Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCα in an aldosterone-sensitive manner

Aldosterone is a major regulator of epithelial Na+ absorption and acts in large part through induction of the epithelial Na+ channel (ENaC) gene in the renal collecting duct. We previously identified Dot1a as an aldosterone early repressed gene and a repressor of ENaC alpha transcription through mediating histone H3 Lys-79 methylation associated with the ENaC alpha promoter. Here, we report a novel aldosterone-signaling network involving AF9, Dot1a, and ENaC alpha. AF9 and Dot1a interact in vitro and in vivo as evidenced in multiple assays and colocalize in the nuclei of mIMCD3 renal collecting duct cells. Overexpression of AF9 results in hypermethylation of histone H3 Lys-79 at the endogenous ENaC alpha promoter at most, but not all subregions examined, repression of endogenous ENaC alpha mRNA expression and acts synergistically with Dot1a to inhibit ENaC alpha promoter-luciferase constructs. In contrast, RNA interference-mediated knockdown of AF9 causes the opposite effects. Chromatin immunoprecipitation assays reveal that overexpressed FLAG-AF9, endogenous AF9, and Dot1a are each associated with the ENaC alpha promoter. Aldosterone negatively regulates AF9 expression at both mRNA and protein levels. Thus, Dot1a-AF9 modulates histone H3 Lys-79 methylation at the ENaC alpha promoter and represses ENaC alpha transcription in an aldosterone-sensitive manner. This mechanism appears to be more broadly applicable to other aldosterone-regulated genes because overexpression of AF9 alone or in combination with Dot1a inhibited mRNA levels of three other known aldosterone-inducible genes in mIMCD3 cells.