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Regulation of ENaC Transcription

ENaC 转录的调控

基本信息

批准号：
8531222
负责人：
BRUCE C. KONE
金额：
$ 29.74万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-04-04 至 2015-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8531222
关键词：
Abbreviations Accounting Address Aldosterone Altitude Anabolism Androgen Receptor Architecture BCL1 Oncogene Benzofurans Binding Sites Biological Assay Blood Pressure Cell Nucleus Cells Cessation of life Chromatin Chromatin Loop Chromosomes Chromosomes, Human, Pair 9 Clinical Colon Complex Cystic Fibrosis DNA Methyltransferase DNA Modification Methylases Disease Distal Duct (organ) structure Edema Elements Environment Epigenetic Process Epithelial Equilibrium Esters Estrogens Eukaryotic Cell Event Exocrine Glands Fluid Balance Functional disorder Funding Gatekeeping Gene Expression Genes Genetic Genetic Transcription Glucocorticoids Goals Histone Deacetylase Histone H3 Histone-Lysine N-Methyltransferase Histones Homeostasis Hypertension Immunoprecipitation Inflammatory Intestines Kidney Lead Ligands Liquid substance Lung Lysine MLL gene MLLT3 gene Maintenance Mass Spectrum Analysis Measures Mediating Mediator of activation protein Methylation Methyltransferase Mineralocorticoid Receptor Modeling Modification Molecular Molecular Conformation Mus Nephrons Nuclear Nuclear Hormone Receptors Nuclear Receptors Nucleosomes Outcome Pathologic Pathway interactions Phosphotransferases Physiology Play Potassium Potassium Deficiency Protein Binding Domain Proteins Pulmonary Edema Recruitment Activity Regulation Renal tubule structure Reporter Repression Research Response Elements Role SBFI-AM Series Serum Signal Transduction Sodium Sodium Channel Sodium Chloride Testing Transactivation Transcriptional Activation Transcriptional Regulation Transgenic Mice Water Work absorption base blood pressure regulation body system clinical practice clinically relevant epithelial Na+ channel gain of function genetic regulatory protein histone acetyltransferase histone modification innovation insight loss of function mutation novel prevent promoter protein complex public health relevance receptor respiratory distress syndrome response restoration transcription factor

项目摘要

DESCRIPTION (provided by applicant): There is a large gap in understanding how a constrained, but favorable transcriptional ground state for the epithelial sodium channel ??subunit (??ENaC) gene is maintained, and how complex interactions between multiple regulatory proteins, and between these protein complexes and the chromatin template, result in aldosterone induction of ??ENaC transcription. Given its clinical importance as a key regulator of sodium balance and blood pressure, closing this gap is imperative. During the current project period, we discovered and characterized novel nuclear represor complexes containing the histone lysine-79 methyltransferase Dot1a together with either AF9 or Sirt1. These complexes associate with targeted regions of the ??ENaC promoter to constrain basal ??ENaC transcription in the collecting duct, and are downregulated by aldosterone, leading to de-repression. The long-term goal is to characterize novel transcriptional control mechanisms of aldosterone-inducible genes, using ??ENaC to model these events in the collecting duct. The objective of this application is to characterize the mechanisms underlying the gatekeeper and aldosterone-dependent activation functions, and how they are fine-tuned and integrated in the ??ENaC chromatin environment. The central hypothesis is that these responses depend on the dynamic and orchestrated action of key transcription factors and changes in the profile of chromatin modifiers and modifications that ultimately lead to expression of ??ENaC. The rationale for the proposal is that it will establish the mechanistic basis for the Dot1a heterocomplexes as basal and aldosterone-sensitive regulators of ??ENaC transcription, and for the integration of de-repression and mineralocorticoid receptor transactivation of ??ENaC in mediating aldosterone induction. Guided by substantial advances during the current funding period, this hypothesis will be tested in three specific aims: 1) Define the components, functions, and interplay of the Dot1a-AF9 and Dot1a-Sirt1 complexes in repressing ??ENaC transcription in colecting duct; 2) Define the dynamics and mediators of aldosterone- induced reprogramming at the ??ENaC promoter; and 3) Determine the dynamics, mediators, and roles of chromatin architecture in aldosterone-induced ??ENaC transcription. Immunoprecipitation/mass spectroscopy, quantitative ChIP assays, chromosome conformation capture, and promoter-reporter assays will be used in cultured collecting duct cells and mouse kidneys to examine the aldosterone-induced dynamics of the Dot1 heterocomplexes at the ??ENaC promoter, interactions with novel factors, and changes in histone modifications and promoter methylation. Studies in BAC transgenic mice will test whether the candidate AF9 binding site is critical for the responses of the endogenous ??ENaC gene. The approach is innovative because it represents a major departure from the classical model of mineralocorticoid receptor trans-activation of ?? ENaC. The proposed research is significant because it will advance our understanding of transcriptional de- repression, nuclear receptor-mediated transcriptional activation, and aldosterone signaling through chromatin. PUBLIC HEALTH RELEVANCE: Sodium reabsorption by the epithelial sodium channel in the kidney tubules is an essential mechanism involved in the regulation of sodium balance, fluid volume, and blood pressure. Abnormal expression levels of this protein can result in hypertension, potassium deficiency, and edema. This proposal examines a novel network of interacting proteins in the cell nucleus that regulate transcription, the initial molecular step in generating this protein.

描述（由申请人提供）：对于如何维持上皮钠通道亚基（ENaC）基因受限制但有利的转录基态，以及多个调节蛋白之间以及这些蛋白复合物与染色质模板之间的复杂相互作用如何导致醛固酮诱导 ENaC 转录，在理解上存在很大差距。鉴于其作为钠平衡和血压关键调节剂的临床重要性，缩小这一差距势在必行。在当前项目期间，我们发现并表征了包含组蛋白赖氨酸 79 甲基转移酶 Dot1a 以及 AF9 或 Sirt1 的新型核抑制复合物。这些复合物与 ENaC 启动子的目标区域结合，限制集合管中基础 ENaC 转录，并被醛固酮下调，从而导致去抑制。长期目标是利用 ENaC 来模拟集合管中的这些事件，从而表征醛固酮诱导基因的新型转录控制机制。该应用的目的是表征看门人和醛固酮依赖性激活功能的潜在机制，以及它们如何在 ENaC 染色质环境中进行微调和集成。核心假设是，这些反应取决于关键转录因子的动态和精心策划的作用以及染色质修饰剂和修饰的变化，最终导致 ENaC 的表达。该提案的基本原理是，它将为 Dot1a 异质复合物作为 ENaC 转录的基础和醛固酮敏感调节剂，以及 ENaC 的去抑制和盐皮质激素受体反式激活在介导醛固酮诱导中的整合奠定机制基础。在当前资助期间取得的重大进展的指导下，该假设将在三个具体目标上得到检验：1）定义Dot1a-AF9和Dot1a-Sirt1复合物在抑制输尿管中ENaC转录中的成分、功能和相互作用； 2）定义醛固酮诱导的ENaC启动子重编程的动力学和介质； 3) 确定醛固酮诱导的 ENaC 转录中染色质结构的动力学、介质和作用。免疫沉淀/质谱、定量 ChIP 测定、染色体构象捕获和启动子报告基因测定将用于培养的集合管细胞和小鼠肾脏，以检查 ENaC 启动子处 Dot1 杂合物的醛固酮诱导动力学、与新因子的相互作用以及组蛋白修饰和启动子甲基化的变化。 BAC 转基因小鼠的研究将测试候选 AF9 结合位点对于内源性 ENaC 基因的反应是否至关重要。该方法具有创新性，因为它代表了与盐皮质激素受体反式激活的经典模型的重大背离。 ENaC。这项研究意义重大，因为它将增进我们对转录去抑制、核受体介导的转录激活以及通过染色质的醛固酮信号传导的理解。公共卫生相关性：肾小管上皮钠通道的钠重吸收是调节钠平衡、液体容量和血压的重要机制。该蛋白的异常表达水平可导致高血压、缺钾和水肿。该提案研究了细胞核中调节转录的相互作用蛋白质的新型网络，转录是生成该蛋白质的初始分子步骤。