Regulation of ENaC Transcription

ENaC 转录的调控

• 批准号: 7456744
• 负责人: BRUCE C. KONE
• 金额: $ 29.16万
• 依托单位: UNIVERSITY OF FLORIDA
• 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/7456744
• 关键词: 5' Flanking Region; Abbreviations; Affinity; Aldosterone; Apanteles kariyai growth blocking peptide; Binding; Biological Assay; Blood Pressure; Calcium-Binding Proteins; Cell Nucleus; Cells; Chromatin; Complex; Condition; Deacetylase; Deacetylation; Duct (organ) structure; Elements; Enzymes; Epigenetic Process; Epithelial; Event; Figs - dietary; Gene Activation; Gene Expression Regulation; Gene Silencing; Gene Targeting; Genes; Genetic Transcription; Glucocorticoids; Goals; Histone Deacetylase; Histone Deacetylation; Histone H3; Histones; Hyperaldosteronism; Hypermethylation; In Vitro; Kidney; Ligands; Link; Mediating; Mineralocorticoid Receptor; Mineralocorticoids; Modeling; Modification; Molecular; Monitor; Mus; Nuclear Protein; Nuclear Proteins; Nuclear Receptors; Nucleosomes; PCAF gene; Pathway interactions; Pattern; Phosphorylation; Phosphotransferases; Play; Protein Subunits; Protein-Arginine N-Methyltransferase; Proteins; RNA Interference; Rate; Regulation; Regulatory Element; Reporter; Repression; Research; Research Personnel; Response Elements; Role; Scheme; Sequence-Specific DNA Binding Protein; Series; Serum; Signal Pathway; Signal Transduction; Silencing Mediator of Retinoid Thyroid Receptor; Site; Sodium Channel; Testing; Transcriptional Activation; Transcriptional Regulation; Transfection; Transgenic Mice; absorption; arginine methyltransferase; chromatin immunoprecipitation; chromatin remodeling; combinatorial; epithelial Na+ channel; gene repression; genetic regulatory protein; histone acetyltransferase; histone methyltransferase; hormone response element; in vivo; insight; novel; p300/CBP-Associated Factor; programs; promoter; research study; response; transcription factor

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.

这项研究的主要目标是确定转录控制的分子机制， 肾脏上皮Na+通道α亚单位（ENaCa）。ENaC是一种多亚基蛋白质， 在控制上皮Na+转运、血压和对醛固酮增多症的反应中起作用。的 ENaCa亚单位在集合管中由醛固酮诱导，并且似乎是ENaC的速率限制 在这一环节的活动。尽管它的重要性，控制ENaCa转录及其 醛固酮诱导作用还没有完全确定。我们发现了一种新的组蛋白 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，形成一个甲基转移酶。 在小鼠ENaCa基因的5 '-侧翼区域中的染色质相关阻遏物复合物。通过 SGK 1介导的AF 9a磷酸化，醛固酮破坏复合物，解除对AF 9a的抑制。 ENaCa和激活其转录，独立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-报告子瞬时 转染试验，以跟踪特异性转录因子和共调节蛋白与细胞的结合。 在ENaCa启动子处的AF 9a-Dot 1复合物，以定义结合模式，以测试关于 这些因素之间的相互作用，并监测共价组蛋白修饰相关的变化， ENaCa基因在基础条件下的转录激活和对醛固酮的应答。的 将在共表达中测试确定的核蛋白改变ENaCa启动子的反式能力， RNA干扰实验。在转基因小鼠中的研究将测试候选AF 9a调节基因是否能够在转基因小鼠中表达。 鉴定的元件对于内源性ENaCa基因的应答的忠实复制是关键的。要求1 将检验AF 9a是结合ENaCa的5'侧翼区的转录阻遏物的假设 并使特异性辅阻遏物和染色质重塑蛋白有序地募集到细胞中， ENaCa启动子。目的2将检验AF 9a、Dot 1、SIRT 1、Rad 6和其他共调节因子 蛋白质促进组蛋白超甲基化和脱乙酰化以及ENaCa的基础抑制。目标3将测试 醛固酮和SGK 1促进顺序和组合募集和释放假说 在AF 9a元件位点的共调节蛋白，指示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ENaCa基因调控， 肾，并提供重要的见解转录控制的复杂基因和分子 盐皮质激素的作用。拟议的研究还将定义SGK 1，Dot 1， 和AF 9a，其可以更广泛地应用于其它靶蛋白和一般的基因调控。