Aldosterone-induced Sgk1 relieves Dot1a-Af9–mediated transcriptional repression of epithelial Na+ channel α
Wenzheng Zhang, Xuefeng Xia, Mary Rose Reisenauer, Timo Rieg, Florian Lang, Dietmar Kuhl, Volker Vallon, Bruce C. Kone
Wenzheng Zhang, Xuefeng Xia, Mary Rose Reisenauer, Timo Rieg, Florian Lang, Dietmar Kuhl, Volker Vallon, Bruce C. Kone
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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.