Transcriptional Control of the Nfatc1 Gene in vivo

Nfatc1 基因的体内转录控制

• 批准号: 262975079
• 负责人: Professor Dr. Edgar Serfling
• 金额: --
• 依托单位: Labor für Molekulare Pathologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/262975079?language=en
• 关键词: Transcriptional; Control; Nfatc1; Gene; vivo

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.

NFATc转录因子是淋巴细胞中抗原受体信号的重要介质。Nfatc 1基因的一个特殊性质是短亚型NFATc 1 α A在效应淋巴细胞中的诱导表达。NFATc 1 alphaA与其他NFATc蛋白的不同之处在于其生存功能。我们以前已经表明，在淋巴细胞中的Nfatc 1 P1启动子是不足以将这种诱导转移到一个报告基因。然而，通过创建表达Nfatc 1-Egfp BAC转基因的小鼠，我们现在表明NFATc 1诱导的所有元件都存在于210 kb BAC DNA中。通过测试两个高度保守的DNase I超敏位点，指定为E1和E2，从Nfatc 1基因的最后一个内含子10，我们确定了E2元件作为转录增强子，用于在T细胞中诱导Nfatc 1基因。在报告基因测定中，该增强子赋予Nfatc 1启动子类似于内源基因的诱导行为。目前，E2增强子元件的活性在携带Nfatc 1基因座的转基因Nfatc 1-Egfp小鼠中检测，其中该元件已被删除。本项目将在体内ChIP试验中详细分析Nfatc 1基因的启动子和E2增强子区域与调控转录因子如NFAT、NF-kB等的结合，并检测因子结合的功能。我们还将研究表观遗传标记，特别是组蛋白修饰，这是在外周淋巴细胞的个别子集的活性和抑制转录的指示。进一步的目标将是通过3C、染色体构象捕获、测定和在转基因小鼠模型中的测试从Nfatc 1基因中鉴定额外的调控元件。Nfatc 1/Egfp在携带突变增强子元件的现有创始人外周血淋巴细胞中的表达表明存在比内含子10更多的增强子，这些增强子似乎在淋巴细胞和非淋巴细胞中具有活性。为此目的，我们计划产生BAC转基因小鼠携带Nfatc 1基因座，其中E2段已被删除。在将这些Nfatc 1 deltaE 2 BAC tg小鼠与Nfatc 1 fl/fl x cre小鼠杂交以灭活T或B细胞中的整个Nfatc 1基因后，我们将能够研究E2增强子在这些细胞中的体内作用。如果失调，会导致人类自身免疫性疾病。