Host Cell Factor-1 Interacts with and Antagonizes Transactivation by the Cell Cycle Regulatory Factor Miz-1
2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês
10.1074/jbc.m206226200
ISSN1083-351X
AutoresDavid Piluso, Patricia Bilan, John P. Capone,
Tópico(s)Herpesvirus Infections and Treatments
ResumoHuman host cell factor-1 (HCF-1) is essential for cell cycle progression and is required, in conjunction with the herpes simplex virus transactivator VP16, for induction of viral immediate-early gene expression. We show here that HCF-1 directly binds to the Myc-interacting protein Miz-1, a transcription factor that induces cell cycle arrest at G1, in part by directly stimulating expression of the cyclin-dependent kinase inhibitor p15INK4b. A domain encompassing amino acids 750–836, contained within a subregion of HCF-1 required for cell cycle progression, was sufficient to bind Miz-1. Conversely, HCF-1 interacted with two separate regions in Miz-1: the N-terminal POZ domain and a C-terminal domain (residues 637–803) previously shown to harbor determinants for interaction with c-Myc and the coactivator p300. The latter functioned as a potent transactivation domain when tethered to DNA, indicating that HCF-1 targets a transactivation function in Miz-1. HCF-1 or a Miz-1-binding fragment of HCF-1 repressed transactivation by Gal4-Miz-1 in transfection assays. Moreover, HCF-1 repressed Miz-1-mediated transactivation of a reporter gene linked to the p15INK4b promoter. Protein/protein interaction studies and transient transfection assays demonstrated that HCF-1 interferes with recruitment of p300 to Miz-1, similar to what has been reported with c-Myc. Our findings identify Miz-1 as a novel HCF-1-interacting partner and illustrate cross-talk between these two proteins that may be of consequence to their respective functions in gene regulation and their opposing effects on the cell cycle. Human host cell factor-1 (HCF-1) is essential for cell cycle progression and is required, in conjunction with the herpes simplex virus transactivator VP16, for induction of viral immediate-early gene expression. We show here that HCF-1 directly binds to the Myc-interacting protein Miz-1, a transcription factor that induces cell cycle arrest at G1, in part by directly stimulating expression of the cyclin-dependent kinase inhibitor p15INK4b. A domain encompassing amino acids 750–836, contained within a subregion of HCF-1 required for cell cycle progression, was sufficient to bind Miz-1. Conversely, HCF-1 interacted with two separate regions in Miz-1: the N-terminal POZ domain and a C-terminal domain (residues 637–803) previously shown to harbor determinants for interaction with c-Myc and the coactivator p300. The latter functioned as a potent transactivation domain when tethered to DNA, indicating that HCF-1 targets a transactivation function in Miz-1. HCF-1 or a Miz-1-binding fragment of HCF-1 repressed transactivation by Gal4-Miz-1 in transfection assays. Moreover, HCF-1 repressed Miz-1-mediated transactivation of a reporter gene linked to the p15INK4b promoter. Protein/protein interaction studies and transient transfection assays demonstrated that HCF-1 interferes with recruitment of p300 to Miz-1, similar to what has been reported with c-Myc. Our findings identify Miz-1 as a novel HCF-1-interacting partner and illustrate cross-talk between these two proteins that may be of consequence to their respective functions in gene regulation and their opposing effects on the cell cycle. Human host cell factor-1 (HCF-1) 1The abbreviations used for: HCF, host cell factor; VIC, VP16-induced complex; DBD, DNA-binding domain; AD, activation domain; AAD, acidic activation domain; GST, glutathioneS-transferase. 1The abbreviations used for: HCF, host cell factor; VIC, VP16-induced complex; DBD, DNA-binding domain; AD, activation domain; AAD, acidic activation domain; GST, glutathioneS-transferase. is a transcriptional regulatory protein that was originally identified as an accessory factor required for induction of herpes simplex virus immediate-early genes by the viral transactivator VP16 (1Herr W. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 599-607Crossref PubMed Scopus (43) Google Scholar, 2Xiao P. Capone J.P. Mol. Cell. Biol. 1990; 10: 4974-4977Crossref PubMed Scopus (89) Google Scholar, 3Wilson A.C. LaMarco K. Peterson M.G. Herr W. Cell. 1993; 74: 115-125Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 4Kristie T.M. Sharp P.A. J. Biol. Chem. 1993; 268: 6525-6534Abstract Full Text PDF PubMed Google Scholar, 5Kristie T.M. Pomerantz J.L. Twomey T.C. Parent S.A. Sharp P.A. J. Biol. Chem. 1995; 270: 4387-4394Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Subsequently, findings demonstrated that HCF-1 is an essential cellular protein that is required for cell proliferation (6Goto H. Motomura S. Wilson A.C. Freiman R.N. Nakabeppu Y. Fukushima K. Fujishima M. Herr W. Nishimoto T. Genes Dev. 1997; 11: 726-737Crossref PubMed Scopus (128) Google Scholar). HCF-1 binds directly to VP16 and, in conjunction with the cellular octamer-binding transcription factor Oct-1 promotes the cooperative assembly and stability of a multicomponent protein·DNA transcription complex termed the VP16-induced complex (VIC) on regulatory elements present in the promoter regions of the herpes simplex virus immediate-early genes (reviewed in Refs. 1Herr W. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 599-607Crossref PubMed Scopus (43) Google Scholar and 7Wilson A.C. Cleary M.A. Lai J.S. LaMarco K. Peterson M.G. Herr W. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 167-178Crossref PubMed Scopus (52) Google Scholar). Numerous studies of VP16 and its association with Oct-1, HCF-1, and DNA have provided fundamental insights into mechanisms of transcriptional activation and how combinatorial networks of protein/protein and protein/DNA interactions underpin complexity, specificity, and diversity in transcriptional regulation (1Herr W. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 599-607Crossref PubMed Scopus (43) Google Scholar, 7Wilson A.C. Cleary M.A. Lai J.S. LaMarco K. Peterson M.G. Herr W. Cold Spring Harbor Symp. Quant. Biol. 1993; 58: 167-178Crossref PubMed Scopus (52) Google Scholar). Human HCF-1 is a ubiquitously expressed and evolutionarily conserved protein with a number of unusual properties and features (3Wilson A.C. LaMarco K. Peterson M.G. Herr W. Cell. 1993; 74: 115-125Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 5Kristie T.M. Pomerantz J.L. Twomey T.C. Parent S.A. Sharp P.A. J. Biol. Chem. 1995; 270: 4387-4394Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 8Kristie T.M. J. Biol. Chem. 1997; 272: 26749-26755Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 9Liu Y. Hengartner M.O. Herr W. J. Virol. 1999; 74: 99-109Google Scholar). HCF-1 is synthesized as a 2035-amino acid long precursor protein that is autocatalytically processed at centrally reiterated 26-amino acid repeat elements (HCFPRO repeats) to generate a family of N- and C-terminal polypeptides that remain tightly, but noncovalently, associated with each other (10Wilson A.C. Peterson M.G. Herr W. Genes Dev. 1995; 9: 2445-2458Crossref PubMed Scopus (84) Google Scholar, 11Vogel J.L. Kristie T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9425-9430Crossref PubMed Scopus (26) Google Scholar, 12Hughes T.A. La Boissiere S. O'Hare P. J. Biol. Chem. 1999; 274: 16437-16443Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 13Wilson A.C. Boutros M. Johnson K.M. Herr W. Mol. Cell. Biol. 2000; 20: 6721-6730Crossref PubMed Scopus (39) Google Scholar). VP16 associates with a discrete 380-residue N-terminal modular domain referred to as HCFVIC (also called the kelch domain), which is composed of six repeated copies of a kelch-like sequence proposed to form a barrel-like six-bladed β-propeller (14Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar). The HCFVICdomain is necessary and sufficient to bind VP16, to stabilize VIC, and to promote transactivation (14Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar). In addition to subserving a role in viral gene expression, HCF-1 is essential for normal cell cycle progression (6Goto H. Motomura S. Wilson A.C. Freiman R.N. Nakabeppu Y. Fukushima K. Fujishima M. Herr W. Nishimoto T. Genes Dev. 1997; 11: 726-737Crossref PubMed Scopus (128) Google Scholar). This finding arose from studies of tsBN67 cells, a temperature-sensitive hamster cell line that reversibly arrests at the G0/G1 decision point of the cell cycle at the nonpermissive temperature. The defect in tsBN67 cells is due to a single proline-to-serine missense mutation (P134S) at position 134 in HCF-1 (6Goto H. Motomura S. Wilson A.C. Freiman R.N. Nakabeppu Y. Fukushima K. Fujishima M. Herr W. Nishimoto T. Genes Dev. 1997; 11: 726-737Crossref PubMed Scopus (128) Google Scholar). Interestingly, HCF-1(P134S) also fails to bind to VP16 (14Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar), foretelling the existence of cellular factors that mimic VP16 in their interaction with HCF-1. The first such HCF-1-interacting cellular factor identified was LZIP (also called Luman) (15Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar, 16Lu R. Yang P. O'Hare P. Misra V. Mol. Cell. Biol. 1997; 17: 5117-5126Crossref PubMed Scopus (147) Google Scholar, 17Lu R. Yang P. Padmakumar S. Misra V. J. Virol. 1998; 72: 6291-6297Crossref PubMed Google Scholar), a basic leucine zipper protein belonging to the cAMP-responsive element-binding protein/activating transcription factor family of transcription factors. Like VP16, LZIP and a related protein called Zhangfei (18Lu R. Misra V. Nucleic Acids Res. 2000; 28: 2446-2454Crossref PubMed Google Scholar) target determinants in the HCFVIC domain. More recently, the transcription factors GA-binding protein (19Vogel J.L. Kristie T.M. EMBO J. 2000; 19: 683-690Crossref PubMed Scopus (72) Google Scholar) and Sp1 (20Gunther M. Laithier M. Brison O. Mol. Cell Biochem. 2000; 210: 131-142Crossref PubMed Google Scholar), the nuclear hormone receptor co-regulatory factor PGC-1 (21Lin J. Puigserver P. Donovan J. Tarr P. Spiegelman B. J. Biol. Chem. 2002; 277: 1645-1648Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar), and a protein phosphatase (22Ajuh P.M. Browne G.J. Hawkes N.A. Cohen P.T. Roberts S.G. Lamond A.I. Nucleic Acids Res. 2000; 28: 678-686Crossref PubMed Scopus (31) Google Scholar) have been shown to associate with HCF-1. The foregoing adds to growing evidence that HCF-1 is an essential, multifunctional, co-regulatory protein that plays a global role in coordinating viral and cellular gene regulation and cell proliferation. However, the cellular role and mechanisms of action of HCF-1 and the identity of its cellular gene targets are essentially unknown. Recent findings show that HCF-1 is chromatin-associated, and it has been postulated that HCF-1 is recruited to DNA through its association with sequence-specific DNA-binding proteins, analogous to what occurs with VP16 and Oct-1 (23Wysocka J. Reilly P.T. Herr W. Mol. Cell. Biol. 2001; 21: 3820-3829Crossref PubMed Scopus (154) Google Scholar). The HCFVIC domain is necessary and sufficient for chromatin association, and the P134S mutation renders this association temperature-sensitive, suggesting that this detachment is responsible for the growth arrest phenotype in tsBN67 cells (23Wysocka J. Reilly P.T. Herr W. Mol. Cell. Biol. 2001; 21: 3820-3829Crossref PubMed Scopus (154) Google Scholar). However, the minimal region capable of rescuing tsBN67 cells encompasses residues 1–902, which include the HCFVICdomain and a downstream region rich in basic amino acids (6Goto H. Motomura S. Wilson A.C. Freiman R.N. Nakabeppu Y. Fukushima K. Fujishima M. Herr W. Nishimoto T. Genes Dev. 1997; 11: 726-737Crossref PubMed Scopus (128) Google Scholar, 14Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar). Moreover, the binding of the cellular proteins LZIP and Zhangfei to HCF-1 is not required to rescue tsBN67 cells and to promote cell cycle progression (23Wysocka J. Reilly P.T. Herr W. Mol. Cell. Biol. 2001; 21: 3820-3829Crossref PubMed Scopus (154) Google Scholar, 24Mahajan S.S. Wilson A.C. Mol. Cell. Biol. 2000; 20: 919-928Crossref PubMed Scopus (25) Google Scholar). This indicates that other cellular factors important for cell cycle control that target the N-terminal domain and/or the basic region of HCF-1 may exist. The importance of the basic region in cell proliferation is underscored by recent studies with the HCF-1 family member HCF-2 and the related Caenorhabditis elegans homolog (25Johnson K.M. Mahajan S.S. Wilson A.C. J. Virol. 1999; 73: 3930-3940Crossref PubMed Google Scholar, 26Lee S. Herr W. J. Virol. 2001; 75: 12402-12411Crossref PubMed Scopus (16) Google Scholar). HCF-1, HCF-2, and C. elegansHCF share conserved N- and C-terminal domains; however, HCF-2 andC. elegans HCF lack the basic region and the central HCFPRO repeats. Although HCF-2 and C. elegansHCF are able to support VIC formation, these proteins are unable to rescue the temperature-sensitive cell cycle defect in tsBN67 cells (26Lee S. Herr W. J. Virol. 2001; 75: 12402-12411Crossref PubMed Scopus (16) Google Scholar). Coexpression of HCF-2 inhibits rescue by HCF-1, however, suggesting that the two factors share a common interacting partner(s) (25Johnson K.M. Mahajan S.S. Wilson A.C. J. Virol. 1999; 73: 3930-3940Crossref PubMed Google Scholar). The foregoing indicates that the basic region of HCF-1 provides an additional function that is required, in conjunction with the N-terminal proximal region, to promote cell cycle progression. To shed light on the cellular and functional roles of HCF-1, we carried out yeast two-hybrid interaction cloning with the HCF-1 basic region as bait to identify putative cellular factors that target this region. We show here that HCF-1 interacts physically and functionally with Miz-1, a recently identified cell cycle regulatory factor that was originally isolated by virtue of its ability to interact with the cellular oncoprotein c-Myc (27Peukert K. Staller P. Schneider A. Carmichael G. Hanel F. Eilers M. EMBO J. 1997; 16: 5672-5686Crossref PubMed Scopus (290) Google Scholar, 28Schneider A. Peukert K. Eilers M. Hanel F. Curr. Top. Microbiol. Immunol. 1997; 224: 137-146PubMed Google Scholar, 29Sakamuro D. Prendergast G.C. Oncogene. 1999; 18: 2942-2954Crossref PubMed Scopus (153) Google Scholar). Miz-1 is an 803-amino acid long POZ domain/zinc finger transcription factor that modulates transcription by binding directly to initiator elements of target genes (27Peukert K. Staller P. Schneider A. Carmichael G. Hanel F. Eilers M. EMBO J. 1997; 16: 5672-5686Crossref PubMed Scopus (290) Google Scholar, 30Staller P. Peukert K. Kiermaier A. Seoane J. Lukas J. Karsunky H. Moroy T. Bartek J. Massague J. Hanel F. Eilers M. Nat. Cell. Biol. 2001; 3: 392-399Crossref PubMed Scopus (449) Google Scholar, 31Ziegelbauer J. Shan B. Yager D. Larabell C. Hoffmann B. Tjian R. Mol. Cell. 2001; 8: 339-349Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In contrast to the stimulation of cell cycle progression ascribed to HCF-1, Miz-1 causes cell cycle arrest at G1 (27Peukert K. Staller P. Schneider A. Carmichael G. Hanel F. Eilers M. EMBO J. 1997; 16: 5672-5686Crossref PubMed Scopus (290) Google Scholar). This is manifested in part by Miz-1-mediated activation of the gene encoding the key cell cycle inhibitor p15INK4b, a potent inhibitor of cyclin-dependent kinases (30Staller P. Peukert K. Kiermaier A. Seoane J. Lukas J. Karsunky H. Moroy T. Bartek J. Massague J. Hanel F. Eilers M. Nat. Cell. Biol. 2001; 3: 392-399Crossref PubMed Scopus (449) Google Scholar). Accumulation of p15INK4b at G1 results in cell cycle arrest due to a reduction in cyclin D1-associated kinase activity. Recent studies have shown that transcriptional activation of p15INK4b and the subsequent cell cycle arrest are potentiated by the antimitogenic cytokine transforming growth factor-β and antagonized by the cell proliferation mediator c-Myc through opposing pathways that directly converge on Miz-1 (30Staller P. Peukert K. Kiermaier A. Seoane J. Lukas J. Karsunky H. Moroy T. Bartek J. Massague J. Hanel F. Eilers M. Nat. Cell. Biol. 2001; 3: 392-399Crossref PubMed Scopus (449) Google Scholar, 33Seoane J. Pouponnot C. Staller P. Schader M. Eilers M. Massague J. Nat. Cell. Biol. 2001; 3: 400-408Crossref PubMed Scopus (402) Google Scholar). Thus, c-Myc binds directly to Miz-1 on the p15INK4b initiator element and represses Miz-1 transactivation by abrogating recruitment of the coactivator p300. Conversely, transforming growth factor-β activates SMAD proteins, which relieve c-Myc-mediated repression by causing the dissociation of c-Myc from Miz-1 and which directly transactivate the p15INK4b promoter in cooperation with Miz-1. Miz-1 is thus at the nexus of reciprocal growth regulatory signaling pathways that link p15INK4b to cell cycle control (32Amati B. Nat. Cell. Biol. 2001; 3: E112-E113Crossref PubMed Scopus (36) Google Scholar). We demonstrate here that Miz-1/HCF-1 association is manifested through the basic domain of HCF-1 and by two independent regions of Miz-1: the N-terminal POZ domain and a C-terminal transactivation domain. We further show that HCF-1 represses Miz-1-mediated transactivation of a reporter gene linked to the p15INK4b promoter, likely as a result of HCF-1 interfering with the recruitment of p300 to Miz-1. Thus, HCF-1 functions in a manner analogous to c-Myc in modulating Miz-1 function. Our findings point to a convergence between two regulatory proteins that have opposing roles in cell proliferation and may thus be relevant to the mechanisms by which HCF-1 promotes cell cycle progression. Mammalian expression vectors pCGNHCF and pFLAG-C1, encoding hemagglutinin/c-Myc and FLAG epitope-tagged derivatives of full-length human HCF-1, respectively, have been described (3Wilson A.C. LaMarco K. Peterson M.G. Herr W. Cell. 1993; 74: 115-125Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 18Lu R. Misra V. Nucleic Acids Res. 2000; 28: 2446-2454Crossref PubMed Google Scholar) and were provided by W. Herr and T. Kristie, respectively. pCGNHCF-1FL(P134S) (where FL is full-length) was constructed by isolating a SpeI/XhoI fragment from pCGNHCF and inserting this fragment into pCGNHCF-1-(1–1011)(P134S), supplied by A. Wilson (25Johnson K.M. Mahajan S.S. Wilson A.C. J. Virol. 1999; 73: 3930-3940Crossref PubMed Google Scholar). A mammalian expression vector encoding V5 epitope-tagged full-length Miz-1 was provided by R. Tjian (31Ziegelbauer J. Shan B. Yager D. Larabell C. Hoffmann B. Tjian R. Mol. Cell. 2001; 8: 339-349Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). Mammalian expression vectors for full-length Miz-1 (pPK7) and human c-Myc (pMNBabeIRESc-Myc) were obtained from M. Eilers (27Peukert K. Staller P. Schneider A. Carmichael G. Hanel F. Eilers M. EMBO J. 1997; 16: 5672-5686Crossref PubMed Scopus (290) Google Scholar) and L. Penn (University of Toronto), respectively. pGal4(5X)luc, a luciferase reporter vector that contains five upstream copies of the Gal4-binding site, was obtained from J. Hassell (McMaster University). p15INK4b luc, a luciferase reporter gene that contains sequences spanning −113 to +160 relative to the transcription start site of the p15INK4b promoter, was provided by M. Eilers (30Staller P. Peukert K. Kiermaier A. Seoane J. Lukas J. Karsunky H. Moroy T. Bartek J. Massague J. Hanel F. Eilers M. Nat. Cell. Biol. 2001; 3: 392-399Crossref PubMed Scopus (449) Google Scholar). The in vitro transcription vector pSPUTK was obtained from D. Andrews (McMaster University). The yeast two-hybrid bait plasmid pGBT9-HCF-1-(450–1439), which contains residues 450–1439 of HCF-1 linked to the Gal4 DNA-binding domain (DBD), was constructed by cloning a PCR fragment corresponding to this region into the two-hybrid vector pGBT9 (Clontech). Similarly, pGBT9-HCF-1-(1–380) expresses the HCFVIC subdomain. Deletions of HCF-1 were constructed in pGBT9 by conventional or PCR-based methods using appropriate primers. The Gal4 activation domain (AD)-VP16 fusion protein (residues 1–404 of VP16) was cloned into pGAD424 (Clontech). Mammalian Gal4-DBD-Miz-1 derivatives were derived by subcloning PCR-derivedXhoI/BamHI subfragments into the corresponding sites of the Gal4 fusion protein expression vector pSG424 by standard procedures. A mammalian expression plasmid for HCF-1-(750–902) was generated by amplifying this fragment from pCGNHCF and cloning into the XhoI site of pCMV/Myc/nuc (Invitrogen). This construct expresses HCF-1-(750–902) tagged with a c-Myc epitope and containing a nuclear localization signal at the C terminus. Various glutathioneS-transferase (GST)-HCF-1 and GST-Miz-1 derivatives, as indicated in the figure legends, were constructed by cloning PCR fragments into the EcoRI/SalI sites of pGEX4T1 (Amersham Biosciences). GST-VP16-(1–404) contains residues 1–404 of VP16 cloned into pGEX2T. In vitro transcription/translation vector for full-length Miz-1 was generated by cloning the Miz-1 open reading frame into the NcoI and SalI sites of pGEM5zf (Promega). In vitro expression vectors for Miz-1 derivatives expressing the POZ domain (residues 109–308) and residues 637–803 were generated by cloning PCR fragments into pSPUTK. In vitro expression vectors for HCF-1-(612–902) and HCF-1-(1–902) were constructed by cloning appropriate PCR fragments into pSPUTK and pGEM5zf, respectively. The GST-p300-(1572–2371) plasmid was obtained from R. Eckner, and the pcDNA-Myc vector used for in vitro transcription/translation was provided by M. Eilers. Site-directed mutagenesis of pGEM5zf-HCF-1-(1–902) to incorporate the P134S mutation was performed using the QuikChange site-directed mutagenesis kit (Stratagene) with the following mutagenic primers: 5′-AAAAACGGGCCCCCTTCGTGTCCTCGACTC-3′ and 5′-GAGTCGAGGACACGAAGGGGGCCCGTTTTTG-3′ (altered nucleotides are underlined). The authenticity of all clones constructed above was verified by DNA sequence analysis. Mammalian Gal4-VP16AAD, which expresses the Gal4 DBD linked to the C-terminal acidic activation domain of VP16 has been described (34Popova B. Bilan P. Xiao P. Faught M. Capone J.P. Virology. 1995; 209: 19-28Crossref PubMed Scopus (8) Google Scholar). Two-hybrid analysis was carried out using the Clontech Matchmaker system essentially as described (35Bai C. Elledge S.J. Methods Enzymol. 1997; 283: 141-156Crossref PubMed Scopus (75) Google Scholar, 36$$Google Scholar). Briefly, yeast strain HF7c (MATa, ura3-52, his3-200,ade2-101, lys2-801, trp1-901,leu2-3,112, can r, gal4-542,gal80-538, URA3::GAL1-lacZ) was transformed with pGBT9-HCF-1-(450–1439) and a HeLa cell Matchmaker cDNA library fused to the Gal4 AD (Clontech) by the lithium acetate method (37Elble R. BioTechniques. 1992; 13: 18-20PubMed Google Scholar). Independent transformants (2 × 106) were grown on His−, Leu−, and Trp− plates supplemented with 20 mm3-amino-1,2,4-triazole. Library plasmids from His+ colonies were selectively recovered from yeast following transformation intoEscherichia coli HB101 (leuB −), transformed along with pGBT9-HCF-1-(450–1439) or control Gal4-DBD plasmids into yeast strain Y190 and assayed for β-galactosidase activity using the β-galactosidase overlay assay (36$$Google Scholar). Colonies that scored positive for β-galactosidase activity in the presence of the HCF-1 bait plasmid, but not with control or irrelevant plasmids, were sequenced. One clone containing a partial cDNA encoding amino acids 269–803 of Miz-1 was selected for further studies. Protein binding assays with GST fusion proteins and [35S]methionine-radiolabeled proteins synthesized in vitro using a coupled rabbit reticulocyte transcription/translation system (Promega) were carried out as described previously (38Meertens L.M. Miyata K.S. Cechetto J.D. Rachubinski R.A. Capone J.P. EMBO J. 1998; 17: 6972-6978Crossref PubMed Scopus (69) Google Scholar). Briefly, E. coli BL21 cells harboring expression vectors for GST-Miz-1, GST-HCF-1, or GST alone were grown to an A 600 nm of 0.6–0.8 and induced with isopropyl-β-d-thiogalactopyranoside (Gibco BRL) for 3 h. Bacteria were collected by centrifugation and resuspended in buffer containing 0.5% Nonidet P-40, 1 mm EDTA, 20 mm Tris-Cl (pH 8.0), 100 mm NaCl, and one tablet of mini C protease inhibitor (Roche Molecular Biochemicals)/25 ml of buffer, and cell extracts were prepared by sonication. 50 μl of a 50:50 slurry of glutathione-Sepharose 4B was incubated with clarified cell extracts containing GST or GST fusion protein for 1 h at 4 °C. Beads were collected by centrifugation and washed twice with phosphate-buffered saline, and beads containing equivalent amounts of bound protein (as determined by Coomassie Blue staining of SDS-polyacrylamide gels) were incubated with 10–20 μl of reticulocyte lysate containing radiolabeled translated protein in buffer A (150 mm KCl, 0.02 mg/ml bovine serum albumin, 0.1% Triton X-100, 0.1% Nonidet P-40, 5 mmMgCl2, and 20 mm Hepes (pH 7.9)) for 2–3 h at 4 °C. Beads were washed extensively with buffer A lacking bovine serum albumin, and bound radiolabeled proteins were eluted from the beads by boiling in SDS sample buffer and analyzed by SDS-PAGE. Competition assays were carried out with unlabeled competitor protein synthesized in vitro as detailed in the figure legends. The total amount of rabbit reticulocyte lysate was kept constant by adding unprogrammed lysate to the binding reactions as required. COS-1 cells were cultured at 37 °C in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% l-glutamine, and 1% penicillin/streptomycin. Cells were seeded in six-well plates at a concentration of 2–3 × 105 cells/well 1 day prior to transfection to achieve 70–80% confluence. Transfections were carried out using LipofectAMINE reagent (4–8 μl/well; Invitrogen) as outlined by the manufacturer. Unless indicated otherwise in the figure legends, transfections typically contained 0.5 μg of luciferase reporter plasmid (pGal4(5X)luc or p15INK4b luc) and 0.05–0.5 μg of various effector plasmids (HCF-1, Miz-1, c-Myc, or derivatives thereof). Total DNA and promoter dosage was kept constant with the appropriate amounts of corresponding empty vectors. Luciferase activity was assayed in cell lysates prepared 48 h post-transfection and normalized to protein concentration. Transfections were carried out as described above using expression plasmids (0.5 μg/well) for V5-Miz-1FL and/or FLAG-HCF-1FL. Lysates were prepared 48 h post-transfection using Nonidet P-40 lysis buffer (150 mmNaCl, 50 mm Tris (pH 8.0), 1% Nonidet P-40, and 0.2 mm phenylmethylsulfonyl fluoride). 1 ml of clarified supernatant, normalized for protein concentration, was precleared with 50 μl of protein G-Sepharose (Roche Molecular Biochemicals) and incubated with 1 μg of anti-FLAG antibody (Upstate Biotechnology, Inc.) and 50 μl of protein G-Sepharose for 2 h at 4 °C. Immune complexes were collected, extensively washed, suspended in 50 μl of SDS-polyacrylamide gel sample buffer, and subjected to PAGE. Proteins were transferred to HybondTM-C pure nitrocellulose membrane (Amersham Biosciences) and probed using mouse anti-V5 monoclonal antibody (Invitrogen) as the primary antibody, followed by horseradish peroxidase-coupled sheep anti-mouse polyclonal antibody (Amersham Biosciences) as the secondary antibody. Proteins were detected by enhanced chemiluminescence with a commercially available kit (ECL, Amersham Biosciences) according to the manufacturer's instructions. COS-1 cells were transfected as described above, except that 1 μg of the various Gal4-Miz-1 expression plasmids was used. Preparation of cell extracts for immunoblotting using HybondTM-C pure nitrocellulose membrane was carried out as described (36$$Google Scholar, 38Meertens L.M. Miyata K.S. Cechetto J.D. Rachubinski R.A. Capone J.P. EMBO J. 1998; 17: 6972-6978Crossref PubMed Scopus (69) Google Scholar). Gal4 fusion proteins were detected by ECL as described above using mouse anti-Gal4 DNA-binding domain monoclonal antibody (Santa Cruz Biotechnology) as the primary antibody and horseradish peroxidase-coupled sheep anti-mouse polyclonal antibody as the secondary antibody. We carried out yeast two-hybrid screens to identify novel HCF-1-interacting proteins that may provide insights into the cellular functions, targets, and mechanisms of action of HCF-1. Similar approaches by others (15Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar, 16Lu R. Yang P. O'Hare P. Misra V. Mol. Cell. Biol. 1997; 17: 5117-5126Crossref PubMed Scopus (147) Google Scholar) have used the N-terminal domain (residues 1–450) of HCF-1 as bait because this region is known to be sufficient for interaction with VP16 and for promoting VIC formation (14Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar). We focused our attention on a separate region of HCF-1 (residues 450–1439) that encompasses the basic domain (Fig.1 A) because this region has been shown to be required, along with the HCFVIC domain, to rescue the temperature-sensitive block to cell cycle progression in tsBN67 cells (14Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar). The rationale was that the basic region may target novel HCF-1-interacting proteins that subserve a role in cell cycle regulation and/or other functions of HCF-1 that are independent of the HCFVIC region. Yeast two-hybrid screens using a Gal4-DBD-HCF-1-(450–1439) fusion protein as bait resulted in the identification
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