| First Author | Zhang W | Year | 2013 |
| Journal | Am J Physiol Renal Physiol | Volume | 304 |
| Issue | 4 | Pages | F367-75 |
| PubMed ID | 23152297 | Mgi Jnum | J:193142 |
| Mgi Id | MGI:5467699 | Doi | 10.1152/ajprenal.00537.2011 |
| Citation | Zhang W, et al. (2013) An Af9 cis-element directly targets Dot1a to mediate transcriptional repression of the alphaENaC gene. Am J Physiol Renal Physiol 304(4):F367-75 |
| abstractText | The epithelial Na(+) channel subunit-alpha (alphaENaC) of the distal nephron is essential for salt balance. We previously demonstrated that the histone methyltransferase Dot1a and its protein partner Af9 basally repress alphaENaC transcription in mouse inner medullary collecting duct type 3 (mIMCD3) cells and link aldosterone-elicited chromatin modifications to alphaENaC transcriptional activation. Af9 DNA-binding activity has never been demonstrated, and whether and where Af9 binds to the alphaENaC promoter to target Dot1a are unknown. The present study sought to identify functional Af9 cis-element(s) in the -57/+439 "R3" subregion of alphaENaC, 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 alphaENaC expression in vivo. mIMCD3 cell lines expressing alphaENaC 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 alphaENaC promoter. Gel shift and antibody competition assays using wild-type and mutant oligomers revealed Af9-containing +78/+92 alphaENaC DNA-protein complexes in nuclear extracts of mIMCD3 cells. Mutation of the +78/+92 element resulted in higher basal alphaENaC 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 alphaENaC mRNA levels in kidney compared with control. Thus, we conclude that +78/+92 of alphaENaC represents the primary Af9 binding site involved in recruiting Dot1a to repress basal and aldosterone-sensitive alphaENaC transcription and that Dot1l inactivation promotes alphaENaC mRNA expression by eliminating Dot1a-mediated repression. |
