Regulation of ENaC Transcription

ENaC 转录的调控

• 批准号: 7393666
• 负责人: BRUCE C. KONE
• 金额: $ 28.18万
• 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/7393666
• 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.

本研究的广泛目标是确定潜在的转录控制的分子机制