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 4

Sgk1 associates with and downregulates Dot1a-Af9 interaction at the ENaCα promoter.

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Sgk1 associates with and downregulates Dot1a-Af9 interaction at the ENaC...
(A) Diagram of the ENaCα promoter (8). (B) ChIP assay showing association of WT and kinase-dead mutant Sgk1 with the R0–R3, but not Ra, subregions of the ENaCα promoter. Chromatin was immunoprecipitated by anti-FLAG antibody from mIMCD3 cells transfected with pCMV500, WT pFLAG-Sgk1, or mutant pFLAG-Sgk1 Lys127Met, followed by quantitative real-time PCR with primers amplifying the Ra and R0–R3 subregions of ENaCα promoter as shown in A (n = 3). FLAG-Sgk1 abundance in R0 from the FLAG-Sgk1–transfected cells was assigned as 1. (C–F) ChIP and sequential ChIP assays demonstrating that overexpression of Sgk1 differentially affected the abundance of FLAG-Af9 (C), Dot1a (D), histone H3 Lys79 methylation (E), and FLAG-Af9 interaction with Dot1a (F) at the ENaCα promoter. mIMCD3 cells were cotransfected with pFLAG-Af9 and pcDNA3.1 or its derivatives expressing Sgk1 or its kinase-dead mutant, followed by ChIP with the indicated antibodies. For sequential ChIP (re-ChIP), chromatin was sequentially immunoprecipitated with anti-FLAG and anti-Dot1a antibodies. Relative ChIP or sequential ChIP efficiency was defined as the amount of (re)immunoprecipitated material compared with that of the initial input sample and was assigned as 1 in R0 from vector-transfected cells (n = 3–6). No Ra expression was detected in B, C, or F. *P < 0.05 versus vector control.