epigenetic control of t‑helper‑cell

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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:

[email protected]

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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92 |FEBRUARY 2009 |VOLUME 9 www.nature.com/reviews/immunol

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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)


Permissive

H2aK5, H2bK12, H2bK15,H3K14, H3K18, H4K5and H4K8

KAT3A (CBP) andKAT3B (p300)

HDAC1 and HDAC2(in SIN3A, NURD andCoREST complexes)


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


KMT6 (EZH2) KDM6A and KDM6B(UTX and JMJD3)

Binds or recruits PRC1 complex andDNA methyltransferases

Repressive


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

epigenetic control of t‑helper‑cell

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