Aldosterone-sensitive repression of ENaC by AF9 and Dot1

描述(由申请人提供)： 该研究计划的主要目的是为申请者提供有关肾脏和整个动物生理学研究的新的强化培训，并为阐明控制肾脏生理学的离子转运关键的醛固酮信号通路的出版工作奠定基础。申请者的长期目标是成为肾脏分子和细胞生物学研究的一名成功的独立研究员。为了实现这一目标，申请者将在德克萨斯医疗中心丰富的科学环境中接受培训，培训由这些领域的专家布鲁斯·通内博士密切赞助。该提案的科学目的是模拟醛固酮对集合管上皮Na+通道的分子作用。醛固酮是钠离子和酸碱平衡和血压控制的主要调节器。本研究的具体目的是通过SGK1对AF9a的磷酸化和mDotla对H3K79的甲基化来激活mIMCDS细胞中ENaC的转录，从而定义一个由醛固酮启动的新的信号级联反应，揭示AF9a和mDot1 a是醛固酮在体内调节ENaC转录的新的生理靶点。研究表明，MIMCDS细胞对醛固酮有反应，表达SGK1、AF9a、mDot1a和ENaC。为了实现这些目标，将应用广泛的分子方法，包括染色质免疫沉淀、实时RTqPCFt核连续检测和肾脏生理检测(离子转运和尿电解质)。预计AF9a将通过醛固酮作用被确定为早期抑制的靶点，它是SGK1的新的生理底物，是第一个与mDot1a相互作用的非组蛋白，也是第一个靶向调节H3K79甲基化的蛋白；2)Dot1a将成为第一个作为醛固酮信号通路整合成分的染色质重塑蛋白；3)ENaC将成为AF9a的第一个直接下游靶点。总的来说，这项工作将使申请者成为肾脏信号和离子转运方面的一名创新的独立科学家。

DESCRIPTION (provided by applicant): The major objective of this research program is to provide the applicant with new and intensive training in relavant renal and whole animal physiological studies and a foundation of published work in elucidating aldosterone signaling pathways controlling ion transport criticle to renal physiology. The applicant's long-term goal is to become a successful independent investigator in molecular and cell biological research of the kidney. To achieve this goal, the applicant will be trained in the rich scientific environment of the Texas Medical Center under the close sponsorship of Dr. Bruce Kone, an expert in these areas. The scientific aims of the proposal are to model the molecular action of aldosterone on the epithelial Na+ channel in the collecting duct. Aldosterone is a major regulator of Na+ and acid-base balance and control of blood pressure. The specific aims are to define a novel signaling cascade initiated by aldosterone, via AF9a phosphorylation by SGK1 and H3 K79 methylation by mDotla to transcriptional activation of ENaC in mIMCDS cells and to reveal AF9a and mDotl a as novel physiological targets of aldosterone in regulating ENaC transcription in vivo in mice. mIMCDS cells have been shown to respond to aldosterone and express SGK1, AF9a, mDotl a and ENaC. A wide range of molecular approaches including chromatin immunoprecipitation, real-time RTqPCFt nuclear run-on assays and renal physiological assays (ion transport and urinary electrolyte) will be applied to achieve the goals. It is anticipated that 1) AF9a will identified as an early repressed target by aldosterone action, a novel physiological substrate of SGK1, the first non-histone protein interacting with mDotla and the first protein modulating H3 K79 methylation in a targeted manner; 2) Dotla will become the first chromatin-remodeling protein that serves as an integrate component of aldosterone signaling pathway; 3) ENaC will become the first immediate downstream target of AF9a. Collectively this work will poise the applicant to be an innovative independent scientist in renal signaling and ion transport.