Regulation of ENaC Transcription

描述（由申请人提供）：在理解上皮钠通道的受限但有利的转录基态如何？？亚基(? ?ENaC)基因是如何维持的，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是如何导致醛固酮诱导的？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。在目前的项目期间，我们发现并表征了含有组蛋白赖氨酸-79甲基转移酶Dot1a和AF9或Sirt1的新型核阻遏子复合物。这些复合物与？？ENaC启动子约束基底？ENaC转录在集合管中，并被醛固酮下调，导致去抑制。长期目标是表征醛固酮诱导基因的新型转录控制机制，使用？？ENaC来模拟收集管中的这些事件。本应用程序的目的是表征看门人和醛固酮依赖激活功能的机制，以及它们如何在？？ENaC染色质环境。核心假设是，这些反应依赖于关键转录因子的动态和协调作用，以及染色质修饰物和修饰物的变化，最终导致ENaC的表达。该建议的基本原理是，它将建立Dot1a异质复合物作为基底和醛固酮敏感的调节因子的机制基础。ENaC转录，并用于整合去抑制和矿皮质激素受体的转激活？？ENaC介导醛固酮诱导。在当前资助期内的重大进展的指导下，该假设将在三个具体目标中进行测试：1)定义Dot1a-AF9和Dot1a-Sirt1复合物在抑制？？ENaC在收集管中的转录；2)定义醛固酮诱导的？？博子;3)确定醛固酮诱导的染色质结构的动力学、介质和作用。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分析将用于培养的收集管细胞和小鼠肾脏，以检查醛固酮诱导的Dot1异位复合物在？？ENaC启动子，与新因子的相互作用，组蛋白修饰和启动子甲基化的变化。对BAC转基因小鼠的研究将检验候选AF9结合位点是否对内源性？？即基因。这种方法是创新的，因为它代表了对矿物皮质激素受体反式激活的经典模型的重大背离。钠。这项研究具有重要意义，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醛固酮通过染色质信号传导的理解。

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.