Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN. Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN. Werner syndrome (WS) 3The abbreviations used are: WSWerner syndromeGSTglutathione S-transferaseTSAtrichostatin APBSphosphate-buffered salineBSAbovine serum albuminntnucleotides4NQO4-nitroquinoline-1-oxideIPimmunoprecipitatesiRNAsmall interfering RNA. 3The abbreviations used are: WSWerner syndromeGSTglutathione S-transferaseTSAtrichostatin APBSphosphate-buffered salineBSAbovine serum albuminntnucleotides4NQO4-nitroquinoline-1-oxideIPimmunoprecipitatesiRNAsmall interfering RNA. is a human autosomal recessive disorder that displays symptoms of premature aging, including graying and loss of hair, wrinkling and ulceration of skin, atherosclerosis, osteoporosis, and cataracts. WS patients also exhibit an increased incidence of diabetes mellitus type 2, hypertension, and are highly disposed to cancers (1Martin G.M. Austad S.N. Johnson T.E. Nat. Genet. 1996; 13: 25-34Crossref PubMed Scopus (536) Google Scholar). WS results from mutation of the WRN gene, a member of the RecQ DNA helicase family (2Yu C.E. Oshima J. Fu Y.H. Wijsman E.M. Hisama F. Alisch R. Matthews S. Nakura J. Miki T. Ouais S. Martin G.M. Mulligan J. Schellenberg G.D. Science. 1996; 272: 258-262Crossref PubMed Scopus (1473) Google Scholar). Mutations in other family members, BLM and RECQ4, are responsible for the two other cancer-prone and premature aging syndromes, Bloom (3Ellis N.A. Groden J. Ye T.Z. Straughen J. Lennon D.J. Ciocci S. Proytcheva M. German J. Cell. 1995; 83: 655-666Abstract Full Text PDF PubMed Scopus (1200) Google Scholar) and Rothmund-Thomson (4Kitao S. Shimamoto A. Goto M. Miller R.W. Smithson W.A. Lindor N.M. Furuichi Y. Nat. Genet. 1999; 22: 82-84Crossref PubMed Scopus (565) Google Scholar), respectively. Consistent with other RecQ helicases, WRN protein possesses 3′ to 5′ DNA helicase activity; however, it is the only human RecQ member to also have a 3′ to 5′ exonuclease activity. Although its physiological substrate is not yet clear, WRN appears to preferentially act on replication and recombination structures. Cells from WS patients show premature replicative senescence compared with cells derived from normal individuals (5Martin G.M. Sprague C.A. Epstein C.J. Lab. Investig. 1970; 23: 86-92PubMed Google Scholar). WS cells also show hypersensitivity to selected DNA-damaging agents including 4-nitroquinoline-1-oxide (4NQO; 6Ogburn C.E. Oshima J. Poot M. Chen R. Gollahon K.A. Rabinovitch P.S. Martin G.M. Hum. Genet. 1997; 101: 121-125Crossref PubMed Scopus (162) Google Scholar), topoisomerase inhibitors (7Lebel M. Leder P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13097-13102Crossref PubMed Scopus (254) Google Scholar), and certain DNA cross-linking agents (8Poot M. Yom J.S. Whang S.H. Kato J.T. Gollahon K.A. Rabinovitch P.S. FASEB J. 2001; 15: 1224-1226Crossref PubMed Scopus (132) Google Scholar). Compared with normal cells, WS cells also exhibit increased genomic instability including higher levels of DNA deletions, translocations, and chromosomal breaks (9Fukuchi K. Martin G.M. Monnat R.J. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5893-5897Crossref PubMed Scopus (390) Google Scholar, 10Stefanini M. Scappaticci S. Lagomarsini P. Borroni G. Berardesca E. Nuzzo F. Mutat. Res. 1989; 219: 179-185Crossref PubMed Scopus (42) Google Scholar), suggesting that WRN plays an important role in one or more genome maintenance pathways (11Orren D.K. Front Biosci. 2006; 11: 2657-2671Crossref PubMed Scopus (27) Google Scholar). Werner syndrome glutathione S-transferase trichostatin A phosphate-buffered saline bovine serum albumin nucleotides 4-nitroquinoline-1-oxide immunoprecipitate small interfering RNA. Werner syndrome glutathione S-transferase trichostatin A phosphate-buffered saline bovine serum albumin nucleotides 4-nitroquinoline-1-oxide immunoprecipitate small interfering RNA. WRN protein shows dynamic relocalization within the nucleus under different conditions of growth. The WRN protein localizes to the nucleoli in a variety of cell types (12Marciniak R.A. Lombard D.B. Johnson F.B. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6686-6892Crossref Scopus (179) Google Scholar), and this localization is modulated by DNA damage and cell cycle. Upon serum starvation or treatment with hydroxyurea (HU), aphidicolin, 4NQO, etoposide, or camptothecin, WRN migrates from nucleoli to discrete nuclear foci (13Constantinou A. Tarsounas M. Karow J.K. Brosh Jr., R.M. Bohr V.A. Hickson I.D. West S.C. EMBO Rep. 2000; 1: 80-84Crossref PubMed Scopus (335) Google Scholar, 14Brosh Jr, R.M. Karmakar P. Sommers J.A. Yang Q. Wang X.W. Spillare E.A. Harris C.C. Bohr V.A. J. Biol. Chem. 2001; 276: 35093-35102Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 15Nguyen D.T. Rovira I. Finkel T. FEBS Lett. 2002; 521: 170-174Crossref PubMed Scopus (10) Google Scholar, 16Gray M.D. Wang L. Youssoufian H. Martin G.M. Oshima J. Exp. Cell Res. 1998; 242: 487-494Crossref PubMed Scopus (124) Google Scholar, 17Sakamoto S. Nishikawa K. Heo S.J. Goto M. Furuichi Y. Shimamoto A. Genes Cells. 2001; 6: 421-430Crossref PubMed Scopus (152) Google Scholar). The fact that DNA damage also induces the formation of RPA and RAD51 foci, and these co-localize with WRN almost fully (RPA), or partially (RAD51) (17Sakamoto S. Nishikawa K. Heo S.J. Goto M. Furuichi Y. Shimamoto A. Genes Cells. 2001; 6: 421-430Crossref PubMed Scopus (152) Google Scholar), supports a potential role of the WRN protein in DNA replication and/or DNA recombination. Recent work by Blander et al. (18Blander G. Zalle N. Daniely Y. Taplick J. Gray M.D. Oren M. J. Biol. Chem. 2002; 277: 50934-50940Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) indicated that WRN is acetylated in vivo. Translocation of WRN into nucleoplasmic foci is significantly enhanced by the protein deacetylase inhibitor, trichostatin A (TSA). Moreover, TSA delays the re-localization of WRN back to the nucleolus at late times after DNA damage. WRN acetylation is markedly stimulated by the acetyltransferase p300 in vivo. Importantly, p300 augments the translocation of WRN into nucleoplasmic foci. These findings support the notion that WRN plays a role in the cellular response to DNA damage and suggest that the activity of WRN is modulated by DNA damage-induced acetylation. Acetylation might either decrease the interaction of WRN with protein(s) that retain it in the nucleolus in the absence of genotoxic stress or enhance its interaction with proteins that anchor it to the nucleoplasmic foci (18Blander G. Zalle N. Daniely Y. Taplick J. Gray M.D. Oren M. J. Biol. Chem. 2002; 277: 50934-50940Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Studies on WRN translocation after different DNA-damaging treatments found that acetylation of endogenous WRN is involved in its reversible translocation from nucleoli to nucleoplasm (19Karmakar P. Bohr V.A. Mech. Ageing Dev. 2005; 126: 1146-1158Crossref PubMed Scopus (44) Google Scholar). The yeast silent information regulator 2 (Sir2) protein belongs to a novel family of histone deacetylases, and is involved in gene silencing, telomere position effects, and cellular aging (reviewed in Ref. 20Guarente L. Genes Dev. 2000; 14: 1021-1026PubMed Google Scholar). The Sir2 ortholog in mammals (SIRT1) has also been found to contain NAD-dependent histone deacetylase activity (21Imai S. Armstrong C.M. Kaeberlein M. Guarente L. Nature. 2000; 403: 795-800Crossref PubMed Scopus (2698) Google Scholar). It has been demonstrated that SIRT1 can both deacetylate p53 and attenuate its transcriptional activity (22Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1856) Google Scholar, 23Vaziri H. Dessain S.K. Eaton E.N. Imai S.I. Frye R.A. Pandita T.K. Guarente L. Weinberg R.A. Cell. 2001; 107: 149-159Abstract Full Text Full Text PDF PubMed Scopus (2241) Google Scholar). SIRT1 also regulates premature cellular senescence induced by the tumor suppressors PML and p53 (24Langley E. Pearson M. Faretta M. Bauer U.M. Frye R.A. Minucci S. Pelicci P.G. Ouzarides T. EMBO J. 2002; 21: 2383-2396Crossref PubMed Scopus (739) Google Scholar). SIRT1 deacetylates the DNA repair factor Ku70, causing it to sequester Bax away from mitochondria and inhibiting stress-induced apoptotic cell death (25Cohen H.Y. Miller C. Bitterman K.J. Wall N.R. Hekking B. Kessler B. Howitz K.T. Gorospe M. de Cabo R. Sinclair D.A. Science. 2004; 305: 390-392Crossref PubMed Scopus (1644) Google Scholar). SIRT1 knock-out mice have a severe phenotype, most dying within the first month after birth (26McBurney M.W. Yang X. Jardine K. Hixon M. Boekelheide K. Webb J.R. Lansdorp P.M. Lemieux M. Mol. Cell. Biol. 2003; 23: 38-54Crossref PubMed Scopus (517) Google Scholar, 27Cheng H.L. Mostoslavsky R. Saito S. Manis J.P. Gu Y. Patel P. Bronson R. Appella E. Alt F.W. Chua K.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10794-10799Crossref PubMed Scopus (916) Google Scholar). Consistent with regulation of p53 by SIRT1 in vivo, these knock-out mice have hyperacetylated p53 and exhibit increased apoptosis, at least in thymocytes (27Cheng H.L. Mostoslavsky R. Saito S. Manis J.P. Gu Y. Patel P. Bronson R. Appella E. Alt F.W. Chua K.F. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 10794-10799Crossref PubMed Scopus (916) Google Scholar) and spermatogonia (26McBurney M.W. Yang X. Jardine K. Hixon M. Boekelheide K. Webb J.R. Lansdorp P.M. Lemieux M. Mol. Cell. Biol. 2003; 23: 38-54Crossref PubMed Scopus (517) Google Scholar). In this report, we provide evidence that SIRT1 can interact with WRN both in vitro and in vivo, and this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, while SIRT1 can deacetylate WRN. WRN acetylation inhibits its helicase and exonuclease activities while deacetylation by SIRT1 reverses this inhibition. WRN acetylation regulates its nuclear distribution. Importantly, down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that WRN-mediated cellular responses to DNA damage are regulated by SIRT1 deacetylation. Culture Medium and Reagents—HEK293 and U2OS cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. TSA, nicotinamide, and anti-β-actin monoclonal antibody were purchased from Sigma. Anti-WRN antibodies (H300) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-SIRT1 was made by Covance Inc. (22Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1856) Google Scholar). Anti-acetylated-lysine antibody was purchased from Cell Signaling Technology (Danvers, MA). Anti-acetylated WRN specific antibody was produced by immunizing rabbits with a synthetic acetylated peptide (KLH-coupled) corresponding to residues surrounding Lys-1413 of human WRN and affinity-purified with acetylated peptide after depletion of antibodies to unacetylated WRN by passing through the same peptide without acetylation of the lysine. Transfection of the SIRT1 siRNA (Dharmacon Inc.) was performed using Oligofectamine reagent (Invitrogen) following the manufacturer's instructions with a final oligonucleotide concentration of 20 μm. GST Pulldown Assay—Full-length SIRT1 protein was in vitro expressed in the presence of [35S]methionine by the TnT lysate in vitro translation system (Promega). GST fusion proteins were expressed and purified from BL21 bacterial cells and bound to a GST-agarose column. 35S-radiolabeled SIRT1 and GST fusion WRN proteins were incubated for 2 h in buffer containing 100 mm NaCl, 20 mm Tris-HCl (pH 7.3), 10% glycerol, and 0.2% Triton X-100. Beads were washed five times with the incubation buffer and boiled in the presence of 1× SDS sample buffer before SDS-PAGE. Labeled proteins were visualized by autoradiography. Co-immunoprecipitation Assay—For anti-FLAG M2 immunoprecipitation, cells were harvested 36 h after transfection and were lysed in BC100 buffer (100 mm NaCl, 50 mm Tris-HCl pH 7.3, 10% glycerol, 0.2% Triton X-100) for 1 h. Cell extracts were incubated with anti-FLAG M2 beads (Sigma) at 4 °C overnight. After the beads were washed five times with BC100 buffer, bound proteins were eluted with FLAG peptide (Sigma) for 2 h. Western blots of immunoprecipitated proteins and whole cell extracts were performed with rabbit anti-WRN polyclonal antibody, rabbit anti-SIRT1 polyclonal antibody, mouse anti-acetylated lysine monoclonal antibody. For endogenous protein immunoprecipitation, cells were treated for 6 h with either 20 μm etoposide (Sigma) or 500 μm hydrogen peroxide (SM Biotech, Inc.) before harvest, then lysed with BC100 buffer for 1 h, and cell extracts were incubated for 2 h with either goat anti-WRN polyclonal antibody (C19, Santa Cruz Biotechnology) or normal goat IgG (Santa Cruz Biotechnology). After incubation, protein A/G PLUS-agarose beads (Santa Cruz Biotechnology) were added to the extracts and incubated overnight at 4 °C. Agarose beads were washed five times with BC100 buffer, and bound proteins were eluted by boiling the beads with 1× SDS sample buffer. In Vitro Deacetylation Assay—Acetylated WRN protein was purified by co-transfecting FLAG-WRN and CBP plasmids into HEK293 cells. Cells were treated with TSA and nicotinamide 6 h before harvest. Cell lysates were immunoprecipitated with anti-FLAG M2 beads and eluted with FLAG peptide. Purified acetylated WRN protein was incubated at 30 °C for 1 h with purified SIRT1 protein as indicated. SIRT1 was FLAG-tagged and purified from a stable cell line by immunoprecipitation with anti-FLAG M2 beads and eluted with FLAG peptide. The incubation buffer contained 50 mm Tris-HCl, pH 9), 50 mm NaCl, 4 mm MgCl2, 0.5 mm dithiothreitol, 0.2 mm phenylmethylsulfonyl fluoride, 0.02% Nonidet P-40, and 5% glycerol. When indicated, NAD+ co-factor (Sigma) was added to the reaction at a final concentration of 50 μm. Reaction mixtures were resolved in SDS-PAGE and analyzed by Western blot with monoclonal anti-acetylated lysine antibody. Detecting WRN Acetylation in Cells—In a transiently transfected WRN acetylation assay, HEK293 cells were transfected using the calcium phosphate method with FLAG-WRN, CMV-p300, or CMV-CBP plasmid DNA (10 μg each). At 36 h after transfection, cells were lysed in FLAG lysis buffer (50 mm Tris-HCl, pH 7.8, 137 mm NaCl, 1 mm NaF, 1 mm NaVO3, 1% Triton X-100, 0.2% Sarkosyl, 1 mm dithiothreitol, and 10% glycerol) containing fresh protease inhibitors, 10 μm TSA, and 5 mm nicotinamide. Cell extracts were immunoprecipitated with anti-FLAG monoclonal antibody M2 beads (Sigma). After elution with the FLAG peptide, proteins were resolved by either 8 or 4-20% SDS-PAGE (Invitrogen) and analyzed by Western blot with anti-acetylated lysine or anti-WRN antibodies. For endogenous WRN acetylation assay, U2OS cells were treated with 20 μm etoposide alone or treated with 1 μm TSA and 5 mm nicotinamide for 6 h before harvest. Cell pellets were lysed in radioimmune precipitation assay buffer (10 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Nonidet P-40, 1% deoxycholic acid, 0.025% SDS, and 1 mm phenylmethylsulfonyl fluoride) with mild sonication. The cell extracts were immunoprecipitated with anti-WRN antibody and protein A/G-beads (Santa Cruz Biotechnology). The beads were washed with 0.5 ml of radioimmune precipitation assay buffer five times, and proteins were eluted using SDS sample buffer. The samples were further analyzed by Western blot with anti-acetylated lysine or anti-WRN antibodies. Helicase and Exonuclease Assay—Using the acetylation assay described in Fig. 2a, we made WRN and acetylated WRN proteins from HEK293 cells. To maximally acetylate WRN protein, we treated the cells with TSA and nicotinamide to inhibit cellular deacetylase activity before harvest. Using these proteins, helicase assays were performed on a 3′ overhang partial duplex (21 bp) DNA structure. This substrate was generated by radiolabeling a 21-mer with [γ-32P]ATP and T4 polynucleotide kinase followed by annealing with unlabeled complementary 70-mer. Non-acetylated, acetylated, and deacetylated FLAG-WRN proteins were incubated with substrate for 5 min at 37 °C in WRN reaction buffer (40 mm Tris-HCl, pH 8.0, 4 mm MgCl2, 1 mm ATP, 0.1% Nonidet P-40, 0.1 mg/ml bovine serum albumin (BSA), and 5 mm dithiothreitol). Reactions were stopped by addition of one-sixth volume of loading dyes (30% glycerol, 0.25% bromphenol blue, 0.25% xylene cyanol, 50 mm EDTA, and 0.9% SDS). Samples were subjected to electrophoresis in an 8% neutral gel in 1× Tris borate-EDTA at 100 V for 3 h at room temperature. The gel was vacuum-dried at 80 °C for 1 h, and radioactive DNA was visualized by phosphorimaging. For the exonuclease assay, a partial duplex (35 bp) with a recessed 3′-end was constructed by annealing labeled 35-mer to a 2-fold excess of unlabeled 70-mer, separated by native PAGE (12%), excised, and extracted before use. Exonuclease reactions (10 μl) containing 3′-recessed end substrate (0.1 nm) and FLAG-WRN proteins at the indicated concentrations in WRN reaction buffer minus ATP were preincubated on ice for 5 min, and then transferred to 37 °C for 5 min. Reactions were stopped by the addition of formamide loading buffer (95% formamide, 20 mm EDTA, 0.1% bromphenol blue, and 0.1% xylene cyanol). DNA products were heated at 95 °C and separated by denaturing (14%) PAGE. Digestion of the labeled strand by the 3′ to 5′ exonuclease activity of WRN proteins was visualized by phosphorimaging. Immunofluorescence and Recovery Assays—U2OS cells were seeded onto duplicated sterile coverslips in a 6-well tissue culture plate at 20-30% confluence. Cells were treated with 40 μm etoposide for 2 h and then washed and supplied with fresh medium for an additional 24 h. Nicotinamide (5 mm) was used as indicated in specific wells. For the SIRT1 down-regulation experiment, cells were transfected with siRNA for SIRT1 (or in control experiments, siRNA for GFP) by Oligofectamine reagent (Invitrogen) following the manufacturer's instructions with a final concentration of the siRNA oligonucleotides of 20 μm. The siRNA sequences for SIRT1: AACTTGTACGACGAAGACGAC. The cells were treated with etoposide as above. At each time point, cells were fixed in PBS containing 4% paraformaldehyde for 20 min and permeabilized with PBS containing 1% of BSA and 0.1% Triton X-100 for 5 min. Primary mouse anti-WRN monoclonal antibody (BD Biosciences Pharmingen) was added in 1% BSA/PBS for 45 min at room temperature. After washing with 1% BSA/PBS, FITC-conjugated anti-mouse antibody (1:400; Jackson ImmunoResearch Laboratories) was added and incubated for 30 min at room temperature. Finally, cells were counterstained with DAPI to visualize the nuclei. Coverslips were mounted in Aqua Poly/Mount (Polysciences), and the slides were examined under an Olympus IX71 microscope. SIRT1 Interacts with WRN Both in Vitro and in Vivo—The interaction of SIRT1 and WRN was first tested by a transient transfection assay. We either transfected FLAG-tagged SIRT1 with WRN or FLAG-tagged WRN with SIRT1 into HEK293 cells. After anti-FLAG M2 beads immunoprecipitation, the IP products were subjected to SDS-PAGE and a Western blot with anti-WRN and anti-SIRT1 antibodies. As shown in Fig. 1a, SIRT1 protein was clearly co-immunoprecipitated with FLAG-WRN (lane 1, upper panel), and WRN protein was also clearly co-immunoprecipitated with FLAG-SIRT1 (lane 3, upper panel). We further tested whether endogenous WRN can interact with endogenous SIRT1. HEK293 cells were immunoprecipitated with an anti-WRN polyclonal antibody or control IgG. Endogenous SIRT1 can clearly be co-immunoprecipitated with WRN (Fig. 1c, lane 5 versus lane 8). Interestingly, this interaction was increased in the cells after a 6-h treatment with etoposide or H2O2 (Fig. 1c, lanes 5-7), suggesting that the possible regulation of WRN by SIRT1 is enhanced after DNA damage. Furthermore, we tested whether SIRT1 directly interacted with WRN and the specific region of WRN mediating this interaction. GST fusion proteins were generated for the N-terminal (GST-WRNNT), central (GST-WRNM), and C-terminal (GST-WRNCT) regions of WRN and immobilized on GST-agarose. As shown in Fig. 1b, 35S-labeled in vitro translated SIRT1 strongly bound to immobilized GST-WRNCT but not to immobilized GST alone (lane 3 versus lane 4). Moreover, SIRT1 only bound to the C-terminal domain of WRN (GST-WRNCT), as no binding to the N-terminal domain (GST-WRNNT) and central helicase domain (GST-WRNM) was detected (lanes 1-3). Thus, these findings demonstrate that SIRT1 interacts with WRN both in vitro and in vivo, apparently through binding to the C-terminal region of WRN. SIRT1 Deacetylates WRN Both in Vitro and in Vivo—Blander et al. (18Blander G. Zalle N. Daniely Y. Taplick J. Gray M.D. Oren M. J. Biol. Chem. 2002; 277: 50934-50940Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) have reported that WRN is acetylated in cells by transfecting WRN expression plasmid alone or with p300 expression plasmid into HEK293 cells. After in vivo labeling with radioactive acetate, they detected WRN acetylation, which can be augmented by cotransfecting p300. Using a different approach, we also detected WRN acetylation (Fig. 2a). In our approach, FLAG-tagged WRN expression plasmid was transfected alone or together with p300 or CBP expression vectors. After immunoprecipitation with anti-FLAG M2 beads, we detected WRN acetylation by Western blot with anti-acetylated lysine antibody. As shown in Fig. 2a, we can clearly detect WRN acetylation when WRN was cotransfected with CBP or p300 (lanes 2 and 3). Furthermore, acetylation of endogenous WRN was clearly detectable after cells were treated with 20 μm etoposide (Fig. 2b, lane 2 versus lane 1). Moreover, treating cells with etoposide in combination with the histone deacetylase inhibitors TSA and nicotinamide further increased WRN acetylation (lane 3 versus lane 2). These experiments clearly demonstrate that WRN can be acetylated in vivo in response to DNA damage. We further investigated the consequences of the interaction of WRN and SIRT1 proteins by testing whether SIRT1 can deacetylate WRN protein. Acetylated FLAG-WRN was purified from HEK293 cells, and SIRT1 was stably transfected into a cell line and subsequently purified. This in vitro deacetylation assay was performed by incubating SIRT1 with purified acetylated FLAG-WRN at 30 °C for 1 h followed by SDS-PAGE and Western blot with anti-acetylated lysine antibody. As shown in Fig. 2c, WRN can be deacetylated by SIRT1 (lane 4). The deacetylation is NAD-dependent (lane 4 versus lane 2) as expected for SIRT1-mediated deacetylation. However, the deacetylation activity was completely inhibited in the presence of nicotinamide (lane 5 versus lane 4). We also examined whether SIRT1 could mediate WRN deacetylation in cells. A high level of acetylated WRN was found in HEK293 cells cotransfected with CBP and WRN (Fig. 2d, lane 1); however, WRN acetylation levels were progressively decreased by increasing expression of SIRT1 (Fig. 2d, lanes 2-4). In contrast, this level of WRN acetylation was not reduced by transfection of a deacetylase-deficient SIRT1-363Y point mutant (Fig. 2d, lane 5). These results indicate that WRN is a specific target for SIRT1 deacetylase activity. We also examined the endogenous WRN acetylation status after siRNA-mediated reduction of SIRT1 protein levels in HEK293 cells. After immunoprecipitation with antibody specific for acetylated WRN, we clearly detected a significantly increased level of acetylated WRN when SIRT1 was down-regulated (Fig. 2e, lane 2 versus lane 1) indicating that SIRT1 is the specific deacetylase for the WRN protein. SIRT1 Regulates Both WRN Helicase and Exonuclease Activities—To further address the functional consequences of WRN-SIRT1 interactions, we tested whether the WRN helicase and exonuclease activities were altered by these modifications. We first tested the effect of acetylation on WRN helicase and exonuclease activities. Using the acetylation assay described in Fig. 2a, we expressed and purified the FLAG-WRN and acetylated FLAG-WRN proteins from HEK293 cells. Using these proteins (Fig. 3d, lanes 1 and 2), the helicase assay was performed on a 21-bp partial duplex with a 49-nt 3′ overhang structure. As shown in Fig. 3, a and b, unacetylated WRN was consistently more efficient at unwinding this substrate than acetylated WRN over a range of concentrations (lanes 2-4 versus lanes 5-7). An exonuclease assay was also performed using the same proteins but a different partial duplex substrate with a recessed 3′-end structure. As shown in Fig. 3c, acetylated WRN showed markedly less exonuclease activity than unacetylated WRN (lanes 5-7 versus lanes 2-4), indicating WRN acetylation reduced both helicase activity and exonuclease activity. Furthermore, we tested whether deacetylation of WRN by SIRT1 could reverse the reduction of WRN helicase and exonuclease by acetylation. For this experiment, FLAG-WRN isolated from HEK293 cells cotransfected with FLAG-WRN, CBP, and SIRT1 was compared with unacetylated and acetylated FLAG-WRN. Using these proteins (Fig. 3d, lane 3), we performed the helicase and exonuclease assays on the same 3′ overhang and 3′ recessed end substrates, respectively. Importantly, introduction of SIRT1 deacetylase (that markedly reduces the level of WRN acetylation) restores these helicase and exonuclease activities (Fig. 3a, lanes 8-10 versus lanes 5-7 and Fig. 3b; Fig. 3c, lanes 8-10 versus lanes 5-7), similar to the levels attained with non-acetylated WRN. These data demonstrate that the acetylation state of WRN significantly affects its helicase and exonuclease activities, and suggest that the SIRT1 deacetylation of WRN may play an important role in the regulation of its enzymatic function. SIRT1 Mediates the Reentry of WRN to the Nucleolus after DNA Damage—DNA damage induces WRN translocation from nucleolus to nucleoplasm and acetylation is thought to regulate this translocation (18Blander G. Zalle N. Daniely Y. Taplick J. Gray M.D. Oren M. J. Biol. Chem. 2002; 277: 50934-50940Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 19Karmakar P. Bohr V.A. Mech. Ageing Dev. 2005; 126: 1146-1158Crossref PubMed Scopus (44) Google Scholar). To test the effect of SIRT1 on WRN nuclear translocation, we first tested whether the SIRT1 inhibitor nicotinamide can delay the reentry of WRN into the nucleoli after DNA damage. U2OS cells were first treated with the DNA-damaging agent etoposide. After removing etoposide, cells were treated with nicotinamide, then fixed and immunostained for WRN localization. WRN underwent translocation from the nucleolus to the nucleoplasm after the etoposide treatment (95.8 versus 12.5%, Fig. 4b). The nicotinamide treatment reduced the number of cells that were able to relocate WRN back to the nucleolus after 24 h compared with cells without nicotinamide (85.0 versus 51.5%, Fig. 4b). Representative cells with WRN nuclear and nucleolar localization are shown in Fig. 4a. We further tested whether SIRT1 was involved in regulating WRN translocation after DNA damage. SIRT1 protein was specifically down-regulated by siRNA to SIRT1 in U2OS cells throughout the interval in which WRN localization was measured (Fig. 4e, lanes 2, 4, 6 versus lanes 1, 3, 5). After etoposide treatment, both SIRT1 down-regulated and control cells showed similar WRN translocat
The hairless (HR) protein contains a Jumonji C (JmjC) domain that is conserved among a family of proteins with histone demethylase (HDM) activity. To test whether HR possesses HDM activity, we performed a series of in vitro demethylation assays, which demonstrated that HR can demethylate mono-methylated or dimethylated histone H3 lysine 9 (H3K9mel or me2). Moreover, ectopic expression of wild-type HR, but not JmjC-mutant HR, led to pronounced demethylation of H3K9 in cultured human HeLa cells. We also show that two missense mutations in HR, which we and others described in patients with atrichia with papular lesions, abolished the demethylase activity of HR, demonstrating the role of HR demethylase activity in human disease. By ChlP-Seq analysis, we identified multiple new HR target genes, many of which play important roles in epidermal development, neural function, and transcriptional regulation, consistent with the predicted biological functions of HR Our findings demonstrate for the first time that HR is a H3K9 demethylase that regulates epidermal homeostasis via direct control of its target genes.—Liu, L., Kim, H., Casta, A., Kobayashi, Y., Shapiro, L. S., Christiano, A. M. Hairless is a histone H3K9 demethylase. FASEB J. 28, 1534–1542 (2014). www.fasebj.org
While the European Union is striving to become the 'Innovation Union', there remains a lack of quantifiable indicators to compare and benchmark regional innovation clusters. To address this issue, a HealthTIES (Healthcare, Technology and Innovation for Economic Success) consortium was funded by the European Union's Regions of Knowledge initiative, research and innovation funding programme FP7. HealthTIES examined whether the health technology innovation cycle was functioning differently in five European regional innovation clusters and proposed regional and joint actions to improve their performance. The clusters included BioCat (Barcelona, Catalonia, Spain), Medical Delta (Leiden, Rotterdam and Delft, South Holland, Netherlands), Oxford and Thames Valley (United Kingdom), Life Science Zürich (Switzerland), and Innova Észak-Alföld (Debrecen, Hungary).Appreciation of the 'triple helix' of university-industry-government innovation provided the impetus for the development of two quantifiable innovation indexes and related indicators. The HealthTIES H-index is calculated for disease and technology platforms based on the h-index proposed by Hirsch. The HealthTIES Innovation Index is calculated for regions based on 32 relevant quantitative and discriminative indicators grouped into 12 categories and 3 innovation phases, namely 'Input' (n = 12), 'Innovation System' (n = 9) and 'Output' (n = 11).The HealthTIES regions had developed relatively similar disease and technology platform profiles, yet with distinctive strengths and weaknesses. The regional profiles of the innovation cycle in each of the three phases were surprisingly divergent. Comparative assessments based on the indicators and indexes helped identify and share best practice and inform regional and joint action plans to strengthen the competitiveness of the HealthTIES regions.The HealthTIES indicators and indexes provide useful practical tools for the measurement and benchmarking of university-industry-government innovation in European medical and life science clusters. They are validated internally within the HealthTIES consortium and appear to have a degree of external prima facie validity. Potentially, the tools and accompanying analyses can be used beyond the HealthTIES consortium to inform other regional governments, researchers and, possibly, large companies searching for their next location, analyse and benchmark 'triple helix' dynamics within their own networks over time, and to develop integrated public-private and cross-regional research and innovation strategies in Europe and beyond.
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