Aldosterone-sensitive repression of ENaC by AF9 and Dot1

描述（由申请人提供）： 本研究项目的主要目的是为申请人提供相关肾脏和整体动物生理学研究的新的强化培训，并为阐明醛固酮信号通路控制离子转运至肾脏生理学的基础。申请人的长期目标是成为肾脏分子和细胞生物学研究的成功独立研究者。为了实现这一目标，申请人将在德克萨斯医疗中心丰富的科学环境中接受培训，并得到这些领域专家布鲁斯科内博士的密切赞助。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Na+和酸碱平衡以及血压控制的主要调节剂。具体的目的是定义由醛固酮启动的新的信号级联，通过SGK 1的AF 9a磷酸化和mDotla的H3 K79甲基化，在mIMCDS细胞中转录激活ENaC，并揭示AF 9a和mDotla作为醛固酮在小鼠体内调节ENaC转录的新的生理靶标。已经显示mIMCDS细胞响应于醛固酮并表达SGK 1、AF 9a、mDot 1a和ENaC。将应用广泛的分子方法，包括染色质免疫沉淀、实时RTqPCFt核运行试验和肾生理试验（离子转运和尿电解质）来实现这些目标。预计1）AF 9a将被确定为醛固酮作用的早期抑制靶点、SGK 1的新的生理底物、第一个与mDotla相互作用的非组蛋白蛋白、第一个靶向调节H3 K79甲基化的蛋白; 2）Dotla将成为第一个作为醛固酮信号通路整合组分的染色质重塑蛋白; 3）ENaC将成为AF 9a的第一个直接下游靶标。总的来说，这项工作将使申请人成为肾信号传导和离子转运方面的创新独立科学家。

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.