Research Focus

Transcriptional partners in regulatory T cells: Foxp3,Runx and NFAT

Hui Hu, Ivana Djuretic, Mark S. Sundrud and Anjana Rao

The CBR Institute for Biomedical Research and the Department of Pathology, Harvard Medical School, Boston, MA 02115, USA

Update TRENDS in Immunology Vol.28 No.8

A general theme in gene regulation is that transcriptionfactors never function alone. Recent studies have empha-sized this concept for regulatory T cells, a unique lineageof CD4+ T cells that exert active immune suppression andare essential to maintaining self-tolerance.

Regulatory T cells and Foxp3Cooperation between transcription factors is wellestablished for acute gene regulation in the context oftwo well-studied promoters: the b-interferon promoter,where b-interferon induction is accomplished through inti-mate interactions between interferon regulatory factor andnuclear factor (NF)-kB within the context of a complexenhanceosome [1], and the promoter for interleukin-2(IL-2), where the rapidly induced transcription factorsnuclear factor of activated T cells (NFAT), activatorprotein 1 (AP-1) and NF-kB function cooperatively andsynergistically to switch on IL-2 expression by activatedT cells [2]. The same principles of transcriptional coopera-tion apply during cell lineage specification, a processdriven by nearly simultaneous inputs from strong positivefeedback loops that reinforce the choice of one specific cellfate, and negative feedback loops that suppress alternatechoices [3]. In regulatory T cells, the cooperating transcrip-tion factors are NFAT, the forkhead transcription factorFoxp3, and the runt-related transcription factor Runx [alsoknown as acute myeloid leukemia factor (AML)].

Regulatory T cells come in several ‘flavors’: ‘natural’regulatory T (nTreg) cells, which develop from CD4+ Tcells in the thymus, ‘induced’ regulatory T (iTreg) cells,which are efficiently generated from naive T cells by differ-entiation in the presence of transforming growth factor-band IL-2, and type 1 regulatory cells, which produce IL-10[4] (Figure 1). nTreg and some iTreg lineages character-istically express the transcription factor Foxp3 [4]. Hum-ans with mutations in the X-linked FOXP3 gene develop amultiorgan autoimmune inflammatory disease known asimmunodysregulation, polyendocrinopathy, enteropathy,X-linked syndrome (IPEX), and a spontaneous mutationof the Foxp3 gene in the scurfy mouse strain is alsoassociated with aggressive autoimmune disease [5]. Theconstant presence of nTreg cells in the periphery seems tobe essential because acute ablation of Foxp3+ cells in adultmice results in a similar syndrome of progressive auto-immunity and inflammatory disease [6]. The peripheral

Corresponding author: Rao, A. (arao@cbr.med.harvard.edu).Available online 6 July 2007.

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tolerance mediated by Foxp3+ T cells complements andreinforces ‘central tolerance’, the process by which self-reactive T cell clones are deleted by ‘negative selection’ inthe thymus [7]. Both mechanisms are crucial to preventautoimmune disease.

Foxp3 is a member of the forkhead transcription factorfamily, which has over 40 other members with importantroles in a variety of cellular processes [8]. In the Foxpsubfamily, which has four members, Foxp1 is crucial forearly B cell development, in part because it regulates theexpression of the recombination-activating genesRag1 andRag2 [9], whereas Foxp3 is studied intensively because ofits essential role in nTreg cell development and function[4–6,10–14]. The transcription factors that partner withFoxp1 during B cell development have not yet been definedbut NFAT and Runx1 have been identified as two crucialpartners of Foxp3 [15,16].

NFAT and Runx (AML) transcription factorsThe NFAT family of transcription factors consists of fourproteins (NFAT1–4, also known as NFATc1–c4), whoseactivity is regulated by calcium and the calcium-dependentprotein phosphatase calcineurin [17]. NFAT proteinsregulate cell lineage specification, in addition to acutegene expression in diverse organs and cell types [17]. Theyhave been particularly well studied in cells of the immunesystem [18]. Stimulation of antigen receptors on T and Bcells, and Fc receptors on natural killer cells andmast cells,leads to calcium influx and calcineurin activation; calci-neurin, in turn, dephosphorylates and activates NFAT,which translocates from the cytoplasm to the nucleus,where it controls the inducible expression of many targetgenes [17,18].

Runx proteins are essential for hematopoietic celldifferentiation and fetal development [19,20]. The Runxfamily is composed of three members, Runx1 (also knownas AML1), Runx2 (also known as AML3) and Runx3 (alsoknown as AML2; the Runx nomenclature is used hereafter).Runx proteins are broadly expressed in the hematopoieticsystem, and their expression is regulated in a lineage-and developmental stage-specific manner. In thymocytes,Runx1 is an important transcriptional repressor of CD4expression; it is expressed through all maturation stages,withhighest expression inCD4�CD8�double-negative cells[20]. By contrast, Runx3 regulates CD4 silencing in CD8+

single-positive cells, where it is expressedathigh levels [20].The switch from one Runx protein to another can be abrupt;Runx1 is the predominantRunxprotein innaiveCD4T cellsbut is replaced by Runx3 in differentiated Th1 cells [21].

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Figure 1. Combinatorial regulation of transcription during Treg T cell development. This schematic representation illustrates the putative involvement of multiple

transcription factors throughout thymocyte development and peripheral differentiation of mature T cells. Recent studies implicate key regulatory roles for NFAT and Runx

transcription factors in the thymic generation of nTreg cells and peripheral development of iTreg cells from naive T cell precursors [15,16]. Further study of the molecular

interplay among these transcription factors is warranted. Abbreviations: DN, double negative; DP, double positive; IFN-g, interferon-g.

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BothRunxandNFATproteinsbind inasequence-specificmanner to the promoter and other regulatory elements oftarget genes; both recruit coactivator and corepressorproteins, includinghistone-modifying enzymesand chroma-tin remodeling complexes; and both can function to activateor repress target genes [17,22]. In addition, however, Runxproteins are capable of localizing to the nuclear matrix, asubnuclear structure thought to have a role in gene organ-ization and regulation, through a targeting sequence nearthe C-terminus [23]. These proteins might therefore func-tion not only as classical transcriptional activators andrepressors, but also as scaffolding proteins contributing tothe organization of higher-order nuclear structure.

Transcriptional partners of Foxp3There is structural, biochemical and functional evidence fora cooperative interaction between NFAT and Foxp3 [15].The crystal structures of NFAT–AP-1 and NFAT–Foxpcomplexes on an NFAT–AP-1 composite site in the Il2promoter have been solved; comparison of the structuresshows that Foxp andAP-1 occupy the sameDNA region andinteract with overlapping, but not identical, residues ofNFAT [15] (Figure 2). The NFAT–AP-1 complex activates,but the NFAT–Foxp3 complex represses, IL-2 expression[15]. TheNFAT–Foxp3 complex isnot invariably repressive,however: Foxp3 cooperates with NFAT to upregulateexpression of cytotoxic T lymphocyte-associated antigen-4(CTLA-4) andCD25, two surface receptors highly expressedby Treg cells. Foxp3 proteins withmutations that disruptedthe NFAT–Foxp3 interface were unable to downregulateIL-2 expression, upregulate CTLA-4 or CD25 expression, orsuppress T effector cell function in vivo [15]. Chromatinimmunoprecipitation assays confirmed that NFAT andFoxp3 bind together at the promoters of many target genes,including Il2, Ctla4 and Cd25; notably, Foxp3 binding to

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these sites was enhanced following stimulation, and wasblocked by the calcineurin inhibitor cyclosporin A, as wouldbe expected if bindingwas driven by cooperative interactionwith NFAT [15,24]. The results are consistent with the ideathatTreg cellsneed tobeactivated toexert their suppressiveeffects [5,10], and with microarray data showing that thepromoters of many genes switched on in hybridomas thathave been transduced to express Foxp3 contain both NFATand Foxp3 binding sites [14].

Ono et al. [16] present data supporting a similarphysical and functional interaction between Runx1 andFoxp3. They first showed, without involving Foxp3, thatRunx1 regulates IL-2 expression in Jurkat T cells andprimary mouse CD4+ T cells. Endogenous Runx1 wasfound at the Il2 promoter, even under resting conditions,and three Runx consensus sites in the Il2 promoter boundto Runx1 in vitro and were functional in reporter assays.RNA interference (RNAi)-mediated depletion of Runx1 inJurkat cells, or overexpression of a dominant-interferingversion of Runx1 (a C-terminally truncated proteinthat lacks the main activation and repression domains)in primary CD4+ T cells, led to diminished IL-2 productionafter stimulation, whereas overexpression of Runx1 inprimary CD4+ T cells increased IL-2 production [16]. Inter-estingly, an earlier study by Djuretic et al. [21] showed thata different family member, Runx3, was also a positiveregulator of IL-2 expression in primary murine T cells.Runx3 upregulated IL-2 expression when transduced intoCD4+ T cells, and T cells deficient in Runx3 producedconsiderably less IL-2 than did wild-type T cells [21]. Thesedata support positive roles for both Runx1 and Runx3 asactivators of IL-2 gene expression. Similarly to othermembers of transcription factor families, Runx proteinsdisplay a high degree of sequence similarity, particularly intheir DNA-binding domains, so it is not surprising that

Figure 2. Schematic and structural representations of Runx1 and Foxp3. Ono et al. [16] report a functional interaction between Runx1 and Foxp3, mediated by C-terminal

domains, indicated by asterisks. Crystal structures have been solved of ternary complexes featuring Runx1–CBFb [28] and NFAT–Foxp [15] on DNA as indicated. Putative

Runx and NFAT–Foxp binding sites within the Il2 promoter are shown. The complete functional relevance of both complexes on gene regulation in Treg cells has yet to

be fully understood, and higher-order complexes containing Foxp3, NFAT and Runx proteins are predicted (see the main text). DYK are three amino acids of Foxp3 that are

critical for its interaction with Runx1. Substitution of D, Y and K with V, H and L, respectively, abrogates the Foxp3–Runx1 interaction [16]. Abbreviations: ARRE, antigen-

receptor response element (from the murine Il2 promoter); CBFb, core binding factor b; ID, inhibition domain; L-zip, leucine zipper; ZnF, zinc finger.

Update TRENDS in Immunology Vol.28 No.8 331

different members might have redundant roles in T cellsand Treg cells.

Ono et al. [16] next addressed whether Runx1 was aninteracting partner for Foxp3 in Treg cells. This was some-thing of a conceptual leap because Foxp3 is clearly a repres-sor of IL-2 expression, whereas they had just demonstratedthat Runx1 is a likely activator [16]. They showed thatectopically introduced Runx1 and Foxp3 coimmunoprecipi-tated when overexpressed in cell lines, and used this assayto define the regions of interaction. The interaction seems tobeof relativelyhighaffinitybecause theauthorswereable tocoimmunoprecipitate endogenous Runx1 with endogenousFoxp3 from the lysates of human peripheral blood mono-nuclear cells (PBMCs). The result is remarkable, given thatonly a fraction of resting PBMCs express Foxp3 [5]. Amajortechnical caveat is that any pair of DNA-binding proteinsmight seem to interact if cell lysates are contaminated withgenomic DNA because DNA fragments that happen to con-tain high-affinity binding sites for both proteinswill link theproteins and effect an artefactual ‘coimmunoprecipitation’ –a problem that is best addressed by treating lysates withDNase or conducting the immunoprecipitation in the pre-sence of a DNA intercalator [21]. Ono et al. bypassed thiscaveat but functionally validated the biological importanceof the Runx1–Foxp3 interaction, by constructing a Foxp3mutant with three amino acid substitutions (the 329VHL

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mutant) that was impaired in its ability to interact withRunx1 but not with NFAT. Similarly to the NFAT inter-action mutants of Foxp3 described by Wu et al. [15], the329VHLmutant ofFoxp3wasunable to repress IL-2 expres-sion or upregulate CD25, CTLA4 or glucocorticoid-inducedtumor necrosis factor receptor (GITR) expression whenintroduced into primary CD4+ T cells. A lingering problemwith all mutational analyses is that the mutants areimpaired in some unexamined way – DNA-binding, proteinstability, localization to the correct intracellular compart-ment, or interaction with a protein partner other than theone being examined.

A major remaining question is: which Runx-familyprotein actually partners with Foxp3 in regulatory T cells?Ono et al. [16] focused on Runx1 but their supplementarydata also suggested the possibility that a different familymember might be the predominant partner in Treg cellsunder certain conditions because they showed that bothRunx2 and Runx3 interacted with Foxp3 in overexpressedcell lines. They showed in chromatin immunoprecipitationexperiments that Runx1 binds to Il2, Cd25, Gitr and Ctla4promoters, although these experiments were performed inCD4+ T cells, in which Runx1 is highly expressed, ratherthan in the relevant cell type, Treg cells [16]. Moreover,crossreactivity of the Runx1 antibody with other Runxfamilymemberswas not ruled out. To address this question,

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it will be necessary to compare the expression levels of eachRunx family member in Treg cells, and to examine thephenotype of Treg cells in mice in which individual ormultiple Runx proteins have been conditionally ablated.Runx3 is a particularly attractive candidate for partneringwith Foxp3 in regulatory T cells; Runx3-deficient micedevelop autoimmune colitis, in addition to asthma[25,26], and defects in Treg cell development or functionmight well contribute to these two immunological abnorm-alities.

The big picture – higher-order complexes containingFoxp3?The Foxp3–Runx1 interaction differs from theNFAT–Foxp3 interaction in one essential respect. Foxp3and Runx1 interact through regions distinct from theirDNA-binding domains, on sites that are widely separatedin the genome [16], whereas the cooperative interactionbetween NFAT and Foxp3 involves direct protein–proteincontacts between the DNA-binding domains of these twotranscription factors, at least some of which occur on ‘com-posite’DNAelementswithadjacent binding sites for the twotranscription factors (Figure 2). It should therefore bepossible to assemble larger complexes containing all threeproteins [15] (Figure2).Defining the structural basis for thistripartite interaction will be of enormous interest.

The tripartite interaction is also predicted to involve atranscriptional switch, analogous to that previously pos-tulated for NFAT with AP-1 or Foxp3 [15]. Runx1 is themajor Runx familymember expressed in resting CD4+ cellsand is bound to the Il2 promoter in these cells [16]; itcooperates with NFAT–AP-1 complexes, which bind onlyafter stimulation to activate IL-2 expression. By contrast,in Treg cells, Runx proteins presumably cooperate withNFAT–Foxp3 complexes to repress Il2 but activate Ctla4,Cd25 and Gitr gene expression. Indeed, RNAi-mediateddepletion of Runx1 in human CD25highCD4+ T cellsrelieved their proliferation block and decreased their sup-pressive activity in vitro. NFAT–Foxp3 complexes mighthave a competitive advantage in Treg cells, whichmight bedefective in the signaling pathways needed to induce AP-1[27]; even minimal stimulation would therefore result inNFAT–Foxp3 complexes occupying regulatory elements inDNA [14,15,24]. However, to show definitively whetherRunx1 (or Runx3; see earlier) is required for Foxp3-de-pendent transcriptional programs, and hence for properTreg cell function, future studies on the phenotype ofappropriate Runx-deficient mice are needed.

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