)
Binds or recruits bromodomain-containing proteins (such as TAF1)
Permissive
H4K5, H4K8, H4K12and H4K16
KAT5 (TIP60) HDAC1 and HDAC2(in SIN3A, NURD andCoREST complexes)
Binds or recruits bromodomain-containing proteins (such as TAF1)
Permissive
H2aK5, H2bK12, H2bK15,H3K14, H3K18, H4K5and H4K8
KAT3A (CBP) andKAT3B (p300)
HDAC1 and HDAC2(in SIN3A, NURD andCoREST complexes)
Binds or recruits bromodomain-containing proteins (such as TAF1)
Permissive
Methylation H3K4 monomethylation KMT7 (SET7 orSET9)
KDM1 (LSD1) andKDM5B (JARID1B)
Binds or recruits the WDR5component of the H3K4methyltransferase MLL complex
Permissive
H3K4 dimethylationand trimethylation
KMT2AKMT2E(MLL1MLL5)
KDM1 (LSD1) andKDM5AKDM5D(JARID1AJARID1D)
Binds or recruits chromodomain,PHD- and Tudor-domain-containingproteins (such as TFIID, CHD1, BPTFand WDR5)
Permissive
Unmodified H3K4 Binds or recruits NURD complexes
and DNMT3aDNMT3l
Repressive
H3K27 dimethylationand trimethylation
KMT6 (EZH2) KDM6A and KDM6B(UTX and JMJD3)
Binds or recruits PRC1 complex andDNA methyltransferases
Repressive
H3K9 dimethylationand trimethylation
KMT1B (SUV39H)and KMT1C (G9a)
KDM1 (LSD1) andKDM4AKDM4D(JMJD2AJMJD2D)
Binds or recruits CBX5 (HP1) and DNAmethyltransferases
Repressive
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 oftheir 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, hi stone deacetylase; HP1, heterochromatin protein 1; JARID1, jumonji AT-rich interactive domain 1; JMJD, jumonji-domain-containing protein histone demethylase; KAT, lysine acetyltransferase; KDM, lysine demethylase; KMT, lysine methyltransferase; LSD1, lysine-specifichistone demethylase 1; NURD, nucleosome remodelling and histone deacetylation; PCAF, p300/CBP-associated factor; PHD, plant homeodomain; PRC1, polycombrepressive 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.
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TH2 cell
Naive T cell
STAT5GATA3
STAT6
STAT6 CTCFGATA3
STAT6
GATA3 STAT6
MAF
GATA3
STAT6
CTCF
CTCF
TH1 cell
CTCF
CTCF
CTCF T-bet
RUNX3
enh enhsilenhenhTH2 LCR
Il5 Il13 Il4Rad50 CNS1 CNS2CGRE
140 kilobases
RHS2 RHS3 RHS6 RHS7
RHS2
RHS1 RHS2 RHS3 RHS4
RHS5
RHS6
RHS7
HS1 HSI HSIV
HSIV
HSIV
HSVAHSV
HSII
HSIIIHss1
HS2 Hss2
HS3
Hss3
Hss3
Hss3
RHS3 RHS6?
Permissive histone
modifications
Bivalent histone
modifications
Repressive histone
modifications
CNSDNaseI hypersensitive site
|
pro pro pro pro
Il4and Il13promoters and to HSVA, as well as to RHS6
and RHS7; GATA3 binds to the Il5and Il13promoters,to the first intron of Il4, HSV
Aand RHS7, and to the Il13
HS1CGRE region; and recombination-signal-binding
protein for immunoglobulin- J region (RBPJ), whichis the DNA-binding component of the Notch pathway,binds to HSV60,61. Perhaps surprisingly, none of theseelements, including the T
H2-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 ofIl4,Il5 andIl13mRNA before cell division when activated byTCR ligation62,63. In these cells, the T
H2-cytokine locus
is characterized by a paucity of DNaseI hypersensitivesites and histone modifications, although some are stillpresent (FIG. 3). Specifically, DNaseI hypersensitive sitesare found in the promoter of the constitutively expressed
Rad50 gene, at HSIV (the Il4silencer) and Hss3 (oneof three DNaseI hypersensitive sites that are locatedbetweenIl4 and Il13, which is of unknown function), aswell as at RHS6 in the T
H2-cytokine LCR (although this
was observed in one study64but not another 65). HSIValso has bivalent histone modifications (that is, bothpermissive H3K4 dimethylation and H3 acetylation,and repressive H3K27 trimethylation are detectable)66,67,whereas Hss3 is marked solely by repressive trimethy-lated H3K27. The 3Il4enhancer, Il4CNS2 and the T
H2-
cytokine LCR are marked weakly by dimethylated H3K4and/or H3 acetylation64,67,68.
The expression of TH
2-type cytokines is probablyrestrained in naive T cells by the high degree of CpGmethylation (~90%) at their promoters, CNS1, CNS2and the T
H2-cytokine LCR40,51,53,64,69. The Il4promoter
is less methylated (~60%) than the other promoters
Figure 3 |The T helper 2 cytokine locus in mouse T cells. Naive CD4+T cells have DNaseI hypersensitive sites at
hypersensitive site s3 (Hss3), HSIV, the 5end of the Rad50gene at Rad50hypersensitive site 2 (RHS2) and 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), Il13andIl5, 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 T
H1 cells repressive trimethylated
H3K27 spreads to encompass Il4, Il13and a region that extends from conserved non-coding sequence 1 (CNS1) to CNS2 (to
our knowledge, data for Rad50and Il5are 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), signaltransducer 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 Il4are indicated as HS followed by a
roman numeral (for example, HSIV), sites between Il4 and Il13are indicated as Hss followed by a number (for example, Hss1)
sites in or upstream of Il13are indicated as HS followed by a number (for example, HS1), and sites in or near Rad50are
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.
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and lacks CpG motifs in the 250 base pairs that areproximal to the start site53, which might facilitate theearly low-level expression of IL-4. Therefore, in naiveCD4+ T cells, modest amounts of permissive andrepressive epigenetic marks are focally targeted to asubset of the known regulatory elements in the T
H2-
cytokine locus. This bivalent epigenetic state may helppoise this locus, permitting TCR-induced transcriptionfactors to bind and induce early, low-level expressionof T
H2-type cytokines and providing the potential for this
locus to convert to a fully permissive state during TH2-
cell differentiation or to a silenced state during TH1- and
TH17-cell differentiation5,6.Permissive histone modifications are acquired in the
TH
2-cytokine locus in the first 2448 hours followingactivation of naive CD4+T cells under both T
H1- and
TH
2-cell-polarizing conditions through the actions ofNFAT and other transcription factors that are inducedby TCR signalling70,71. These transcription factors alsoinduce the expression of IL-2, which activates STAT5,which in turn binds to and induces chromatin remodel-
ling at intron 2 of Il4to promote TH2-cell differentia-tion72. However, the continued presence and progressiveincrease in these permissive histone modifications, andthe subsequent acquisition of T
H2-specific DNaseI
hypersensitive sites and DNA demethylation at theT
H2-cytokine locus are unique to T
H2 cells40,6468,70,7274.
The active locus of effector TH
2 cells contains thehypersensitive sites that are found in naive T cells aswell as new sites at the Il4, Il5and Il13promoters, ateach of the known enhancers and at the LCR, althoughthe cells must be restimulated for HSV
Ato be detected.
Permissive H3 and H4 acetylation and H3K4 dimethy-lation are acquired or become more prominent at theseelements, and repressive H3K27 trimethylation islost throughout the locus. These changes are evidentin the first week of mouse T
H2-cell differentiation in
culture. By this time, demethylation of the DNaseIhypersensitive sites has commenced but it progressesslowly by a passive replication-dependent process.By contrast, RHS7 undergoes a more rapid and poten-tially active demethylation40,51,53,64,69,75. With the exceptionof the demethylation of RHS5 and RHS6, these epi-genetic changes are not found in the T
H2-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 TH
2-cell-specific epi-
genetic modifications. It may do so through the director indirect recruitment of histone acetyltransferases(HATs) and histone H3K4 methyltransferases5,56, the dis-placement of MBD2 and associated HDAC-containingcomplexes51, the displacement of DNMT1 and inhibi-tion of DNA methylation53,69, and the recruitment ofchromatin-remodelling complexes to the mouse T
H2-
cytokine locus76. However, the molecular details of themechanism by which GATA3 induces epigenetic modi-fications remain to be fully elucidated. In physiologicalsituations, STAT6 and/or Notch signalling are activatedbefore GATA3 and help to induce GATA3 expressionby binding to, transactivating and recruiting HATs and
other chromatin modifiers to one or both of its promot-ers18,74,77. Once induced, GATA3 binds to its promoter,sustaining its own expression through direct activa-tion and recruitment of the H3K4 methyltransferaseMLL56. STAT6 also facilitates T
H2-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 tothe Il4CNS2 to a co-activator of Il4 in a process thatdepends on Notch signalling61. Therefore, althoughT
H2-cell differentiation can be initiated either through
STAT6-dependent or Notch-dependent pathways, it isstabilized by the autoactivation of GATA3 and by theGATA3-mediated epigenetic modification of Gata3 andthe T
H2-cytokine locus.
IFN and TH1-lineage commitment. IFNG is not clus-
tered with other co-expressed cytokine genes. In all ver-tebrates except rodents, the nearest upstream neighboursof IFNG are IL22 andIL26, which are mainly expressed
by TH17 cells21,31,78, with the housekeeping geneMDM1located further upstream; the nearest downstream geneto IFNGis~500 kilobases away. In mice and rats, com-plex structural rearrangements are evident >70 kilobasesupstream ofIfng,and Il26 is absent; a few remnants ofsequences from Il26remain, which is consistent with thisgene having been lost in rodents as a result of the struc-tural rearrangements79(FIG. 4). Despite these differences,cell-type-specific patterns of IFNGexpression are simi-lar in rodents and humans, suggesting that the essentialregulatory elements and their relationships are conservedand are proximal to these structural changes. Consistentwith this possibility, multiple regulatory elements andCNS have been identified in a region that extends6070 kilobases upstream and downstream of the mouseIfnglocus. These include enhancers at CNS-34, CNS-22,CNS-6, CNS+1820 and CNS+29, as well as a putativeinsulator (BOX 1)at CNS+46, although enhancer functionhas been confirmed in vivofor only CNS-22 (REF. 80) anda previously described enhancer in intron 1 (REF. 81).Recent genome-wide analyses of DNaseI hypersensitivesites and histone modifications in human CD4+T cellssuggest that a similar set of regulatory elements is presentin the human IFNGlocus44,46,82(FIG. 4).
Activated naive CD4+T cells produce low levelsof IFN, indicating that the Ifnglocus is in a poisedstate. DNA at the mouse Ifngpromoter and at CNS-34,
CNS-22, CNS+29 and CNS+46 is demethylated in naiveT cells, and CNS-34 and CNS-22 exhibit low levels ofpermissive H3K4 dimethylation and H4 acetylation38,79,80.Conversely, moderate levels of repressive H3K27 trimethy-lation are present between Ifngand CNS+1820, fromCNS+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 silenc-ing. T
H1-cell differentiation in vitroand in response to
infection in vivoleads to a marked increase in H3K4dimethylation, H3 and/or H4 acetylation, the acquisitionof DNaseI hypersensitive sites at regulatory elementswithin the Ifng locus and a complete loss of repressive
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34 225470 6 +1820 +29 +46 +66
|
TH2 cell
TH1 cell
MouseCNS
CTCF CTCFCTCF
CTCF CTCF
STAT5T-betRUNX3
T-bet T-bet T-bet T-bet T-betSTAT4
T-bet T-betSTAT4
GATA3 GATA3STAT6
insenhenhenhenhenh
BLIMP1
HSIHSII
HSIII
-22 -4-63 -31 -18 +22 +40 +80 +119
HSIHSII
HSIII
Naive T cell
Ifng
enh enh enh enh enh ins
Permissive histonemodifications
Bivalent histonemodifications
Repressive histonemodifications
CNSDNaseI hypersensitive site
HumanCNS
pro
pro
H3K27 trimethylation throughout the locus59,79,80,8385.However, repressive H3K9 dimethylation is induced andretained at specific sites of the Ifng locus in T
H1 cells86,
in which it may help to prevent aberrant transcriptional
initiation33. By contrast, when naive T cells differenti-ate into T
H2 cells, permissive histone modifications are
lost, repressive H3K27 trimethylation across the locusand CpG methylation are increased and NFAT loses theability to bind to the Ifng promoter38,79,83,86.
Similarly to the TH2-cytokine locus, permissive his-
tone modifications are initially acquired at the Ifnglocusunder both T
H1- and T
H2-cell-inducing conditions, but
TH1-cell-specific changes are evident by 17 hours follow-
ing stimulation70. STAT1, STAT4 and STAT5 all contrib-ute to these modifications, although they are not essentialfor T
H1-cell differentiation in vivo14. STAT1 probably pro-
motes the transcription of Ifng through T-bet expression
and has not been shown to bind to the endogenous Ifnggene. As with T
H2-cell differentiation, STAT5 directly
promotes the transcription of Ifngby binding to CNS-6,the Ifng promoter and CNS+1820, thereby facilitating
histone acetylation, chromatin remodelling and T-betbinding to the Ifng promoter87. STAT4 binds to the Ifngpromoter and many other elements, including CNS-22,leading to the induction of permissive epigenetic modifi-cations and the activation of gene expression. In addition,STAT4 recruits BRG1-containing chromatin-remodellingcomplexes to the Ifngpromoter, induces the acquisitionof HSI and HSII and promotes permissive histone modi-fications at this site17,55.
Although STAT4 supports Ifng expression and TH1-
cell differentiation synergistically with T-bet88, theexpression of which is enhanced by STAT4 (REF. 89),T-bet can induce T
H1-cell differentiation and the
Figure 4 |The Ifnglocus 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
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 trimethylatedH3K27 (light blue blocks) at the 3 end of the locus. DNA at CNS-34, CNS-22, the Ifngpromoter (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 IfngCNS-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 cells82and in T
H1 cells (C.B.W. and M.S., unpublished
observations). DNaseI hypersensitive sites in human CNS-4 and CNS+80 of the IFNGlocus 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 theVISTA web site) are shown at the bottom. T-cell subset-specific patterns of DNA methylation at this
locus are described in the text.
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|
Il17a Il17f Mcm3Pkhd1
CNS1 CNS2 CNS3 CNS4 CNS5 CNS6 CNS7 CNS8STAT3 STAT3RORt
200 kilobases
Permissive histone modifications CNS
TH2 cell
TH1 cell
TH17 cell
Naive T cell
pro proproenh
production of IFNin the absence of STAT4 when theexpression of T-bet is forced71,90. However, under physi-ological conditions, STAT4 and IL-12 are required forT
H1-cell differentiation, as their absence substantially
reduces or abolishes this process9193. T-bet directlytransactivates Ifngtranscription and has many addi-tional effects. More specifically, it binds to the Ifngpro-moter and to many enhancers80,94,95 (FIG. 4), it induces theexpression of HLX and RUNX3, it binds with these tran-scription factors to the Ifng promoter and with RUNX3to the Il4silencer12,13,96and it inhibits GATA3 expres-sion and function16,17. T-bet binds to the Ifngpromotereven when its DNA is repressively methylated97, whereit displaces HDAC-containing complexes and possiblyrecruits HATs98. Moreover, the Weinmann laboratory99recently showed that T-bet can directly recruit jumonji-domain-containing protein histone demethylase 3(JMJD3) to remove repressive H3K27 trimethylationand the histone methyltransferases SET7 (also knownas SET9 and KMT7) to induce H3K4 dimethylation,
thereby creating a permissive chromatin state. Together,these functions probably explain how forced expres-sion of T-bet can induce the expression of IFN even incommitted T
H2 cells100.
TH17-cytokine loci and lineage commitment. Much less
is known about the regulatory mechanisms and epige-netic processes that control T
H17-cell differentiation.
IL-17A and IL-17F are typically co-expressed by TH17
cells, and the genes that encode them are co-localizedin mammals, suggesting that they may be coordinatelyregulated by shared regulatory elements (similarly to theT
H2-type cytokines). In mice, eight candidate regulatory
elements have been described in the Il17locus based onsequence conservation101(FIG. 5). In these eight elements,as well as the Il17a and Il17f promoters, permissive H3acetylation is induced or increases solely or to a greaterdegree in naive CD4+T cells that are cultured underT
H17-cell-inducing conditions than in those that are
cultured under TH
1- or TH
2-cell-inducing conditions101.Consistent with its crucial role in T
H17-cell differen-
tiation, STAT3 binds to and induces H3 acetylation atthe Il17a andIl17fpromoters20,21,102. The T
H17-lineage-
specific transcription factors RORt and ROR do notseem to bind to these promoters, but can bind to CNS2,which is a ROR-dependent enhancer that is located justupstream of Il17a24. The transcription factors that bindto the other CNS and their function, if any, are unknown.The genes that encode other T
H17-type cytokines (IL-21,
IL-22 and IL-26) are located on different chromosomesand, curiously, are close to genes that are expressed byT
H1 cells but not by T
H17 cells. Il21is located next to Il2,
and Il22 andIl26 (or its remnants in rodents) are located
next to Ifng. The juxtaposition of TH1-type cytokinegenes to T
H17-type cytokine genes suggests that they
may be regulated in part by competition for or alternativeuse of regulatory elements. However, these possibilities,and the epigenetic processes by which this might beachieved, have not been investigated.
Structuring regulatory relationships
The location of regulatory elements within the genome islinear and fixed, whereas the actual relationships of theseelements to each other and to their cognate genes are non-linear, mobile and dynamic. The three-dimensional struc-ture of chromosomes changes during cell differentiation,
Figure 5 |The Il17aIl17flocus in mouse naive, T helper 1, T helper 2 and T helper 17 cells. Naive mouse CD4+
T cells have weak permissive histone H3 acetylation (light green blocks) at conserved non-coding sequence 1 (CNS1),
CNS5, CNS7 and CNS8, which is reduced in T helper 1 (TH1) and T
H2 cells. By contrast, T
H17 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 Il17aand Il17fpromoters in TH17
cells
20,21,102
and retinoic-acid-receptor-related orphan receptor-t(RORt) has been shown to bind to CNS2, at leastwhen 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.
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such that loops of chromatin containing relevant genescan be extended outside the chromatin territory. Suchchromatin looping can bring distal regulatory elementsin proximity to one another and to the promoters of theirtarget genes, thereby facilitating gene expression103105.
Chromatin looping allows the Il4, Il5and Il13 pro-moters to come into proximity with each other in manycell types, which results in a basal conformation that isnot specific to T cells. However, studies in mice haveshown that the LCR is located close to these promot-ers only in T cells, apparently helping to poise this locusfor subsequent activation106. STAT6, the T
H2 LCR and
probably GATA3 are required to establish this poisedconformation during T-cell development, but once it hasbeen established, the conformation is maintained andis similar in naive, T
H1 and T
H2 cells. T
H2-cell-specific
changes to this conformation are induced in response tocell activation in T
H2-cell-inducing conditions. This trig-
gers the expression of special AT-rich sequence bindingprotein 1 (SATB1; an architectural factor), which bindsto CNS1, CNS2 and other sites across the locus and pro-
motes the formation of additional loops and more inti-mate interactions between regulatory elements and theIl4, Il5 and Il13promoters107. In the absence of SATB1these changes are lost and T
H2-type cytokine expression
is compromised. SATB1-induced chromatin loopingextends to include the flanking Kif3agene, suggestingthat the regulatory domain of the T
H2-cytokine locus
includes this gene.Genome-wide studies suggest that CCCTC-binding
factor (CTCF) may also be involved in the three-dimen-sional organization of the T
H2-cytokine locus. CTCF is a
self-interacting, insulator protein that can mediate chro-matin looping to proximal elements within a locus andcan insulate a locus from surrounding chromatin, nearbygenes and regulatory elements105,108. CTCF co-localizeswith cohesins, which contributes to the CTCF-dependentinsulator function and perhaps to CTCF-mediated chro-matin looping109. In mouse and human T cells, CTCFand cohesins have been shown to strongly bind to sitesthat flank the T
H2-cytokine locus as well as to RHS2 and
Hss3 within the locus46,109.Similar mechanisms may be involved at the Ifngand
Il17loci, which are flanked by sites where CTCF andcohesins are bound46,109. CTCF binds to Ifngin T
H1 cells,
but not TH2 cells109, and may help to induce a T
H1-specific
locus architecture (M.S. and C.B.W., unpublished obser-vations), including T
H1-specific chromatin looping that
brings IfngCNS+1820 close to the Ifngpromoter110.Interchromomal interactions between the Ifng andthe T
H2-cytokine locus in naive T cells, which are lost
after TH1- and T
H2-cell differentiation, have also been
described110, but the molecular basis and functionalimportance of these interactions remain unclear.
Heritability, plasticity and diversity
The TH
1- and TH
2-cell paradigm arose from studiesof long-term T-cell clones1. Commitment to a singlelineage was later shown to be acquired after 4 cyclesof cell division under T-cell-lineage inducing condi-tions in vitro62,63. Indeed, after 4 cell divisions T
H1 or
TH2 cells did not express lineage-inappropriate cytokines
or lose robust expression of lineage-appropriatecytokines when polarizing conditions were removedor switched. Commitment was attributed to heritablealterations to the cytokine genes themselves and/or tothe lineage-specifying transcription factors GATA3 andT-bet. Recent studies have identified the mechanismsthat are involved in lineage commitment.
Silencing and remembering.Silencing of Ifngin TH
2cells occurs at many levels. GATA3 and STAT6 bind tothe Ifngpromoter, and this is associated with bindingof PRC1 and the H3K27 methyltransferase EZH2 tothe Ifng locus, as well as with increased acquisition ofrepressive H3K27 trimethylation, during T
H2-cell dif-
ferentiation86. As a result, IFN expression is repressed,although another report suggests that DNA binding byEZH2 occurs in both T
H1 and T
H2 cells and may have
other effects111. GATA3 also interacts with T-bet fol-lowing its tyrosine phosphorylation by IL-2-inducibleT-cell kinase (ITK) and can inhibit T-bet function16,98;
this may account for the contribution of ITK to TH2-celldifferentiation112. Whether the expression of Tbx21(thegene encoding T-bet) is directly inhibited by GATA3 isnot known. GATA3 also binds HDACs98, which it mayrecruit to Ifng, and inhibits the expression of IL-12R2and STAT4 (REF. 17). T
H2-cell differentiation38and/or
loss of STAT4 signalling113result in the recruitment ofDNMT3a and an associated increase in CpG methyla-tion at the Ifngpromoter, perhaps as an indirect result ofreduced T-bet-mediated H3K4 methylation and bind-ing of DNMT3aDNMT3l complexes to unmodifiedH3K4 (REF. 114). Finally, the transcriptional repressorB-lymphocyte-induced maturation protein 1 (BLIMP1)represses IFN expression in T
H
2 cells, in which it ishighly expressed, possibly by binding to CNS-22 inthe Ifnglocus (FIG. 4)and in many sites near or in theTbx21 gene115.
In addition to silencing Ifng, GATA3 is essential forpromoting the expression of T
H2-type cytokines and
consequently for inducing and, at least partly, maintain-ing the T
H2-cell response68,116,117. Deletion of GATA3 in
TH2 cells that had been generated in vivoin response
to infection with Nippostrongylus brasiliensisor in vitroby culturing naive CD4+T cells for 45 weeks in T
H2-
inducing conditions resulted in reduced numbers ofIL-5- and IL-13-producing cells (but not IL-4-producingcells) and in a reduction in the production of IL-5 and
IL-13, as well as a ~50% reduction in the amount ofIL-4 produced per cell117. Similarly, silencing of Ifngwas markedly impaired when GATA3 was deleted atthe start of T
H2-cell differentiation. Interestingly, how-
ever, when IFNexpression was evaluated after Gata3had been deleted from established T
H2 cells, silencing
of Ifngwas found to be less impaired117. These findingssuggest that GATA3 is not essential for but does helpto maintain the permissive epigenetic state of the T
H2-
cytokine locus and the repressive state of the Ifnglocus.In addition, the data indicate that GATA3 has a modestrole in transactivating Il4 and a more important rolein transactivating Il5 and Il13in committed T
H2 cells.
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Maintenance of this permissive epigenetic state and theexpression of Gata3, Il4, Il5 and Il13 depends on theH3K4 methyltransferase MLL, which binds to Gata3and the T
H2-cytokine locus in memory T
H2 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 impor-
tant for maintaining the differentiation state13,118. Adominant-negative form of T-bet inhibits the expres-sion of IFN and abolishes DNaseI hypersensitive sitesfrom the Ifnglocus in developing T
H1 cells, but not in
long-term TH
1-cell lines. The stability of TH
1 clonescorrelates with marked DNA demethylation within theIfnglocus, which is acquired more slowly than permis-sive chromatin modifications and may contribute to orbe a marker of heritable commitment. Similarly to Il5andIl13in T
H2 cells, the maintenance of the expres-
sion of IL-12R2 and HLX requires T-bet, as theirexpression was lost when T-bet function was blocked.Thus, the full programme of T
H1- and T
H2-cell-specific
gene expression cannot be sustained without contin-
ued expression of T-bet and GATA3, respectively. Andalthough the expression of Ifngin T
H1 cells and Il4
in TH
2 cells can be partly sustained through herit-able epigenetic modifications in the absence of theselineage-specifying transcription factors, in physiologicalcontexts the expression of Ifngand Il4 is collabora-tively sustained by these transcription factors and byepigenetic 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 forsilencing the T
H2-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 TH
2-cytokine locus is heritable and sustained in the absenceof T-bet has not to our knowledge been determined. Inaddition, T-bet is thought to be required for the silencingof Il4in T
H1 cells mainly, although not completely, by
cooperatively binding with RUNX3 to the Il4silencer,which is located 3of Il4at HSIV12,96. RUNX proteinsinduce heritable silencing in other contexts119, but themechanism by which they do so at the Il4silencer is notclear. The Il4silencer is crucial for silencing the expres-sion of IL-4 in T
H1 cells120, possibly through a switch that
allows constitutively bound EZH2 to generate repressivetrimethylated H3K27 across this locus67.
TH
17-cell differentiation is strongly inhibited by
IL-4, IL-27 and IFN, attenuated by IL-2, more read-ily achieved in T-bet-deficient cells and repressed byforced expression of T-bet20,21,121. The inhibitory effectsof IL-2 and IL-27 on the differentiation of this lineagedepend on STAT5 and STAT1, respectively. STAT1,STAT5 and STAT6 bind to the Il17apromoter, wherethey may compete with STAT3 for binding121. Inhibitionby STAT5 may also be mediated by the induction ofFOXP3, which binds to and inhibits RORt22. Whetherany of these STAT-dependent effects contribute directlyto the silencing of IL-17 expression and the mechanismsby which T
H17-cell differentiation is stably repressed are
not known.
During the induction of TH17 cells, the expression
of TH1- and T
H2-type cytokines is repressed by TGF,
which inhibits T-bet and GATA3 expression122,123.TGF-induced SMAD (mothers against decapenta-plegic homologue) proteins can bind to the promotersand probably repress Tbx21and Ifngin T
H17 cells124.
However, there is currently no evidence suggesting thatthese effects are heritable in the absence of TGF, orthat T
H17-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 areoften co-expressed by cells in vivoand that T-bet mayhelp to induce the expression of IL-23R and be inducedby IL-23 in T
H17 cells125.
Forgetting or disregarding. A limitation of the TH1, T
H2,
TH17 paradigm and most of the studies cited above is
that the systems that were used in each study were oftendesigned for the purpose of showing what is possiblerather than seeking to recapitulate what actually occurs
in vivo. Although the relevance of these subsets to hostdefence, autoimmunity and allergy is clear, mutuallyexclusive, canonical phenotypes and irreversible commit-ment are often violated in vivo. CD4+T cells that expressIFN plus IL-17 and/or IL-22 are commonly generatedin vivo78,126131. And although T
H17 and T
H1 cells typi-
cally express either CC-chemokine receptor 6 (CCR6) orCXC-chemokine receptor 3 (CXCR3), respectively, cellsthat produce both T
H1- and T
H17-type cytokines typically
express both receptors31,78,128,132. Memory CD4+T cellsthat produce IFN in conjunction with IL-10, produceIL-17 and IL-10 or produce IFN in conjunction withIL-4 have been described in mice and humans133139.
Thein vivoinduction of cells that co-express cytokinesof mixed lineages and the lack of irreversible silencing oflineage-inappropriate cytokines in some T
Hcells prob-
ably reflect the more diverse and resource-limited natureof life in vivoas opposed to the homogeneous environ-ments that are constructed in vitro140. Heterogeneity inthe mix and abundance of cytokines, Notch ligands,peptideMHC complexes and co-stimulatory molecules,asymmetric cell division and variation in the number oftimes cells have divided provide ample opportunities fordiversification and retention of plasticity or heritability.
At present, little is known about the mechanismsby which this diversity is achieved and whether suchdiversity is itself heritable, reflecting a permanent
dtente that is achieved through the loss of cross-lineagecounter-regulatory mechanisms. The complex networkof epigenetic processes and feed-forward networks thatare evident in canonical T
H-cell subsets indicate that lin-
eage fidelity could be tweaked at many points to achievediversity and maintain a desirable degree of plasticity.
Concluding remarks
There is now a compelling body of evidence showing thatthe state of chromatin and DNA methylation at lineage-restricted cytokine and transcription factor genes, aswell as their regulatory elements in T
Hcells, both reflects
and affects their functions in transcription. There is also
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increasing clarity regarding the specific epigenetic modi-fications that are associated with active and accessible,inactive but poised, and silenced loci, but the extent towhich these modifications work in a combinatorialmanner to encode and finely tune transcription is notyet completely clear.
A future challenge will be to determine with greaterclarity how specific combinations of epigenetic modifi-cations are established by networks of lineage-specifyingtranscription factors, and whether, when and how theycan later be removed or selectively modified to achieveor alter T
H-lineage specification. Such analyses should
help to unravel the basis for the non-canonical pat-terns of cytokines that are expressed by T
Hcells in vivo,
and whether these patterns reflect residual plasticity ofcells within the main T
Hlineages, stable subsets within
these lineages or a snapshot in time of cells in the proc-ess of shifting from one lineage to another. New, high-resolution and comprehensive approaches provide toolsto probe for these mechanisms. Initial insights from suchstudies show that total human resting CD4+T cells exhibit
molecular signatures of open chromatin at the Ifnglocus(FIG. 4)that resemble those found in mouse and humanT
H1 cells82. This indicates that resting memory T cells
maintain epigenetic signatures of previous events while
retaining pla