Differential Distribution of Unmodified and Phosphorylated Histone Deacetylase 2 in Chromatin

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 mm mm and and were 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). immunoprecipitation with serum was as to for was carried out as described previously V.A. J.M. S. Davie J.R. 1998; Google Scholar). human HDAC1 HDAC2 Sp1 Sp3 and were of the immunoprecipitation were with the supplier's instructions. was from HDAC activity were as described previously G.P. Davie J.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). and DNA or cisplatin 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, V.A. Davie J.R. The 2002; J. Google Scholar, V.A. Davie J.R. The 2002; J. Google Scholar). cross-linked to DNA were by were and proteins were The proteins were IP buffer for immunoprecipitation or using the kit for Immunoprecipitation of was the MCF-7 cells, grown to were in and for or with mm at The was by to a of 10 mm and the cells for at The cells were lysed by in buffer (50 mm mm mm and Four of were with or complexes were by protein and with buffer and with buffer (50 mm mm mm and and with The proteins were for in buffer at a of 5% the on to and as of cross-linked or proteins was 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). was as described previously H.Y. J.M. Davie J.R. Biochem. J. 1996; PubMed Scopus Google Scholar, Davie J.R. Google Scholar, V.A. S. Davie J.R. Google Scholar). of cisplatin cross-linked proteins or from MCF-7 cells were on and the were and on a the proteins were with or Immunoprecipitation were on MCF-7 cells as described previously with V.A. J.M. Davie J.R. 2003; PubMed Scopus Google Scholar). MCF-7 cells were in and were to or with formaldehyde The cells to were first with mm or mm for at was to a of and the cells were for 10 min. with to a of mm and of the cells, the chromatin was to of to in buffer mm mm mm and bovine serum and by incubation with of protein chromatin were with μg of complexes were by incubation with protein with of yeast and were with of buffer I 2 mm mm and mm buffer 2 mm mm and mm and buffer m mm and 10 mm and with mm 10 mm chromatin complexes were from the with of buffer m of the at DNA was from the using a PCR kit The ChIP and DNA were determined with the kit of ChIP and were by using the two pairs of 1; forward, and reverse, to a in the promoter region of the and forward, and reverse, to a in the gene The DNA were according to a M.A. PubMed Scopus Google Scholar). and immature chromatin was prepared as described previously (10Delcuve G.P. Davie J.R. Biochem. J. 1989; 263: 179-186Crossref PubMed Scopus (50) Google Scholar). was on a (10Delcuve G.P. Davie J.R. Biochem. J. 1989; 263: 179-186Crossref PubMed Scopus (50) Google Scholar). The were collected and DNA from were extracted with on a and with HDAC2, 2 from were by and with chromatin immunoprecipitation was carried out as described previously C. J. PubMed Scopus Google Scholar). Approximately 2 of were with or ChIP with formaldehyde were carried out on of were at for 10 with or formaldehyde. was by the of to a of and were buffer and to Immunoprecipitation with DNA and were as described the the were with buffer (50 mm mm mm mm and mm buffer mm mm mm mm and mm and buffer mm mm mm mm and mm at °C was for of ChIP and were by PCR using and to a in the promoter region of the and to a in the coding region and to a in the promoter and and to a in the coding region The DNA were as described HDAC2 the phosphorylation states of HDAC1 and HDAC2 in breast cancer MCF-7 cells, we of MCF-7 and that HDAC1 and HDAC2 were as that most of proteins were not modified in MCF-7 cells. However, we previously 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, J.M. V.A. Chen H.Y. J. Davie J.R. Cell PubMed Scopus Google Scholar). MCF-7 cells were with cisplatin, which proteins to DNA but not proteins with The proteins cross-linked to nuclear DNA in were by and by that HDAC1 was as a that most of the HDAC1 was in unmodified that the cisplatin not affect the of HDAC proteins. In the population of HDAC2 that was cross-linked to DNA a in di-, and of the cisplatin cross-linked protein with in the of the HDAC2 and the of the form of HDAC2 evidence that phosphorylation was the modification in the of the HDAC2 the HDAC2 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 are transcription factors to transcription or dynamic by recruiting HDAC1 and 2 to specific promoters Sin3 NuRD complexes T. Biochem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. A.J. PubMed Scopus Google Scholar, Seto E. PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). transcription factors preferentially recruited phosphorylated HDAC2, we carried out of MCF-7 with to transcription factors. and were on a and by for HDAC1 and 2 that HDAC2 form with on a was preferentially associated with transcription factors p53, Rb, YY1, and the and Our studies with Sp1 and Sp3 that the of HDAC2 in this was to phosphorylation as of the with in the of the form and of the (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). HDAC1 was found associated with of transcription factors. These results that a of transcription factors recruit phosphorylated HDAC2 in Several of the transcription factors recruit the corepressor complexes Sin3 and NuRD, which HDAC1 and 2 M. J. A.J. PubMed Scopus Google Scholar). The of phosphorylated HDAC2 with transcription factors as and Sp1 that phosphorylated HDAC2 is a of the Sin3 and NuRD corepressor complexes. the HDAC2 form associated with the endogenous Sin3 and NuRD corepressor complexes, we a MCF-7 with and and collected the and that the phosphorylated of HDAC2 were in the with HDAC1 and were that proteins are components of the Sin3 and NuRD complexes. of phosphorylated HDAC2 was in the MCF-7 using from the NuRD and the in Sin3 and NuRD complexes HDAC1 was of the complexes by and of the from MCF-7 cells in a of HDAC2, evidence that the of HDAC2 was to phosphorylation that the of phosphorylated HDAC2 with the Sin3 and NuRD complexes was found in HeLa cells. and were in phosphorylated Our the that transcription factors recruit the Sin3 or NuRD corepressor complexes associated with phosphorylated HDAC2 and unmodified We previously reported that the protein kinase 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). we was a of the Sin3 and NuRD corepressor complexes. MCF-7 was with and the was by with and that was a of complexes it was to from Sin3 and from NuRD, as as being to HDAC1 and the other which to complexes HDAC1 and HDAC2, was not found associated with in MCF-7 cells. is a core of the Sin3 and NuRD corepressor complexes. with HDAC2 and HDAC1, MCF-7 cells were with DSP, a and the cross-linked proteins were from the with or were to proteins that were not cross-linked to the and the cross-linked proteins were identified by that in the of HDAC2 was not associated with the of the incubation with DSP, was cross-linked to HDAC2 to the protein was primarily the phosphorylated form were required to of in the HDAC1 These provide evidence that phosphorylated HDAC2 with out the of phosphorylation for binding of HDAC2 to we determined associate with HDAC2 in which the serine phosphorylation were to or with a HDAC2 in which the were to glutamic or aspartic HEK293 cells were transfected with FLAG-HDAC2 the HDAC2 or a HDAC2 that phosphorylation from serine to The were with and the were by for the of the protein the transcription factors Sp1 and and that Sp1, and Sp3 were associated with the HDAC2 the form of HDAC2 was of proteins were by In HDAC1 was associated with is that HDAC2 was to to Sp1 or Sp3 of a of by the serine to to the of this the phosphorylation were from serine to aspartic or glutamic acid have been previously to phosphorylated serine residues (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). that aspartic acid and glutamic acid for phosphorylated not HDAC2 to with but enzymatic activity, which was the phosphorylation were with These that HDAC2 phosphorylation is required for with and the formation of Sin3 and NuRD complexes. Unmodified HDAC2 to but to DNA by phosphorylated form of HDAC2 was preferentially cross-linked to DNA in with formaldehyde and These results suggested that phosphorylated HDAC2 was associated with chromatin, the question as to the unmodified HDAC2 was this we determined the of HDAC2 in chromatin in transcriptionally active and histones G.P. Davie J.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). immature m and to be in active and in HDAC2 enzymatic activity were G.P. Davie J.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The chromatin were on a were with and the and that are in transcriptionally active genes. that the and to a the most of the HDAC2 from the of HDAC2 in and was not phosphorylated. The of HDAC2 in the and with the enzymatic activity previously G.P. Davie J.R. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that this HDAC2 was to binding to chromatin and not to a of HDAC2 chicken immature nuclei were to the HDAC2 was from the that HDAC2 was associated with chromatin. this we carried out a native ChIP on the using the to preferentially chromatin associated with active using a described previously C. J. PubMed Scopus Google Scholar). proteins were on a and by that HDAC2 was associated with the transcriptionally active gene chromatin from and was not phosphorylated. The that and to phosphorylated of HDAC2 was the to unmodified the HDAC2 cross-linked to with formaldehyde was preferentially phosphorylated, was to with and the proteins to DNA were by the form of HDAC2 and the phosphorylated were found in the DNA cross-linked The unmodified form of HDAC2, the majority of the HDAC2 was found in the and was not cross-linked to DNA by formaldehyde. a we the nuclear extract on that not to These that the phosphorylated and more abundant HDAC2 are associated with chromatin. However, it is the phosphorylated HDAC2 form that is cross-linked to DNA with formaldehyde. The of HDAC2 with the suggested that of unmodified HDAC2 to chromatin required the of the linker this were with to chromatin and the resulting was on the and of the and that the HDAC2, that HDAC2 with chromatin requires the linker DNA HDAC2 with that unmodified HDAC2 was associated with chromatin to that unmodified HDAC2 was associated with coding whereas phosphorylated HDAC2 through with corepressor complexes was recruited to promoters and The results in with a to this we between the phosphorylated and unmodified of HDAC2 by a and a Although of HDAC2 be identified in the phosphorylated HDAC2 was cross-linked to DNA and in the and were with from the chicken immature The was this was primarily of with a DNA of which to promoter from coding of the gene in ChIP the in the promoter and coding that were in PCR are by more the HDAC2 was to the two of ChIP using the which results in the immunoprecipitation of chromatin associated with phosphorylated and unmodified HDAC2, the promoter and coding were in the complexes. the immunoprecipitation of DNA cross-linked to the phosphorylated of HDAC2 of the promoter region that was that the of the coding region in the complexes These results provide evidence that the phosphorylated of HDAC2 were primarily recruited to the promoter whereas the unmodified form of HDAC2 was associated with coding the of we on the promoter and coding of the active gene in chicken immature erythrocytes Davie J.R. PubMed Scopus Google Scholar). The in the PCR to DNA from the promoter or coding were by more that results with the were to with with the of the promoter region being in and whereas the of the coding region was in the in the We the of HDAC2 with the gene in MCF-7 cells. We the promoter it is associated with Sp1 or which the Sin3 HDAC Mol. PubMed Scopus Google Scholar). In we and have that the promoter is associated with Sp1 or Sp3 as as HDAC2 and in the or of J.M. V.A. Chen H.Y. J. Davie J.R. Cell PubMed Scopus Google Scholar, S. M. S. PubMed Scopus Google Scholar). study that the phosphorylated of HDAC2 were preferentially cross-linked to we that it was the phosphorylated of HDAC2 that were at the promoter. we have that the promoter and coding were associated with histones that are in dynamic acetylation J.M. V.A. Chen H.Y. J. Davie J.R. Cell PubMed Scopus Google Scholar). and J. that HDACs are associated with the of HDAC2 with the promoter and coding we used a in which was to formaldehyde Chen S.L. PubMed Scopus Google Scholar). 10 that MCF-7 cells were with formaldehyde resulting in the of the phosphorylated of HDAC2 to the promoter not the coding was in the HDAC2 the other MCF-7 cells were with the protein to with the coding region was in the HDAC2 DNA These results are with the that the unmodified form of HDAC2 was cross-linked to DNA by the of and formaldehyde. results were protein was used of DSP, the of the was that of not These results that the of the phosphorylated and unmodified of HDAC2 at promoter coding of is in and human cells. We have the of and phosphorylated HDAC2 in corepressor complexes and in chromatin. Our results are with HDAC2 being in MCF-7 cells at and the phosphorylated residues (8Tsai S.C. Seto E. J. Biol. Chem. 2002; 277: 31826-31833Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). the mono-, di-, and of HDAC2 were preferentially cross-linked to Our results that phosphorylation of HDAC2 is required for binding to which in the for other proteins to form the Sin3 or NuRD corepressor complexes. factors recruit corepressor complexes the phosphorylated HDAC2 complexes, the of phosphorylated HDAC2 with a host of transcription factors and In it was that to the HDAC2 and T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (31) Google Scholar). is that the region from to is between and human HDAC2, whereas the region from to two acid However, HDAC2 with are from the region the phosphorylation These results that not to the HDAC2 region the phosphorylated is that the phosphorylation of HDAC2 a protein that the to phosphorylated HDAC2 but not unmodified HDAC2 was cross-linked to DNA with cisplatin or formaldehyde. and S. PubMed Scopus Google reported that the of histones and DNA by formaldehyde was the of DNA to the DNA-binding protein and was not that the of formaldehyde was Our results that and DNA associated with complexes like Sin3 or NuRD phosphorylated However, chromatin associated with the more abundant unmodified form of HDAC2 not be the of most ChIP results be as a of chromatin with unmodified Our results show that unmodified HDAC2 is associated with chromatin. the of or with and we that unmodified HDAC2 is associated with coding of transcribed genes, to the of dynamic acetylation in has been that the use of cross-linkers as and formaldehyde is more formaldehyde to proteins associated with DNA Chen S.L. PubMed Scopus Google Scholar). The of the unmodified HDAC2 associated with chromatin to be results show that the linker DNA is required for HDAC2 to associate with chromatin. Further, the majority of HDAC2 is associated with HDAC1 as by and by studies that two HDACs are in the of MCF-7 cells S. J.M. Davie J.R. Mol. Biol. PubMed Scopus Google Scholar). The complexes associated with phosphorylated and unmodified HDAC2 and their in transcribed are to the of yeast and T. S. J. Full Text Full Text PDF PubMed Scopus Google Scholar). In to in mammalian and cells phosphorylation of HDAC2 the in the and chromatin Our results a in which phosphorylation of HDAC2 results in with and the formation of Sin3 and NuRD HDAC1/2 complexes. The HDAC complexes phosphorylated HDAC2 are recruited by transcription factors Sp3 recruitment of the Sin3 HDAC to the The recruited HDAC2 the proteins at promoters and Dynamic histone acetylation of coding of transcribed is by HDAC complexes unmodified The phosphorylated HDAC2 corepressor complexes were associated with protein kinase is that activities of protein kinase and HDAC2 the of phosphorylated HDAC2 for the HDAC2 complexes Sin3 and We the of the for at the of Cell We Dr. Edward Seto for the gift of the FLAG-HDAC2 and for and Dr. for of the

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