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• W2113252276 abstract "Background & Aims: Histone deacetylase inhibitors (HDACi) induce growth arrest, apoptosis, and differentiation, particularly in colon cancer cells where they are potential chemopreventive agents. HDACi induction of the cyclin-dependent kinase inhibitor p21waf1 has been shown to require ataxia-telangiectasia mutated (ATM). Nevertheless, how ATM participates in p21waf1 gene expression has not been defined. Methods: In vivo protein complexes forming in response to butyrate were studied using co-immunoprecipitation and mass spectroscopy. DNA elements in the p21waf1 promoter were analyzed in vivo by chromatin immunoprecipitation and in vitro DNA affinity precipitation assays. The expression of p21waf1 was analyzed by immunoblots and reporter assays. Results: Reduction of ZBP-89 or ATM with small interfering RNAs blocked HDACi-induced p21waf1 expression. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that both ZBP-89 and ATM are recruited to the GC-rich DNA elements of the p21waf1 promoter with HDACi treatment. Co-immunoprecipitation revealed that ATM associates with ZBP-89 in an HDACi-dependent manner. Serial deletions revealed that ATM interacts with both the N-terminal and DNA binding domains of ZBP-89. Moreover, we found that immunodepletion of ZBP-89 prevented recruitment of ATM to the p21waf1 promoter in vitro. Silencing of ZBP-89 expression blocked HDACi-induced phosphorylation of ATMSer1981 and p53Ser15. ATMSer1981 phosphorylation in the colons of mutant mice expressing an N-terminally truncated form of ZBP-89 was not observed after ingestion of dextran sodium sulfate and correlated with exacerbation of the mucosal injury. Conclusions:ZBP-89 interacts with ATM in a butyrate-dependent manner and is essential for colonic homeostasis in the setting of acute mucosal injury. Background & Aims: Histone deacetylase inhibitors (HDACi) induce growth arrest, apoptosis, and differentiation, particularly in colon cancer cells where they are potential chemopreventive agents. HDACi induction of the cyclin-dependent kinase inhibitor p21waf1 has been shown to require ataxia-telangiectasia mutated (ATM). Nevertheless, how ATM participates in p21waf1 gene expression has not been defined. Methods: In vivo protein complexes forming in response to butyrate were studied using co-immunoprecipitation and mass spectroscopy. DNA elements in the p21waf1 promoter were analyzed in vivo by chromatin immunoprecipitation and in vitro DNA affinity precipitation assays. The expression of p21waf1 was analyzed by immunoblots and reporter assays. Results: Reduction of ZBP-89 or ATM with small interfering RNAs blocked HDACi-induced p21waf1 expression. Chromatin immunoprecipitation and DNA affinity precipitation assays showed that both ZBP-89 and ATM are recruited to the GC-rich DNA elements of the p21waf1 promoter with HDACi treatment. Co-immunoprecipitation revealed that ATM associates with ZBP-89 in an HDACi-dependent manner. Serial deletions revealed that ATM interacts with both the N-terminal and DNA binding domains of ZBP-89. Moreover, we found that immunodepletion of ZBP-89 prevented recruitment of ATM to the p21waf1 promoter in vitro. Silencing of ZBP-89 expression blocked HDACi-induced phosphorylation of ATMSer1981 and p53Ser15. ATMSer1981 phosphorylation in the colons of mutant mice expressing an N-terminally truncated form of ZBP-89 was not observed after ingestion of dextran sodium sulfate and correlated with exacerbation of the mucosal injury. Conclusions:ZBP-89 interacts with ATM in a butyrate-dependent manner and is essential for colonic homeostasis in the setting of acute mucosal injury. Short-chain fatty acids (SCFAs), mainly butyrate, acetate, and propionate, are produced in the large bowel in millimolar concentrations by bacterial fermentation of undigested dietary carbohydrates and fiber polysaccharides. These SCFAs play an important role in the maintenance of colonic homeostasis and suppression of intestinal inflammation.1Treem W.R. Ahsan N. Shoup M. Hyams J.S. Fecal short-chain fatty acids in children with inflammatory bowel disease.J Pediatr Gastroenterol Nutr. 1994; 18: 159-164Crossref PubMed Scopus (120) Google Scholar, 2Chapman M.A. The role of the colonic flora in maintaining a healthy large bowel mucosa.Ann R Coll Surg Engl. 2001; 83: 75-80PubMed Google Scholar, 3Galvez J. Rodriguez-Cabezas M.E. Zarzuelo A. Effects of dietary fiber on inflammatory bowel disease.Mol Nutr Food Res. 2005; 49: 601-608Crossref PubMed Scopus (194) Google Scholar In particular, butyrate is considered the major energy source for colonocytes.4Roediger W.E. Utilization of nutrients by isolated epithelial cells of the rat colon.Gastroenterology. 1982; 83: 424-429Abstract Full Text PDF PubMed Scopus (907) Google Scholar, 5Davie J.R. Inhibition of histone deacetylase activity by butyrate.J Nutr. 2003; 133: 2485S-2493SPubMed Google Scholar In addition, numerous studies have shown that butyrate can induce cell growth arrest and stimulate apoptosis and differentiation in colon cancer–derived cell lines and other transformed cell types.5Davie J.R. Inhibition of histone deacetylase activity by butyrate.J Nutr. 2003; 133: 2485S-2493SPubMed Google Scholar, 6Boffa L.C. Vidali G. Mann R.S. Allfrey V.G. Suppression of histone deacetylation in vivo and in vitro by sodium butyrate.J Biol Chem. 1978; 253: 3364-3366Abstract Full Text PDF PubMed Google Scholar, 7Sealy L. Chalkley R. The effect of sodium butyrate on histone modification.Cell. 1978; 14: 115-121Abstract Full Text PDF PubMed Scopus (560) Google Scholar, 8Kruh J. Effects of sodium butyrate, a new pharmacological agent, on cells in culture.Mol Cell Biochem. 1982; 42: 65-82PubMed Google Scholar, 9Hassig C.A. Tong J.K. Schreiber S.L. Fiber-derived butyrate and the prevention of colon cancer.Chem Biol. 1997; 4: 783-789Abstract Full Text PDF PubMed Scopus (82) Google Scholar In animal models, butyrate represses the growth of carcinogen-induced colonic tumors.10McIntyre A.G.P. Young G.P. Butyrate production from dietary fibre and protection against large bowel cancer in a rat model.Gut. 1993; 34: 386-391Crossref PubMed Scopus (498) Google Scholar, 11Medina V.A.J. Alvarez-Arguelles H. Hernandez C. Gonzalez F. Sodium butyrate inhibits carcinoma development in a 1,2-dimethylhydrazine-induced rat colon cancer.JPEN J Parenter Enteral Nutr. 1998; 22: 14-17Crossref PubMed Scopus (83) Google Scholar, 12Kameue C.T.T. Yamada K. Koyama H. Iwasaki Y. Nakayama K. Ushida K. Dietary sodium gluconate protects rats from large bowel cancer by stimulating butyrate production.J Nutr. 2004; 134: 940-944PubMed Scopus (42) Google Scholar The association between ulcerative colitis (UC) and an increased risk for colon cancer has been well established. Oxidative DNA damage in the mucosa of UC patients contributes to colonic dysplasia.13Itzkowitz S.H. Yio X. Inflammation and cancer IV Colorectal cancer in inflammatory bowel disease: the role of inflammation.Am J Physiol. 2004; 287: G7-G17Crossref PubMed Scopus (1051) Google Scholar, 14D’Inca R. Cardin R. Benazzato L. Angriman I. Martines D. Sturniolo G.C. Oxidative DNA damage in the mucosa of ulcerative colitis increases with disease duration and dysplasia.Inflamm Bowel Dis. 2004; 10: 23-27Crossref PubMed Scopus (88) Google Scholar Butyrate effectively suppresses colonic inflammation and is currently in clinical trials for the treatment of inflammatory bowel disease (IBD).3Galvez J. Rodriguez-Cabezas M.E. Zarzuelo A. Effects of dietary fiber on inflammatory bowel disease.Mol Nutr Food Res. 2005; 49: 601-608Crossref PubMed Scopus (194) Google Scholar Butyrate is a potent inhibitor of histone deacetylases (HDACs)15Candido E.P. Reeves R. Davie J.R. Sodium butyrate inhibits histone deacetylation in cultured cells.Cell. 1978; 14: 105-113Abstract Full Text PDF PubMed Scopus (816) Google Scholar and its action is mimicked by trichostatin A (TSA), a structurally unrelated HDAC inhibitor (HDACi). Moreover, overexpression of HDACs block the effects of butyrate.16Archer S.Y. Meng S. Shei A. Hodin R.A. p21WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells.Proc Natl Acad Sci U S A. 1998; 95: 6791-6796Crossref PubMed Scopus (504) Google Scholar Therefore, it is generally accepted that butyrate functions mainly by inhibiting HDAC activity, which leads to hyperacetylation of histones and chromatin remodeling. Recently, HDAC inhibition by TSA was shown by Bakkenist and Kastan17Bakkenist C.J. Kastan M.B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.Nature. 2003; 421: 499-506Crossref PubMed Scopus (2725) Google Scholar to activate ataxia-telangiectasia mutated (ATM), a key mediator of DNA damage signals. This result suggested that hyperacetylation and subsequently chromatin remodeling alone are sufficient to activate ATM signaling in the absence of DNA damage.17Bakkenist C.J. Kastan M.B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.Nature. 2003; 421: 499-506Crossref PubMed Scopus (2725) Google Scholar However, the mechanisms by which HDACi activate ATM were not explored. Chromatin becomes more accessible to transcription factors with HDAC inhibition resulting in the activation of approximately 2% of all genes and, subsequently, growth arrest.18Van Lint C. Emiliani S. Verdin E. The expression of a small fraction of cellular genes is changed in response to histone hyperacetylation.Gene Expr. 1996; 5: 245-253PubMed Google Scholar The GC-rich elements within these genes are typically the DNA sequences responsive to chromatin remodeling.5Davie J.R. Inhibition of histone deacetylase activity by butyrate.J Nutr. 2003; 133: 2485S-2493SPubMed Google Scholar, 19Park C. Chamberlin M.E. Pan C.J. Chou J.Y. Differential expression and butyrate response of human alkaline phosphatase genes are mediated by upstream DNA elements.Biochemistry. 1996; 35: 9807-9814Crossref PubMed Scopus (12) Google Scholar, 20Koyama N. Hoelzer D. Ottmann O.G. Regulation of human IL-18 gene expression: interaction of PU.1 with GC-box binding protein is involved in human IL-18 expression in myeloid cells.Eur J Immunol. 2004; 34: 817-826Crossref PubMed Scopus (20) Google Scholar p21waf1 is a cyclin-dependent kinase inhibitor with a GC-rich promoter that plays a critical role in cell growth arrest.21Boulaire J. Fotedar A. Fotedar R. The functions of the cdk-cyclin kinase inhibitor p21WAF1.Pathol Biol (Paris). 2000; 48: 190-202PubMed Google Scholar It has been shown previously that p21waf1 is an HDACi-responsive gene required for butyrate-mediated growth inhibition in human colon cancer cell lines.16Archer S.Y. Meng S. Shei A. Hodin R.A. p21WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells.Proc Natl Acad Sci U S A. 1998; 95: 6791-6796Crossref PubMed Scopus (504) Google Scholar In addition, increased p21waf1 protein levels in the colonic crypts are associated with active UC.22Hofseth L.J. Saito S. Hussain S.P. Espey M.G. Miranda K.M. Araki Y. Jhappan C. Higashimoto Y. He P. Linke S.P. Quezado M.M. Zurer I. Rotter V. Wink D.A. Appella E. Harris C.C. Nitric oxide-induced cellular stress and p53 activation in chronic inflammation.Proc Natl Acad Sci U S A. 2003; 100: 143-148Crossref PubMed Scopus (295) Google Scholar The GC-rich DNA elements of the proximal promoter are critical for HDACi-induced expression of p21waf1.23Huang L. Sowa Y. Sakai T. Pardee A.B. Activation of the p21WAF1/CIP1 promoter independent of p53 by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) through the Sp1 sites.Oncogene. 2000; 19: 5712-5719Crossref PubMed Scopus (199) Google Scholar, 24Xiao H. Hasegawa T. Isobe K. p300 collaborates with Sp1 and Sp3 in p21(waf1/cip1) promoter activation induced by histone deacetylase inhibitor.J Biol Chem. 2000; 275: 1371-1376Crossref PubMed Scopus (159) Google Scholar, 25Bai L. Merchant J.L. Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21waf1 transcription in human cells.J Biol Chem. 2000; 275: 30725-30733Crossref PubMed Scopus (98) Google Scholar Recently, HDACi activation of p21waf1 gene expression was shown to require ATM.26Ju R. Muller M.T. Histone deacetylase inhibitors activate p21(WAF1) expression via ATM.Cancer Res. 2003; 63: 2891-2897PubMed Google Scholar However, because ATM is a phosphatidylinositol kinase and not a DNA binding protein, the nature of the requirement is not known. Previously, we have shown that the HDACi induction of p21waf1 is enhanced by the transcription factor zinc-finger (DNA) binding protein (ZBP-89) (ZNF148, Zfp148, and BFCOL1).25Bai L. Merchant J.L. Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21waf1 transcription in human cells.J Biol Chem. 2000; 275: 30725-30733Crossref PubMed Scopus (98) Google Scholar ZBP-89 is a DNA binding protein that regulates a variety of genes through direct binding to GC-rich elements that also are bound by Sp1 and Sp3.27Merchant J.L. Bai L. Okada M. ZBP-89 mediates butyrate regulation of gene expression.J Nutr. 2003; 133: 2456S-2460SPubMed Google Scholar In addition to direct regulation of gene expression, ZBP-89 also stabilizes p53 protein28Bai L. Merchant J.L. ZBP-89 promotes growth arrest through stabilization of p53.Mol Cell Biol. 2001; 21: 4670-4683Crossref PubMed Scopus (90) Google Scholar and is required for p53 phosphorylation at Ser15, the same site targeted by ATM kinase.29Takeuchi A. Mishina Y. Miyaishi O. Kojima E. Hasegawa T. Isobe K.I. Heterozygosity with respect to Zfp148 causes complete loss of fetal germ cells during mouse embryogenesis.Nat Genet. 2003; 33: 172-176Crossref PubMed Scopus (93) Google Scholar Collectively, this evidence suggests that p21waf1 activation by HDACi requires the assembly of multiple factors at the p21waf1 promoter. We hypothesized that ZBP-89 might play a central role in the assembly of these factors. Indeed, we show here that ATM associates with ZBP-89 in an HDACi-dependent manner and we show a role for the ATM/ZBP-89 complex in the activation of p21waf1 gene expression and in colonic homeostasis. Generation of rabbit anti–ZBP-89 polyclonal antibody has been described previously.30Taniuchi T. Mortensen E.R. Ferguson A. Greenson J. Merchant J.L. Overexpression of ZBP-89, a zinc finger DNA binding protein, in gastric cancer.Biochem Biophys Res Commun. 1997; 233: 154-160Crossref PubMed Scopus (38) Google Scholar Polyclonal antibodies against Ac-H2A, H2A, Lamin B, and monoclonal antibodies against phospho-ATMSer1981 and phospho-p53Ser15 were obtained from Cell Signaling (Beverly, MA). Monoclonal antibodies against p21waf1, ATM, and p300, rabbit polyclonal antibodies against phospho-p53Ser15, Sp1, Sp3, and p300, and control scrambled small interfering RNA (siRNA) sequences were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-ATM 3E8 and 2C1 were obtained from GeneTex (San Antonio, TX), and rabbit polyclonal antibodies against phospho-ATMSer1981, ATM, and fluorescein isothiocyanate–conjugated phospho-ATMSer1981 were obtained from Rockland (Gilbertsville, PA). The goat ATM polyclonal antibodies were purchased from Bethyl Laboratories (Montgomery, TX). Mouse anti-p21waf1 monoclonal antibody was purchased from DAKO (Carpinteria, CA). Alexa Fluor 568 goat anti-mouse immunoglobulin (Ig)G antibody was obtained from Molecular Probes/Invitrogen (Eugene, OR). ATM siRNAs were purchased from Ambion (Austin, TX). Monoclonal Flag M2 antibody, sodium butyrate, TSA, and FLAG peptide were from Sigma (St. Louis, MO). Dextran sodium sulfate (DSS) (average molecular weight, 40 kilodaltons) was purchased from ICN Biomedical Inc. (Aurora, OH). The ZBP-89ΔN/ΔN mouse model, which expresses truncated ZBP-89 without the N-terminal p300-interaction domain (PID) (1–128 amino acids), was generated by targeting exon 4 of mouse ZBP-89 by homologous recombination.31Law D.J. Labut E.M. Adams R.D. Merchant J.L. An isoform of ZBP-89 predisposes the colon to colitis.Nucleic Acids Res. 2006; 34: 1342-1350Crossref PubMed Scopus (19) Google Scholar All the animals were housed in microisolator, solid-bottomed polycarbonate cages in nonbarrier mouse rooms (conventional housing). The study was performed with the approval of the University of Michigan Animal Care and Use Committee, which maintains an American Association for the Assessment and Accreditation of Laboratory Animal Care facility. To generate deletion mutants of ZBP-89, site-directed mutagenesis was used to introduce SacII sites into sequences flanking the regions to be deleted on pcDNA3–Flag–ZBP-89. After successful introduction of SacII sites, the plasmids were cut with SacII and religated with T4 DNA ligase. The deletion was verified by DNA sequencing. The expression of mutant Flag–ZBP-89 was verified by immunoblots. The following primers were used for site-directed mutagenesis of the indicated ZBP-89 residues: 6 forward: 5′-GAACATTGACGACAACCGCGGAAGGATTGTTTC-3′; 6 backward: 5′-GAAACAATCCTTCCGCGGTGTCGTCAATGTTC-3′; 180 forward: 5′-GCACTGCAACGCTGCCGCGGGAACGAACTATCACTTACAG-3′; 180 backward: 5′-CTGTAAGTGATAGTTCGTTCCCGCGGCAGCGTTGCAGTGC-3′; 298 forward: 5′-GGTGGCCTTCTGACATCCGCGGAAGATTCTGGCTTTTC-3′; 298 backward: 5′-GAAAAGCCAGAATCTTCCGCGGATGTCAGAAGGCCACC-3′; 447 forward: 5′-GTGCTGACATCGATCCCGCGGATAACTTGCAGGAG-3′; and 447 backward: 5′-CTCCTGCAAGTTATCCGCGGGATCGATGTCAGCAC-3′. The A549 (human lung cancer cell line), U2OS, and HeLa cell lines were purchased from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco’s modified Eagle medium with 10% fetal bovine serum. GM00637I and GM05849D, human fibroblast cell lines, were purchased from Coriell Cell Repository (Camden, NJ) and cultured in modified Eagle’s medium containing 10% fetal bovine serum. HCT116 and HCT116 p53 (−/−) cells were kindly provided by Dr Bert Vogelstein (John Hopkins University, Baltimore, MD) and cultured in McCoy’s 5A medium with 10% fetal bovine serum. To generate stable cell lines that express full-length or mutant forms of ZBP-89, HCT116 cells were transfected with the pcDNA3 vector or different forms of ZBP-89 expression vectors then selected with G418 at 1 mg/mL for 1–2 weeks. Pooled G418-resistant cells that expressed different forms of ZBP-89 were used for further analyses. Cells were irradiated at room temperature at a dose rate of 1–2 Gy/min using an AECL Theratron 80 (AECL/Theratonics International Ltd, Ottawa, Ontario, Canada) (60Cobalt source). RNA interference of ZBP-89 with specific siRNA has been described before.32Bai L. Merchant J.L. Transcription factor ZBP-89 is required for STAT1 constitutive expression.Nucleic Acids Res. 2003; 31: 7264-7270Crossref PubMed Scopus (28) Google Scholar For reporter assay, cells cultured in 48-well plates were first transfected with siRNAs for 1 day, and then with the p21waf1 0-Luc plasmid, which contains 2.4 kb of the human p21waf1 promoter using FUGENE 6 (Roche, Indianapolis, IN). The transfection efficiency was normalized to β-galactosidase activity expressed from the pCMV–β-gal reporter. The cells were treated with 2.5 mmol/L sodium butyrate for 20 hours before performing luciferase reporter assays that were normalized to protein.25Bai L. Merchant J.L. Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21waf1 transcription in human cells.J Biol Chem. 2000; 275: 30725-30733Crossref PubMed Scopus (98) Google Scholar The Student t test was performed to determine statistical significance. A P value of less than .05 was considered significant. The DNA affinity precipitation assay (DAPA) was performed essentially as described before.25Bai L. Merchant J.L. Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21waf1 transcription in human cells.J Biol Chem. 2000; 275: 30725-30733Crossref PubMed Scopus (98) Google Scholar A biotin-labeled DNA element from the p21waf1 proximal promoter located at −227 to −198 was used as the probe. For immunodepletion, 50 μg of nuclear extracts were first incubated with the indicated polyclonal antibodies and protein A–agarose for 16 hours. The immunodepleted nuclear extracts then were pre-incubated with protein A–agarose for another 2 hours and the supernatant was used for DAPA. Lamin B was used to confirm the absence of chromatin contamination. The chromatin immunoprecipitation (ChIP) assays were performed using the ChIP Assay Kit (Upstate, Charlottesville, VA) according to the manufacturer’s protocol. HCT116 cells grown at approximately 75% confluence were treated with 2.5 mmol/L sodium butyrate for 1 hour before the addition of the cross-linker. The rabbit anti–ZBP-89 polyclonal antibody was used for immunoprecipitation. The following primers were used for polymerase chain reaction: −515 forward: 5′-GGGATCAGTGGGAATAGAGGTG-3′; −227 backward: 5′-CGCCTGCGTTGGTGCGCTGG-3′; −246 forward: 5′-CCAGCGCACCAACGCAGGCG-3′; +46 backward: 5′-CCGGCTCCACAAGGAACTGAC-3′; +26 forward: 5′-GTCAGTTCCTTGTGGAGCCGG-3′; +355 backward: 5′-CTGTACTTGTAATCCCGCTCTCC-3′; +332 forward: 5′-GGAGAGCGGGATTACAAGTACAGG-3′; and +582 backward: 5′-GGATCCCAGAGCCACCCGGG-3′. HCT116 cells were transfected with pcDNA3-Flag–ZBP-89 expression vector or the empty pcDNA3 vector and selected with G418 at 1 mg/mL for 1 week. The G418-resistant cells, which express low levels of Flag–ZBP-89, were treated with 2.5 mmol/L sodium butyrate for 16 hours. For affinity purification, nuclear extracts were prepared and incubated with M2-agarose beads (Sigma) at 4°C for 6 hours. After extensive rinsing with the wash buffer (20 mmol/L HEPES, pH 7.9, 300 mmol/L KCl, 0.2 mmol/L ethylenediaminetetraacetic acid [EDTA], 0.5 mmol/L dithiothreitol, 20% glycerol, and 0.2% NP-40), associated complexes were eluted from the beads by incubating at 4°C for 60 minutes with the elution buffer (20 mmol/L HEPES, pH 7.9, 300 mmol/L KCl, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 20% glycerol, 0.2% NP-40, 0.5 mg/mL FLAG peptide). The eluted proteins were separated on a Novex 3%–8% Tris-acetate gel (Invitrogen, Carlsbad, CA) and stained with Coomassie Brilliant Blue. Protein bands were cut off from the gel and the proteins were eluted from the gel and digested with trypsin. The identities of the proteins were identified by MALDI-TOF (matrix assisted laser desorption/ionization-time of flight) mass spectrometry. Colitis was induced over 5 days by oral administration of 4% DSS dissolved in the drinking water as described previously.31Law D.J. Labut E.M. Adams R.D. Merchant J.L. An isoform of ZBP-89 predisposes the colon to colitis.Nucleic Acids Res. 2006; 34: 1342-1350Crossref PubMed Scopus (19) Google Scholar Antigen retrieval was performed by microwaving the deparaffinized and rehydrated sections in 10 mmol/L citrate buffer (pH 6.0) for 10 minutes. The sections were blocked by incubating in phosphate-buffered saline with 10% normal goat serum and 10% normal donkey serum for 1 hour before a 4°C overnight incubation with fluorescein-conjugated phospho-ATMSer1981 antibody (1:100). For double staining, the sections were first incubated with mouse anti-p21waf1 monoclonal antibody (1:50; DAKO) for 2 hours at room temperature, followed by incubation with Alexa Fluor 568 goat anti-mouse IgG antibody (1:500) for 30 minutes. After 3 washes in phosphate-buffered saline with 0.2% Triton X-100, the sections then were incubated with fluorescein-conjugated phospho-ATMSer1981 antibody (1:100) at 4°C overnight. After 4 washes in phosphate-buffered saline with 0.2% Triton X-100, sections were mounted in aqueous mounting medium with 1 nmol/L 4′,6-diamidino-2-phenylindole. Sections also were stained with H&E. All sections were imaged using an Olympus BX60 microscope (Center Valley, PA) equipped with a SPOT camera and software (Diagnostic Instruments, Sterling Heights, MI). Chromatin remodeling by HDACi regulates growth-related genes primarily through GC-rich elements, although the mechanism remains poorly defined. We have shown previously that expression of ZBP-89, a zinc finger transcription factor, is induced by butyrate and binds to a GC-rich element within the p21waf1 promoter.25Bai L. Merchant J.L. Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21waf1 transcription in human cells.J Biol Chem. 2000; 275: 30725-30733Crossref PubMed Scopus (98) Google Scholar Therefore, because it is a DNA binding protein regulated by HDACs, we used this protein as bait to identify proteins that participate in chromatin remodeling. First, to show that ZBP-89 is essential for expression of p21waf1, ZBP-89 protein levels were reduced by siRNA before immunoblot analysis of p21waf1 protein levels. The results showed that p21waf1 induction was abolished without affecting histone acetylation (Figure 1A). The requirement for ZBP-89 was independent of p53 status because the same result was obtained in p53 null cells (Figure 1B) and also was not limited to colon cell lines because p21waf1 induction also was blocked by reduced levels of ZBP-89 in a human small-cell lung cancer (A549) and a cervical carcinoma (HeLa) cell line (Figure 1C). Therefore, ZBP-89 appeared to be essential for induction of endogenous p21waf1 by HDACi and exerted its effect distal to histone hyperacetylation. Next, we examined whether endogenous ZBP-89 binds the p21waf1 promoter by ChIP. We found that ZBP-89 binds to the p21waf1 5′ genomic DNA in 2 locations (Figure 2A). The binding site between −246 and +46 of the proximal promoter was reported previously to be bound by Sp1, Sp3, and ZBP-89 in electrophoretic mobility shift assays and DNase I footprinting.24Xiao H. Hasegawa T. Isobe K. p300 collaborates with Sp1 and Sp3 in p21(waf1/cip1) promoter activation induced by histone deacetylase inhibitor.J Biol Chem. 2000; 275: 1371-1376Crossref PubMed Scopus (159) Google Scholar, 25Bai L. Merchant J.L. Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21waf1 transcription in human cells.J Biol Chem. 2000; 275: 30725-30733Crossref PubMed Scopus (98) Google Scholar, 33Hasegawa T. Xiao H. Isobe K.-I. Cloning of a GADD34-like gene that interacts with the zinc-finger transcription factor which binds to the p21WAF promoter.Biochem Biophys Res Commun. 1999; 256: 249-254Crossref PubMed Scopus (37) Google Scholar The second site within an intron between +332 and +582 had not been recognized previously, but was not inducible. The DNA element within the intron also contained multiple GC-rich ZBP-89 and Sp1/Sp3 consensus binding elements (Figure 2B). The inducible recruitment of ZBP-89 to the p21waf1 promoter was confirmed using a ZBP-89/Sp1/Sp3 DNA binding element from the p21waf1 proximal promoter at −227 for the DAPA (Figure 2C). We found that Sp1 also was recruited to the same element with butyrate treatment (Figure 2C). Thus, we concluded that butyrate promotes both ZBP-89 and Sp1 binding to the p21waf1 promoter. Having established a critical role for ZBP-89 in the activation of p21waf1 gene expression, we used ZBP-89 as bait to identify interacting proteins in response to chromatin remodeling agents. We performed co-immunoprecipitation using Flag-tagged ZBP-89–expressing HCT116 cells and found that a high molecular weight (∼310–350 kilodaltons) protein associated with ZBP-89 in a butyrate-dependent manner (Figure 3A). Mass spectrometry followed by immunoprecipitation and immunoblotting identified this band as the protein ATM (Figure 3B). To confirm that the interaction between ZBP-89 and ATM occurs endogenously, HCT116 cells were treated with butyrate or TSA for 1 hour. Co-immunoprecipitation was performed with anti–ZBP-89 IgG. Endogenous ATM was detected in the ZBP-89–immunoprecipitated protein complex (Figure 3C). Moreover, the association was enhanced with either butyrate or TSA treatment, confirming the requirement for HDAC inhibition. Because ZBP-89 is recruited to the p21waf1 promoter by butyrate and ATM is associated with ZBP-89 in response to butyrate, we assessed whether ATM also is recruited to the p21waf1 promoter with butyrate treatment. ChIP assays showed that ATM is recruited to the proximal p21waf1 promoter in vivo in response to butyrate (Figure 3D). p300 in many instances provides the histone acetyltransferase activity activated once HDACs are inhibited and we have shown previously that ZBP-89 associates with p300 through its N-terminal domain.25Bai L. Merchant J.L. Transcription factor ZBP-89 cooperates with histone acetyltransferase p300 during butyrate activation of p21waf1 transcription in human cells.J Biol Chem. 2000; 275: 30725-30733Crossref PubMed Scopus (98) Google Scholar Therefore, we considered that p300 might associate with Sp1/Sp3 at the p21waf1 promoter during induction by HDACi.24Xiao H. Hasegawa T. Isobe K. p300 collaborates with Sp1 and Sp3 in p21(waf1/cip1) promoter activation induced by histone deacetylase inhibitor.J Biol Chem. 2000; 275: 1371-1376Crossref PubMed Scopus (159) Google Scholar Depletion of ZBP-89 from nuclear extracts blocked the recruitment of ATM to the p21waf1 promoter, but only modestly reduced the recruitment of p300 (Figure 3E). However, when ATM was immunodepleted from nuclear extracts, we found that p300 did not precipitate with the −227 GC-rich element bound by ZBP-89 and Sp1/Sp3. Conversely, when p300 was immunodepleted from the extracts, ATM no longer precipitated with the GC-rich element in the DAPA assay (Figure 3E), suggesting cooperation between ATM and p300 during the response to butyrate. Although Sp1 inducibly binds to the p21waf1 promoter with HDACi treatment (Figure 2C), depletion of Sp1 or Sp3 had no significant effect on the recruitment of ATM or p300. Thus, alth" @default.
• W2113252276 created "2016-06-24" @default.
• W2113252276 creator A5003693281 @default.
• W2113252276 creator A5042603101 @default.
• W2113252276 creator A5049458323 @default.
• W2113252276 creator A5055596211 @default.
• W2113252276 date "2006-09-01" @default.
• W2113252276 modified "2023-10-13" @default.
• W2113252276 title "Recruitment of Ataxia-Telangiectasia Mutated to the p21waf1 Promoter by ZBP-89 Plays a Role in Mucosal Protection" @default.
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