Transcriptional Control of the Nfatc1 Gene in vivo

NFATc转录因子是淋巴细胞抗原受体信号的重要介导者。NFATc1基因的一个特性是在效应淋巴细胞中诱导表达短的异构体NFATc1αA。NFATc1αA与其他NFATc蛋白的生存功能不同。我们以前已经证明，在淋巴细胞中，NFATc1 P1启动子不足以将这种诱导作用转移到报告基因。然而，通过创建表达NFATc1-EGFP BAC转基因的小鼠，我们现在表明，NFATc1诱导的所有元素都存在于210kb的BAC DNA中。通过检测NFATc1基因最后一个内含子10的两个高度保守的DNase I超敏位点，分别命名为E1和E2，我们确定了E2元件是在T细胞中诱导NFATc1基因的转录增强子。在报告基因分析中，这种增强子赋予NFATc1启动子类似于内源基因的诱导行为。目前，E2增强子元件的活性是在带有缺失该元件的NFATc1-EGFP转基因小鼠中检测的，本项目将在体内芯片实验中详细分析NFATc1基因的启动子和E2增强子区域与NFAT、NF-kB等调节转录因子的结合，并测试因子结合的功能。我们还将研究表观遗传标记，特别是组蛋白修饰，这表明外周淋巴细胞个别亚群中转录的活跃和抑制。进一步的目标将是通过3C、染色体构象捕获、分析以及它们在转基因小鼠模型中的测试来识别NFATc1基因的其他调控元件。在现有创始人的外周淋巴细胞中表达的NFATc1/EGFP带有突变的增强子元件，这表明存在比内含子10更多的增强子，似乎在淋巴和非淋巴细胞中具有活性。最后，我们打算测试E2增强子对淋巴细胞分化和功能的活性。为此，我们计划产生BAC转基因小鼠，携带一个E2片段已被删除的NFATc1基因座。将这些NFATc1 deltaE2 BAC TG小鼠与NFATc1fl/fl x cre小鼠杂交，以灭活T或B细胞中完整的NFATc1基因，我们将能够在体内研究E2增强子在这些细胞中的作用。综上所述，我们的研究将提供关于淋巴细胞中NFATc1基因表达调控的全面综述，在我们看来，这对免疫反应具有重要意义，如果DYS调控，则对人类自身免疫性疾病的发生具有重要意义。

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.