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• W2084538148 abstract "Histone deacetylase 2 (HDAC2) is one of the histone-modifying enzymes that regulate gene expression by remodeling chromatin structure. Along with HDAC1, HDAC2 is found in the Sin3 and NuRD multiprotein complexes, which are recruited to promoters by DNA-binding proteins. In this study, we show that the majority of HDAC2 in human breast cancer cells is not phosphorylated. However, the minor population of HDAC2, preferentially cross-linked to DNA by cisplatin, is mono-, di-, or tri-phosphorylated. Furthermore, HDAC2 phosphorylation is required for formation of Sin3 and NuRD complexes and recruitment to promoters by transcription factors including p53, Rb, YY1, NF-κB, Sp1, and Sp3. Unmodified HDAC2 requires linker DNA to associate with chromatin but is not cross-linked to DNA by formaldehyde. We provide evidence that unmodified HDAC2 is associated with the coding region of transcribed genes, whereas phosphorylated HDAC2 is primarily recruited to promoters. Histone deacetylase 2 (HDAC2) is one of the histone-modifying enzymes that regulate gene expression by remodeling chromatin structure. Along with HDAC1, HDAC2 is found in the Sin3 and NuRD multiprotein complexes, which are recruited to promoters by DNA-binding proteins. In this study, we show that the majority of HDAC2 in human breast cancer cells is not phosphorylated. However, the minor population of HDAC2, preferentially cross-linked to DNA by cisplatin, is mono-, di-, or tri-phosphorylated. Furthermore, HDAC2 phosphorylation is required for formation of Sin3 and NuRD complexes and recruitment to promoters by transcription factors including p53, Rb, YY1, NF-κB, Sp1, and Sp3. Unmodified HDAC2 requires linker DNA to associate with chromatin but is not cross-linked to DNA by formaldehyde. We provide evidence that unmodified HDAC2 is associated with the coding region of transcribed genes, whereas phosphorylated HDAC2 is primarily recruited to promoters. Histone-modifying enzymes and ATP-dependent chromatin-remodeling complexes affect gene expression by altering the compaction level of chromatin and the accessibility of DNA to binding proteins. Histone hyperacetylation is generally associated with chromatin decondensation and increased transcriptional activity, whereas histone hypoacetylation contributes to chromatin condensation and transcriptional repression (1Wang X. He C. Moore S.C. Ausio J. J. Biol. Chem. 2001; 276: 12764-12768Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 2Tse C. Sera T. Wolffe A.P. Hansen J.C. Mol. Cell Biol. 1998; 18: 4629-4638Crossref PubMed Scopus (482) Google Scholar). Dynamic histone acetylation at transcriptionally active genes, resulting from the opposing activities of histone deacetylases (HDACs) 2The abbreviations used are: HDAC, histone deacetylase; DSP, dithiobis(succinimidyl)propionate; TFF1, trefoil factor 1; ChIP, chromatin immunoprecipitation; N-ChIP, native ChIP; X-ChIP, ChIP with formaldehyde cross-linking. and histone acetyltransferases, leads to a rapid oscillation between condensed and decondensed chromatin states (3Spencer V.A. Davie J.R. J. Biol. Chem. 2001; 276: 34810-34815Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 4Sun J.M. Chen H.Y. Davie J.R. J. Biol. Chem. 2001; 276: 49435-49442Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Four classes of HDACs have been identified in mammalian cells (5Glozak M.A. Seto E. Oncogene. 2007; 26: 5420-5432Crossref PubMed Scopus (815) Google Scholar). HDAC1 and 2 belong to class I and are homologous to yeast RPD3. Both HDACs are core components of multi-protein corepressor complexes like Sin3 and NuRD, in which their activities are modulated through interactions with other proteins while being recruited by transcription factors to specific promoters (6De Ruijter A.J. Van Gennip A.H. Caron H.N. Kemp S. Van Kuilenburg A.B. Biochem. J. 2003; 370: 737-749Crossref PubMed Scopus (2494) Google Scholar). HDAC1 and 2 are phosphoproteins, and this post-translational modification enhances their enzymatic activity (7Pflum M.K. Tong J.K. Lane W.S. Schreiber S.L. J. Biol. Chem. 2001; 276: 47733-47741Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 8Tsai S.C. Seto E. J. Biol. Chem. 2002; 277: 31826-31833Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 9Sun J.M. Chen H.Y. Moniwa M. Litchfield D.W. Seto E. Davie J.R. J. Biol. Chem. 2002; 277: 35783-35786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In studies with exogenously expressed tagged HDAC1 and 2, HDAC phosphorylation appeared to be a pre-requisite to form the corepressor complexes. However, studies characterizing endogenous HDAC corepressor complexes are lacking. The role of phosphorylation in the recruitment of HDAC2 by transcription factors has not yet been investigated. Several transcription factors repress gene expression by recruiting HDAC1/2 corepressor complexes to the promoters that they affect. Further, pending the promoter context, transcription factors recruit HDAC1 and 2 corepressor complexes to mediate dynamic deacetylation of histones and non-histone chromosomal proteins associated with or close to the promoter. We have previously reported that the Sp1 and Sp3 transcription factors are associated with phosphorylated HDAC2 in breast cancer cells. Although most HDAC2 in breast cancer cells was not phosphorylated, it was the phosphorylated form of HDAC2 that was cross-linked to chromatin by the cross-linkers formaldehyde and cisplatin (9Sun J.M. Chen H.Y. Moniwa M. Litchfield D.W. Seto E. Davie J.R. J. Biol. Chem. 2002; 277: 35783-35786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). These results suggested that it was the phosphorylated form of HDAC2 that was principally associated with chromatin, posing the question as to the role and location of unmodified HDAC2 in chromatin. In this study we determined which form of HDAC2 was recruited by a variety of transcription factors and which form of HDAC was associated with chromatin. Our results provide evidence that endogenous protein kinase CK2-phosphorylated HDAC2 is associated with the Sin3 and NuRD corepressor complexes and is recruited to promoters by a host of transcription factors. The more abundant unmodified HDAC2 requires linker DNA to associate with chromatin and locates to the coding region of transcribed genes. Cell Culture and Nuclear Extraction—Human breast cancer MCF-7 cells, HeLa cells and human embryonic kidney HEK293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum as described previously (4Sun J.M. Chen H.Y. Davie J.R. J. Biol. Chem. 2001; 276: 49435-49442Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 9Sun J.M. Chen H.Y. Moniwa M. Litchfield D.W. Seto E. Davie J.R. J. Biol. Chem. 2002; 277: 35783-35786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Chicken immature erythrocytes were prepared and stored at -80 °C until use as described previously (10Delcuve G.P. Davie J.R. Biochem. J. 1989; 263: 179-186Crossref PubMed Scopus (50) Google Scholar). Isolated nuclei from chicken immature erythrocytes were extracted by incubation on ice with 0.3 m NaCl for 10 min. The nuclear extract was the supernatant collected after centrifugation (11Sun J.M. Chen H.Y. Litchfield D.W. Davie J.R. J. Cell Biochem. 1996; 62: 454-466Crossref PubMed Scopus (13) Google Scholar). Plasmids and Transfection—Plasmid FLAG-HDAC2 was a gift from Dr. Edward Seto (8Tsai S.C. Seto E. J. Biol. Chem. 2002; 277: 31826-31833Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Plasmids FLAG-HDAC2-M3A (S394A/S422A/S424A), FLAG-HDAC2-M3D (S394D/S422D/S424D), and FLAG-HDAC2-M3E (S394E/S422E/S424E) were constructed in two steps by PCR using a site-directed mutagenesis kit (Stratagene, Cedar Creek, TX). The first step consisted of mutating serine residues 422 and 424 into alanine, aspartic acid, or glutamic acid residues and was carried out with plasmid FLAG-HDAC2 as template, using the primer pairs 2A, forward, 5′-GATGAAGAATTCGCAGATGCTGAGGATGAAGGAGGTCGA-3′, and reverse, 5′-TCGACCTCCTTCATCCTCAGCATCTGCGAATTCTTCATC-3′; 2D, forward, 5′-GATGAAGAATTCGATGATGATGAGGATGAAGGAGAAGGA-3′, and reverse, 5′-TCCTTCTCCTTCATCCTCATCATCATCGAATTCTTCATC-3′; 2E, forward, 5′-GATGAAGAATTCGAAGATGAAGAGGATGAAGGAGAAGGA-3′, and reverse, 5′-TCCTTCTCCTTCATCCTCTTCATCTTCGAATTCTTCATC-3′ to generate plasmids FLAG-HDAC2-M2A, -M2D or -M2E. The second step used the FLAG-HDAC2-M2 plasmids as templates with the primer pairs 3A, forward, 5′-GCTGTTCATGAAGACGCTGGAGATGAAGATGGAGAAGAT-3′, and reverse, 5′-ATCTTCTCCATCTTCATCTCCAGCGTCTTCATGAACAGC-3′; 3D, forward, 5′-GCTGTTCATGAAGACGATGGAGATGAAGATGGAGAAGAT-3′, and reverse, 5′-ATCTTCTCCATCTTCATCTCCATCGTCTTCATGAACAGC-3′; 3E, forward, 5′-GCTGTTCATGAAGACGAAGGAGATGAAGATGGAGAAGAT-3′, and reverse, 5′-ATCTTCTCCATCTTCATCTCCTTCGTCTTCATGAACAGC-3′ to produce a substitution of alanine, aspartic acid, or glutamic acid for serine in position 394, resulting in plasmids FLAG-HDAC2-M3A, FLAG-HDAC2-M3D, or FLAG-HDAC2-M3E, respectively. The constructed plasmids were verified by DNA sequencing (310 Genetic Analysis, ABI). Two μg of FLAG-HDAC2 or FLAG-HDAC2-M3 plasmids were transfected into HEK293 cells using the Lipofectamine™ 2000 transfection reagent (Invitrogen, Carlsbad, CA) according to the supplier's instructions. Approximately 24 h after transfection, the cells were harvested for immunoprecipitation or storage at -80 °C. Immunoprecipitation and Immunoblotting—MCF-7 and HeLa cells were lysed in IP buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40) containing phosphatase and protease inhibitors, and immunoprecipitations were done as described previously (9Sun J.M. Chen H.Y. Moniwa M. Litchfield D.W. Seto E. Davie J.R. J. Biol. Chem. 2002; 277: 35783-35786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). For each cell lysate, an immunoprecipitation with preimmune serum was also performed as negative control to check for nonspecific immunoprecipitation. Immunoblot analysis was carried out as described previously (12Samuel S.K. Spencer V.A. Bajno L. Sun J.M. Holth L.T. Oesterreich S. Davie J.R. Cancer Res. 1998; 58: 3004-3008PubMed Google Scholar). Polyclonal antibodies against human HDAC1 (Affinity BioReagents, Golden, CO), HDAC2 (Affinity BioReagents), Sp1 (Upstate, Charlottesville, VA), Sp3 (Upstate), Sin3A (Santa Cruz Biotechnology Inc., Santa Cruz, CA), MBD3B (Santa Cruz Biotechnology Inc.), Mi2 (Santa Cruz Biotechnology Inc.), RbAp48 (GeneTex Inc., San Antonio, TX), CK2α (Santa Cruz Biotechnology Inc.), p53 (Santa Cruz Biotechnology Inc.), Rb (Santa Cruz Biotechnology Inc.), YY1 (Santa Cruz Biotechnology Inc.), NF-κB p65 (Santa Cruz Biotechnology Inc.), NF-κB p50 (Santa Cruz Biotechnology Inc.), and hyperacetylated H4 (Upstate) were used. For some of the immunoprecipitation experiments, antibodies were conjugated with CNBr-activated Sepharose (Sigma) following the supplier's instructions. Anti-FLAG M2 agarose affinity gel was obtained from Sigma. HDAC Activity Assay—HDAC activity assays were performed as described previously (13Hendzel M.J. Delcuve G.P. Davie J.R. J. Biol. Chem. 1991; 266: 21936-21942Abstract Full Text PDF PubMed Google Scholar). Formaldehyde and Cisplatin DNA Cross-linking—Formaldehyde or cisplatin cross-linking was carried out as described previously (9Sun J.M. Chen H.Y. Moniwa M. Litchfield D.W. Seto E. Davie J.R. J. Biol. Chem. 2002; 277: 35783-35786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 14Spencer V.A. Davie J.R. The Protein Protocols Handbook. 2002; (Walker, J. M., ed) pp. Humana Press, Totowa, NJ: 753-757Crossref Google Scholar, 15Spencer V.A. Davie J.R. The Protein Protocols Handbook. 2002; (Walker, J. M., ed) pp. Humana Press, Totowa, NJ: 747-751Crossref Google Scholar). Proteins cross-linked to DNA were isolated by hydroxyapatite column chromatography. DNA-protein cross-links were reversed, and proteins were isolated. The proteins were either dialyzed against IP buffer for further immunoprecipitation or precleaned using the ReadyPrep two-dimensional clean-up kit (Bio-Rad) for further two-dimensional electrophoresis. Immunoprecipitation of DSP Cross-linked Proteins—DSP cross-linking was performed following the manufacturer's instructions (Pierce). Briefly, MCF-7 cells, grown to 80% confluence, were washed in phosphate-buffered saline and incubated for 0, 5, 10, or 20 min with 1 mm DSP at room temperature. The reaction was stopped by adding Tris-HCl, pH 7.5, to a final concentration of 10 mm and incubating the cells for 15 min at room temperature. The cells were lysed by sonication in low stringency buffer (50 mm Tris-HCl, pH 7.5, 120 mm NaCl, 0.5 mm EDTA, 0.5% Nonidet P-40) containing phosphatase and protease inhibitors. Four A260 of cell lysate were incubated with anti-HDAC2, anti-HDAC1 or anti-RbAp48 antibodies. Immune complexes were recovered by adding protein A-Sepharose and washing three times with low stringency buffer containing phosphatase and protease inhibitors, three times with high stringency buffer (50 mm Tris-HCl, pH 7.5, 500 mm NaCl, 0.5 mm EDTA, 0.5% Nonidet P-40, 0.5% SDS) containing phosphatase and protease inhibitors, and once with phosphate-buffered saline. The proteins were boiled for 5 min in SDS loading buffer containing β-mercaptoethanol at a final concentration of 5% (to reverse the DSP cross-linking), separated on SDS-10% polyacrylamide gels, transferred to nitrocellulose membranes, and immunochemically stained as indicated. Alkaline Phosphatase Digestion—Alkaline phosphatase digestion of cross-linked or immunoprecipitated proteins was done as described previously (9Sun J.M. Chen H.Y. Moniwa M. Litchfield D.W. Seto E. Davie J.R. J. Biol. Chem. 2002; 277: 35783-35786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Two-dimensional Electrophoresis—Two dimensional electrophoresis was done as described previously (16Chen H.Y. Sun J.M. Hendzel M.J. Rattner J.B. Davie J.R. Biochem. J. 1996; 320: 257-265Crossref PubMed Scopus (25) Google Scholar, 17Samuel S.K. Minish T.M. Davie J.R. Cancer Res. 1997; 57: 147-151PubMed Google Scholar, 18Spencer V.A. Samuel S. Davie J.R. Cancer Res. 2000; 60: 288-292PubMed Google Scholar). Twenty micrograms of cisplatin cross-linked proteins or total cell lysate from MCF-7 cells were loaded on isoelectric focusing strips (pH 3-10) and electrophoresed following the manufacturer's instructions (Bio-Rad). Isoelectric focusing strips were then transferred and electrophoresed on SDS-10% polyacrylamide gels. After transfer onto a nitrocellulose membrane, the proteins were immunochemically stained with anti-HDAC1 or anti-HDAC2 antibodies. Chromatin Immunoprecipitation (ChIP)—ChIP assays were done on MCF-7 cells as described previously with some modifications (19Spencer V.A. Sun J.M. Li L. Davie J.R. Methods. 2003; 31: 67-75Crossref PubMed Scopus (147) Google Scholar). MCF-7 cells were resuspended in phosphate-buffered saline and were either subjected to dual cross-linking or treated with formaldehyde alone. The cells subjected to dual cross-linking were first incubated with 1 mm DSP or 1 mm ethylene glycolbis(succinimidylsuccinate) for 30 min at room temperature. Formaldehyde was then added to a final concentration of 1%, and the cells were incubated for 10 min. After quenching with glycine to a final concentration of 125 mm and lysis of the cells, the chromatin was sheared to an average fragment size of 500 base pairs, diluted to 4 A260 units/ml in dilution buffer (16.7 mm Tris-HCl, pH 8.1, 1.2 mm EDTA, 167 mm NaCl, 1.1% Triton X-100, 0.01% SDS, and 0.5 mg/ml bovine serum albumin), and precleared by incubation with 60 μl/ml of protein A/G-agarose beads. Cross-linked chromatin fragments (1 ml) were incubated with 5 μg of anti-HDAC2 (Affinity BioReagents). Immunoprecipitated complexes were recovered by an incubation with protein A/G-agarose (pretreated with 500 μg/ml of yeast tRNA) and were serially washed with 1 ml of washing buffer I (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris, pH 8.1, and 150 mm NaCl), washing buffer II (0.1% SDS, 1% Triton X-100, 2 mm EDTA, 20 mm Tris, pH 8.1, and 500 mm NaCl), and washing buffer III (0.25 m LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mm EDTA, and 10 mm Tris, pH 8.1) and then washed twice with 1 mm EDTA, 10 mm Tris-HCl, pH 8.0. Precipitated chromatin complexes were eluted from the beads with 100 μl of elution buffer (1% SDS, 0.1 m NaHCO3). After reversal of the cross-linking at 65 °C, DNA was isolated directly from the agarose slurry using a QIAQuick PCR purification kit (Qiagen). The ChIP and input DNA concentrations were determined with the Quant-iT Picogreen dsDNA kit (Invitrogen). Equal amounts (2 ng) of ChIP and input DNAs were quantitated by real time PCR, using the two following pairs of primers: pair 1; forward, 5′-GACGGAATGGGCTTCATGAGC-3′, and reverse, 5′-GATAACATTTGCCTAAGGAGG-3′ to amplify a 386-bp fragment in the promoter region of the TFF1 gene, and pair 2; forward, 5′-TTGTGGTTTTCCTGGTGTCA-3′, and reverse, 5′-GGAGGGACGTCGATGGTAT-3′ to amplify a 114-bp fragment in the TFF1 gene exon 2. The enrichment values (ChIP DNA versus input DNA) were calculated according to a published formula (20Ciccone D.N. Morshead K.B. Oettinger M.A. Methods Enzymol. 2004; 376: 334-348Crossref PubMed Scopus (24) Google Scholar). Chromatin Fractionation and Fraction Immunoprecipitation—Chicken immature erythrocyte salt-soluble chromatin S150 was prepared as described previously (10Delcuve G.P. Davie J.R. Biochem. J. 1989; 263: 179-186Crossref PubMed Scopus (50) Google Scholar). S150 (200-300 A260 units) was loaded on a gel exclusion chromatography Bio-Gel A1.5m column (Bio-Rad) (10Delcuve G.P. Davie J.R. Biochem. J. 1989; 263: 179-186Crossref PubMed Scopus (50) Google Scholar). The fractions were collected and pooled. To check DNA size, 20 μl from each fraction were extracted with phenol/chloroform, electrophoresed on a 1% agarose gel, and stained with ethidium bromide. To analyze HDAC2, 2 A260 from each fraction were trichloroacetic acid-precipitated, resolved by SDS-PAGE, and immunoblotted with anti-HDAC2 antibodies. Native chromatin immunoprecipitation (N-ChIP) was carried out as described previously (21Hebbes T.R. Thorne A.W. Crane Robinson C. EMBO J. 1988; 7: 1395-1402Crossref PubMed Scopus (715) Google Scholar). Approximately 2 A260 of polynucleosome fraction (F1) were incubated overnight with Sepharose-conjugated anti-hyperacetylated H4 antibodies (Upstate). N-ChIP or ChIP with formaldehyde cross-linking (X-ChIP) assays were carried out on fraction F2. Equal amounts of F2 (∼50 A260) were incubated at room temperature for 10 min with (X-ChIP) or without (N-ChIP) 1% formaldehyde. Then cross-linking was quenched by the addition of glycine to a final concentration of 0.125 m, and both F2 samples were dialyzed against dilution buffer (described above) and concentrated to about 8 A260/ml. Immunoprecipitation with anti-HDAC2 antibodies (Affinity BioReagents), DNA recovery, and quantitation were done as described above. For the N-ChIP samples, the beads were washed with wash buffer A (50 mm NaCl, 20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 20 mm butyrate, and 0.1 mm phenylmethyl sulfonyl fluoride), wash buffer B (100 mm NaCl, 20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 20 mm butyrate, and 0.1 mm phenylmethyl sulfonyl fluoride), and wash buffer C (150 mm NaCl, 20 mm Tris-HCl, pH 7.5, 5 mm EDTA, 20 mm butyrate, and 0.1 mm phenylmethyl sulfonyl fluoride). Incubation at 65 °C was omitted for N-ChIP samples. Equal amounts (1 ng) of ChIP and input DNAs were analyzed by real time PCR using forward 5′-GGTGTGCTGGGAGGAAGGA-3′ and reverse 5′-CCAAACCACAGCACTCTGCAT-3′ primers to amplify a 78-bp fragment in the promoter region of the βA-globin gene, forward 5′-CTGTGAAGAACCTGGACAAC-3′ and reverse 5′-AAGTTCTCGGGGTCCACATG-3′ primers to amplify a 88-bp fragment in the βA-globin coding region (exon 2), forward 5′-CGTGTGCAAAGGAAAAACAG-3′ and reverse 5′-CGCCCCGTACTGATTTGA-3′ primers to amplify a 112-bp fragment in the H2A.F promoter region, and forward 5′-CCCCACATCCACAAGTCTCT-3′ and reverse 5′-GGGGAACACAAAAGCCATAG-3′ primers to amplify a 174-bp fragment in the H2A.F coding region (exon 5). The enrichment values (ChIP DNA versus input DNA) were calculated as described above. HDAC2 Phosphorylated Forms—To assess the phosphorylation states of HDAC1 and HDAC2 in breast cancer MCF-7 cells, we conducted two-dimensional electrophoretic analyses of MCF-7 cell lysates. Fig. 1 (B and D) shows that HDAC1 and HDAC2 were both immunodetected as single spots, demonstrating that most of these proteins were not modified in MCF-7 cells. However, we had previously showed that phosphorylated HDAC2 was preferentially associated with the Sp1 and Sp3 transcription factors and preferentially cross-linked to DNA (9Sun J.M. Chen H.Y. Moniwa M. Litchfield D.W. Seto E. Davie J.R. J. Biol. Chem. 2002; 277: 35783-35786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 22Sun J.M. Spencer V.A. Li L. Chen H.Y. Yu J. Davie J.R. Exp. Cell Res. 2005; 302: 96-107Crossref PubMed Scopus (45) Google Scholar). Therefore, MCF-7 cells were incubated with cisplatin, which cross-links proteins directly to DNA but unlike formaldehyde, does not cross-link proteins with each other. The proteins cross-linked to nuclear DNA in situ were isolated by hydroxyapatite column chromatography and separated by two-dimensional PAGE. Fig. 1C shows that HDAC1 was again immunodetected as a single spot, demonstrating that most of the DNA-bound HDAC1 was in an unmodified form. This also indicates that the cisplatin cross-linking itself did not affect the electrophoretic behavior of HDAC proteins. In contrast, the population of HDAC2 that was cross-linked to DNA showed a marked enrichment in tri-, di-, and mono-phosphorylated forms (Fig. 1A). Digestion of the cisplatin cross-linked protein fraction with alkaline phosphatase resulted in the loss of the HDAC2 forms and the appearance of the parent form of HDAC2 (Fig. 1E), providing evidence that phosphorylation was the modification involved in the genesis of the HDAC2 forms. Transcription Factors Recruit the Phosphorylated HDAC2 Forms—We had previously reported that Sp1 and Sp3 preferentially associated with phosphorylated HDAC2 (9Sun J.M. Chen H.Y. Moniwa M. Litchfield D.W. Seto E. Davie J.R. J. Biol. Chem. 2002; 277: 35783-35786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Rb, p53, YY1, and NF-κB (p50, p65 subunits) are transcription factors known to achieve transcription silencing or dynamic acetylation/deacetylation by recruiting HDAC1 and 2 to specific promoters via Sin3 and/or NuRD complexes (23Brehm A. Kouzarides T. Trends Biochem. Sci. 1999; 24: 142-145Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 24Murphy M. Ahn J. Walker K.K. Hoffman W.H. Evans R.M. Levine A.J. George D.L. Genes Dev. 1999; 13: 2490-2501Crossref PubMed Scopus (394) Google Scholar, 25Thomas M.J. Seto E. Gene (Amst.). 1999; 236: 197-208Crossref PubMed Scopus (417) Google Scholar, 26Lee S.K. Kim J.H. Lee Y.C. Cheong J. Lee J.W. J. Biol. Chem. 2000; 275: 12470-12474Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). To determine whether these transcription factors preferentially recruited phosphorylated HDAC2, we carried out immunoprecipitations of MCF-7 cell lysates with antibodies to these transcription factors. Total cell lysates and immunoprecipitated fractions were electrophoresed on a SDS-10% polyacrylamide gel and analyzed by immunoblot for HDAC1 and HDAC2. Fig. 2 shows that an HDAC2 form with reduced mobility on a SDS gel was preferentially associated with transcription factors p53, Rb, YY1, and the NF-κB subunits p50 and p65. Our previous studies with Sp1 and Sp3 showed that the reduced mobility of HDAC2 in this SDS gel system was due to phosphorylation as treatment of the immunoprecipitate with alkaline phosphatase resulted in the disappearance of the slow migrating form and appearance of the faster migrating parent band (9Sun J.M. Chen H.Y. Moniwa M. Litchfield D.W. Seto E. Davie J.R. J. Biol. Chem. 2002; 277: 35783-35786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Nonphosphorylated HDAC1 was found associated with all of these transcription factors. These results demonstrate that a multitude of transcription factors selectively recruit phosphorylated HDAC2 in situ. Several of the transcription factors recruit the corepressor complexes Sin3 and NuRD, which contain HDAC1 and 2 (24Murphy M. Ahn J. Walker K.K. Hoffman W.H. Evans R.M. Levine A.J. George D.L. Genes Dev. 1999; 13: 2490-2501Crossref PubMed Scopus (394) Google Scholar). The association of phosphorylated HDAC2 with transcription factors such as p53 and Sp1 may reflect that phosphorylated HDAC2 is a component of the Sin3 and NuRD corepressor complexes. To determine the HDAC2 form associated with the endogenous Sin3 and NuRD corepressor complexes, we incubated a MCF-7 cell lysate with anti-Sin3A and anti-Mi2 antibodies and collected the immunoprecipitated and immunodepleted fractions. Fig. 3A shows that the phosphorylated forms of HDAC2 were highly enriched in the immunoprecipitated fractions with both antibodies. Nonphosphorylated HDAC1 and CK2 were also coimmunoprecipitated, confirming that these proteins are components of the Sin3 and NuRD complexes. A marked enrichment of phosphorylated HDAC2 was also observed in the MCF-7 cell immunoprecipitated fractions using antibodies against MBD3 from the NuRD complex and the histone-binding protein, RbAp48, present in both Sin3 and NuRD complexes (Fig. 3B). Nonphosphorylated HDAC1 was also part of the complexes immunoprecipitated by anti-MBD3 and anti-RbAp48 (Fig. 3B). Alkaline phosphatase treatment of the RbAp48 immunoprecipitated fraction from MCF-7 cells resulted in a faster electrophoretic migration of HDAC2, providing evidence that the reduced mobility of HDAC2 was due to phosphorylation (Fig. 3C). Fig. 3D shows that the preferential association of phosphorylated HDAC2 with the Sin3 and NuRD complexes was also found in HeLa cells. Indeed, MBD3 and Sin3A immunoprecipitated fractions were highly enriched in phosphorylated HDAC2. Our data support the idea that several transcription factors recruit either the Sin3 or NuRD corepressor complexes associated with phosphorylated HDAC2 and unmodified HDAC1. We had previously reported that the protein kinase CK2 was associated with HDAC1 and HDAC2 (9Sun J.M. Chen H.Y. Moniwa M. Litchfield D.W. Seto E. Davie J.R. J. Biol. Chem. 2002; 277: 35783-35786Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Here we tested whether CK2 was a component of the Sin3 and NuRD corepressor complexes. A MCF-7 cell lysate was incubated with anti-CK2α antibodies, and the immunoprecipitated fraction was analyzed by immunoblotting with anti-Sin3A and anti-MBD3B antibodies. Fig. 4 shows that CK2 was a component of both complexes because it was bound to Sin3A from Sin3 and MBD3B from NuRD, as well as being bound to HDAC1 and HDAC2. On the other hand, HDAC3, which belongs to different complexes than HDAC1 and HDAC2, was not found associated with CK2 in MCF-7 cells. RbAp48 is a core component of both the Sin3 and NuRD corepressor complexes. To determine whether RbAp48 directly interacts with HDAC2 and HDAC1, MCF-7 cells were treated with DSP, a homobifunctional, thiol-cleavable, primary amine-reactive cross-linker, and the cross-linked proteins were immunoprecipitated from the lysate with anti-HDAC2 or anti-HDAC1 antibodies. Immunoprecipitates were washed under stringent conditions to remove proteins that were not cross-linked to the immunoprecipitated protein, and the cross-linked proteins were identified by immunoblotting. Fig. 5A shows that in the absence of DSP incubation, HDAC2 was not associated with RbAp48 following the stringent washes of the immunoprecipitates. Following 5-min incubation with DSP, RbAp48 was cross-linked to HDAC2. HDAC2 crosslinked to the RbAp48 immunoprecipitated protein was primarily the slow migrating phosphorylated form (Fig. 5B). Longer cross-linking times were required to observe retention of RbAp48 in the HDAC1 immunoprecipitate (Fig. 5C). These observations provide evidence that phosphorylated HDAC2 interacts directly with RbAp48. To find out the requirement of phosphorylation for binding of HDAC2 to RbAp48, we determined whether RbAp48 would associate with HDAC2 in which the three serine phosphorylation sites were mutated to alanine or with a potential HDAC2 phosphomimic in which the serines were mutated to glutamic or aspartic acid. HEK293 cells were transiently transfected with vectors expressing FLAG-HDAC2 fusion proteins, either the wild type HDAC2 or a triple mutant HDAC2 that had its three phosphorylation sites changed from serine to alanine (Fig. 6A). The lysates were immunoprecipitated with anti-FLAG, and the immunoprecipitated fractions were tested by immunoblot analysis for the presence of the histone-binding protein RbAp48, the transcription factors Sp1 and Sp3, and HDAC1. Fig. 6B shows that RbAp48, Sp1, and Sp3 were associated with the wild type HDAC2 construct. When the mutated form of HDAC2 was expressed, none of these three proteins were coimmunoprecipitated by FLAG. In contrast, HDAC1 was associated with mutant FLAG-HDAC2. It is possible that mutated HDAC2 was unable to bind to RbAp48, Sp1 or Sp3 because of a change of structure caused by the serine to alanine mutations rather than to the absence of phosphorylation. To test this possibility, the three phosphorylation sites were mutated from serine to aspartic or glutamic acid because these amino acids have been previously shown to mimic phosphorylated serine residues (7Pflum M.K. Tong J.K. Lane W.S. Schreiber S.L. J. Biol. C" @default.
• W2084538148 created "2016-06-24" @default.
• W2084538148 creator A5017330034 @default.
• W2084538148 creator A5028813507 @default.
• W2084538148 creator A5048849645 @default.
• W2084538148 date "2007-11-01" @default.
• W2084538148 modified "2023-10-01" @default.
• W2084538148 title "Differential Distribution of Unmodified and Phosphorylated Histone Deacetylase 2 in Chromatin" @default.
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