Regulation of ENaC Transcription

ENaC 转录的调控

• 批准号: 7081856
• 负责人: BRUCE C. KONE
• 金额: $ 36.25万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-04 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7081856
• 关键词: RNA interference; acetylation; aldosterone; chromatin immunoprecipitation; clinical research; gene induction /repression; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; genetically modified animals; hormone regulation /control mechanism; kidney; laboratory mouse; methylation; nuclear proteins; protein binding; protein protein interaction; protein structure; reporter genes; sodium channel; transcription factor

DESCRIPTION (provided by applicant): The broad goal of this research is to identify the molecular mechanisms underlying transcriptional control of the epithelial Na+ channel a-subunit (ENaCa) in kidney. ENaC .is a multi-subunit protein that plays a major role in control of epithelial Na+ transport, blood pressure, and the response to hyperaldosteronism. The ENaCa subunit is induced by aldosterone in the collecting duct, and appears to be rate-limiting for ENaC activity in this segment. Despite its importance, the mechanisms controlling ENaCa transcription and its induction by aldosterone are incompletely defined. We have characterized a novel histone methyltransferase, disrupter of telomeric silencing (Dot1), and show that it interacts with AF9a to form a chromatin-associated represser complex in the 5'-flanking region of the mouse ENaCa gene. Through SGK1-mediated phosphorylation of AF9a, aldosterone disrupts the complex, relieving the repression on the ENaCa and activating its transcription, independent of actions of the mineralocorticoid receptor. We now propose to use quantitative chromatin immunoprecipitation assays and promoter-reporter transient transfection assays to follow association of specific transcription factors and coregulatory proteins with the AF9a-Dot1 complex at the ENaCa promoter, to define patterns of binding, to test hypotheses regarding interactions among these factors, and to monitor changes in covalent histone modifications associated with transcriptional activation of the ENaCa gene under basal conditions and in response to aldosterone. The ability of defined nuclear proteins to alter the ENaCa promoter in trans will be tested in coexpression and RNA interference experiments. Studies in transgenic mice will test whether the candidate AF9a regulatory element identified is critical for faithful replication of the responses of the endogenous ENaCa gene. Aim 1 will test the hypothesis that AF9a is a transcriptional represser that binds the 5' flanking region of ENaCa and nucleates the ordered recruitment of specific corepressors and chromatin remodeling proteins to the ENaCa promoter. Aim 2 will test the hypothesis that AF9a, Dot1, SIRT1, Rad6 and other coregulator proteins promote histone hypermethylation and deacetylation and basal repression of ENaCa. Aim 3 will test the hypothesis that aldosterone and SGK1 promote sequential and combinatorial recruitment and dismissal of coregulatory proteins at the AF9a element locus, dictating ENaCa gene activation. These studies will allow us to construct a novel, dynamic regulatory network to the overall model of ENaCa gene regulation in kidney, and to provide important insights into transcriptional control of complex genes and the molecular actions of mineralocorticoids. The proposed studies will also define new modes of function for SGK1, Dot1, and AF9a that may be more broadly applicable to other target proteins and to gene regulation in general.

描述（由申请人提供）：本研究的主要目标是确定肾脏中上皮 Na+ 通道 a 亚基 (ENaCa) 转录控制的分子机制。 ENaC 是一种多亚基蛋白，在控制上皮 Na+ 转运、血压和醛固酮增多症反应中发挥重要作用。 ENaCa 亚基由集合管中的醛固酮诱导，并且似乎是该部分 ENaC 活性的速率限制。尽管其重要性，但控制 ENaCa 转录及其由醛固酮诱导的机制尚未完全确定。我们鉴定了一种新型组蛋白甲基转移酶，即端粒沉默干扰剂 (Dot1)，并表明它与 AF9a 相互作用，在小鼠 ENaCa 基因的 5' 侧翼区域形成染色质相关阻遏复合物。通过 SGK1 介导的 AF9a 磷酸化，醛固酮破坏复合物，解除对 ENaCa 的抑制并激活其转录，不依赖于盐皮质激素受体的作用。我们现在建议使用定量染色质免疫沉淀测定和启动子报告基因瞬时转染测定来跟踪特定转录因子和共调节蛋白与 ENaCa 启动子处的 AF9a-Dot1 复合物的关联，定义结合模式，测试有关这些因子之间相互作用的假设，并监测与 ENaCa 基因转录激活相关的共价组蛋白修饰的变化。 基础条件和对醛固酮的反应。确定的核蛋白反式改变 ENaCa 启动子的能力将在共表达和 RNA 干扰实验中进行测试。转基因小鼠的研究将测试所确定的候选 AF9a 调控元件对于内源 ENaCa 基因反应的忠实复制是否至关重要。目标 1 将检验以下假设：AF9a 是一种转录阻遏蛋白，它结合 ENaCa 的 5' 侧翼区域，并使特定辅阻遏物和染色质重塑蛋白有序招募到 ENaCa 启动子上。目标 2 将检验以下假设：AF9a、Dot1、SIRT1、Rad6 和其他辅助调节蛋白促进组蛋白高甲基化和脱乙酰化以及 ENaCa 的基础抑制。目标 3 将检验以下假设：醛固酮和 SGK1 促进 AF9a 元件位点处的共调节蛋白的顺序和组合招募和消除，从而决定 ENaCa 基因激活。这些研究将使我们能够构建一个新颖的动态调控网络来实现肾脏中 ENaCa 基因调控的整体模型，并为复杂基因的转录控制和盐皮质激素的分子作用提供重要的见解。拟议的研究还将定义 SGK1、Dot1 和 AF9a 的新功能模式，这些模式可能更广泛地适用于其他靶蛋白和一般基因调控。