Analysis of the mouse Scnn1a promoter in cortical collecting duct cells and in transgenic mice

Kohler, Stefan A.; Pradervand, Sylvain; Verdumo, Chantal; Mérillat, Anne-Marie; Bens, Marcelle; Vandewalle, Alain; Beermann, Friedrich; Hummler, Edith

doi:10.1016/s0167-4781(01)00228-7

Cited by 14 publications

(18 citation statements)

References 18 publications

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“…The present study, identifying for the first time Af9 binding to a gene promoter and recruitment of Dot1a, adds new mechanistic evidence. Previous structure-function analysis of the mouse ␣ENaC gene revealed that high basal activity is retained with promoter-reporter fragments containing the 76 bp immediately downstream of the major transcription start site (12). Whether the proximity of the ϩ78/ϩ92 element to the transcription start site confers hindrance to the RNA polymerase II transcriptional machinery is not known.…”

Section: Discussionmentioning

confidence: 99%

“…It had been assumed that this response was solely due to the action of aldosterone, liganded to the mineralocorticoid receptor (MR), acting at one or more hormone response elements in the ␣ENaC promoterenhancer. Indeed, promoter-reporter studies of the murine ␣ENaC gene in CD cells revealed the functional importance of a glucocorticoid-responsive element (GRE) at Ϫ811 of the ␣ENaC gene in the aldosterone response (12). However, mice with CNT/CD-specific knockout of the MR did not develop the severe salt-wasting phenotype (21) observed with targeted ablation of ␣ENaC in these same segments (5), suggesting the importance of MR-independent pathways in ␣ENaC regulation.…”

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

Zhang

et al. 2013

American Journal of Physiology-Renal Physiology

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The epithelial Na ϩ channel subunit-␣ (␣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 ␣ENaC transcription in mouse inner medullary collecting duct type 3 (mIMCD3) cells and link aldosterone-elicited chromatin modifications to ␣ENaC transcriptional activation. Af9 DNA-binding activity has never been demonstrated, and whether and where Af9 binds to the ␣ENaC promoter to target Dot1a are unknown. The present study sought to identify functional Af9 cis-element(s) in the Ϫ57/ϩ439 "R3" subregion of ␣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 ␣ENaC expression in vivo. mIMCD3 cell lines expressing ␣ENaC promoter-reporter constructs harboring deletion of ϩ74/ϩ107 demonstrated greatly reduced association of Af9 and Dot1a by ChIP/qPCR. Aldosterone treatment resulted in further decrements in Af9 and Dot1a association with the ␣ENaC promoter. Gel shift and antibody competition assays using wild-type and mutant oligomers revealed Af9-containing ϩ78/ ϩ92 ␣ENaC DNA-protein complexes in nuclear extracts of mIMCD3 cells. Mutation of the ϩ78/ϩ92 element resulted in higher basal ␣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 ␣ENaC mRNA levels in kidney compared with control. Thus, we conclude that ϩ78/ϩ92 of ␣ENaC represents the primary Af9 binding site involved in recruiting Dot1a to repress basal and aldosterone-sensitive ␣ENaC transcription and that Dot1l inactivation promotes ␣ENaC mRNA expression by eliminating Dot1a-mediated repression. chromatin; transcription; collecting duct; transcription factor; gene expression THE EPITHELIAL SODIUM CHANNEL (ENaC) plays a central role in collecting duct (CD) Na ϩ reabsorption, and hence the regulation of extracellular fluid volume and blood pressure (1). ENaC is expressed in the apical membrane of salt-absorbing epithelia of kidney, distal colon, and lung, where it constitutes the rate-limiting step in active Na ϩ and fluid absorption. ENaC consists of three subunits -␣, ␤, and ␥-encoded by the Scnn1a (referred to as "␣ENaC" in this report), Scnn1b, and Scnn1c genes. ENaC is also an important molecular target of aldosterone. In the CD, aldosterone administration or hyperaldosteronism induced by a low-Na ϩ diet increases ␣ENaC gene transcription, without increasing ␤-or ␥-subunit expression or ␣ENaC mRNA turnover (14), and this response appears to be rate-limiting for ENaC activity in this segment. The physiological importance of ␣ENaC to overall salt balance is highlighted by the finding that targeted inactivation of ␣ENaC in the connecting tubule (CNT)/CD of mice results in severe renal salt wasting characteristic of a pseudohypoaldosteronism type...

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Section: Discussionmentioning

confidence: 99%

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

Zhang

et al. 2013

American Journal of Physiology-Renal Physiology

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“…The 5Ј-flanking regions of human, mouse, and rat ENaC␣ contain a highly conserved imperfect glucocorticoid-response element (GRE) that is essential for GR-or mineralocorticoid receptor-mediated trans-activation in the presence of glucocorticoid or aldosterone (10,12,13). Cell-specific expression and corticosteroid-mediated regulation of murine ENaC␣ requires the proximal 1.56-kb of 5Ј-upstream sequence that harbors the conserved GRE and multiple GRE half-sites (12). Before interacting with the cognate hormone response element, the ligand-receptor complex must have accessibility to the DNA, which is compacted in chromatin.…”

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Dot1a-AF9 Complex Mediates Histone H3 Lys-79 Hypermethylation and Repression of ENaCα in an Aldosterone-sensitive Manner

Zhang¹,

Xia²,

Reisenauer³

et al. 2006

Journal of Biological Chemistry

153

231

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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␣ transcription through mediating histone H3 Lys-79 methylation associated with the ENaC␣ promoter. Here, we report a novel aldosterone-signaling network involving AF9, Dot1a, and ENaC␣. 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␣ promoter at most, but not all subregions examined, repression of endogenous ENaC␣ mRNA expression and acts synergistically with Dot1a to inhibit ENaC␣ 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␣ promoter. Aldosterone negatively regulates AF9 expression at both mRNA and protein levels. Thus, Dot1a-AF9 modulates histone H3 Lys-79 methylation at the ENaC␣ promoter and represses ENaC␣ 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.The epithelial sodium channel (ENaC) 2 is a heteromultimeric protein composed of three partially homologous subunits (␣, ␤, and ␥) that is expressed in the apical membrane of salt-absorbing epithelia of kidney, colon, and lung where it constitutes the rate-limiting steps in active Na ϩ and fluid absorption. ENaC plays a major role in the regulation of salt homeostasis and blood pressure as evidenced by the fact that ENaC mutations are associated with genetic hypertensive and hypotensive diseases, such as Liddle's syndrome (1) and pseudohypoaldosteronism type 1 (2), and the fact that it is subject to tight and complex regulation by aldosterone. Aldosterone is a major regulator of epithelial Na ϩ absorption and acts in large part through ENaC induction in the renal collecting duct (3, 4). Aldosterone administration or hyperaldosteronism induced by a low-Na ϩ diet increases ENaC␣ gene transcription, without increasing ␤-or ␥-subunit expression (5-9), and without a separate effect on ENaC␣ mRNA turnover (10) in this segment. Although ENaC␣ synthesis is believed to be the rate-limiting step in Na ϩ channel formation in the collecting duct, only limited information exists regarding the specific mechanisms governing transcriptional regulation of this gene, in particular epigenetic mechanisms exerting such controls.Traditional models of aldosterone trans-activation of target genes, including ENaC␣, have emphasized interaction of the liganded mineralocorticoid receptor or g...

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“…A promoter-reporter study (11) of the ␣ENaC gene in CD cells revealed the involvement of a glucocorticoid responsive element at Ϫ811 in the aldosterone response, leading to the assumption that aldosterone activation of ␣ENaC gene transcription was solely due to the action of aldosterone, liganded to the mineralocorticoid receptor (MR), acting at this glucocorticoid responsive element. However, mice with CNT/CDspecific MR inactivation (19) failed to develop the severe salt-wasting phenotype observed with CNT/CD-specific ablation of ␣ENaC in these same segments (5), indicating the large contribution of MR-independent pathways in ␣ENaC gene regulation.…”

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confidence: 99%

“…Aldosterone, through suppression of Dot1a, Af9, and Sirt1 abundance and through serum-and glucocorticoid-induced kinase-1-mediated phosphorylation of Af9 and consequent disruption of the Dot1/Af9 complex at the ␣ENaC promoter, results in the derepression of ␣ENaC transcription in a MR-independent manner (28). At the same time, aldosterone enhances Sp1- (23) and MR-mediated (11,25) trans-activation in CD cells. Interestingly, aldosterone enhances Sp1 occupation of the ␣ENaC promoter without changing the nuclear abundance of the transcription factor (23), suggesting that other context-dependent factors influence its binding and/or activity at the ␣ENaC promoter.…”

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confidence: 99%

Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting cuct

Kong

Kone

2013

American Journal of Physiology-Renal Physiology

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Yu Z, Kong Q, Kone BC. Aldosterone reprograms promoter methylation to regulate ␣ENaC transcription in the collecting cuct. Am J Physiol Renal Physiol 305: F1006 -F1013, 2013. First published August 7, 2013 doi:10.1152/ajprenal.00407.2013.-Aldosterone increases tubular Na ϩ absorption largely by increasing ␣-epithelial Na ϩ channel (␣ENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal ␣ENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated ␣ENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2=-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the ␣ENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal ␣ENaC transcription, indicating that promoter methylation suppresses basal ␣ENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the ␣ENaC promoter, consistent with active demethylation. 5-AzaCdR mimicked aldosterone by enhancing Sp1 binding to the ␣ENaC promoter. We conclude that DNMT3b-and MBD4-dependent methylation of the ␣ENaC promoter limits basal ␣ENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the ␣ENaC promoter to induce ␣ENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced ␣ENaC transcription that adds to previously described epigenetic controls exerted by histone modifications. epigenetic; chromatin; transcription factor; methylcytosine; methyltransferase; epithelial Na ϩ channel THE EPITHELIAL NA ϩ CHANNEL (ENaC) mediates Na ϩ entry across the apical membranes of salt-absorbing epithelia of the kidney, distal colon, and lung and functions as the rate-limiting step in active transepithelial Na ϩ and fluid absorption. In serving this role, ENaC plays important roles in the regulation of extracellular fluid volume and blood pressure (1,20). Of the three ENaC subunits (␣, ␤, and ␥), ␣ENaC appears to be critical to the overall salt balance, as evidenced by the finding that mice with targeted inactivation of ␣ENaC in the connecting tubule (CNT)/collecting duct (CD) exhibit severe renal salt wasting characteristic of a pseudohypoaldosteronism type I phenotype (5). ␣ENaC is also a molecular target of aldosterone, which stimulates its transcription in a manner that is rate l...

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Analysis of the mouse Scnn1a promoter in cortical collecting duct cells and in transgenic mice

Cited by 14 publications

References 18 publications

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

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

Dot1a-AF9 Complex Mediates Histone H3 Lys-79 Hypermethylation and Repression of ENaCα in an Aldosterone-sensitive Manner

Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting cuct

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