Sirtuin 1 Functionally and Physically Interacts with Disruptor of Telomeric Silencing-1 to Regulate α-ENaC Transcription in Collecting Duct
2009; Elsevier BV; Volume: 284; Issue: 31 Linguagem: Inglês
10.1074/jbc.m109.020073
ISSN1083-351X
AutoresDongyu Zhang, Shiyu Li, Pedro E. Cruz, Bruce C. Kone,
Tópico(s)Epigenetics and DNA Methylation
ResumoAldosterone increases renal tubular Na+ absorption in large part by increasing transcription of the epithelial Na+ channel α-subunit (α-ENaC) expressed in the apical membrane of collecting duct principal cells. We recently reported that a complex containing the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) associates with and represses the α-ENaC promoter in mouse inner medullary collecting duct mIMCD3 cells, and that aldosterone acts to disrupt this complex and its inhibitory effects (Zhang, W., Xia, X., Reisenauer, M. R., Rieg, T., Lang, F., Kuhl, D., Vallon, V., and Kone, B. C. (2007) J. Clin. Invest. 117, 773–783). Here we demonstrate that the NAD+-dependent deacetylase sirtuin 1 (Sirt1) functionally and physically interacts with Dot1 to enhance the distributive activity of Dot1 on H3K79 methylation and thereby represses α-ENaC transcription in mIMCD3 cells. Sirt1 overexpression inhibited basal α-ENaC mRNA expression and α-ENaC promoter activity, surprisingly in a deacetylase-independent manner. The ability of Sirt1 to inhibit α-ENaC transcription was retained in a truncated Sirt1 construct expressing only its N-terminal domain. Conversely, Sirt1 knockdown enhanced α-ENaC mRNA levels and α-ENaC promoter activity, and inhibited global H3K79 methylation, particularly H3K79 trimethylation, in chromatin associated with the α-ENaC promoter. Sirt1 and Dot1 co-immunoprecipitated from mIMCD3 cells and colocalized in the nucleus. Sirt1 immunoprecipitated from chromatin associated with regions of the α-ENaC promoter known to associate with Dot1. Aldosterone inhibited Sirt1 association at two of these regions, as well as Sirt1 mRNA expression, in a coordinate manner with induction of α-ENaC transcription. Overexpressed Sirt1 inhibited aldosterone induction of α-ENaC transcription independent of effects on mineralocorticoid receptor trans-activation. These data identify Sirt1 as a novel modulator of α-ENaC, Dot1, and the aldosterone signaling pathway. Aldosterone increases renal tubular Na+ absorption in large part by increasing transcription of the epithelial Na+ channel α-subunit (α-ENaC) expressed in the apical membrane of collecting duct principal cells. We recently reported that a complex containing the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) associates with and represses the α-ENaC promoter in mouse inner medullary collecting duct mIMCD3 cells, and that aldosterone acts to disrupt this complex and its inhibitory effects (Zhang, W., Xia, X., Reisenauer, M. R., Rieg, T., Lang, F., Kuhl, D., Vallon, V., and Kone, B. C. (2007) J. Clin. Invest. 117, 773–783). Here we demonstrate that the NAD+-dependent deacetylase sirtuin 1 (Sirt1) functionally and physically interacts with Dot1 to enhance the distributive activity of Dot1 on H3K79 methylation and thereby represses α-ENaC transcription in mIMCD3 cells. Sirt1 overexpression inhibited basal α-ENaC mRNA expression and α-ENaC promoter activity, surprisingly in a deacetylase-independent manner. The ability of Sirt1 to inhibit α-ENaC transcription was retained in a truncated Sirt1 construct expressing only its N-terminal domain. Conversely, Sirt1 knockdown enhanced α-ENaC mRNA levels and α-ENaC promoter activity, and inhibited global H3K79 methylation, particularly H3K79 trimethylation, in chromatin associated with the α-ENaC promoter. Sirt1 and Dot1 co-immunoprecipitated from mIMCD3 cells and colocalized in the nucleus. Sirt1 immunoprecipitated from chromatin associated with regions of the α-ENaC promoter known to associate with Dot1. Aldosterone inhibited Sirt1 association at two of these regions, as well as Sirt1 mRNA expression, in a coordinate manner with induction of α-ENaC transcription. Overexpressed Sirt1 inhibited aldosterone induction of α-ENaC transcription independent of effects on mineralocorticoid receptor trans-activation. These data identify Sirt1 as a novel modulator of α-ENaC, Dot1, and the aldosterone signaling pathway. Epithelial Na+ channel (ENaC) 2The abbreviations used are: ENaCepithelial sodium channelDot1disruptor of telomeric silencingAf9ALL1-fused gene from chromosome 9Sir2silent information regulator 2Sgk1serum- and glucocorticoid-induced kinase 1MRmineralocorticoid receptorGREglucocorticoid-response elementEGFPenhanced green fluorescent proteinRFPred fluorescent proteinsiRNAsmall interfering RNARTreverse transcriptaseChIPchromatin immunoprecipitationqPCRquantitative. 2The abbreviations used are: ENaCepithelial sodium channelDot1disruptor of telomeric silencingAf9ALL1-fused gene from chromosome 9Sir2silent information regulator 2Sgk1serum- and glucocorticoid-induced kinase 1MRmineralocorticoid receptorGREglucocorticoid-response elementEGFPenhanced green fluorescent proteinRFPred fluorescent proteinsiRNAsmall interfering RNARTreverse transcriptaseChIPchromatin immunoprecipitationqPCRquantitative. is composed of three genetically distinct subunits (α, β, and γ), and is expressed in the apical membrane of salt-absorbing epithelia of kidney, colon, and lung (1Rossier B.C. Pradervand S. Schild L. Hummler E. Annu. Rev. Physiol. 2002; 64: 877-897Crossref PubMed Scopus (318) Google Scholar). ENaC expressed in the distal nephron constitutes the rate-limiting step for renal Na+ and fluid reabsorption and therefore is essential for physiological control of blood pressure and electrolyte composition. Genetic disorders of ENaC activity, such as pseudohypoaldosteronism type 1 and Liddle syndrome, are characterized by hypotension and hypertension, respectively (2Schild L. Nephrologie. 1996; 17: 395-400PubMed Google Scholar). The number of channels present at the plasma membrane is highly regulated through channel biosynthesis and delivery to the plasma membrane and by channel retrieval from the plasma membrane (4Husted R.F. Volk K.A. Sigmund R.D. Stokes J.B. Am. J. Physiol. Renal Physiol. 2007; 293: F813-F820Crossref PubMed Scopus (17) Google Scholar, 5Zhang W. Hayashizaki Y. Kone B.C. Biochem. J. 2004; 377: 641-651Crossref PubMed Google Scholar, 6Zhang W. Xia X. Jalal D.I. Kuncewicz T. Xu W. Lesage G.D. Kone B.C. Am. J. Physiol. Cell Physiol. 2006; 290: C936-C946Crossref PubMed Scopus (54) Google Scholar). Of the three ENaC subunits, it is synthesis of α-ENaC subunits that appears to be rate-limiting for the formation of new ENaC multimers. Thus mechanisms controlling α-ENaC gene transcription are central to the understanding of channel biosynthesis. epithelial sodium channel disruptor of telomeric silencing ALL1-fused gene from chromosome 9 silent information regulator 2 serum- and glucocorticoid-induced kinase 1 mineralocorticoid receptor glucocorticoid-response element enhanced green fluorescent protein red fluorescent protein small interfering RNA reverse transcriptase chromatin immunoprecipitation quantitative. epithelial sodium channel disruptor of telomeric silencing ALL1-fused gene from chromosome 9 silent information regulator 2 serum- and glucocorticoid-induced kinase 1 mineralocorticoid receptor glucocorticoid-response element enhanced green fluorescent protein red fluorescent protein small interfering RNA reverse transcriptase chromatin immunoprecipitation quantitative. Aldosterone, a key regulator of epithelial Na+ absorption, induces transepithelial Na+ transport in the collecting duct in large part through activation of α-ENaC gene transcription. Aldosterone administration or secondary hyperaldosteronism caused by salt restriction enhances α-ENaC gene transcription without increasing β or γ subunit expression or altering α-ENaC mRNA turnover (3Mick V.E. Itani O.A. Loftus R.W. Husted R.F. Schmidt T.J. Thomas C.P. Mol. Endocrinol. 2001; 15: 575-588Crossref PubMed Scopus (107) Google Scholar). The biological actions of aldosterone on target gene transcription have generally been believed to be mediated principally or exclusively through the mineralocorticoid receptor (MR), a member of the nuclear hormone receptor family. Aldosterone has both immediate ( 3 h) that involve the synthesis of new ENaC subunits (1Rossier B.C. Pradervand S. Schild L. Hummler E. Annu. Rev. Physiol. 2002; 64: 877-897Crossref PubMed Scopus (318) Google Scholar, 2Schild L. Nephrologie. 1996; 17: 395-400PubMed Google Scholar, 15Avalos J.L. Bever K.M. Wolberger C. Mol. Cell. 2005; 17: 855-868Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, 18Zschoernig B. Mahlknecht U. Biochem. Biophys. Res. Commun. 2009; 381: 372-377Crossref PubMed Scopus (58) Google Scholar, 33Altaf M. Utley R.T. Lacoste N. Tan S. Briggs S.D. Côté J. Mol. Cell. 2007; 28: 1002-1014Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). A highly conserved imperfect glucocorticoid-response element (GRE) has been identified in the 5′-flanking regions of the human, mouse, and rat α-ENaC genes and found to be necessary for trans-activation (12van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar, 17Finnin M.S. Donigian J.R. Pavletich N.P. Nat. Struct. Biol. 2001; 8: 621-625Crossref PubMed Scopus (315) Google Scholar, 35Ronzaud C. Loffing J. Bleich M. Gretz N. Gröne H.J. Schütz G. Berger S. J. Am. Soc. Nephrol. 2007; 18: 1679-1687Crossref PubMed Scopus (98) Google Scholar). Although heterologous expression of human α-ENaC in murine M1 collecting duct cells suggests that increased α-ENaC expression alone may be insufficient to increase ENaC activity under basal conditions, increased α-ENaC expression does appear to be necessary for full aldosterone induction of ENaC function (4Husted R.F. Volk K.A. Sigmund R.D. Stokes J.B. Am. J. Physiol. Renal Physiol. 2007; 293: F813-F820Crossref PubMed Scopus (17) Google Scholar). In prior work, we identified in the mouse inner medullary collecting duct cell line mIMCD3 a novel aldosterone-sensitive nuclear repressor complex, comprised of Dot1 and Af9 (5Zhang W. Hayashizaki Y. Kone B.C. Biochem. J. 2004; 377: 641-651Crossref PubMed Google Scholar). Under basal conditions, this complex associates with chromatin, and Dot1 hypermethylates histone H3K79 associated with four specific regions of the α-ENaC 5′-flanking region, thereby repressing α-ENaC transcription (6Zhang W. Xia X. Jalal D.I. Kuncewicz T. Xu W. Lesage G.D. Kone B.C. Am. J. Physiol. Cell Physiol. 2006; 290: C936-C946Crossref PubMed Scopus (54) Google Scholar, 7Zhang W. Xia X. Reisenauer M.R. Hemenway C.S. Kone B.C. J. Biol. Chem. 2006; 281: 18059-18068Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 8Zhang W. Xia X. Reisenauer M.R. Rieg T. Lang F. Kuhl D. Vallon V. Kone B.C. J. Clin. Invest. 2007; 117: 773-783Crossref PubMed Scopus (132) Google Scholar). Aldosterone inhibited Dot1 and Af9 expression and their interaction, leading to hypomethylation of histone H3K79 at specific regions of the α-ENaC 5′-flanking region and release of the basal transcriptional repression of α-ENaC (7Zhang W. Xia X. Reisenauer M.R. Hemenway C.S. Kone B.C. J. Biol. Chem. 2006; 281: 18059-18068Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 8Zhang W. Xia X. Reisenauer M.R. Rieg T. Lang F. Kuhl D. Vallon V. Kone B.C. J. Clin. Invest. 2007; 117: 773-783Crossref PubMed Scopus (132) Google Scholar). We further determined that Dot1, like α-ENaC, was expressed in collecting duct principal cells (6Zhang W. Xia X. Jalal D.I. Kuncewicz T. Xu W. Lesage G.D. Kone B.C. Am. J. Physiol. Cell Physiol. 2006; 290: C936-C946Crossref PubMed Scopus (54) Google Scholar). Dot1 was originally identified as a gene affecting telomeric silencing in Saccharomyces cerevisiae (9Singer M.S. Kahana A. Wolf A.J. Meisinger L.L. Peterson S.E. Goggin C. Mahowald M. Gottschling D.E. Genetics. 1998; 150: 613-632Crossref PubMed Google Scholar), and it is distinguished from other histone lysine methyltransferases by the absence of a Su(var)3–9, Enhancer-of-zeste, Trithorax (SET) domain, and by the fact that it introduces multiple methyl groups on H3K79 via a “distributive” (also termed “nonprocessive”) kinetic mechanism (10Frederiks F. Tzouros M. Oudgenoeg G. van Welsem T. Fornerod M. Krijgsveld J. van Leeuwen F. Nat. Struct. Mol. Biol. 2008; 15: 550-557Crossref PubMed Scopus (127) Google Scholar). Dot1 can methylate 3 methyl groups of Lys-79 in the core domain of histone H3, so that monomethylated (H3K79me1), dimethylated (H3K79me2), and trimethylated (H3K79me3) forms of H3K79 can be observed in chromatin (11Lacoste N. Utley R.T. Hunter J.M. Poirier G.G. Côte J. J. Biol. Chem. 2002; 277: 30421-30424Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 12van Leeuwen F. Gafken P.R. Gottschling D.E. Cell. 2002; 109: 745-756Abstract Full Text Full Text PDF PubMed Scopus (664) Google Scholar). In contrast to the independent generation of multiple methylation states via a processive mechanism, the lower methylation states catalyzed by Dot1 are necessary transient intermediates for the synthesis of higher methylation states. Thus, the relative abundance of the methylation states is expected to depend on Dot1 activity, and the global H3K79 methylation appears to be functionally more important than a specific H3K79 methylation state in yeast (10Frederiks F. Tzouros M. Oudgenoeg G. van Welsem T. Fornerod M. Krijgsveld J. van Leeuwen F. Nat. Struct. Mol. Biol. 2008; 15: 550-557Crossref PubMed Scopus (127) Google Scholar). Indeed, functional roles of the different methylated states or of particular patterns of H3K79me1, H3K79me2, and H3K79me3 have yet to be demonstrated. Sirtuins comprise the family of Sir2 orthologs in mammals. Of the seven human sirtuins, Sirt1 is most similar to the yeast Sir2 protein (13Liou G.G. Tanny J.C. Kruger R.G. Walz T. Moazed D. Cell. 2005; 121: 515-527Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 14Michishita E. Park J.Y. Burneskis J.M. Barrett J.C. Horikawa I. Mol. Biol. Cell. 2005; 16: 4623-4635Crossref PubMed Scopus (1055) Google Scholar). Sirt1 functions as a class III histone deacetylase, binding NAD+ and acetyllysine within protein targets and generating lysine, 2′-O-acetyl-ADP-ribose, and nicotinamide as enzymatic products. Nicotinamide, by promoting a base-exchange reaction at the expense of deacetylation, serves as a noncompetitive inhibitor of Sirt1 (15Avalos J.L. Bever K.M. Wolberger C. Mol. Cell. 2005; 17: 855-868Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar). Sirt1 is a modular protein with three partially characterized domains that contain putative or experimentally confirmed motifs. The N-terminal domain (amino acids 1–210) includes a nuclear localization signal (16Tanno M. Sakamoto J. Miura T. Shimamoto K. Horio Y. J. Biol. Chem. 2007; 282: 6823-6832Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar) and multiple phosphorylation motifs. The central region (amino acids 210–500) contains a conserved catalytic domain for deacetylation (17Finnin M.S. Donigian J.R. Pavletich N.P. Nat. Struct. Biol. 2001; 8: 621-625Crossref PubMed Scopus (315) Google Scholar), and a C-terminal region that may be regulated by phosphorylation (18Zschoernig B. Mahlknecht U. Biochem. Biophys. Res. Commun. 2009; 381: 372-377Crossref PubMed Scopus (58) Google Scholar, 19Pandithage R. Lilischkis R. Harting K. Wolf A. Jedamzik B. Lüscher-Firzlaff J. Vervoorts J. Lasonder E. Kremmer E. Knöll B. Lüscher B. J. Cell Biol. 2008; 180: 915-929Crossref PubMed Scopus (164) Google Scholar). Through its deacetylase activity, Sirt1 regulates diverse biological processes, including gene silencing, stress resistance, metabolism, differentiation, and aging (20Michan S. Sinclair D. Biochem. J. 2007; 404: 1-13Crossref PubMed Scopus (1372) Google Scholar). In vitro, Sirt1 preferentially deacetylates histones H4K16 and H3K9, interacts with and deacetylates histone H1 at Lys-26, and contributes to heterochromatin formation (21Vaquero A. Scher M.B. Lee D.H. Sutton A. Cheng H.L. Alt F.W. Serrano L. Sternglanz R. Reinberg D. Genes Dev. 2006; 20: 1256-1261Crossref PubMed Scopus (473) Google Scholar). In addition, more than a dozen nonhistone proteins, including transcription factors and transcriptional coregulatory proteins, serve as substrates for Sirt1 (22Feige J.N. Auwerx J. Curr. Opin. Cell Biol. 2008; 20: 303-309Crossref PubMed Scopus (168) Google Scholar). However, no previous study has implicated sirtuins in aldosterone signaling or the regulation of a membrane transport protein, and deacetylase-independent actions of Sirt1 on biological processes have not been fully explored. Given the well described functional interplay in gene silencing between Sir proteins and Dot1 in yeast (23Yang B. Britton J. Kirchmaier A.L. J. Mol. Biol. 2008; 381: 826-844Crossref PubMed Scopus (26) Google Scholar), we hypothesized that Sirt1 might participate in chromatin modifications important for the Dot1-mediated repression of α-ENaC gene transcription in murine collecting duct cells. Here, we report that Sirt1 is indeed expressed along with α-ENaC in collecting duct cells, forms an aldosterone-sensitive complex with Dot1 in chromatin associated with specific regions of the α-ENaC promoter that results in global H3K79 hypermethylation, and also serves to suppress α-ENaC gene transcription. Surprisingly, however, we found that these effects of Sirt1 are independent of its deacetylase activity and apparently do not involve Af9 interactions. These results disclose novel actions of Sirt1, a physical and functional interaction of Sirt1 and Dot1, and detail further complexity in the regulation of epithelial Na+ transport and aldosterone signaling. LipofectamineTM 2000 reagent (Invitrogen), aldosterone (Sigma), spironolactone (Sigma), nicotinamide (Sigma), and the Dual Luciferase kit (Promega) were purchased and used according to the manufacturer's instructions. An antibody directed against enhanced green fluorescent protein (EGFP) was from Clontech. Antibodies directed against murine Sirt1, acetylated or total histone H4K5, H4K8, H4K12, and H4K16 were from Cell Signaling, and antibodies directed against H3K79me1, H3K79me2, and H3K79me3 were from Abeam. The plasmids EGFP-Dot1a, FLAG-Af9, and pGL3Zeocin-1.3α-ENaC-Luc (termed α-ENaC-Luc) have been described (6Zhang W. Xia X. Jalal D.I. Kuncewicz T. Xu W. Lesage G.D. Kone B.C. Am. J. Physiol. Cell Physiol. 2006; 290: C936-C946Crossref PubMed Scopus (54) Google Scholar). pGRE-Luc, a plasmid containing a 4× GRE concatamer coupled to a TATA box upstream of the firefly luciferase gene, was purchased from Stratagene. The murine Sirt1 cDNA was purchased from Upstate. The deacetylase-deficient murine Sirt1 mutant plasmid pcDNA3.1-Sirt1(H355Y) was constructed by using a PCR-directed mutagenesis method (Stratagene). RFP-tagged murine Sirt1 fusion protein (pRFP-Sirt1) was constructed by cloning the Sirt1 cDNA into the XhoI site of pDsRed-monomer-C1 (Clontech). DNA encoding the N-terminal (amino acids 1–240) and central (amino acids 210–500) domains of Sirt1 were cloned into pcDNA3.1 to generate expression plasmids pSirt1-(1–240) and pSirt1-(210–500). The authentic expression of these mutant Sirt1 proteins was verified by immunoblotting of extracts of transiently transfected mIMCD3 cells. All inserts in the constructs were verified by DNA sequencing. mIMCD3 cell culture, aldosterone treatment, and transient transfections using the LipofectamineTM 2000 reagent were performed as described (8Zhang W. Xia X. Reisenauer M.R. Rieg T. Lang F. Kuhl D. Vallon V. Kone B.C. J. Clin. Invest. 2007; 117: 773-783Crossref PubMed Scopus (132) Google Scholar). Briefly, cells were cultured in Dulbecco's modified Eagle's medium/F-12 plus 10% fetal bovine serum. Before adding 1 μm aldosterone or 0.01% ethanol as vehicle control at different time points, the cell medium was changed to Dulbecco's modified Eagle's medium/F-12 with 10% charcoal-stripped fetal bovine serum for at least 24 h. All cells were then harvested at the same time point. To knockdown Sirt1 mRNA levels by RNA interference, murine Sirt1 ON-TARGETplus SMARTPOOL siRNA or the negative control siRNA (Dharmacon) was transiently transfected into mIMCD3 cells using the DharmaFECT transfection reagent. The efficiency of silencing Sirt1 expression was measured by quantitative RT-PCR and determined by comparison to RNA harvested from cells transfected with the negative control plasmid. Total RNA of mIMCD3 cells was purified using the miniRNA kit plus DNase I treatment (Qiagen). The cDNA synthesis and PCR amplification were then carried out using the iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad), and primers specific for Sirt11, α-ENaC, or β-actin (used as a reference standard) according to the manufacturer's instructions. The copy number of Sirt1 or α-ENaC transcripts was normalized to that of β-actin from the same sample. The sequences of all primers are available from the authors upon request. Their specificity was analyzed by agarose gel and melting curve analysis. Co-immunoprecipitation and immunoblotting were performed as we previously described (8Zhang W. Xia X. Reisenauer M.R. Rieg T. Lang F. Kuhl D. Vallon V. Kone B.C. J. Clin. Invest. 2007; 117: 773-783Crossref PubMed Scopus (132) Google Scholar). Briefly, whole cell lysates of HEK 293 cells transfected with an empty vector, pEGFP-Dot1, or pFLAG-Af9 were prepared by using low-salt lysis buffer (50 mm HEPES, pH 7.6, 150 mm NaCl, 1.5 mm MgCl2, 1 mm EDTA, 1% Triton X-100, and 10% glycerol). After preclearing with IgG plus protein A/G-Sepharose beads, the lysates were incubated with anti-Sirt1 antibody, anti-Af9 antibody, or isotype control IgG at 4 °C for 4 h on a rotator, and then the protein A/G-Sepharose beads were added to the lysates for an additional 1 h rotation at 4 °C. The beads were washed 6 times with low salt lysis buffer, and the precipitates were eluted with sample buffer, resolved by SDS-PAGE, and further analyzed by immunoblot with antibody against EGFP or FLAG to detect immunoreactivity for Dot1 and Af9, respectively. mIMCD3 cells grown on glass coverslips were transiently co-transfected with pEGFP-Dot1 and pRFP-Sirt1. The cells were then fixed in buffered 1% formaldehyde for 10 min. After 3 washes in phosphate-buffered saline, slides were stained with 4′,6-diamino-2-diphenylindole, mounted with UltraCruz Mounting medium (Santa Cruz) and visualized. Sections were examined under a confocal laser scanning microscope (LSM, 510 UV; Carl Zeiss). Images were acquired with a Plan-Neofluor ×40 (numerical aperture = 0.75) objective and evaluated with Zeiss LSM 510 imaging software including three-dimensional reconstruction. ChIP and ChIP-qPCR assays were performed according to the manufacturer's instructions (Upstate) and as previously described (8Zhang W. Xia X. Reisenauer M.R. Rieg T. Lang F. Kuhl D. Vallon V. Kone B.C. J. Clin. Invest. 2007; 117: 773-783Crossref PubMed Scopus (132) Google Scholar). The eluted DNA fragments were purified to serve as templates for qPCR with primers constructed to amplify the regions Ra (−1327/−965), R0 (−988/−713), R1 (−735/−415), R2 (−414/+80), and R3 (−57/+494) from α-ENaC. qPCRs were run in triplicate and the values were transferred into copy numbers of DNA using a standard curve of genomic DNA with known copy numbers. The resulting transcription values were then normalized for primer pair amplification efficiency using the qPCR values obtained with input DNA. Quantitative data are presented as the mean ± S.D. Unpaired Student's t test was performed and significance was set at p < 0.05. All experiments were replicated a minimum of three times, with each independent observation representing a single n in the figure legends. The duplicates or triplicates in RT-qPCR or qPCR experiments were counted as a single n because they were derived from a single sample, and their average was used to represent the corresponding sample to calculate the final average and standard deviation, which were further normalized to the control as indicated in the figure legend. To test whether Sirt1 regulates basal α-ENaC transcription, we manipulated the Sirt1 expression level and then measured the effects on endogenous α-ENaC mRNA expression, and, in separate experiments, on the activity of an α-ENaC promoter-Luc construct stably incorporated in mIMCD3 cells. mIMCD3 cells exhibit many of the phenotypic properties of the IMCD in vivo, and contain all of the components of the Dot1, Af9, Sgk1, and aldosterone signaling pathways. As seen in the representative immunoblot (Fig. 1A), robust Sirt1 overexpression was achieved by transient transfection and resulted in ∼35–40% lower endogenous α-ENaC mRNA expression (Fig. 1B) and α-ENaC promoter-Luc activity (Fig. 1C). To determine whether the inhibitory effect of Sirt1 was partially synergistic or additive with that of Dot1, co-transfection experiments were performed. Co-transfection of Dot1 with Sirt1 resulted in greater inhibition of α-ENaC promoter-Luc activity compared with transfection of either encoding DNA alone, suggesting functional interplay of the two proteins (Fig. 1D). In reciprocal experiments, Sirt1 knockdown was accomplished by RNA interference. qRT-PCR determinations indicated that Sirt1 mRNA expression in Sirt1 siRNA-transfected cells was ∼50% less than in cells transfected with the control siRNA (Fig. 2A), and this was accompanied by a substantial decrease in Sirt1 protein expression (Fig. 2A). Transfection of Sirt1 siRNA resulted in ∼40% greater levels of endogenous α-ENaC mRNA compared with controls (Fig. 2B). Transfection of the stable cell lines expressing α-ENaC promoter-Luc with the Sirt1 siRNA resulted in a nearly 2-fold increase in basal promoter activity (Fig. 2C). We previously established that Dot1 and Af9 co-immunoprecipitate from mouse kidney lysates (6Zhang W. Xia X. Jalal D.I. Kuncewicz T. Xu W. Lesage G.D. Kone B.C. Am. J. Physiol. Cell Physiol. 2006; 290: C936-C946Crossref PubMed Scopus (54) Google Scholar, 7Zhang W. Xia X. Reisenauer M.R. Hemenway C.S. Kone B.C. J. Biol. Chem. 2006; 281: 18059-18068Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 8Zhang W. Xia X. Reisenauer M.R. Rieg T. Lang F. Kuhl D. Vallon V. Kone B.C. J. Clin. Invest. 2007; 117: 773-783Crossref PubMed Scopus (132) Google Scholar). To determine whether endogenous Sirt1 interacts with Dot1, co-immunoprecipitation experiments with anti-Sirt1 antibody or isotype control IgG were performed in HEK 293 cells that had been transiently transfected with EGFP-Dot1a. HEK 293 cells were used as host cells because of their high efficiency of transient transfection and because they are kidney epithelial cells. The precipitated immune complex was further analyzed by immunoblotting with anti-EGFP antibody to identify Dot1 in the complex. As shown in Fig. 3A, EGFP-Dot1 was immunoprecipitated by the anti-Sirt1 antibody but not with nonimmune IgG. Similar experiments were performed with overexpression of FLAG-Af9, and subsequent co-immunoprecipitation with anti-Sirt1 antibody followed by immunoblotting with anti-FLAG antibody. However, Af9 immunoreactivity was not detected in the immunoprecipitates under these conditions (not shown). We attempted glutathione S-transferase pulldown assays with Sirt1 and Dot1 fusion proteins, but we could not consistently generate the full-length Sirt1 fusion protein, presumably because of its size. Moreover, using fragments of the two proteins, we did not reproducibly detect binding. This negative result could mean that the conformations of the full-length proteins are important for the interaction, or that the two proteins do not directly interact, but do so through a bridging protein. Sirt1 has been shown to reside in the cytoplasm and nucleus depending on the cell type. We previously demonstrated that Dot1 and Af9 colocalize in the nucleus when their epitope-tagged coding DNAs are cotransfected in HEK 293 cells (6Zhang W. Xia X. Jalal D.I. Kuncewicz T. Xu W. Lesage G.D. Kone B.C. Am. J. Physiol. Cell Physiol. 2006; 290: C936-C946Crossref PubMed Scopus (54) Google Scholar, 7Zhang W. Xia X. Reisenauer M.R. Hemenway C.S. Kone B.C. J. Biol. Chem. 2006; 281: 18059-18068Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 8Zhang W. Xia X. Reisenauer M.R. Rieg T. Lang F. Kuhl D. Vallon V. Kone B.C. J. Clin. Invest. 2007; 117: 773-783Crossref PubMed Scopus (132) Google Scholar). We sought to determine whether Sirt1 is also expressed in the nucleus of mIMCD3 cells. EGFP-Dot1 was co-expressed with RFP-tagged Sirt1 by transient transfection, and the expressed proteins analyzed by laser scanning confocal microscopy. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole. In these studies, EGFP-Dot1 was used, because the available Dot1 antibodies were not suitable for immunolocalization studies. As shown in Fig. 3B, EGFP-Dot1 and RFP-tagged Sirt1 displayed exclusively nuclear co-localization. The neighboring, unsuccessfully transfected cells, identified by 4′,6-diamidino-2-phenylindole (blue) staining of their nuclei, showed no fluorescence for either protein (Fig. 3B). Previously, we demonstrated in ChIP assays with an antibody directed against H3 K79me2 that Dot1 bound under basal conditions to each of 5 partially overlapping subregions (Ra, −1327/−965; R0, −988/−713; R1, −735/−415; R2, −414/+80; and R3, −57/+494) spanning the 1.3-kb α-ENaC 5′-flanking region in mIMCD3 cells (Fig. 4), and that this was correlated with histone H3K79me2 hypermethylation at these sites (7Zhang W. Xia X. Reisenauer M.R. Hemenway C.S. Kone B.C. J. Biol. Chem. 2006; 281: 18059-18068Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 8Zhang W. Xia X. Reisenauer M.R. Rieg T. Lang F. Kuhl D. Vallon V. Kone B.C. J. Clin. Invest. 2007; 117: 773-783Crossref PubM
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