Regulation of ENaC Transcription

ENaC 转录的调控

• 批准号: 7616173
• 负责人: BRUCE C. KONE
• 金额: $ 6.8万
• 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 2009-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7616173
• 关键词: 5' Flanking Region; Abbreviations; Affinity; Aldosterone; Binding; Biological Assay; Blood Pressure; Calcium-Binding Proteins; Cell Nucleus; Cells; Chromatin; Complex; 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; 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; 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 modification; chromatin remodeling; combinatorial; epithelial Na+ channel; gene repression; genetic regulatory protein; histone acetyltransferase; histone methyltransferase; histone modification; 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 肾脏中的上皮 Na+ 通道 a 亚基 (ENaCa)。 ENaC 是一种多亚基蛋白质，在 在控制上皮 Na+ 转运、血压和醛固酮增多症反应中发挥作用。这 ENaCa 亚基由集合管中的醛固酮诱导，并且似乎是 ENaC 的速率限制 该部分的活动。尽管它很重要，但控制 ENaCa 转录的机制及其 醛固酮诱导的定义不完全。我们已经表征了一种新颖的组蛋白 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' 侧翼区域的染色质相关阻遏物复合物。通过 SGK1 介导的 AF9a 磷酸化，醛固酮破坏复合物，解除对 AF9a 的抑制 ENaCa 并激活其转录，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-报告基因瞬态分析 转染测定跟踪特定转录因子和共调节蛋白与 ENaCa 启动子上的 AF9a-Dot1 复合物，定义结合模式，测试有关的假设 这些因素之间的相互作用，并监测与相关的共价组蛋白修饰的变化 ENaCa 基因在基础条件下和醛固酮反应下的转录激活。这 确定的核蛋白反式改变 ENaCa 启动子的能力将在共表达中进行测试， RNA干扰实验。转基因小鼠研究将测试候选 AF9a 是否具有调节作用 鉴定出的元件对于忠实复制内源 ENaCa 基因的反应至关重要。目标1 将检验 AF9a 是结合 ENaCa 5' 侧翼区域的转录抑制子的假设 并将特定辅阻遏物和染色质重塑蛋白有序招募到 ENaCa 启动子。目标 2 将测试 AF9a、Dot1、SIRT1、Rad6 和其他核心调节器的假设 蛋白质促进组蛋白高甲基化和脱乙酰化以及 ENaCa 的基础抑制。目标 3 将进行测试 醛固酮和 SGK1 促进顺序和组合招募和解散的假设 AF9a 元件位点的辅助调节蛋白的数量，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调控网络来构建 ENaCa 基因调控的整体模型 肾，并为复杂基因的转录控制和分子生物学提供重要见解 盐皮质激素的作用。拟议的研究还将定义 SGK1、Dot1、 AF9a 可能更广泛地适用于其他靶蛋白和一般基因调控。