An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the alpha ENaC gene

作者：Zhang Wenzheng; Yu Zhiyuan; Wu Hongyu; Chen Lihe; Kong Qun; Kone Bruce C^*

来源：American Journal of Physiology - Renal Fluid and Electrolyte Physiology, 2013, 304(4): F367-F375.

DOI：10.1152/ajprenal.00537.2011

• chromatin
• transcription
• collecting duct
• transcription factor
• gene expression

Zhang W, Yu Z, Wu H, Chen L, Kong Q, Kone BC. An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the alpha ENaC gene. Am J Physiol Renal Physiol 304: F367-F375, 2013. First published November 14, 2012; doi:10.1152/ajprenal.00537.2011.-The epithelial Na+ channel subunit-alpha (alpha ENaC) of the distal nephron is essential for salt balance. We previously demonstrated that the histone methyltransferase Dot1a and its protein partner Af9 basally repress alpha ENaC transcription in mouse inner medullary collecting duct type 3 (mIMCD3) cells and link aldosterone-elicited chromatin modifications to alpha ENaC transcriptional activation. Af9 DNA-binding activity has never been demonstrated, and whether and where Af9 binds to the alpha ENaC promoter to target Dot1a are unknown. The present study sought to identify functional Af9 cis-element(s) in the -57/+439 %26quot;R3%26quot; subregion of alpha ENaC, the principal site for Dot1a-Af9 interaction, in mIMCD3 cells. We also exploited connecting tubule/collecting duct-specific Dot1l-deficient mice (Dot1l(AC)) to determine the impact of Dot1l inactivation on renal alpha ENaC expression in vivo. mIMCD3 cell lines expressing alpha ENaC promoter-reporter constructs harboring deletion of +74/+107 demonstrated greatly reduced association of Af9 and Dot1a by ChIP/qPCR. Aldosterone treatment resulted in further decrements in Af9 and Dot1a association with the alpha ENaC promoter. Gel shift and antibody competition assays using wild-type and mutant oligomers revealed Af9-containing +78/+92 alpha ENaC DNA-protein complexes in nuclear extracts of mIMCD3 cells. Mutation of the +78/+92 element resulted in higher basal alpha ENaC promoter activity and impaired Dot1a-mediated inhibition in trans-repression assays. In agreement, mice with connecting tubule/collecting duct-specific knockout of Dot1l exhibited greater alpha ENaC mRNA levels in kidney compared with control. Thus, we conclude that +78/+92 of alpha ENaC represents the primary Af9 binding site involved in recruiting Dot1a to repress basal and aldosterone-sensitive alpha ENaC transcription and that Dot1l inactivation promotes alpha ENaC mRNA expression by eliminating Dot1a-mediated repression.

• 出版日期2013-2