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 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.