Title: HCF-1 Functions as a Coactivator for the Zinc Finger Protein Krox20
Abstract: HCF-1 is a transcriptional cofactor required for activation of herpes simplex virus immediate-early genes by VP16 as well as less clearly defined roles in cell proliferation, cytokinesis, and spliceosome formation. It is expressed as a large precursor that undergoes proteolysis to yield two subunits that remain stably associated. VP16 uses a degenerate 4-amino acid sequence, known as the HCF-binding motif, to bind to a six-bladed β-propeller domain at the N terminus of HCF-1. Functional HCF-binding motifs are also found in LZIP and Zhangfei, two cellular bZIP transcription factors of unknown function. Here we show that the HCF-binding motif occurs in a wide spectrum of DNA-binding proteins and transcriptional cofactors. Three well characterized examples were further analyzed for their ability to use HCF-1 as a coactivator. Krox20, a zinc finger transcription factor required for Schwann cell differentiation, and E2F4, a cell cycle regulator, showed a strong requirement for functional HCF-1 to activate transcription. In contrast, activation by estrogen receptor-α did not display HCF dependence. In Krox20, the HCF-binding motif lies within the N-terminal activation domain and mutation of this sequence diminishes both transactivation and association with the HCF-1 β-propeller. The activation domain in the C-terminal subunit of HCF-1 contributes to activation by Krox20, possibly through recruitment of p300. These results suggest that HCF-1 is recruited by many different classes of cellular transcription factors and is therefore likely to be required for a variety of cellular processes including cell cycle progression and development. HCF-1 is a transcriptional cofactor required for activation of herpes simplex virus immediate-early genes by VP16 as well as less clearly defined roles in cell proliferation, cytokinesis, and spliceosome formation. It is expressed as a large precursor that undergoes proteolysis to yield two subunits that remain stably associated. VP16 uses a degenerate 4-amino acid sequence, known as the HCF-binding motif, to bind to a six-bladed β-propeller domain at the N terminus of HCF-1. Functional HCF-binding motifs are also found in LZIP and Zhangfei, two cellular bZIP transcription factors of unknown function. Here we show that the HCF-binding motif occurs in a wide spectrum of DNA-binding proteins and transcriptional cofactors. Three well characterized examples were further analyzed for their ability to use HCF-1 as a coactivator. Krox20, a zinc finger transcription factor required for Schwann cell differentiation, and E2F4, a cell cycle regulator, showed a strong requirement for functional HCF-1 to activate transcription. In contrast, activation by estrogen receptor-α did not display HCF dependence. In Krox20, the HCF-binding motif lies within the N-terminal activation domain and mutation of this sequence diminishes both transactivation and association with the HCF-1 β-propeller. The activation domain in the C-terminal subunit of HCF-1 contributes to activation by Krox20, possibly through recruitment of p300. These results suggest that HCF-1 is recruited by many different classes of cellular transcription factors and is therefore likely to be required for a variety of cellular processes including cell cycle progression and development. HCF-1 (also known as C1 factor) is a heterodimeric nuclear protein composed of a family of polypeptides generated from a 2035-amino acid precursor by site-specific proteolysis (1Kristie 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 (92) Google Scholar, 2Wilson A.C. LaMarco K. Peterson M.G. Herr W. Cell. 1993; 74: 115-125Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 3Wilson A.C. Peterson M.G. Herr W. Genes Dev. 1995; 9: 2445-2458Crossref PubMed Scopus (84) Google Scholar). After cleavage of the precursor, the N- and C-terminal processing products remain as a stable complex through two matching sets of self-interaction domains (3Wilson A.C. Peterson M.G. Herr W. Genes Dev. 1995; 9: 2445-2458Crossref PubMed Scopus (84) Google Scholar, 4Wilson A.C. Boutros M. Johnson K.M. Herr W. Mol. Cell. Biol. 2000; 20: 6721-6730Crossref PubMed Scopus (39) Google Scholar). HCF-1 is expressed and processed in most cell types and with the exception of unstimulated sensory neurons, is predominantly nuclear and tightly associated with chromatin (3Wilson A.C. Peterson M.G. Herr W. Genes Dev. 1995; 9: 2445-2458Crossref PubMed Scopus (84) Google Scholar, 5Frattini A. Faranda S. Redolfi E. Zucchi I. Villa A. Patrosso M.C. Strina D. Susani L. Vezzoni P. Genomics. 1994; 23: 30-35Crossref PubMed Scopus (16) Google Scholar, 6Kristie T.M. Vogel J.L. Sears A.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1229-1233Crossref PubMed Scopus (99) Google Scholar, 7Lu R. Yang P. O'Hare P. Misra V. Mol. Cell. Biol. 1997; 17: 5117-5126Crossref PubMed Scopus (147) Google Scholar, 8Wysocka J. Reilly P.T. Herr W. Mol. Cell. Biol. 2001; 21: 3820-3829Crossref PubMed Scopus (157) Google Scholar). The two HCF-1 subunits are composed of multiple functional domains. The N-terminal subunit includes a six-bladed β-propeller domain characteristic of proteins in the kelch superfamily and an adjacent basic region (9LaBoissière S. Walker S. O'Hare P. Mol. Cell. Biol. 1997; 17: 7108-7118Crossref PubMed Scopus (44) Google Scholar, 10Simmen K.A. Newell A. Robinson M. Mills J.S. Canning G. Handa R. Parkes K. Borkakoti N. Jupp R. J. Virol. 1997; 71: 3886-3894Crossref PubMed Google Scholar, 11Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar). The C-terminal subunit includes a transcriptional activation domain (HCF-1AD), a pair of degenerate fibronectin type III repeats, and a bipartite nuclear localization signal (4Wilson A.C. Boutros M. Johnson K.M. Herr W. Mol. Cell. Biol. 2000; 20: 6721-6730Crossref PubMed Scopus (39) Google Scholar, 12LaBoissière S. Hughes T. O'Hare P. EMBO J. 1999; 18: 480-489Crossref PubMed Scopus (80) Google Scholar, 13Luciano R.L. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13403-13408Crossref PubMed Scopus (37) Google Scholar). The cellular functions of HCF-1 appear to be multifaceted and are still only partially understood. The most clearly established role is in regulation of viral and cellular transcription. This was first described in the context of the herpes simplex virus (HSV) 1The abbreviations used are: HSVherpes simplex virusHBMHCF-binding motifERαestrogen receptor αbFGF-2basic fibroblast growth factor 2ADactivation domainlucluciferaseWTwild type. transactivator VP16 (reviewed in Refs. 14O'Hare P. Semin. Virol. 1993; 4: 145-155Crossref Scopus (134) Google Scholar and 15Wysocka J. Herr W. Trends Biochem. Sci. 2003; 28: 294-304Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). HCF-1 is necessary for the recruitment of VP16 to HSV immediate-early gene promoters and for transcriptional activation by the assembled VP16-induced complex (13Luciano R.L. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13403-13408Crossref PubMed Scopus (37) Google Scholar). In addition, both subunits of HCF-1 have been shown to interact with cellular transcriptional activators and repressors (7Lu R. Yang P. O'Hare P. Misra V. Mol. Cell. Biol. 1997; 17: 5117-5126Crossref PubMed Scopus (147) Google Scholar, 16Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar, 17Gunther M. Laithier M. Brison O. Mol. Cell. Biochem. 2000; 210: 131-142Crossref PubMed Google Scholar, 18Lu R. Misra V. Nucleic Acids Res. 2000; 28: 2446-2454Crossref PubMed Google Scholar, 19Piluso D. Bilan P. Capone J.P. J. Biol. Chem. 2002; 277: 46799-46808Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 20Scarr R.B. Sharp P.A. Oncogene. 2002; 21: 5245-5254Crossref PubMed Scopus (44) Google Scholar, 21Vogel J.L. Kristie T.M. EMBO J. 2000; 19: 683-690Crossref PubMed Scopus (73) Google Scholar). Analysis of a temperature-sensitive hamster cell line (tsBN67) carrying a missense mutation in the HCF-1 β-propeller has revealed a role in cell cycle progression and cytokinesis (22Goto 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 (129) Google Scholar, 23Reilly P.T. Herr W. Exp. Cell Res. 2002; 277: 119-130Crossref PubMed Scopus (22) Google Scholar). Last, HCF-1 is incorporated into spliceosome complexes and may participate in mRNA splicing as well as transcription (24Ajuh P. Chusainow J. Ryder U. Lamond A.I. EMBO J. 2002; 21: 6590-6602Crossref PubMed Scopus (19) Google Scholar). herpes simplex virus HCF-binding motif estrogen receptor α basic fibroblast growth factor 2 activation domain luciferase wild type. The N-terminal β-propeller domain is central to most HCF-1 functions. The 380-amino acid domain encompasses six kelch (HCFKEL) repeats, which are predicted to form the six blades of the propeller structure. The domain is sufficient to bind VP16 and assemble a VP16-induced complex on DNA in association with the cellular POU protein Oct-1 (11Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar). In the HCF-1 protein encoded by the tsBN67 cell line, there is a missense mutation in the β-propeller that changes proline 134 to serine (11Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar, 22Goto 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 (129) Google Scholar). At the non-permissive temperature, HCF-1 is unable to support transactivation by VP16 and is released from the cellular chromatin (8Wysocka J. Reilly P.T. Herr W. Mol. Cell. Biol. 2001; 21: 3820-3829Crossref PubMed Scopus (157) Google Scholar, 22Goto 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 (129) Google Scholar). Yeast interaction screens using the β-propeller domain identified two ATF/cAMP-response element-binding protein-like basic leucine zipper proteins known as LZIP/Luman and Zhangfei (7Lu R. Yang P. O'Hare P. Misra V. Mol. Cell. Biol. 1997; 17: 5117-5126Crossref PubMed Scopus (147) Google Scholar, 16Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar, 18Lu R. Misra V. Nucleic Acids Res. 2000; 28: 2446-2454Crossref PubMed Google Scholar). Sequence comparison with VP16 revealed a 4-amino acid sequence motif, termed the HCF-binding motif (HBM), that is used by all three proteins to recognize the HCF-1 β-propeller (16Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar, 25Lu R. Yang P. Padmakumar S. Misra V. J. Virol. 1998; 72: 6291-6297Crossref PubMed Google Scholar). This motif ((D/E)HXY) consists of an acidic residue (aspartic acid or glutamic acid) followed by an invariant histidine, any residue (X), and then an invariant tyrosine. The HBM is an integral part of the LZIP transactivation domain, and recruitment of HCF-1 is required for activation by LZIP (26Luciano R.L. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10757-10762Crossref PubMed Scopus (33) Google Scholar). Recently, functional HBM sequences have been identified in the peroxisome proliferator-activated receptor γ-coactivator PGC-1β and a novel nuclear export factor termed HPIP (27Lin J. Puigserver P. Donovan J. Tarr P. Spiegelman B.M. J. Biol. Chem. 2002; 277: 1645-1648Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 28Mahajan S.S. Little M.M. Vazquez R. Wilson A.C. J. Biol. Chem. 2002; 277: 44292-44299Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). As a starting point for study of defined cellular processes regulated by HCF-1, we searched the protein sequence data bases to identify known transcription factors that contain potential HBMs. Many candidates were identified in this way, and in several the HBM lies in a region of the protein already known to be involved in gene activation. Three candidates, Krox20 (also known as EGR2, NGF1-B), E2F4, and estrogen receptor α (ERα), representing well studied members of unrelated protein families, were selected for further analysis. Krox20 is a member of the Krüpple-like zinc finger protein family and plays a critical role in hindbrain development and myelination of the peripheral nervous system (29Nagarajan R. Svaren J. Le N. Araki T. Watson M. Milbrandt J. Neuron. 2001; 30: 355-368Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). E2F4 plays a key role in regulating the cell cycle as well as differentiation processes such as adipogenesis (30Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 11-20Crossref PubMed Scopus (966) Google Scholar). ERα is a steroid hormone receptor that stimulates gene expression in a ligand-dependent manner (31McDonnell D.P. Norris J.D. Science. 2002; 296: 1642-1644Crossref PubMed Scopus (491) Google Scholar). We found that transactivation by Krox20 and E2F4 but not ERα was dependent on the presence of functional HCF-1. Mutation of the HBM or deletion of the activation domain of HCF-1 significantly reduced the ability of Krox20 to stimulate transcription from a synthetic promoter composed of a GC-rich Krox20 binding site or a natural promoter from the human bFGF-2 gene. The results of these experiments suggest that HCF-1 plays multiple roles in the regulation of cellular transcription. In addition to ubiquitous processes such as promoting cell proliferation, HCF-1 is likely to be important for tissue-specific events including myelination by Schwann cells and development of the central nervous system. Data Base Analysis—The SWISS-PROT and Translated EMBL (TrEMBL) data bases were searched using ScanProSite, a web-based motif search tool provided by the Expert Protein Analysis System (ExPASy) 2us.expasy.org. proteomics server of the Swiss Institute of Bioinformatics (32Gattiker A. Bienvenut W. Bairoch A. Gasteiger E. Proteomics. 2002; 10: 1435-1444Crossref Scopus (84) Google Scholar). Using the query "[DE]HxY," searches were restricted to mammalian proteins and individual entry annotations were used to identify transcription factors. Expression Plasmids—The full-length murine Krox20 open reading frame was amplified by PCR from a cDNA clone provided by Dr. Jeffrey Milbrandt (Washington University, St. Louis, MO), and subcloned into the cytomegalovirus enhancer-driven mammalian expression vectors pCGT and pCGN, thereby adding N-terminal T7 and hemagglutinin-epitope tags (11Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar). Note that the mouse Krox20 protein was used in these experiments. Mouse and human proteins are highly homologous (overall 89% identical and 95% similar), especially within the activation and DNA-binding domains. In addition, Krox20 sequences (full-length or the first 170 residues) were subcloned into pCGNGal4, creating a hemagglutinin-tagged fusion to the yeast Gal4 DNA-binding domain (residues 1 to 94). All PCR amplified fragments were confirmed by DNA sequencing. Alanine substitution mutagenesis of the Krox20 HBM (residues 162 to 165) was performed by QuikChange™ (Stratagene) and verified by DNA sequencing. A diagnostic PstI site was incorporated into the changes to identify mutants. The plasmid encoding Gal4-E2F4-(240-412) was kindly provided by Dr. David Johnson (University of Texas M. D. Anderson Cancer Center, Houston, TX) (33Wang D. Russell J.L. Johnson D.G. Mol. Cell. Biol. 2000; 20: 3417-3424Crossref PubMed Scopus (82) Google Scholar). The Gal4-responsive luciferase reporter plasmid used in this study was p5xGal4-E1B-luc and contains five tandem Gal4-binding sites (CGGAGTACTGTCCTCCG) (34Sun P. Enslen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (650) Google Scholar). The plasmid encoding the estrogen receptor (CMV-ER) and estrogen response element-luc reporter were kind gifts of Dr. Michael Garabedian (NYU School of Medicine) (35Su L.F. Knoblauch R. Garabedian M.J. J. Biol. Chem. 2001; 276: 3231-3237Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The bFGF-2 promoter and GC-luciferase reporter plasmid (GCGGGGGCG-luc) were provided by Drs. Jeffrey Milbrandt and John Svaren (University of Washington, St. Louis, MO) (36Biesiada E. Razandi M. Levine E.R. J. Biol. Chem. 1996; 271: 18576-18581Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 37Russo M.W. Matheny C. Milbrandt J. Mol. Cell. Biol. 1993; 13: 6858-6865Crossref PubMed Scopus (89) Google Scholar). Cell Culture and Protein Expression Assays—Maintenance and electroporation of 293T, HeLa, and tsBN67-derived cells, luciferase reporter assays, extract preparation, and immunoblotting have been described previously (2Wilson A.C. LaMarco K. Peterson M.G. Herr W. Cell. 1993; 74: 115-125Abstract Full Text PDF PubMed Scopus (213) Google Scholar, 13Luciano R.L. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13403-13408Crossref PubMed Scopus (37) Google Scholar, 26Luciano R.L. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10757-10762Crossref PubMed Scopus (33) Google Scholar). For temperature shift experiments, tsBN67 cells were grown at 33.5 °C. After electroporation, the transfected cells were plated and allowed to recover at 33.5 °C for 2 h before transfer to 39.5 °C. Candidate HCF-binding Motifs Are Found in Many Cellular Transcription Factors—To identify cellular proteins that can interact with HCF-1, we searched the SWISS-PROT and EMBL protein data bases for sequences matching the HBM consensus ((D/E)HXY) using the web-based ScanProsite search tool. As anticipated, this short and degenerate motif was present in a large number of mammalian proteins of different functions and cellular localization. SWISS-PROT alone yielded more than 370 unique hits. Given the previously established role of HCF-1 in regulation of gene expression, and the predominantly nuclear localization of the protein, we focused on known or suspected DNA-binding proteins and transcriptional co-regulators (see Fig. 1 for a listing). As expected, the search identified previously known HBM-containing proteins. These include HSV VP16 and its homologues from related α-herpesviruses as well as the cellular proteins LZIP, Zhangfei, HPIP, and PGC-1α and -β (7Lu R. Yang P. O'Hare P. Misra V. Mol. Cell. Biol. 1997; 17: 5117-5126Crossref PubMed Scopus (147) Google Scholar, 16Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar, 18Lu R. Misra V. Nucleic Acids Res. 2000; 28: 2446-2454Crossref PubMed Google Scholar, 27Lin J. Puigserver P. Donovan J. Tarr P. Spiegelman B.M. J. Biol. Chem. 2002; 277: 1645-1648Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, 28Mahajan S.S. Little M.M. Vazquez R. Wilson A.C. J. Biol. Chem. 2002; 277: 44292-44299Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). The sequences flanking the core motif show no obvious homology, although there is evidence that flanking sequences do influence HBM function (38Wu T.J. Monokian G. Mark D.F. Wobbe C.R. Mol. Cell. Biol. 1994; 14: 3484-3493Crossref PubMed Google Scholar). In terms of the candidate HBM-containing proteins, we noted that in several examples, the HBM-like sequence is conserved in non-human counterparts and/or falls within a region of the protein known to contain an activation domain. A clear example is the Krox20/EGR2 protein (Fig. 2A). Orthologs from other vertebrates preserve the putative HBM, although in birds, fish, and amphibians the first position is changed from an aspartic acid to a glutamic acid. In fish and amphibians the variable third position is also changed from leucine to isoleucine that preserves the hydrophobic nature of the side group. The activation domain of Krox20 has been mapped to the N terminus of the protein (residues 1-184) and encompasses the HBM-like sequence (residues 162-165) (39Vesque C. Charnay P. Nucleic Acids Res. 1992; 20: 2485-2492Crossref PubMed Scopus (35) Google Scholar). This arrangement is strongly reminiscent of LZIP in which the N-terminal activation domain includes the HBM (26Luciano R.L. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10757-10762Crossref PubMed Scopus (33) Google Scholar). Transcriptional Activation by Krox20 and E2F4 Is Temperature-sensitive in tsBN67 Cells—To determine whether any of these sequence matches represent functional HBMs, we selected three candidates, Krox20, E2F4, and ERα, and tested them for HCF dependence. Each activator was analyzed by transient transfection of tsBN67, a derivative of BHK21 that carries a temperature-sensitive version of HCF-1. At the permissive temperature of 33.5 °C, HCF-1 is functional and the cells proliferate normally, whereas at the non-permissive temperature (39.5 °C), the HCF-1 β-propeller domain is inactivated and the cells asynchronously arrest (11Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar, 22Goto 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 (129) Google Scholar). Transactivation by Krox20 was examined in two contexts (Fig. 2, B and C). In the first (Fig. 2B), full-length Krox20 (Krox20FL) was cotransfected into tsBN67 cells together with a Krox20-responsive luciferase reporter (GC-luc) composed of two Krox20-binding sites (5′-GCGGGGGCG-3′) placed upstream of the prolactin core promoter. Pools of transfected cells were split into halves and incubated for 40 h at either the permissive or non-permissive temperature, and then assayed for luciferase activity. Expression of Krox20 gave a 7-fold stimulation at the permissive temperature but this was abolished when cells were cultured at 39.5 °C. The reporter alone showed a similar reduction reflecting the activity of low levels of endogenous Krox20 protein. The result of this experiment suggests that Krox20 requires HCF-1 to activate transcription. In the second approach (Fig. 2B), we tested an N-terminal fragment of Krox20 (residues 2-170) corresponding to the activation domain (39Vesque C. Charnay P. Nucleic Acids Res. 1992; 20: 2485-2492Crossref PubMed Scopus (35) Google Scholar). This fragment was fused to the Gal4 DNA-binding domain (Gal4DBD) and cotransfected with a luciferase reporter gene (5xGal4-E1B-luc) containing five Gal4-binding sites upstream of the adenovirus E1B TATA box. Gal4-Krox20N170 activated transcription 5-fold at the permissive temperature and this activation was reduced to only 2-fold at the non-permissive temperature. This result substantiates the temperature-dependent activation observed using full-length Krox20 and argues that HCF-1 contributes to transcriptional activation rather than recognition of the GC-rich Krox20-binding site. We also tested a C-terminal fragment from human E2F4 (residues 240-412) fused to the Gal4DBD (Gal4-E2F4-(240-412)) (Fig. 2D). This fragment includes the activation and pocket protein-binding domains but not the DNA-binding or dimerization domains (33Wang D. Russell J.L. Johnson D.G. Mol. Cell. Biol. 2000; 20: 3417-3424Crossref PubMed Scopus (82) Google Scholar). Expression of Gal4-E2F4-(240-412) at the permissive temperature increased transcription of the report by ∼8-fold and this was significantly reduced in cells maintained at the non-permissive temperature. In contrast to Krox20 and E2F4, activation by the full-length estrogen receptor (ERα) using a reporter linked to an estrogen response element showed little difference at the two temperatures (Fig. 2E). Under these conditions, ERα is activated by ligand present in the calf sera. Addition of the ligand estradiol (100 nm) to the culture media gave similar results (data not shown). These results indicate that not all of the activators containing HBM-like sequences require HCF-1 to activate transcription, thus highlighting the role of HCF-1 in specific programs of gene expression. Mutation of the HBM Prevents Transactivation by Krox20— To determine whether the HBM-like sequence in Krox20 contributes to activation domain function, we generated a substitution mutant (Krox20 HBMKO, Fig. 2A) simultaneously changing all four residues of the motif to alanine. HeLa cells were cotransfected with the wild type and mutant versions of full-length Krox20 together with the GC-luc reporter (Fig. 3A). Wild type Krox20 (WT) activated transcription 6-fold, whereas the mutant (HBMKO) activated transcription only 1.5-fold. This indicates that the HBM identified through a sequence data base search is an important component of the Krox20 activation domain. We also examined the consequence of overexpressing HCF-1 (Fig. 3B). HeLa cells were transfected with expression plasmids encoding full-length Krox20 and HCF-1. Coexpression of both proteins resulted in a greater level of activation than with Krox20 alone. This stimulation was not observed when HCF-1 was expressed on its own. This suggests that HCF-1 can only contribute to activation when brought to the promoter via a DNA-binding protein such as Krox20. HCF-1 Is Required for Activation of the bFGF-2 Promoter— The bFGF-2 promoter (shown schematically in Fig. 3C) contains multiple GC-rich elements and at least two of these (boxes i and iii) have been shown to bind Krox20 (or the related Krox24/EGR1) protein in vitro, leading to promoter activation (36Biesiada E. Razandi M. Levine E.R. J. Biol. Chem. 1996; 271: 18576-18581Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 40Svaren J. Sevetson B.R. Golda T. Stanton J.J. Swirnoff A.H. Milbrandt J. EMBO J. 1998; 17: 6010-6019Crossref PubMed Scopus (69) Google Scholar). To address the role of HCF-1 in regulation of the bFGF-2 promoter, we cotransfected 293T cells with increasing amounts of expression plasmids encoding wild type and HBMKO Krox20 together with the full-length bFGF-2 promoter driving the luciferase reporter gene (41Shibata F. Baird A. Florkiewicz R.Z. Growth Factors. 1991; 4: 277-287Crossref PubMed Scopus (81) Google Scholar). The promoter was activated in a dose-dependent manner by wild type Krox20, but showed a significantly reduced response to Krox20 HBMKO (Fig. 3D). As shown in Fig. 3E, the experiment was repeated using 500 ng of each Krox20 expression plasmid and extracts were assayed for protein expression by immunoblotting with an antibody against the T7 epitope-tagged Krox20 proteins as well as for luciferase activity. Activation of the bFGF-2 promoter was significantly reduced with the HBMKO mutant compared with wild type, although there was little detectable difference in protein expression. These results show that mutation of the candidate HBM in Krox20 reduces its ability to activate transcription from a natural promoter. The HCF-1 β-Propeller Associates with the Activation Domain of Krox20—By analogy to VP16, LZIP, and HPIP, it is possible that the HCF-1 β-propeller alone is sufficient for recognition of the HBM in Krox20 (11Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar, 16Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar, 26Luciano R.L. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10757-10762Crossref PubMed Scopus (33) Google Scholar, 28Mahajan S.S. Little M.M. Vazquez R. Wilson A.C. J. Biol. Chem. 2002; 277: 44292-44299Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). To date, we have been unable to detect a stable interaction between Krox20 and HCF-1 using gel mobility shift or in vitro coimmunoprecipitation assays, suggesting the interaction is weak or stabilized by other factors present in vivo (data not shown). Instead, we have used a mammalian one-hybrid recruitment assay in transfected 293T cells to characterize the interaction between the β-propeller of HCF-1 and Krox20. Gal4-HCF-1N380WT was co-expressed with Krox20FL and the Gal4-responsive reporter (Fig. 4A). The β-propeller of HCF-1 did not activate the reporter gene unless coexpressed with Krox20FL. The specificity of this interaction was verified using the tsBN67 version of the HCF-1 β-propeller. This is identical to wild type except that proline 134 has been changed to serine (P134S). Previous studies have shown that this single substitution is sufficient to prevent interaction with VP16 and LZIP (11Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar, 16Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar, 25Lu R. Yang P. Padmakumar S. Misra V. J. Virol. 1998; 72: 6291-6297Crossref PubMed Google Scholar). As expected, activation was not observed using the P134S mutant, indicating that recruitment of Krox20FL to the promoter was dependent on a functional β-propeller domain. To better define sequences in Krox20 required for this interaction, the recruitment assay was repeated using Krox20N170 in place of Krox20FL (Fig. 4B). Again Krox20N170 was able to activate the reporter in the presence of Gal4-HCF-1N380WT, but not the P134S mutant. This result shows that the Krox20 activation domain was sufficient for interaction with the HCF-1 β-propeller domain. The HCF-1 C-terminal Activation Domain Is Important for Krox20 Function—HCF-1 contains a potent activation domain (HCF-1AD) located in a domain termed the