Project Summary The transcription factor STAT5 plays key roles in governing the development of multiple CD4+ T cell lineages, including regulatory T cells (Tregs) and T follicular helper cells (TFH). STAT5 does this by both initiating and inhibiting gene transcription. However, the molecular mechanisms that determine whether STAT5 binding leads to gene transcription or gene silencing remain largely unknown. STAT5 has been shown to interact with the co-activators EP300 and CREBBP and this has been suggested to modulate STAT5 function; however, co- repressors that functionally modulate STAT5 function remain essentially undefined. Equally unclear is whether STAT5:co-repressor complexes are preferentially targeted to STAT5-repressed genes, and if so, what the mechanism is that accounts for this differential targeting. Thus, key gaps in our current knowledge of STAT5 function in T cells include: (i) identification of biologically relevant STAT5:co-repressor complexes that affect T cell development, (ii) information about how STAT5:co-repressor complexes affect gene expression in T cells, and (iii) molecular insights into how STAT5 differentially targets co- repressor complexes to STAT5-repressed genes. Our preliminary data strongly supports a key role for the co-repressors NCOR1 and NCOR2 in regulating STAT5-dependent T cell differentiation. Deletion of either Ncor1 or Ncor2 had relatively mild effects on T cell development. In contrast, deletion of both Ncor1 and Ncor2 had dramatic impacts on T cell differentiation in the thymus. These impacts included a striking block in the development of FOXP3+ regulatory T cells in the thymus, and a clear increase in T follicular helper cells in Peyers patches. Thus, loss of NCOR1/2 in T cells parallels several of the phenotypes observed in STAT5 knockout mice. Our overarching hypothesis is that STAT5 interactions with the co-repressors NCOR1 and NCOR2 play a critical role in T cell development. We further propose that STAT5/NCOR complexes are targeted to a unique subset of STAT5-repressed genes via sequence-specific DNA binding sites or interactions with other transcription factors that also help recruit NCOR co-repressor complexes. Successful completion of these aims will provide insights into the mechanism by which STAT5 alters gene transcription and chromatin structure to promote T cell differentiation. It will also provide fundamental insights into the mechanisms by which transcription factors can selectively repress transcription of specific genes without blocking transcription of other genes induced by that same transcription factor.