We study the transcriptional control of T cell development and function. T cells are essential for immune responses. Most of them recognize peptide antigens presented by class I (MHC-I) or class II (MHC-II) classical Major Histocompatibility Complex molecules, and express either of two surface glycoproteins (called coreceptors) that contribute to antigen recognition: CD4, which binds MHC-II, or CD8, which binds MHC-I. Coreceptor expression on mature T cells is mutually exclusive and is essentially dictated by MHC specificity. That is, the general rule is that MHC I-specific T cells are CD4-CD8+ (CD8 cells), whereas MHC II-specific T cells are CD4+CD8- (CD4 cells). In addition, CD4 and CD8 T cells perform distinct functions upon antigen encounter: whereas CD8 T cells typically differentiate into cytotoxic effectors, CD4 T cells provide help to other components of the immune system (and of mucosal barriers) and have essential regulatory functions. CD4 and CD8 T cells differentiate in the thymus from precursors that express both CD4 and CD8 ('double-positive', DP), and this is accompanied by their pre-programming for helper and cytotoxic functions, respectively. Because of their central role in immune responses, CD4 T cells are essential to control infections (they are the key target of the human immunodeficiency virus HIV), and their development in the thymus has remained a key area of focus in the laboratory. Our previous studies had shown that the transcription factor Thpok is required for CD4 T cell differentiation in the thymus and notably represses CD8-lineage gene expression and CD8 T cell differentiation. We have also shown that the combined activities of Thpok and the related transcription factor LRF (Leukemia-Lymphoma Related Factor) are essential for the emergence of helper effector functions in MHC II-restricted thymocytes and for the maintenance of such functions in post-thymic CD4 T cells. Disruption of these factors alters the functional responses of CD4 T cells, as assessed by their cytokine production or expression of surface molecules needed for helper functions; we are currently analyzing the function of these factors in immune responses in vivo. In addition, we are continuing investigations of the transcriptional circuitry driving CD4-lineage differentiation in the thymus by interrogating the impact of Thpok on gene expression and chromatin organization. Epigenetic mechanisms, involving chromatin structure and covalent DNA or histone modifications, are essential for proper control of gene expression in association with transcription factors. Particular attention has focused on histone modifications, including methylation of specific lysine residues. Notably, trimethylation of histone H3 lysine 27 (H3K27Me3) at gene promoters is associated with transcriptional repression and has been involved in the control of gene expression in tumor cells. H3K27Me3 can be demethylated by Utx and Jmjd3, two enzymes which are part of a large family of histone demethylases sharing a similar JmjC catalytic domain. In studies initiated in collaboration with John O'Shea's laboratory (NIAMS), we had found that the Thpok locus was highly 'decorated' with H3K27Me3 in DP thymocytes, which do not express Thpok, whereas the mark was entirely removed in CD4-lineage thymocytes or T cells, which express Thpok. This observation suggested that H3K27Me3 removal was important for Thpok expression, prompting us to investigate the control of H3K27Me3 homeostasis in developing T cells. To test this hypothesis, we generated mice with T cell specific-disruption of Jmjd3 and Utx. Analyses of these animals showed that these enzymes are not required for early T cell development, up to the DP stage of differentiation. Furthermore, and unexpectedly, we found that the removal of the H3K27Me3 mark at the Thpok promoter required neither Jmjd3 not Utx, suggesting the involvement of additional, so far unknown, demethylases, or of histone replacement mechanisms. Accordingly, neither Thpok expression nor the differentiation of DP cells into CD4-lineage thymocytes were affected by the disruption of both Jmjd3 and Utx. In contrast, these enzymes were needed, in a partly redundant manner, for the terminal maturation of such CD4-lineage thymocytes. Specifically, we found that Jmjd3 and Utx promote H3K27Me3 removal at, and expression of S1pr1, encoding a sphingosine phosphate receptor needed for thymic egress, and Klf2, a transcription factor needed for S1pr1 expression. In addition, we have preliminary evidence that these enzymes contribute to the effector differentiation of mature T cells (including expression of cytokine genes and migration to tissues). Accordingly, we had found that Jmjd3 and Utx are required for the differentiation of iNK T cells, a subset of 'innate-like' lymphocytes that recognize CD1d-bound lipid molecules and undergo effector differentiation in the thymus. Collaborative work in the laboratory of Sasha Tarakhosky (Rockefeller University) found evidence that H3K27 methylation-demethylation contributes to controlling the promoter of the gene encoding PLZF, a transcription factor required for iNK T cell development. These studies identify critical but unexpected functions of H3K27Me3 demethylases Jmjd3 and Utx in CD4+ T cell development. They further demonstrate that these enzymes are required for H3K27Me3 homeostasis, although their highly specific impact, even in non-dividing cells, suggests that alternative mechanisms contribute to this process.