Regulation of ENaC Transcription

描述（由申请人提供）：在理解上皮钠通道的受约束但有利的转录基态如何？亚单位（？？ENaC）基因的维持，以及多种调节蛋白之间以及这些蛋白复合物与染色质模板之间的复杂相互作用如何导致醛固酮诱导？ENaC转录。鉴于其作为钠平衡和血压的关键调节剂的临床重要性，缩小这一差距势在必行。在当前的项目期间，我们发现并表征了含有组蛋白赖氨酸-79甲基转移酶Dot 1a以及AF 9或Sirt 1的新型核阻遏物复合物。这些复合物与靶向区域的？ENaC启动子限制基础？ENaC在集合管中转录，并被醛固酮下调，导致去抑制。长期的目标是表征新的转录控制机制的醛固酮诱导基因，使用？ENaC来模拟集合管中的这些事件。本应用程序的目的是表征看门人和醛固酮依赖性激活功能的潜在机制，以及它们如何微调和整合到？？ENaC染色质环境中心假设是，这些反应依赖于关键转录因子的动态和协调作用，以及染色质修饰剂和修饰的变化，最终导致？ENaC。该提案的基本原理是，它将建立Dot 1a杂合物作为基础和醛固酮敏感性调节剂？？ENaC转录，并整合去阻遏和盐皮质激素受体的反式激活？ENaC介导醛固酮诱导。在当前资助期间取得的重大进展的指导下，这一假设将在三个具体目标中进行测试：1）定义Dot 1a-AF 9和Dot 1a-Sirt 1复合物在抑制？ENaC在结肠导管中的转录; 2）确定醛固酮诱导的重编程的动力学和介质在？ENaC启动子; 3）确定醛固酮诱导的？？ENaC转录。免疫沉淀/质谱，定量ChIP分析，染色体构象捕获，和启动子-报告基因分析将用于培养的集合管细胞和小鼠肾脏检查醛固酮诱导的动力学的Dot 1杂合物在？ENaC启动子，与新因子的相互作用，以及组蛋白修饰和启动子甲基化的变化。在BAC转基因小鼠中的研究将测试候选AF 9结合位点是否对内源性？？ENaC基因。这种方法是创新的，因为它代表了一个重大的偏离经典模型的盐皮质激素受体反式激活？ENaC。这项研究意义重大，因为它将促进我们对转录去抑制、核受体介导的转录激活和通过染色质的醛固酮信号传导的理解。 公共卫生相关性：肾小管上皮细胞钠通道对钠的重吸收是调节钠平衡、体液容量和血压的重要机制。这种蛋白质的异常表达水平可导致高血压、钾缺乏和水肿。该提案研究了细胞核中调节转录的相互作用蛋白质的新网络，这是产生这种蛋白质的最初分子步骤。

DESCRIPTION (provided by applicant): There is a large gap in understanding how a constrained, but favorable transcriptional ground state for the epithelial sodium channel ??subunit (??ENaC) gene is maintained, and how complex interactions between multiple regulatory proteins, and between these protein complexes and the chromatin template, result in aldosterone induction of ??ENaC transcription. Given its clinical importance as a key regulator of sodium balance and blood pressure, closing this gap is imperative. During the current project period, we discovered and characterized novel nuclear represor complexes containing the histone lysine-79 methyltransferase Dot1a together with either AF9 or Sirt1. These complexes associate with targeted regions of the ??ENaC promoter to constrain basal ??ENaC transcription in the collecting duct, and are downregulated by aldosterone, leading to de-repression. The long-term goal is to characterize novel transcriptional control mechanisms of aldosterone-inducible genes, using ??ENaC to model these events in the collecting duct. The objective of this application is to characterize the mechanisms underlying the gatekeeper and aldosterone-dependent activation functions, and how they are fine-tuned and integrated in the ??ENaC chromatin environment. The central hypothesis is that these responses depend on the dynamic and orchestrated action of key transcription factors and changes in the profile of chromatin modifiers and modifications that ultimately lead to expression of ??ENaC. The rationale for the proposal is that it will establish the mechanistic basis for the Dot1a heterocomplexes as basal and aldosterone-sensitive regulators of ??ENaC transcription, and for the integration of de-repression and mineralocorticoid receptor transactivation of ??ENaC in mediating aldosterone induction. Guided by substantial advances during the current funding period, this hypothesis will be tested in three specific aims: 1) Define the components, functions, and interplay of the Dot1a-AF9 and Dot1a-Sirt1 complexes in repressing ??ENaC transcription in colecting duct; 2) Define the dynamics and mediators of aldosterone- induced reprogramming at the ??ENaC promoter; and 3) Determine the dynamics, mediators, and roles of chromatin architecture in aldosterone-induced ??ENaC transcription. Immunoprecipitation/mass spectroscopy, quantitative ChIP assays, chromosome conformation capture, and promoter-reporter assays will be used in cultured collecting duct cells and mouse kidneys to examine the aldosterone-induced dynamics of the Dot1 heterocomplexes at the ??ENaC promoter, interactions with novel factors, and changes in histone modifications and promoter methylation. Studies in BAC transgenic mice will test whether the candidate AF9 binding site is critical for the responses of the endogenous ??ENaC gene. The approach is innovative because it represents a major departure from the classical model of mineralocorticoid receptor trans-activation of ?? ENaC. The proposed research is significant because it will advance our understanding of transcriptional de- repression, nuclear receptor-mediated transcriptional activation, and aldosterone signaling through chromatin. PUBLIC HEALTH RELEVANCE: Sodium reabsorption by the epithelial sodium channel in the kidney tubules is an essential mechanism involved in the regulation of sodium balance, fluid volume, and blood pressure. Abnormal expression levels of this protein can result in hypertension, potassium deficiency, and edema. This proposal examines a novel network of interacting proteins in the cell nucleus that regulate transcription, the initial molecular step in generating this protein.