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上皮Na+通道α-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、血压和对醛固酮增多症的反应中起主要作用。ENaCa亚单位在集合管中由醛固酮诱导，并且似乎是ENaC在该段中活性的速率限制。尽管它的重要性，控制ENaCa转录和其诱导醛固酮的机制是不完全确定的。我们已经表征了一种新的组蛋白甲基转移酶，端粒沉默的破坏者（Dot 1），并表明它与AF 9a相互作用，在小鼠ENaCa基因的5 '侧翼区域形成染色质相关的阻遏物复合物。通过SGK 1介导的AF 9a磷酸化，醛固酮破坏复合物，解除对ENaCa的抑制并激活其转录，而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分析和启动子-报告基因瞬时转染分析来跟踪特定转录因子和共调节蛋白与ENaCa启动子处的AF 9a-Dot 1复合物的结合，以确定结合模式，测试关于这些因子之间相互作用的假设，并监测与基础条件下ENaCa基因的转录激活相关的共价组蛋白修饰的变化和对醛固酮的应答。将在共表达和RNA干扰实验中测试确定的核蛋白改变反式ENaCa启动子的能力。在转基因小鼠中的研究将测试所鉴定的候选AF 9a调节元件是否对内源性ENaCa基因的应答的忠实复制至关重要。目的1将检验以下假设：AF 9a是转录阻遏物，其结合ENaCa的5'侧翼区，并使特异性辅阻遏物和染色质重塑蛋白有序募集到ENaCa启动子成核。目的2验证AF 9a、Dot 1、SIRT 1、Rad 6等辅助调节蛋白促进组蛋白高甲基化和去乙酰化以及ENaCa基础阻遏的假说。目的3将测试的假设，醛固酮和SGK 1促进顺序和组合的招聘和解雇的辅调节蛋白在AF 9a元件基因座，决定ENaCa基因激活。这些研究将使我们能够构建一个新的，动态的调控网络的整体模型的ENaCa基因调控在肾脏，并提供重要的见解复杂基因的转录控制和盐皮质激素的分子作用。拟议的研究还将定义SGK 1、Dot 1和AF 9a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的基因调控。