Regulation of ENaC Transcription

描述(申请人提供)：这项研究的广泛目标是确定肾脏上皮细胞Na+通道a亚单位(ENaCa)转录控制的分子机制。ENAC是一种多亚基蛋白质，在控制上皮钠离子转运、血压和对高醛固酮的反应中起主要作用。ENaCa亚基是由集合管中的醛固酮诱导的，在这一段中ENaC的活动似乎是限速的。尽管它很重要，但控制ENaCa转录和醛固酮诱导ENaCa转录的机制尚不完全清楚。我们鉴定了一种新的组蛋白甲基转移酶，端粒沉默干扰物(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的抑制物复合体。通过SGK1介导的AF9a的磷酸化，醛固酮破坏该复合体，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的转录因子和共调节蛋白与ENaCa启动子上的AF9a-Dot1复合体的关联，定义结合模式，检验有关这些因素之间相互作用的假设，并监测与基础条件下ENaCa基因转录激活相关的共价组蛋白修饰的变化以及对醛固酮的响应。确定的核蛋白在反式中改变ENaCa启动子的能力将在共表达和RNA干扰实验中进行测试。对转基因小鼠的研究将测试所确定的候选AF9a调控元件是否对内源性ENaCa基因的反应的忠实复制至关重要。目的1验证AF9a是一个转录抑制因子的假设，它结合ENaCa的5‘侧翼区，并将特定的辅阻遏子和染色质重塑蛋白有序地募集到ENaCa启动子上。目的2验证AF9a、Dot1、SIRT1、Rad6等辅调节蛋白促进组蛋白高甲基化、去乙酰化和ENaCa基础抑制的假说。目的3验证一种假说，即醛固酮和SGK1促进AF9a元件基因座上协同调节蛋白的顺序和组合募集和释放，从而决定ENaCa基因的激活。这些研究将使我们能够构建一个新颖的、动态的调控网络来构建肾脏ENaCa基因调控的整体模型，并为复杂基因的转录调控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的基因调控。

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.