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.

这项研究的广泛目标是确定转录控制的分子机制 肾脏中的上皮Na+通道A-Subunit（ENACA）。 ENAC是一种多用基蛋白，可发挥作用 在控制上皮Na+转运，血压和对甲醛烷酸酯的反应中的作用。这 ENACA亚基是由醛固酮在收集管中诱导的，并且似乎是ENAC的速率限制 该细分市场的活动。尽管其重要性，但控制ENACA转录的机制及其 醛固酮的诱导未完全定义。我们已经描述了一个新型组蛋白 甲基转移酶，端粒沉默（DOT1）的distrupter，并表明它与AF9A相互作用以形成A 小鼠ENACA基因的5'-边缘区域中与染色质相关的抑制剂复合物。通过 SGK1介导的AF9A磷酸化，醛固酮破坏了复合物，减轻了对 ENACA并激活其转录，与矿物皮质激素受体的作用无关。我们现在 提议使用定量染色质免疫沉淀测定法和启动子瞬态 转染测定法，以遵循特定转录因子和核子调节蛋白与 ENACA启动子处的AF9A-DOT1复合物来定义结合模式，以检验有关的假设 这些因素之间的相互作用，并监视与相关的共价组蛋白修饰的变化 ENACA基因在基础条件下并响应醛固酮的转录激活。这 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。转基因小鼠的研究将测试候选AF9A调节 确定的元素对于忠实地复制内源性ENACA基因的反应至关重要。目标1 将检验以下假设，即AF9A是一个结合ENACA侧翼区域的转录阻遏物 并将特定的核压和染色质重塑蛋白的有序募集成核 ENACA启动子。 AIM 2将检验以下假设：AF9A，DOT1，SIRT1，RAD6和其他COREGULATOR 蛋白质可促进组蛋白高甲基化和脱乙酰基化和基础抑制ENACA。 AIM 3将测试 醛固酮和SGK1促进顺序和联合招募和解雇的假设 AF9A元素基因座的核调节蛋白的决定，指示ENACA基因激活。这些研究会 允许我们构建一个新颖的动态调节网络，以构建ENACA基因调节的整体模型 肾脏，并为复杂基因的转录控制和分子提供重要见解 矿物皮质激素的作用。拟议的研究还将定义SGK1，DOT1的新功能模式 和AF9A可能更广泛地适用于其他靶蛋白和基因调节。