Dot1a-AF9 complex mediates histone H3 Lys-79 hypermethylation and repression of ENaCalpha 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 ENaCalpha transcription through mediating histone H3 Lys-79 methylation associated with the ENaCalpha promoter. Here, we report a novel aldosterone-signaling network involving AF9, Dot1a, and ENaCalpha. 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 ENaCalpha promoter at most, but not all subregions examined, repression of endogenous ENaCalpha mRNA expression and acts synergistically with Dot1a to inhibit ENaCalpha 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 ENaCalpha promoter. Aldosterone negatively regulates AF9 expression at both mRNA and protein levels. Thus, Dot1a-AF9 modulates histone H3 Lys-79 methylation at the ENaCalpha promoter and represses ENaCalpha 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.
Zhang
Wenzheng
W
Department of Internal Medicine, Health Science Center, The University of Texas Medical School at Houston, 6431 Fannin, Houston, TX 77030, USA.
Xia
Xuefeng
X
Reisenauer
Mary Rose
MR
Hemenway
Charles S
CS
Kone
Bruce C
BC
D000450:Aldosterone / Q000494:pharmacology / Q000502:physiology; D000818:Animals; D002460:Cell Line; D019175:DNA Methylation; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D053503:Epithelial Sodium Channels; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D011495:Histone-Lysine N-Methyltransferase; D006657:Histones / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology; D008239:Lysine / Q000378:metabolism; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000502:physiology; D034741:RNA, Small Interfering; D015222:Sodium Channels / Q000235:genetics*; D020798:Two-Hybrid System Techniques; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D053503:Epithelial Sodium Channels; D006657:Histones; C489902:Mllt3 protein, mouse; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D015222:Sodium Channels; D000450:Aldosterone; C481527:Dot1l protein, mouse; D008780:Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; D008239:Lysine