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.
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.
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