Transcriptional Control of the Nfatc1 Gene in vivo

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NFATc transcription factors are important mediators of antigen receptor signals in lymphocytes. A specific property of the Nfatc1 gene is the inducible expression of the short isoform NFATc1 alphaA in effector lymphocytes. NFATc1 alphaA differs from other NFATc proteins by its survival function. We have shown previously that in lymphocytes the Nfatc1 P1 promoter is insufficient to transfer this inducibility to a reporter gene. However, by creating mice expressing an Nfatc1-Egfp BAC-transgene we showed now that all elements of NFATc1 induction are present within the 210 kb BAC DNA. By testing two highly conserved DNase I hypersensitive sites, designated as E1 and E2, from the last intron 10 of the Nfatc1 gene we identified the E2 element as a transcriptional enhancer for the induction of the Nfatc1 gene in T cells. In reporter gene assays, this enhancer confers an induction behavior similar to that of the endogenous gene to the Nfatc1 promoters. Currently, the activity of the E2 enhancer element is tested in transgenic Nfatc1-Egfp mice bearing an Nfatc1 locus in which the element has been deleted.In this project the promoter and E2 enhancer regions of Nfatc1 gene shall be analyzed in detail for the binding of regulatory transcription factors, such as NFAT, NF-kB and others, in ChIP assays in vivo, and the function of factor binding will be tested. We will also investigate epigenetic marks, in particular histone modifications, which are indicative for active and suppressed transcription in individual subsets of peripheral lymphocytes. A further goal will be the identification of additional regulatory elements from the Nfatc1 gene by 3C, Chromosome Conformation Capture, assays and their testing in transgenic mouse models. The Nfatc1/Egfp expression in peripheral lymphocytes from existing founders bearing mutated enhancer elements suggests the existence of more enhancers than that in intron 10 which appear to be active in lymphoid and non-lymphoid cells.Finally, we intend to test the activity of E2 enhancer on lymphocyte differentiation and function. For this purpose we plan to generate BAC transgenic mice bearing an Nfatc1 locus in which the E2 segment has been deleted. Upon crossing of those Nfatc1 deltaE2 BAC tg mice with Nfatc1fl/fl x cre mice for the inactivation of entire Nfatc1 gene in T or B cells we will be able to study the effect of E2 enhancer in vivo in these cells.Collectively, our studies will provide a comprehensive view on the regulation of Nfatc1 gene expression in lymphoid cells which, in our view, is of seminal importance for immune reactions and, if dys-regulated, for the generation of human autoimmune diseases.