Aldosterone-induced Sgk1 relieves Dot1a-Af9–mediated transcriptional repression of epithelial Na+ channel α
Wenzheng Zhang, … , Volker Vallon, Bruce C. Kone
Wenzheng Zhang, … , Volker Vallon, Bruce C. Kone
Published March 1, 2007
Citation Information: J Clin Invest. 2007;117(3):773-783. https://doi.org/10.1172/JCI29850.
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Research Article

Aldosterone-induced Sgk1 relieves Dot1a-Af9–mediated transcriptional repression of epithelial Na+ channel α

Abstract

Aldosterone plays a major role in the regulation of salt balance and the pathophysiology of cardiovascular and renal diseases. Many aldosterone-regulated genes — including that encoding the epithelial Na+ channel (ENaC), a key arbiter of Na+ transport in the kidney and other epithelia — have been identified, but the mechanisms by which the hormone modifies chromatin structure and thus transcription remain unknown. We previously described the basal repression of ENaCα by a complex containing the histone H3 Lys79 methyltransferase disruptor of telomeric silencing alternative splice variant a (Dot1a) and the putative transcription factor ALL1-fused gene from chromosome 9 (Af9) as well as the release of this repression by aldosterone treatment. Here we provide evidence from renal collecting duct cells and serum- and glucocorticoid-induced kinase–1 (Sgk1) WT and knockout mice that Sgk1 phosphorylated Af9, thereby impairing the Dot1a-Af9 interaction and leading to targeted histone H3 Lys79 hypomethylation at the ENaCα promoter and derepression of ENaCα transcription. Thus, Af9 is a physiologic target of Sgk1, and Sgk1 negatively regulates the Dot1a-Af9 repressor complex that controls transcription of ENaCα and likely other aldosterone-induced genes.

Authors

Wenzheng Zhang, Xuefeng Xia, Mary Rose Reisenauer, Timo Rieg, Florian Lang, Dietmar Kuhl, Volker Vallon, Bruce C. Kone

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Figure 5

Af9 Ser435Ala and Ser435Asp mutations impair Af9 overexpression–dependent histone H3 Lys79 hypermethylation and ENaCα promoter repression.

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Af9 Ser435Ala and Ser435Asp mutations impair Af9 overexpression–dependen...
(A) ChIP assay showing that Af9 Ser435Ala and Ser435Asp mutations decreased Dot1a-Af9–mediated histone H3 Lys79 methylation, but not Af9 association with the ENaCα promoter, in mIMCD3 cells. pFLAG-Af9, pFLAG-Af9 Ser435Ala, and pFLAG-Af9 Ser435Asp were used, and ChIP assays were performed with the indicated antibodies (n = 3–4). The relative histone H3 Lys79 methylation of R0 from the vector-transfected cells was assigned as 1. Representative agarose gel analyses of the final quantitative PCR products are shown to verify the quantification determined by quantitative real-time PCR for each sample. (B) Luciferase assay indicating that the Ser435Ala mutation impaired Af9-dependent repression of ENaCα promoter activity. mIMCD3 cells stably carrying pGL3Zeocin-1.3ENaCα (12) were transiently transfected with pCMV500, pFLAG-Af9, or pFLAG-Af9 Ser435Ala, followed by luciferase assay (n = 4). *P < 0.05 versus vector control. (C) Quantitative RT-PCR (qRT-PCR) demonstrating that the Ser435Ala mutation partially relieves Af9-dependent repression of endogenous ENaCα transcription. As in B, except that total RNA was analyzed by quantitative real-time RT-PCR with primers specific for ENaCα or β-actin as control and that whole-cell lysates were examined by IP with the indicated antibodies. The relative level of ENaCα mRNA was normalized to actin in the same sample, and that of vector-transfected cells was assigned as 1. The means of 3 independent measurements with SE less than 20% are shown.