The Mammalian YL1 Protein Is a Shared Subunit of the TRRAP/TIP60 Histone Acetyltransferase and SRCAP Complexes
The multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase (HAT) complex performs critical functions in a variety of cellular processes including transcriptional activation, double strand DNA break repair, and apoptosis. We previously isolated the TRRAP/TIP60 complex from HeLa cells (Cai, Y., Jin, J., Tomomori-Sato, C., Sato, S., Sorokina, I., Parmely, T. J., Conaway, R. C., and Conaway, J. W. (2003) J. Biol. Chem. 278, 42733–42736). Analysis of proteins present in preparations of the TRRAP/TIP60 complex led to the identification of several new subunits, as well as several potential subunits including the YL1 protein. Here we present evidence that the YL1 protein is a previously unrecognized subunit of the TRRAP/TIP60 HAT complex. In addition, we present evidence that YL1 is also a component of a novel mammalian multiprotein complex that includes the SNF2-related helicase SRCAP and resembles the recently described Saccharomyces cerevisiae SWR1 chromatin remodeling complex. Taken together, our findings identify the YL1 protein as a new subunit of the TRRAP/TIP60 HAT complex, and they suggest that YL1 plays multiple roles in chromatin modification and remodeling in cells. The multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase (HAT) complex performs critical functions in a variety of cellular processes including transcriptional activation, double strand DNA break repair, and apoptosis. We previously isolated the TRRAP/TIP60 complex from HeLa cells (Cai, Y., Jin, J., Tomomori-Sato, C., Sato, S., Sorokina, I., Parmely, T. J., Conaway, R. C., and Conaway, J. W. (2003) J. Biol. Chem. 278, 42733–42736). Analysis of proteins present in preparations of the TRRAP/TIP60 complex led to the identification of several new subunits, as well as several potential subunits including the YL1 protein. Here we present evidence that the YL1 protein is a previously unrecognized subunit of the TRRAP/TIP60 HAT complex. In addition, we present evidence that YL1 is also a component of a novel mammalian multiprotein complex that includes the SNF2-related helicase SRCAP and resembles the recently described Saccharomyces cerevisiae SWR1 chromatin remodeling complex. Taken together, our findings identify the YL1 protein as a new subunit of the TRRAP/TIP60 HAT complex, and they suggest that YL1 plays multiple roles in chromatin modification and remodeling in cells. The NuA4 complex is a well characterized Saccharomyces cerevisiae histone acetyltransferase (HAT) 1The abbreviations used are: HAT, histone acetyltransferase; BAF53, Brg-associated factor; CBP, CREB-binding protein; CREB, cyclic AMP response element-binding protein; DMAP1, DNA methyltransferase 1-associated protein; GAS41, glioma-amplified sequence-41; HPLC, high pressure liquid chromatography; ING, inhibitor of growth; MRG15, mortality factor on chromosome 4-related gene on chromosome 15; MRGX, mortality factor on chromosome 4-related gene on chromosome X; MRGBP, MRG-binding protein; MYST, MOZ-YBF2/SAS3-SAS2-TIP60; NuA4, nucleosome acetyltransferase of H4; SRCAP, SWI2/SNF2-related CBP activator protein; TRRAP, transformation/transcription domain-associated protein; TIP49a and TIP49b, TATA-binding protein interacting 49-kDa proteins a and b; TIP60, Tat interactive 60-kDa protein; Tricine, N-[2-hydroxyl-1,1-bis(hydroxymethyl)ethyl]glycine; ZnF/HIT1, zinc finger/histidine triad domain-containing protein 1; MudPIT, multidimensional protein identification technology. 1The abbreviations used are: HAT, histone acetyltransferase; BAF53, Brg-associated factor; CBP, CREB-binding protein; CREB, cyclic AMP response element-binding protein; DMAP1, DNA methyltransferase 1-associated protein; GAS41, glioma-amplified sequence-41; HPLC, high pressure liquid chromatography; ING, inhibitor of growth; MRG15, mortality factor on chromosome 4-related gene on chromosome 15; MRGX, mortality factor on chromosome 4-related gene on chromosome X; MRGBP, MRG-binding protein; MYST, MOZ-YBF2/SAS3-SAS2-TIP60; NuA4, nucleosome acetyltransferase of H4; SRCAP, SWI2/SNF2-related CBP activator protein; TRRAP, transformation/transcription domain-associated protein; TIP49a and TIP49b, TATA-binding protein interacting 49-kDa proteins a and b; TIP60, Tat interactive 60-kDa protein; Tricine, N-[2-hydroxyl-1,1-bis(hydroxymethyl)ethyl]glycine; ZnF/HIT1, zinc finger/histidine triad domain-containing protein 1; MudPIT, multidimensional protein identification technology. that acetylates the N-terminal tails of nucleosomal histones H2A and H4. NuA4 plays diverse roles in transcriptional regulation, double strand DNA break repair, and apoptosis (reviewed in Ref. 1.Doyon Y. Cote J. Curr. Opin. Genet. Dev. 2004; 14: 147-154Crossref PubMed Scopus (278) Google Scholar). The NuA4 complex is composed of multiple subunits including ataxia telangiectasia mutated/phosphatidyl inositol 3-kinase family member Tra1, enhancer of polycomb-like protein Epl1, Eaf2, p33ING3 tumor suppressor-related protein Yng2, actin-related protein ARP4, chromodomain-containing protein Eaf3, Yaf9, Eaf7, and Esa1, a HAT belonging to the MYST family of acetyltransferases. A NuA4-like HAT complex referred to as the TRRAP/TIP60 HAT complex has been identified in Drosophila melanogaster (2.Kusch T. Florens L. Macdonald W.H. Swanson S.K. Glaser R.L. Yates J.R. Abmayr S.M. Washburn M.P. Workman J.L. Science. 2004; 306: 2084-2087Crossref PubMed Scopus (549) Google Scholar) and mammalian cells (3.Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 4.Doyon Y. Selleck W. Lane W.S. Cote J. Mol. Cell. Biol. 2004; 24: 1884-1896Crossref PubMed Scopus (426) Google Scholar, 5.Ikura T. Ogryzko V. Gigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (859) Google Scholar). Studies by our laboratory and others have shown that the multiprotein TRRAP/TIP60 complex includes apparent orthologs of many NuA4 subunits including ataxia telangiectasia mutated/phosphatidyl inositol 3-kinase family member TRRAP (transcription/transformation domain-associated protein), enhancer of polycomb proteins EPC1 and EPC-like, Eaf2-like protein DMAP1, Yng2-like protein p33ING3, Arp4-like protein BAF53a, Eaf3-like proteins MRG15 and MRGX, Yaf9-like protein GAS41, Eaf7-like protein MRGBP, and TIP60, an Esa1-like HAT belonging to the MYST family (2.Kusch T. Florens L. Macdonald W.H. Swanson S.K. Glaser R.L. Yates J.R. Abmayr S.M. Washburn M.P. Workman J.L. Science. 2004; 306: 2084-2087Crossref PubMed Scopus (549) Google Scholar, 3.Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 4.Doyon Y. Selleck W. Lane W.S. Cote J. Mol. Cell. Biol. 2004; 24: 1884-1896Crossref PubMed Scopus (426) Google Scholar, 5.Ikura T. Ogryzko V. Gigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (859) Google Scholar). Notably, the mammalian TRRAP/TIP60 HAT complex includes an additional set of subunits not present in the S. cerevisiae NuA4 complex. Among these proteins are the SNF2-related helicase p400, the bromodomain-containing TRCp120 coactivator, and the AAA ATPases TIP49a and TIP49b (3.Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 4.Doyon Y. Selleck W. Lane W.S. Cote J. Mol. Cell. Biol. 2004; 24: 1884-1896Crossref PubMed Scopus (426) Google Scholar, 5.Ikura T. Ogryzko V. Gigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (859) Google Scholar). In this report, we identify the YL1 protein (6.Horikawa I. Tanaka H. Yuasa Y. Suzuki M. Shimizu M. Oshimura M. Exp. Cell Res. 1995; 220: 11-17Crossref PubMed Scopus (10) Google Scholar, 7.Horikawa I. Tanaka H. Yuasa Y. Suzuki M. Oshimura M. Biochem. Biophys. Res. Commun. 1995; 208: 999-1007Crossref PubMed Scopus (17) Google Scholar) as a subunit of the mammalian TRRAP/TIP60 HAT complex. In addition, we demonstrate that the YL1 protein is also a subunit of a novel complex that contains the SNF2-related helicase SRCAP (SWI2/SNF2-related CBP activator protein) (8.Johnston H. Kneer J. Chackalaparampil I. Yaciuk P. Chrivia J. J. Biol. Chem. 1999; 274: 16370-16376Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 9.Monroy M.A. Ruhl D.D. Xu X. Granner D.K. Yaciuk P. Chrivia J.C. J. Biol. Chem. 2001; 276: 40721-40726Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 10.Monroy M.A. Schott N.M. Cox L. Chen J.D. Ruh M. Chrivia J.C. Mol. Endocrinol. 2003; 17: 2519-2528Crossref PubMed Scopus (35) Google Scholar) and resembles the recently described S. cerevisiae SWR1 chromatin remodeling complex, which catalyzes incorporation of the histone variant Htz1 (H2AZ) into nucleosomes (11.Kobor M.S. Venkatasubrahmanyam S. Meneghini M.D. Gin J.W. Jennings J.L. Link A.J. Madhani H.D. Rine J. PLoS Biol. 2004; 2: E131Crossref PubMed Scopus (459) Google Scholar, 12.Krogan N.J. Baetz K. Keogh M.C. Datta N. Sawa C. Kwok T.C. Thompson N.J. Davey M.G. Pootoolal J. Hughes T.R. Emili A. Buratowski S. Hieter P. Greenblatt J.F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13513-13518Crossref PubMed Scopus (195) Google Scholar, 13.Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (975) Google Scholar). Materials—Anti-FLAG (M2)-agarose, anti-FLAG (M2) monoclonal antibodies, and anti-FLAG peptide were purchased from Sigma. Anti-TRRAP (T-17) antibodies were obtained from Santa Cruz Biotechnology. Anti-TIP60 antibodies were from Upstate-Cell Signaling Solutions. Anti-DMAP1 antibodies were purchased from Affinity BioReagents. Anti-MRGBP, anti-TIP49a, anti-TIP49b, anti-YL1, anti-TRCp120, and anti-BAF53a rabbit polyclonal antibodies were raised against recombinant proteins expressed in Escherichia coli (Cocalico Biologicals, Inc.). Anti-p400 antibodies were from Abcam, Inc. Tissue Culture—cDNAs encoding N-terminally FLAG-tagged human MRGBP, YL1, and ZnF/HIT1 were subcloned into pcDNA3.1 and stably transfected into HeLa S3 cells. Parental and stably transformed HeLa cells were maintained in Dulbecco's modified Eagle's medium with 5% glucose and 10% fetal bovine serum. For large scale cultures, HeLa cells were grown in spinner culture in Joklik medium with 5% calf serum. N-terminally FLAG-tagged human H2AZ was subcloned into pcDNA5/FRT and introduced into 293/FRT cells using the Invitrogen Flp-in System. Parental and stably transformed 293/FRT cells were grown in Dulbecco's modified Eagle's medium with 5% glucose and 10% fetal bovine serum. Anti-FLAG-agarose Chromatography—Nuclear extracts were prepared according to the method of Dignam et al. (14.Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar). Nuclear extracts were adjusted to 0.3 m KCl and 0.5% Triton X-100 and centrifuged at 40,000 rpm for 30 min at 4 °C in a Ti-45 rotor. Following centrifugation, the supernatants were mixed with anti-FLAG (M2)-agarose beads in a ratio of 100 μl of packed beads/6 ml of nuclear extract and gently rocked for 4 h at 4 °C. The beads were washed 3 times with an ∼50-fold excess of buffer containing 50 mm Hepes-NaOH (pH 7.9), 0.25 m KCl, 0.1% Triton X-100, and 10% (v/v) glycerol and then eluted with 100 μl of the same buffer containing 0.1 m KCl and 0.2 mg/ml FLAG peptide. Fractionation of YL1-containing Species by Phosphocellulose Chromatography—Approximately 35 ml of nuclear extract prepared according to the method of Dignam et al. (14.Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9132) Google Scholar) from HeLa cells stably expressing FLAG-YL1 were dialyzed against Buffer A (40 mm Hepes-NaOH (pH 7.9), 0.1 mm EDTA, 1 mm dithiothreitol, and 10% (v/v) glycerol) containing 1 mm phenylmethylsulfonyl fluoride to a conductivity equivalent to that of Buffer A containing 0.1 m KCl. Following centrifugation for 30 min at 40,000 rpm in a Ti-45 rotor, the resulting supernatant, which contained ∼200 mg of protein, was applied at 2 packed column volumes/h to a phosphocellulose column (∼10 mg of protein/ml packed column in Buffer A containing 0.1 m KCl. The column was washed at the same with Buffer A containing 0.1 m KCl, and proteins that the column at 0.1 m KCl were proteins were eluted at the same with Buffer A containing and m KCl. column were and from were of the Phosphocellulose 0.1 m KCl The phosphocellulose 0.1 m KCl, mg of protein) was centrifuged at for min at 4 °C. a HPLC, the resulting was applied at 1 to a column in Buffer mm Hepes-NaOH (pH 7.9), mm EDTA, 1 mm dithiothreitol, and 10% (v/v) glycerol) containing m KCl. The column was washed at the same with Buffer containing m KCl and then eluted at 1 with a from to m KCl in Buffer were HAT acetyltransferase were as described A. S. Workman J.L. 2004; Scopus Google Scholar). containing 50 mm (pH 50 mm KCl, 0.1 mm EDTA, 1 mm dithiothreitol, 5% (v/v) 1 mm phenylmethylsulfonyl mm of A 3 of prepared as described T. S. Cote J. Workman J.L. Mol. Biol. 1999; Google and prepared from FLAG-YL1 expressing HeLa cells were at 30 °C. 30 of were to were by and proteins were by using of proteins was using a modification of the multidimensional protein identification described by Washburn et al. M.P. Yates J.R. 2001; PubMed Scopus Google Scholar). proteins were in 30 μl of 100 mm m were with mm with mm and with for at h at °C. were then in 100 mm (pH and at °C with modified in the of were a column W.H. R. T.J. Yates J.R. J. Scopus Google Scholar) of a packed with of by 4 of by 2 of in 5% 0.1% column was then with a was used to the from 0.1 to were were Buffer Buffer 0.1% and Buffer m 5% 0.1% In of are eluted from the to the by in Buffer by in Buffer The in a high with Buffer by the The of a the into a with a Full were on the a by in a on the and from the functions and were by the The R.G. J. A. H. Yates J.R. J. Res. PubMed Scopus Google Scholar) was used to and to of Yates J.R. J. PubMed Scopus Google Scholar) was used to to in a containing human protein from on The of was using the and in were they a in of at and of for for and for In addition, the to at W.H. Yates J.R. J. Res. PubMed Scopus Google Scholar) was used to and this from multiple were using W.H. Yates J.R. J. Res. PubMed Scopus Google Scholar). The YL1 a of the TRRAP/TIP60 HAT previously described of the multiprotein TRRAP/TIP60 HAT complex from HeLa nuclear extracts (3.Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). of the human TRRAP/TIP60 complex led to the identification of the DMAP1, MRG15, MRGX, and proteins as new subunits of the complex. In addition, of proteins present in our preparations of the TRRAP/TIP60 complex by identified the YL1 protein as a potential subunit of the complex. The YL1 gene was isolated by as a gene of the of cells (6.Horikawa I. Tanaka H. Yuasa Y. Suzuki M. Shimizu M. Oshimura M. Exp. Cell Res. 1995; 220: 11-17Crossref PubMed Scopus (10) Google Scholar, 7.Horikawa I. Tanaka H. Yuasa Y. Suzuki M. Oshimura M. Biochem. Biophys. Res. Commun. 1995; 208: 999-1007Crossref PubMed Scopus (17) Google Scholar). The human YL1 gene a protein that to from to The A. C. S. J.D. C. T. R. V. S. Wang Y. Nucleic Acids Res. 2003; PubMed Scopus Google Scholar) identified the human YL1 protein as a potential of the S. cerevisiae protein. Notably, the protein was recently identified as a subunit of the S. cerevisiae SWR1 chromatin remodeling complex, which catalyzes incorporation of histone variant Htz1 (H2AZ) into nucleosomes (11.Kobor M.S. Venkatasubrahmanyam S. Meneghini M.D. Gin J.W. Jennings J.L. Link A.J. Madhani H.D. Rine J. PLoS Biol. 2004; 2: E131Crossref PubMed Scopus (459) Google Scholar, 12.Krogan N.J. Baetz K. Keogh M.C. Datta N. Sawa C. Kwok T.C. Thompson N.J. Davey M.G. Pootoolal J. Hughes T.R. Emili A. Buratowski S. Hieter P. Greenblatt J.F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13513-13518Crossref PubMed Scopus (195) Google Scholar, 13.Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (975) Google Scholar). to the that the YL1 protein is a subunit of the TRRAP/TIP60 HAT complex, we a HeLa stably expressing YL1 with an N-terminal FLAG and YL1 and proteins by We then the nucleosomal HAT of from HeLa cells stably expressing FLAG-YL1 to that of the TRRAP/TIP60 HAT complex, which been by from nuclear extracts of HeLa cells stably expressing the TRRAP/TIP60 subunit with an N-terminal FLAG (3.Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). shown in from and HeLa cells HAT that nucleosomal histones H2A and with the MRGBP, YL1 is a subunit of the TRRAP/TIP60 complex. with this of that from FLAG-YL1 expressing HeLa cells to a set of proteins to in preparations of in the protein of the preparations were apparent 2 and FLAG-YL1 contained of of to MRGBP, MRGX, and MRG15, with our that these proteins are as of the TRRAP/TIP60 complex and as of and and MRG15, (3.Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In addition, FLAG-YL1 contained a of a of to TRCp120 (3.Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). YL1 also to with a of in the of the TRRAP/TIP60 complex and from we of M.P. Yates J.R. 2001; PubMed Scopus Google Scholar, Washburn M.P. Yates J.R. Chem. 2001; PubMed Scopus Google Scholar). is a method for proteins present in complex In MudPIT, a of proteins is into which are then by and and by Analysis by of proteins present in TRRAP/TIP60 by from HeLa cells identified the YL1 protein 2 and as well as of the previously described subunits of the TRRAP/TIP60 complex (3.Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 4.Doyon Y. Selleck W. Lane W.S. Cote J. Mol. Cell. Biol. 2004; 24: 1884-1896Crossref PubMed Scopus (426) Google Scholar, 5.Ikura T. Ogryzko V. Gigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (859) Google Scholar). The of the YL1 protein in TRRAP/TIP60 from HeLa cells was by an of proteins present in from HeLa cells by 2 and and by identified as proteins subunits of the mammalian TRRAP/TIP60 complex. with the of the HAT of these that the YL1 protein is a subunit of the TRRAP/TIP60 of proteins with and by Anti-FLAG-agarose of nuclear extracts from and cells was as described of were by in and proteins were identified by with antibodies that the proteins YL1 also with an the S. cerevisiae SWR1 from the of the of of from HeLa cells identified several proteins not in the TRRAP/TIP60 complex, the that the YL1 protein is present in cells not as a subunit of the TRRAP/TIP60 complex also as a component of additional Among the additional proteins with YL1 were the SNF2-related helicase SRCAP, the actin-related protein and a previously protein by an described in the protein as ZnF/HIT1 SRCAP is a member of the family of SRCAP was identified by to with of the transcriptional and histone acetyltransferase CBP in a (8.Johnston H. Kneer J. Chackalaparampil I. Yaciuk P. Chrivia J. J. Biol. Chem. 1999; 274: 16370-16376Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). SRCAP was to as a for nuclear and to with CBP in this M.A. Ruhl D.D. Xu X. Granner D.K. Yaciuk P. Chrivia J.C. J. Biol. Chem. 2001; 276: 40721-40726Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 10.Monroy M.A. Schott N.M. Cox L. Chen J.D. Ruh M. Chrivia J.C. Mol. Endocrinol. 2003; 17: 2519-2528Crossref PubMed Scopus (35) Google Scholar). SRCAP with the helicase subunit of the TRRAP/TIP60 complex by et al. (11.Kobor M.S. Venkatasubrahmanyam S. Meneghini M.D. Gin J.W. Jennings J.L. Link A.J. Madhani H.D. Rine J. PLoS Biol. 2004; 2: E131Crossref PubMed Scopus (459) Google SRCAP and are to the SNF2-related helicase subunit of the S. cerevisiae SWR1 complex. The SWR1 complex includes potential orthologs of a of TRRAP/TIP60 subunits including DMAP1, TIP49b, BAF53a, and In addition, the SWR1 complex includes the the potential YL1 and which in zinc a with the of the ZnF/HIT1 protein Taken together, these findings that the YL1 protein a subunit of the TRRAP/TIP60 HAT complex and a complex that resembles the S. cerevisiae SWR1 chromatin remodeling complex. this we to nuclear extracts prepared from HeLa cells stably expressing FLAG-YL1 to the TRRAP/TIP60 complex from potential YL1-containing that present in these Nuclear extracts from HeLa cells were applied to a phosphocellulose column at 0.1 m KCl and eluted with and m KCl. shown in FLAG-YL1 was by in the 0.1 m KCl from as well as in that eluted with and m KCl. In the HAT subunit of the TRRAP/TIP60 complex was by in the 0.3 m KCl phosphocellulose identify proteins in the phosphocellulose was to and were by In from the phosphocellulose 0.3 m KCl we from the same set of proteins in from nuclear extracts of cells. In we from a of these proteins in anti-FLAG from of the with the that YL1 is a component of multiple proteins in the phosphocellulose 0.1 m KCl SRCAP, DMAP1, and BAF53a, and in this we to from the subunit of the TRRAP/TIP60 complex and to from the TRRAP, and EPC-like, TIP60, MRG15, MRGX, MRGBP, and subunits of the complex. The that the YL1 protein is present in this in a that is from TRRAP/TIP60 complex and that resembles the recently identified S. cerevisiae SWR1 complex. this the phosphocellulose was to and Notably, YL1 eluted from the column in a which was then to of proteins present in prepared from identified the SRCAP, YL1, DMAP1, BAF53a, TIP49b, GAS41, and proteins column and that the YL1 protein is a component not of the TRRAP/TIP60 HAT complex also of a multiprotein complex containing the SRCAP protein. The ZnF/HIT1 with the SRCAP of to the subunit of the S. cerevisiae SWR1 complex, we to the ZnF/HIT1 protein is a subunit of the SRCAP complex. we a HeLa stably expressing the ZnF/HIT1 protein with an N-terminal FLAG Nuclear extracts prepared from HeLa cells stably expressing were to shown in the of from HeLa cells to a of proteins in from and cells. of proteins present in from HeLa cells by 2 and and by in to the ZnF/HIT1 protein, the SRCAP, DMAP1, BAF53a, TIP49b, GAS41, and YL1 that ZnF/HIT1 is a subunit of the SRCAP complex. In addition, we that subunits of the TRRAP/TIP60 complex were by in from HeLa for the that the YL1 protein is present in in the TRRAP/TIP60 HAT complex and an complex the S. cerevisiae SWR1 complex. The SRCAP and to the H2A our findings that human YL1 protein is a subunit of at multiprotein the TRRAP/TIP60 HAT complex and an complex in subunit to the S. cerevisiae SWR1 complex. In the SWR1 complex is of and to the histone H2A variant Htz1 and of incorporation into in to from H2AZ the mammalian of were by in containing the SRCAP complex. In to from H2AZ were in the SRCAP complex, as from HeLa cells. suggest the S. cerevisiae SWR1 complex, the human SRCAP complex this we a human stably expressing human histone variant H2AZ with an N-terminal FLAG and we proteins by shown in the of 4 and from cells to a set of proteins to in containing the SRCAP complex from cells. of proteins present in from cells by 2 and and by the SRCAP, DMAP1, BAF53a, TIP49b, GAS41, ZnF/HIT1, and YL1 that the SRCAP complex stably to histone variant In addition, we that subunits of the TRRAP/TIP60 complex were by in from cells. the S. cerevisiae SWR1 complex, the SRCAP complex to to histone variant and in this we present evidence that the YL1 protein is a subunit of the TRRAP/TIP60 HAT complex. In addition, we present evidence that the YL1 protein is also present in cells as a subunit of the previously mammalian SRCAP complex, which a to the recently described S. cerevisiae SWR1 chromatin remodeling complex and is composed of the SNF2-related SRCAP ZnF/HIT1, and a of proteins including YL1, DMAP1, TIP49b, BAF53a, and GAS41, which are also subunits of the TRRAP/TIP60 HAT complex. are with the findings of et al. Y. Selleck W. Lane W.S. Cote J. Mol. Cell. Biol. 2004; 24: 1884-1896Crossref PubMed Scopus (426) Google recently that is present in cells as a subunit in additional that are from the TRRAP/TIP60 complex and SRCAP and we the mammalian SRCAP complex has a subunit to that of the S. cerevisiae SWR1 complex and histone variant we have not in SRCAP incorporation of H2AZ into in the of the TRRAP/TIP60 complex. we that HeLa nuclear extracts incorporation of H2AZ into nucleosomal in not we have to our preparations of the SRCAP complex incorporation of H2AZ into nucleosomes in is the S. cerevisiae SWR1 complex, the SRCAP complex is not for H2AZ in mammalian cells as that the SRCAP complex in H2AZ in mammalian cells from as present in nuclear with