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• W2170354958 abstract "HDAC1 is a member of the histone deacetylase family, which plays an important role in modulating the eukaryotic chromatin structure. Numerous studies have demonstrated its involvement in transcription and in tumorigenesis. To better understand the functions and regulation of HDAC1, a yeast two-hybrid screening approach was chosen to identify novel interactions involving HDAC1. Human HDAC1 was found to interact specifically in yeast, mammalian cells, and in vitro with the human Hus1 gene product, whose Schizosaccharomyces pombe homolog has been implicated in G2/M checkpoint control. Both HDAC1 and Hus1 proteins localize to the nuclei. Furthermore, HDAC1 and Hus1 were found to exist in a complex with Rad9, a known Hus1-interacting factor. In addition, bioinformatics analysis of the protein sequences of Hus1, Rad1, and Rad9, three checkpoint Rad proteins that form a complex, revealed that they all contain a putative proliferating cell nuclear antigen (PCNA) fold, raising the possibility that these factors may bind to DNA in a PCNA-like ring structure. The results reported in this study strongly suggest a novel pathway involving HDAC1 in G2/M checkpoint control through the interaction with a functional Rad complex that may utilize a PCNA-like structure. Therefore, physically and functionally similar apparatus may function during G2/M checkpoint and DNA replication. HDAC1 is a member of the histone deacetylase family, which plays an important role in modulating the eukaryotic chromatin structure. Numerous studies have demonstrated its involvement in transcription and in tumorigenesis. To better understand the functions and regulation of HDAC1, a yeast two-hybrid screening approach was chosen to identify novel interactions involving HDAC1. Human HDAC1 was found to interact specifically in yeast, mammalian cells, and in vitro with the human Hus1 gene product, whose Schizosaccharomyces pombe homolog has been implicated in G2/M checkpoint control. Both HDAC1 and Hus1 proteins localize to the nuclei. Furthermore, HDAC1 and Hus1 were found to exist in a complex with Rad9, a known Hus1-interacting factor. In addition, bioinformatics analysis of the protein sequences of Hus1, Rad1, and Rad9, three checkpoint Rad proteins that form a complex, revealed that they all contain a putative proliferating cell nuclear antigen (PCNA) fold, raising the possibility that these factors may bind to DNA in a PCNA-like ring structure. The results reported in this study strongly suggest a novel pathway involving HDAC1 in G2/M checkpoint control through the interaction with a functional Rad complex that may utilize a PCNA-like structure. Therefore, physically and functionally similar apparatus may function during G2/M checkpoint and DNA replication. histone deacetylase(s) glutathione S-transferase proliferating cell nuclear antigen polymerase chain reaction hemagglutinin green fluorescent protein phosphate-buffered saline amino acids germ cell-deficient Gal4 DNA binding domain Gal4 transcription activation domain Histone deacetylases (HDACs)1 play an important role in modulating chromatin structure. Several HDACs have been discovered in mammalian cells (1Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1530) Google Scholar, 2Emiliani S. Fischle W. Van Lint C. Al-Abed Y. Verdin E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2795-2800Crossref PubMed Scopus (277) Google Scholar, 3Verdel A. Khochbin S. J. Biol. Chem. 1999; 274: 2440-2445Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 4Grozinger C.M. Hassig C.A. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4868-4873Crossref PubMed Scopus (652) Google Scholar, 5Fischle W. Emiliani S. Hendzel M.J. Nagase T. Nomura N. Voelter W. Verdin E. J. Biol. Chem. 1999; 274: 11713-11720Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, 6Yang W.-M. Inouye C. Zeng Y.Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12845-12850Crossref PubMed Scopus (482) Google Scholar). They can be divided further into two classes. Class I contains HDAC1, 2, and 3, which have a single deacetylase domain at the N termini and diversified C-terminal regions (1Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1530) Google Scholar, 2Emiliani S. Fischle W. Van Lint C. Al-Abed Y. Verdin E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2795-2800Crossref PubMed Scopus (277) Google Scholar, 6Yang W.-M. Inouye C. Zeng Y.Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12845-12850Crossref PubMed Scopus (482) Google Scholar). Class II includes recently identified HDAC4, 5, and 6, with a deacetylase domain at a more C-terminal position (3Verdel A. Khochbin S. J. Biol. Chem. 1999; 274: 2440-2445Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 4Grozinger C.M. Hassig C.A. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4868-4873Crossref PubMed Scopus (652) Google Scholar, 5Fischle W. Emiliani S. Hendzel M.J. Nagase T. Nomura N. Voelter W. Verdin E. J. Biol. Chem. 1999; 274: 11713-11720Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). In addition, HDAC6 contains a second N-terminal deacetylase domain (3Verdel A. Khochbin S. J. Biol. Chem. 1999; 274: 2440-2445Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 4Grozinger C.M. Hassig C.A. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4868-4873Crossref PubMed Scopus (652) Google Scholar), which can function independently of its C-terminal counterpart (4Grozinger C.M. Hassig C.A. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4868-4873Crossref PubMed Scopus (652) Google Scholar). Identified and cloned first (1Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1530) Google Scholar), HDAC1 is also the most extensively studied. It is involved in transcription regulation as an essential component of cofactor complexes that mediate transcription repression by cellular factors such as nonliganded hormone receptors (7Heinzel T. Lavinsky R.M. Mullen T.M. Soderstrom M. Laherty C.D. Torchia J. Yang W.M. Brard G. Ngo S.D. Davie J.R. Seto E. Eisenman R.N. Rose D.W. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 43-48Crossref PubMed Scopus (1082) Google Scholar, 8Nagy L. Kao H.Y. Chakravarti D. Lin R.J. Hassig C.A. Ayer D.E. Schreiber S.L. Evans R.M. Cell. 1997; 89: 373-380Abstract Full Text Full Text PDF PubMed Scopus (1104) Google Scholar), MeCP2, a repressor that binds to methylated DNA (9Nan X. Ng H.H. Johnson C.A. Laherty C.D. Turner B.M. Eisenman R.N. Bird A. Nature. 1998; 393: 386-389Crossref PubMed Scopus (2770) Google Scholar, 10Jones P.L. Veenstra G.J. Wade P.A. Vermaak D. Kass S.U. Landsberger N. Strouboulis J. Wolffe A.P. Nat. Genet. 1998; 19: 187-191Crossref PubMed Scopus (2237) Google Scholar) and the Max-Mad complex (11Sommer A. Hilfenhaus S. Menkel A. Kremmer E. Seiser C. Loidl P. Luscher B. Curr. Biol. 1997; 7: 357-365Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). HDAC1 also interacts directly with other transcription repressors, including all three of the pocket proteins, Rb, p107 and p130 (12Ferreira R. Magnaghi-Jaulin L. Robin P. Harel-Bellan A. Trouche D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10493-10498Crossref PubMed Scopus (224) Google Scholar, 13Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (838) Google Scholar) and YY1 (14Yang W.M. Yao Y.L. Sun J.M. Davie J.R. Seto E. J. Biol. Chem. 1997; 272: 28001-28007Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). It is generally believed that sequence-specific repressors bind to DNA and target HDAC1 to promoter regions through various protein-protein interactions. HDAC1 is thought to achieve transcription repression by locally deacetylating histones, leading to a compact nucleosomal structure that prevents transcription factors from accessing DNA to promote transcription. HDAC activity has also been implicated in cell cycle regulation and cancer development. Transcription repression of E2F-responsive genes by Rb or Rb-like pocket proteins has been at least partially attributed to HDAC1 (13Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (838) Google Scholar, 15Brehm A. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. Nature. 1998; 391: 597-601Crossref PubMed Scopus (1075) Google Scholar, 16Magnaghi-Jaulin L. Groisman R. Naguibneva I. Robin P. Lorain S. Le Villain J.P. Troalen F. Trouche D. Harel-Bellan A. Nature. 1998; 391: 601-605Crossref PubMed Scopus (803) Google Scholar). The promoter for p21, a cyclin-dependent kinase inhibitor, is activated when cellular HDAC activity is inhibited (17Xiao H. Hasegawa T. Miyaishi O. Ohkusu K. Isobe K. Biochem. Biophys. Res. Commun. 1997; 237: 457-460Crossref PubMed Scopus (99) Google Scholar, 18Sowa Y. Orita T. Minamikawa S. Nakano K. Mizuno T. Nomura H. Sakai T. Biochem. Biophys. Res. Commun. 1997; 241: 142-150Crossref PubMed Scopus (287) Google Scholar, 19Sambucetti L.C. Fischer D.D. Zabludoff S. Kwon P.O. Chamberlin H. Trogani N. Xu H. Cohen D. J. Biol. Chem. 1999; 274: 34940-34947Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). This activation is independent of p53, a known activator of p21 gene transcription. Furthermore, evidence exists to indicate the role of HDAC1 in the development of acute promyelocytic leukemia (20Lin R.J. Nagy L. Inoue S. Shao W. Miller Jr., W.H. Evans R.M. Nature. 1998; 391: 811-814Crossref PubMed Scopus (978) Google Scholar, 21Grignani F. De Matteis S. Nervi C. Tomassoni L. Gelmetti V. Cioce M. Fanelli M. Ruthardt M. Ferrara F.F. Zamir I. Seiser C. Grignani F. Lazar M.A. Minucci S. Pelicci P.G. Nature. 1998; 391: 815-818Crossref PubMed Scopus (939) Google Scholar, 22He L.Z. Guidez F. Tribioli C. Peruzzi D. Ruthardt M. Zelent A. Pandolfi P.P. Nat. Genet. 1998; 18: 126-135Crossref PubMed Scopus (506) Google Scholar). These results suggest a role for HDAC activity in cell cycle regulation and cancer development. Almost all interactions involving HDAC1 known so far were discovered during studies of its interacting factors. In the present study, we asked which novel interactions involving HDAC1 could be identified which might regulate HDAC1 functions. To achieve this goal, we adopted a yeast two-hybrid screening protocol, using a portion of human HDAC1 (amino acids 53–285 out of a total of 482 amino acids) as the bait and a HeLa cDNA library. This resulted in the identification of several clones that contain the full-length cDNA of the human Hus1(hHus1) gene. In addition to the specific interaction observed between HDAC1 and hHus1 in yeast, the proteins interacted specifically in an in vitro GST pull-down assay and in mammalian cells. We also observed that like HDAC1, hHus1 was localized to the nuclei. These results suggested that hHus1 interacted with HDAC1. This interaction has not been reported previously. The Hus1 protein of Schizosaccharomyces pombe is a member of the cell cycle checkpoint Rad proteins that are involved in the mitotic checkpoint induced by either DNA damage or DNA replication block (23Kostrub C.F. al-Khodairy F. Ghazizadeh H. Carr A.M. Enoch T. Mol. Gen. Genet. 1997; 254: 389-399PubMed Google Scholar, 24Kostrub C.F. Knudsen K. Subramani S. Enoch T. EMBO J. 1998; 17: 2055-2066Crossref PubMed Scopus (100) Google Scholar). Although it is not an essential gene for normal growth, the loss of functional hus1 gene leads to cell mortality when DNA damage or replication blocks are induced in S. pombe. The same phenotype is observed when the other checkpointrad genes are mutated (23Kostrub C.F. al-Khodairy F. Ghazizadeh H. Carr A.M. Enoch T. Mol. Gen. Genet. 1997; 254: 389-399PubMed Google Scholar). It is believed that therad gene products are responsible for relaying a signal from damaged DNA and/or blocked DNA replication to the cell cycle regulatory machinery to induce cell cycle arrest and to prevent premature cell division. The exact molecular mechanism of this signaling pathway remains unclear. The immediate downstream effectors of therad genes have been postulated to be the chk andcds1 gene products. Both are protein kinases capable of phosphorylating and deactivating a phosphatase, cdc25p; cdc25 activity is required to activate cdc2p, a cyclin-dependent kinase, which controls cell cycle progress through mitosis. By deactivating cdc25 protein, cdc2p is maintained in its phosphorylated state, and mitosis is not initiated (for review, see Ref. 25Russell P. Trends Biochem. Sci. 1998; 23: 399-402Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The S. pombe Rad proteins discovered so far include Rad1p, Rad3p, Rad9p, Rad17p, Rad26p, and Hus1p. Human homologs have been identified for all of these genes except for rad26 (24Kostrub C.F. Knudsen K. Subramani S. Enoch T. EMBO J. 1998; 17: 2055-2066Crossref PubMed Scopus (100) Google Scholar,26Cimprich K.A. Shin T.B. Keith C.T. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2850-2855Crossref PubMed Scopus (226) Google Scholar, 27Lieberman H.B. Hopkins K.M. Nass M. Demetrick D. Davey S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13890-13895Crossref PubMed Scopus (107) Google Scholar, 28Parker A.E. Van de Weyer I. Laus M.C. Oostveen I. Yon J. Verhasselt P. Luyten W. J. Biol. Chem. 1998; 273: 18332-18339Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 29Udell C.M. Lee S.K. Davey S. Nucleic Acids Res. 1998; 26: 3971-3976Crossref PubMed Scopus (35) Google Scholar). Among them, Rad3p was found to be a protein kinase, whose human homologs include ATM (30Savitsky K. Bar-Shira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenzai M. Pecker I. Frydman M. Harnik R. Patanjali S. Simmons A. Clines G. Sartiel A. Gatti R. Chessa L. Sanal O. Lavin M. Jaspers N. Taylor A. Arlett C. Miki T. Weissmasn S. Lovett M. Collins F. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2370) Google Scholar) and ATR (26Cimprich K.A. Shin T.B. Keith C.T. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2850-2855Crossref PubMed Scopus (226) Google Scholar). Mutations in theATM gene, which is responsible for the severe congenital disease ataxia-telagiectasia (30Savitsky K. Bar-Shira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenzai M. Pecker I. Frydman M. Harnik R. Patanjali S. Simmons A. Clines G. Sartiel A. Gatti R. Chessa L. Sanal O. Lavin M. Jaspers N. Taylor A. Arlett C. Miki T. Weissmasn S. Lovett M. Collins F. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2370) Google Scholar), render cells unable to maintain proper checkpoints upon DNA damage (31Lavin M.F. Shiloh Y. Annu. Rev. Immunol. 1997; 15: 177-202Crossref PubMed Scopus (539) Google Scholar). Human ATR has also been shown to be involved in the G2/M DNA damage checkpoint (32Cliby W.A. Roberts C.J. Cimprich K.A. Stringer C.M. Lamb J.R. Schreiber S.L. Friend S.H. EMBO J. 1998; 17: 159-169Crossref PubMed Scopus (483) Google Scholar). The Rad17 protein has extensive homology with the so-called clamp loaders, such as human RF-C factor (33Griffiths D.J. Barbet N.C. McCready S. Lehmann A.R. Carr A.M. EMBO J. 1995; 14: 5812-5823Crossref PubMed Scopus (179) Google Scholar), known to facilitate the loading of the proliferating cell nucleus antigen (PCNA) onto DNA (34Mossi R. Hubscher U. Eur. J. Biochem. 1998; 254: 209-216PubMed Google Scholar). PCNA trimers are assembled into a ring-like configuration that clamp and slide along the DNA double-helix and are thought to help tether DNA polymerase to DNA. Interestingly, sequence analysis revealed that yeast Rad1 and Hus1 protein and their homologs in higher eukaryotic organisms may all contain the so-called PCNA fold (43Aravind L. Walker D.R. Koonin E.V. Nucleic Acids Res. 1999; 27: 1223-1242Crossref PubMed Scopus (484) Google Scholar, 44Thelen M.P. Venclovas C. Fidelis K. Cell. 1999; 96: 769-770Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and Rad1p, Hus1p, and Rad9p form a DNA damage-responsive complex (24Kostrub C.F. Knudsen K. Subramani S. Enoch T. EMBO J. 1998; 17: 2055-2066Crossref PubMed Scopus (100) Google Scholar, 35Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 36St. Onge R.P. Udell C.M. Casselman R. Davey S. Mol. Biol. Cell. 1999; 10: 1985-1995Crossref PubMed Scopus (129) Google Scholar). Furthermore, based on similarity searches, we suggest in this study that the Rad9 proteins may also contain the PCNA domain, and we demonstrate that HDAC1 could form a complex with Rad9, probably via Hus1. In summary, we report in this study findings suggesting a novel pathway involving HDAC1 in G2/M checkpoint control through the interaction with a functional Rad complex that may utilize a PCNA-like structure. Therefore, physically and functionally similar apparatus may function during G2/M checkpoint and DNA replication. The region of HDAC1 between amino acids 53 and 285 was PCR cloned using the primers 5′-GAAAAATGGAATTCTATCGC-3′ and 5′-GACAAAGTCGACACACTTGG-3′. The amplified fragment was digested withEcoRI/SalI and cloned into pAS2-1 (CLONTECH, Palo Alto, CA) digested withEcoRI/SalI to make our bait construct pAS2-HDAC1-53-285. pHM6-hHus1 expresses hHus1 with an N-terminally fused HA epitope tag. hHus1 cDNA was PCR amplified from the original yeast two-hybrid construct YYN0048 (see below) with GAL4AD LD primers (CLONTECH). The product was cloned into pCR2.1 (Invitrogen, Carlsbad, CA) resulting in pCR2.1-hHus1. TheEcoRI fragment from pCR2.1-hHus1 was cloned into CH2 vector (from the Department of Oncology, Novartis, Summit, NJ) digested withEcoRI to generate CH2-hHus1, from which theKpnI/NotI fragment containing hHus1 cDNA was cloned into pHM6 (Roche Molecular Biochemicals, Basel, Switzerland) digested with KpnI/NotI. pcDNA3-hHus1-HA expresses hHus1 with a C-terminally fused HA epitope tag. It was cloned by inserting into the vector pcDNA3.1 (Invitrogen) the PCR product using pCR2.1-hHus1 as the template and two oligonucleotides, 5′-GCCACCATGAAGTTTCGGGCCAAGATCG-3′ and 5′-GAATTCTTATGCATAGTCTGGAACGTCATATGGATACCCGGACAGTGCAGGGATGAAATACTGAAG-3′. pGEX-2TK-hHus1 is a bacterial construct that, upon isopropyl-1-thio-β-d-galactopyranoside induction, expresses GST-hHus1 fusion protein. It was cloned by inserting theEcoRI/NotI fragment from CH2-hHus1 into pGEX-2TK (Promega, Madison, WI) digested with SmaI. pcDNA3-HDAC1 contains the human HDAC1 cDNA in the pcDNA3.1 vector (a gift from Dr. Sambucetti, Department of Oncology, Novartis). pHA-1–179, pHA-179-482, pHA-241–363, and pHA-364–482 express fusion proteins between HA epitope tag and regions of HDAC1 from amino acids 1–179, 179-482, 241–363, and 364–482, respectively. To clone pHA-1–179, the BamHI/NcoI fragment of pcDNA3-HDAC1 was inserted into KpnI-digested CH2. pHA-179-482 was cloned by inserting the region C-terminal to theNcoI site in HDAC1 cDNA into EcoRI-digested CH2. To clone pHA-241–363, the region between HDAC1 amino acids 241 and 363 was PCR amplified using primers 5′-TTCAAGCCGGTCATGTCCAAAG-3′ and 5′-TTTGATCTTCTCCAGGTACTC-3′. The product was cloned into pCR2.1 to generate pTA-241–363, whose EcoRI fragment was cloned intoNotI-digested pHM6. To clone pHA-364–482, the region between HDAC1 amino acids 364 and 482 was PCR amplified using primers 5′-CAGCGACTGTTTGAGAACCTTAG-3′ and 5′-GCTGGAGAGGTCCATTCAGGC-3′. The product was cloned into pCR2.1 to generate pTA-364–482, whose EcoRI fragment was cloned intoNotI-digested pHM6. pEGFP-hHus1 expresses a fusion protein between green fluorescent protein (GFP) and hHus1, with GFP at the N terminus of the fusion protein. To clone it, theEcoRI/ApaI fragment from pHM6-hHus1 was inserted into pEGFP-C3 (CLONTECH) digested withEcoRI and ApaI. The bait used is described above. A HeLa cDNA library was purchased fromCLONTECH, which fused HeLa cDNAs to GAL4 transcription activation domain (GAL4AD). TheCLONTECH protocol was followed. 12 million transformants were first screened for His+ phenotype, followed by the filter β-galactosidase assay. Sequencing all of the clones (ACGT Inc., Northbrook, IL) after these two steps revealed the cDNA sequence of the hHus1 gene. The cDNA in one clone (YYN0048) was sequenced to completion, and an open reading frame was identified. The putative peptide sequence was subject to a BLAST search against sequences in public data bases. Expression of GST, GST-hHus1, or GST-p21 that fused GST to the cyclin-dependent kinase inhibitor p21 was induced by isopropyl-1-thio-β-d-galactopyranoside from pGEX-2TK-hHus1-, pGEX-2TK-p21-, or pGEX-2TK-transformed bacteria. The proteins were purified and bound to glutathione-Sepharose beads following the protocol from Promega. The amounts of the three proteins on the beads were estimated by subjecting small portions of the samples to SDS-polyacrylamide gel electrophoresis. An equal amount of each protein was used in the binding assays, while glutathione-Sepharose beads were added to the binding mixtures to achieve equal amount of beads in the binding reactions. HDAC1 or deletion mutants of HDAC1 were in vitro translated using T7 TnT kit (Promega) and [35S]methionine (NEN Life Science Products). The binding mixtures contained immobilized GST, GST-p21, or GST-hHus1, in vitro translated HDAC1 or one of the HDAC1 deletion mutants in binding buffer (50 mmHepes, pH 7.9, 250 mm NaCl, 0.5 mm EDTA, 1 mm dithiothreitol, 10% glycerol, 0.1% Triton X-100, and 0.1% SDS). After a 1-h rotation at 4 °C, the beads were washed three times with binding buffer. Captured, radioactively labeled peptides were separated by SDS-polyacrylamide gel electrophoresis and analyzed by a PhosphoImager. COS-7 cells were transfected by electroporation with Gene Pulser II (Bio-Rad) with settings at 0.25 kV and 950 microfarads. 2 days after transfection, the cells were collected and lysed in radioimmune precipitation buffer (RIPA). Immunoprecipitation of HDAC1-FLAG fusion protein used anti-FLAG M2 antibody conjugated to agarose beads (Sigma). Peptides used for competition were included in the indicated immunoprecipitation reactions at a concentration of 200 μg/ml. The assays were stopped by washing the beads with radioimmune precipitation buffer three times. Immunoprecipitated materials were separated on acrylamide gels and examined by Western blot with anti-HA monoclonal antibody 12CA5 (Boehringer Mannheim), anti-GFP antibody (CLONTECH), or anti-Rad9 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Immunoprecipitation assays involving endogenous proteins used nuclear extracts prepared with a simplified Dignam protocol. Briefly, K562 cells, a human chronic myelogenous leukemia cell line, were collected and incubated in Triton-lysis buffer (150 mm NaCl, 1 mm MgCl2, 1 mm Tris, pH 8.0, and 0.08% Triton X-100) on ice for 5 min. The nuclei were collected after a 5-min centrifugation and resuspended in lysis buffer (50 mm Hepes, 1% Triton X-100, 10 mm NaF, 30 mmNa4P2O7, 150 mm NaCl, 1 mm EDTA, 10 mm β-glycerophosphate, 1 mm Na3VO4, and protease inhibitors) and incubated for 45 min on ice. The extracts were obtained by centrifugation for 15 min after the incubation. Immunoprecipitations were carried out for 1 h with the appropriate antibodies and protein A-Sepharose beads. Each reaction was washed three times with the lysis buffer, and immunoprecipitated materials were examined by Western blots. All of the antibodies were from Santa Cruz Biotechnology. H1299 cells, a human lung carcinoma, were plated onto coverslips and transfected with either pcDNA3-hHus1-HA or pcDNA3-HDAC1-FLAG. 2 days after transfection, the cells were fixed with a paraformaldehyde (Sigma) solution (4% paraformaldehyde in PBS) for 15 min followed by a 30-min incubation in −20 °C methanol. The cells were subsequently blocked with 1% bovine serum albumin in PBST (PBS with 0.1% Triton X-100 and 0.05% Tween 20). After blocking, anti-HA monoclonal antibody or anti-FLAG M2 antibody (Kodak) was diluted with PBST, added to the cells, and incubated at room temperature for 1 h. The cells were then washed extensively with PBST and incubated with a PBST solution that contained RNase A, propidium iodide, and anti-mouse IgG antibody conjugated with fluorescein for 30 min. The cells were washed with PBST and mounted for viewing. Iterative data base similarity searches were performed with PSI-BLAST (37Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59759) Google Scholar) using all of the available protein sequences from the Hus1, Rad1, and Rad9 families (i.e. human, mouse, Caenorhabditis elegans, S. pombe, Saccharomyces cerevisiae, and Drosophila melanogaster). After two or three cycles, these homology searches retrieved a few members of the DNA-sliding clamp family including PCNA and polymerase III (β-subunit). Currently, no single computational algorithm guarantees the optimal detection of divergent members in protein families. Thus, to investigate further a possible evolutionary connection among Hus1, Rad1, Rad9, and members of the PCNA family, we have used three additional complementary approaches. First, ungapped conserved regions (or blocks) in Hus1, Rad1, and Rad9 were converted to position-specific scoring matrices (or profiles) using BlockMaker and subsequently submitted as probes in MAST searches (38Henikoff, S., Henikoff, J. G., Alford, W. J., and Pietrokovski, S. (1995) Gene (Amst.)163, GC 17–26Google Scholar, 39Bailey T.L. Gribskov M. J. Comput. Biol. 1997; 4: 45-59Crossref PubMed Scopus (58) Google Scholar). Second, a combination of secondary structure prediction methods (40Cuff J.A. Clamp M.E. Siddiqui A.S. Finlay M. Barton G.J. Bioinformatics. 1998; 14: 892-893Crossref PubMed Scopus (916) Google Scholar) was used with three separate multiple sequence alignments generated by ClustalW (41Higgins D.G. Thompson J.D. Gibson T.J. Methods Enzymol. 1996; 266: 383-402Crossref PubMed Scopus (1288) Google Scholar). In addition, a fold recognition method (GenThreader) that measures the compatibility of a primary sequence with a library of tertiary templates was applied to each of the 15 sequences (42Jones D.T. J. Mol. Biol. 1999; 287: 797-815Crossref PubMed Scopus (784) Google Scholar). To identify novel HDAC1-interacting proteins, we performed a yeast two-hybrid screen using a HeLa cDNA library. Our initial attempt to use the full-length human HDAC1 as the bait failed, indicating that overexpression of this protein might be toxic to the yeast cells. Instead, the region between amino acids 53 and 285 was used. It is part of the conserved N-terminal domain among the different HDACs (1Taunton J. Hassig C.A. Schreiber S.L. Science. 1996; 272: 408-411Crossref PubMed Scopus (1530) Google Scholar, 2Emiliani S. Fischle W. Van Lint C. Al-Abed Y. Verdin E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2795-2800Crossref PubMed Scopus (277) Google Scholar, 6Yang W.-M. Inouye C. Zeng Y.Y. Bearss D. Seto E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12845-12850Crossref PubMed Scopus (482) Google Scholar). Among the 12 million clones screened, several demonstrated specific interaction with the bait. Fig. 1 shows the results of β-galactosidase assays for one of the clones, YYN0048. When YYN0048 was present in yeast with the bait, positive β-galactosidase activity was observed (Fig.1 A), whereas there was no β-galactosidase activity when YYN0048 was present together with either one of two nonspecific baits, a fusion between GAL4BD and the human lamin C protein (Fig.1 B) or GAL4BD alone (Fig. 1 C). All of the positive clones contained cDNA sequence for one gene. The full-length cDNA in YYN0048 revealed a prominent open reading frame. The translated amino acid sequence was subjected to a BLAST search against sequences in public data bases and identified as a human homolog to a fission yeast protein, Hus1p (23Kostrub C.F. al-Khodairy F. Ghazizadeh H. Carr A.M. Enoch T. Mol. Gen. Genet. 1997; 254: 389-399PubMed Google Scholar). They share about 30% identity over the length of both peptides. Based on this homology, we named the human cDNA clone hHus1 for human Hus1. While our study was under way, other groups also cloned hHus1 (24Kostrub C.F. Knudsen K. Subramani S. Enoch T. EMBO J. 1998; 17: 2055-2066Crossref PubMed Scopus (100) Google Scholar, 35Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). To assay forin vitro interaction between HDAC1 and hHus1, in vitro translated HDAC1 using [35S]methionine was mixed with immobilized, bacterially expressed GST-hHus1. Bound HDAC1 was visualized by electrophoresis and a PhosphoImager. As negative controls, GST alone or GST-p21 fusion protein was used in similar binding reactions (Fig. 2 A). GST-hHus1 pulled down HDAC1 (lane 1), but neither GST-p21 fusion protein (lane 2) nor GST alone (lane 3) pulled down any appreciable amounts of HDAC1, indicating specificin vitro interactions between GST-hHus1 and HDAC1. Further characterization of the in vitro interaction using several HDAC1 deletion mutants in similar binding assays suggested that the region between amino acids 1 and 240 is involved in the interaction with GST-hHus1. This was demonstrated by the ability of mutants aa1–179 and aa179-482 to bind to GST-hHus1 (lanes 4and 7), in contrast to the weak or the lack of binding of the mutants aa241–363 and aa364–482 (lanes 10 and13). Based on these results, which are summarized in Fig.2 B and also supported by the yeast two-hybrid experiments, we propose that HDAC1 putative interaction domain with hHus1 is between amino acids 53" @default.
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• W2170354958 title "HDAC1, a Histone Deacetylase, Forms a Complex with Hus1 and Rad9, Two G2/M Checkpoint Rad Proteins" @default.
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