Immune homeostasis is dependent on tight control over the size of a population of regulatory T (T reg ) cells capable of suppressing over-exuberant immune responses. The T reg cell subset is comprised of cells that commit to the T reg lineage by upregulating the transcription factor Foxp3 either in the thymus (tT reg ) or in the periphery (iT reg ) 1,2 . Considering a central role for Foxp3 in T reg cell differentiation and function 3,4 , we proposed that conserved non-coding DNA sequence (CNS) elements at the Foxp3 locus encode information defining the size, composition and stability of the T reg cell population. Here we describe the function of three Foxp3 CNS elements (CNS1-3) in T reg cell fate determination in mice. The pioneer element CNS3, which acts to potently increase the frequency of T reg cells generated in the thymus and the periphery, binds c-Rel in in vitro assays. In contrast, CNS1, which contains a TGF-β-NFAT response element, is superfluous for tT reg cell differentiation, but has a prominent role in iT reg cell generation in gut-associated lymphoid tissues. CNS2, although dispensable for Foxp3 induction, is required for Foxp3 expression in the progeny of dividing T reg cells. Foxp3 binds to CNS2 in a Cbf-β-Runx1 and CpG DNA demethylationdependent manner, suggesting that Foxp3 recruitment to this 'cellular memory module' facilitates the heritable maintenance of the active state of the Foxp3 locus and, therefore, T reg lineage stability.Together, our studies demonstrate that the composition, size and maintenance of the T reg cell population are controlled by Foxp3 CNS elements engaged in response to distinct cell-extrinsic orintrinsic cues.To determine cis-elements that are potentially involved in the control of T reg cell fate, we first examined permissive (mono-methylated histone H3 at Lys4 (H3K4me1), di-methylated H3K4 (H3K4me2), H3K4me3, H3K36me3, and acetylated H3K9/14 (H3K9/14Ac)) and nonpermissive (H3K9me2, H3K9me3 and H3K27me3) modifications of histone H3 bound to three Foxp3 CNS elements (CNS1-3; Fig. 1a) in CD4 + CD25 − Foxp3 − naive T cells (T N ),

In the course of infection or autoimmunity, particular transcription factors orchestrate the differentiation of T H 1, T H 2 or T H 17 effector cells, the responses of which are limited by a distinct lineage of suppressive regulatory T cells (T reg ). T reg cell differentiation and function are guided by the transcription factor Foxp3, and their deficiency due to mutations in Foxp3 results in aggressive fatal autoimmune disease associated with sharply augmented T H 1 and T H 2 cytokine production [1][2][3] . Recent studies suggested that Foxp3 regulates the bulk of the Foxp3-dependent transcriptional program indirectly through a set of transcriptional regulators serving as direct Foxp3 targets 4,5 . Here we show that in mouse T reg cells, high amounts of interferon regulatory factor-4 (IRF4), a transcription factor essential for T H 2 effector cell differentiation, is dependent on Foxp3 expression. We proposed that IRF4 expression endows T reg cells with the ability to suppress T H 2 responses. Indeed, ablation of a conditional Irf4 allele in T reg cells resulted in selective dysregulation of T H 2 responses, IL4-dependent immunoglobulin isotype production, and tissue lesions with pronounced plasma cell infiltration, in contrast to the mononuclear-cell-dominated pathology typical of mice lacking T reg cells. Our results indicate that T reg cells use components of the transcriptional machinery, promoting a particular type of effector CD4 + T cell differentiation, to efficiently restrain the corresponding type of the immune response.T reg cell deficiency results in activation and expansion of CD4 + and CD8 + T cells, dendritic cells, granulocytes and macrophages, and greatly increased production of a wide range of cytokines including interleukin (IL)-2, T H 1 and T H 2 cytokines 6,7 . Expression of Foxp3 is required for the establishment and maintenance of T reg lineage identity and suppressor function [8][9][10][11] . Our recent study suggested that in T reg cells Foxp3 might regulate expression of IRF4 (refs 12 -14) a transcription factor that is indispensable for T H 2 effector cell differentiation 15,16 . Furthermore, a recent study suggested a prominent role for IRF4 in T H 17Correspondence and requests for materials should be addressed to A.Y.R. (rudenska@mskcc.org). † Present address: Department of Immunology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Information Reprints and permissions information is available at www.nature.com/reprints. Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature. Fig. 1a, b). Irf4 messenger RNA was increased in thymic and peripheral Foxp3 + T reg cells in comparison to CD25 − Foxp3 − CD4 + T cells (data not shown) 8 . Furthermore, Foxp3 knockdown using a retrovirally encoded Foxp3-specific short hairpin RNA resulted in a marked diminution in Irf4 mRNA ( Supplementary Fig. 1c 1...