Regulation of ENaC Transcription

这项研究的主要目标是确定转录调控的分子机制。 肾脏上皮细胞Na+通道a亚单位(ENaCa)。ENAC是一种多亚基蛋白质，它在 在控制上皮细胞Na+转运、血压和对高醛固酮反应方面的作用。这个 ENaCa亚基是由集合管中的醛固酮诱导的，对ENaC似乎是限速的 此细分市场中的活动。尽管它很重要，但控制ENaCa转录和它的 对醛固酮诱导作用的定义还不完全。我们描述了一种新奇的组蛋白 甲基转移酶，端粒沉默的破坏者(Dot1)，并表明它与AF9a相互作用形成 小鼠ENaCa基因5‘侧翼区的染色质相关抑制物复合体。穿过 SGK1介导的AF9a，醛固酮的磷酸化破坏了复合体，解除了对 ENaCa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-报告瞬时 转染试验跟踪特定的转录因子和共调节蛋白与 在ENaCa启动子上的AF9a-Dot1复合体，以定义结合模式，检验关于 这些因素之间的相互作用，并监测与以下相关的共价组蛋白修饰的变化 ENaCa基因在基础条件下和对醛固酮的反应中的转录激活。这个 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。对转基因小鼠的研究将测试候选AF9a是否调节 确定的元件对于内源性ENaCa基因的响应的忠实复制至关重要。目标1 将检验AF9a是一个转录抑制因子，它结合了ENaCa5‘侧翼区的假设 并使特定的辅抑制子和染色质重塑蛋白的有序募集到 ENaCa启动子。目标2将检验AF9a、Dot1、SIRT1、Rad6和其他辅助调节因子的假设 蛋白质促进组蛋白的高甲基化和去乙酰化以及ENaCa的基础抑制。AIM 3将测试 醛固酮和SGK1促进顺序和组合招募和解雇的假说 AF9a元件基因座上的共调控蛋白，决定了ENaCa基因的激活。这些研究将 允许我们构建一个新颖的、动态的调控网络来构建ENaCa基因调控的整体模型 肾脏，并为复杂基因和分子的转录调控提供重要的见解 矿物皮质激素的作用。拟议的研究还将定义SGK1、Dot1、 和AF9a，可能更广泛地适用于其他靶蛋白和一般的基因调控。

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.