Regulation of ENaC Transcription

ENaC 转录的调控

基本信息

批准号：
8323932
负责人：
BRUCE C. KONE
金额：
$ 30.81万
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 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8323932
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）：了解上皮钠通道的受约束但有利的转录基态是很大的差距。鉴于其作为钠平衡和血压的关键调节剂的临床重要性，因此必须缩小此差距。在当前的项目时期，我们发现并表征了包含组蛋白赖氨酸79甲基转移酶DOT1A的新型核代表复合物以及AF9或SIRT1。这些复合物与启动子的靶向区域相关联，以限制收集管中的基础eNAC转录，并被醛固酮下调，从而导致抑制。长期目标是使用eNAC在收集管中对这些事件进行建模，以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守门者和醛固酮依赖性激活功能的机制，以及它们如何微调和集成在?? ENAC染色质环境中。中心假设是这些响应取决于关键转录因子的动态和策划作用，以及染色质修饰剂和修饰的变化，最终导致了?? ENAC的表达。该提案的理由是，它将建立DOT1A杂音复合物的机械基础，作为’enac转录的基底和醛固酮敏感的调节剂，以及在中间醛固酮诱导中的脱抑制和矿物皮质激素受体对eNAC的脱抑制和矿物皮质类型受体反式化的整合。在当前资金期间的实质性进步的指导下，该假设将以三个具体的目的进行检验：1）定义DOT1A-AF9和DOT1A-SIRT1复合物在重新定位导管中重新转录时的组件，功能和相互作用； 2）定义醛固酮诱导的重新编程在eNAC启动子上的动力学和介体； 3）确定染色质结构在醛固酮诱导的ENAC转录中的动力学，介体和作用。免疫沉淀/质谱，定量芯片测定，染色体构象捕获和启动子培养剂测定将用于培养的收集导管细胞和小鼠肾脏，以检查醛固酮诱导的DOT1 Heetocomplexes的动力学，在eNAC启动子上，与新颖的启动子相互作用，与新颖的因子和启动剂中的变化相互作用。在BAC转基因小鼠中的研究将测试候选AF9结合位点是否对内源性ENAC基因的反应至关重要。该方法具有创新性，因为它代表了矿物皮质受体反式激活的经典模型？ ENAC。拟议的研究很重要，因为它将提高我们对转录抑制，核受体介导的转录激活和醛固酮信号传导的理解。公共卫生相关性：肾小管中上皮钠通道的钠吸收是涉及钠平衡，液体体积和血压调节的基本机制。该蛋白质的异常表达水平可导致高血压，钾缺乏症和水肿。该建议研究了调节转录的细胞核中相互作用蛋白质的新型网络，这是产生该蛋白质的初始分子步骤。