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）：这项研究的广泛目标是确定肾脏上皮Na+通道A-Subunit（ENACA）的转录控制的分子机制。 ENAC是一种多生产蛋白，在控制上皮Na+转运，血压和对醛固酮降酸的反应中起着重要作用。 ENACA亚基是由醛固酮在收集管中诱导的，并且似乎是该节段中的ENAC活性的速率限制。尽管其重要性，但控制ENACA转录及其醛固酮诱导的机制仍未完全定义。我们已经表征了一种新型的组蛋白甲基转移酶，端粒沉默（DOT1）的dist虫，并表明它与AF9A相互作用，在小鼠ENACA基因的5'-芬兰区域中形成与染色质相关的抑制剂复合物。通过SGK1介导的AF9A的磷酸化，醛固酮破坏了复合物，缓解了对ENACA的抑制并激活其转录，与盐皮质激素受体的作用无关。现在，我们建议使用定量染色质免疫沉淀测定法和启动子捕集蛋白瞬态转染测定法，以遵循特定的转录因子和核心调节蛋白与ENACA启动子处的AF9A-DOT1复合物与AF9A-DOT1复合体的关联，以定义与这些因素的相互作用的相互作用，以定义与这些因素之间的相互作用，并在这些因素之间进行转换，并在这些因素之间进行转换，并在这些因素之间进行转换，并在这些因素之间进行转换。基础条件和对醛固酮的响应。定义的核蛋白改变反式中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候选AF9A调节元件是否对忠实复制内源性ENACA基因的反应至关重要。 AIM 1将检验以下假设：AF9A是一种转录阻遏物，它结合了ENACA的5'侧翼区域，并将特定核压蛋白和染色质重塑蛋白与eNACA启动子的有序募集成核。 AIM 2将检验以下假设：AF9A，DOT1，SIRT1，RAD6和其他核心控蛋白可促进组蛋白高甲基化和脱乙酰基化和基础抑制ENACA。 AIM 3将检验以下假设：醛固酮和SGK1在AF9A元素基因座上促进了顺序和联合募集以及解散核调节蛋白，从而决定了ENACA基因的激活。这些研究将使我们能够为肾脏中ENACA基因调节的整体模型构建一个新型的动态调节网络，并为复杂基因的转录控制和矿物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，这些功能可能更广泛地适用于其他靶蛋白，并且通常适用于基因调节。