Regulation of ENaC Transcription

描述(由申请人提供)：在理解上皮钠通道亚单位(ENAC)基因如何维持受限制但良好的转录基态，以及多个调节蛋白之间以及这些蛋白质复合体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转录方面，存在着很大的差距。鉴于其作为钠平衡和血压关键调节器的临床重要性，缩小这一差距势在必行。在本项目期间，我们发现并表征了包含组蛋白赖氨酸-79甲基转移酶Dot1a和AF9或Sirt1的新型核表达复合体。这些复合体与ENAC启动子的靶向区域结合，在集合管中抑制基本的ENAC转录，并被醛固酮下调，导致抑制。长期的目标是通过使用？？ENAC来模拟收集管道中的这些事件，来表征醛固酮诱导基因的新的转录调控机制。本申请的目的是描述看门人和依赖于醛固酮的激活功能的潜在机制，以及它们如何在ENAC染色质环境中进行微调和整合。核心假设是，这些反应依赖于关键转录因子的动态和精心安排的作用，以及染色质修饰物和修饰的变化，最终导致？？enac的表达。这一建议的基本原理是，它将为Dot1a异源复合体作为eNAc转录的基础和醛固酮敏感调节因子，以及在介导醛固酮诱导过程中解抑和盐皮质激素受体反式激活的整合奠定机制基础。在当前资助期间取得的实质性进展的指导下，这一假说将在三个具体目标上得到验证：1)确定Dot1a-AF9和Dot1a-Sirt1复合体在抑制结肠导管内enac转录方面的组成、功能和相互作用；2)确定醛固酮诱导的enac启动子重编程的动力学和介体；以及3)确定染色质结构在醛固酮诱导的enac转录中的动力学、介体和作用。免疫沉淀/质谱学、定量芯片分析、染色体构象捕获和启动子-报告分析将在培养的集合管细胞和小鼠肾脏中被用来检测醛固酮诱导的dot1异源复合体在enac启动子上的动力学，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AF9结合位点是否对内源性ENAC基因的反应至关重要。这种方法是创新的，因为它与经典的盐皮质激素受体反式激活模型有很大的不同。埃纳克。这项拟议的研究具有重要意义，因为它将促进我们对转录抑制、核受体介导的转录激活以及通过染色质传递醛固酮信号的理解。 公共卫生相关性：肾小管上皮性钠通道的钠重吸收是调节钠平衡、液体容量和血压的重要机制。这种蛋白的异常表达水平可能会导致高血压、缺钾和水肿。这项建议研究了细胞核中一个新的相互作用的蛋白质网络，它调节转录，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.