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• W1980316914 abstract "The mammalian transcriptional coactivator HCF-1 is a critical component of the multiprotein herpes simplex virus immediate early gene enhancer core complex. The protein has also been implicated in basic cellular processes such as cell-cycle progression, transcriptional coactivation, and mRNA processing. Functions have been attributed to HCF-1 primarily from analyses of protein-protein interactions and from the cell-cycle-arrested phenotype of an HCF-1 temperature-sensitive mutant. However, neither the mechanisms involved nor specific cellular transcriptional targets have been identified. As the protein is essential for cell viability and proliferation, a genetic system was developed to specifically sequester the nuclear factor in the cell cytoplasm in a regulated manner. This approach exhibits no significant cell toxicity yet clearly demonstrates the requirement of available nuclear HCF-1 for herpes simplex virus immediate early gene expression during productive infection. Additionally, cellular transcriptional events were identified that contribute to understanding the functions ascribed to the protein and implicate the protein in events that impact the regulation of critical cellular processes. The mammalian transcriptional coactivator HCF-1 is a critical component of the multiprotein herpes simplex virus immediate early gene enhancer core complex. The protein has also been implicated in basic cellular processes such as cell-cycle progression, transcriptional coactivation, and mRNA processing. Functions have been attributed to HCF-1 primarily from analyses of protein-protein interactions and from the cell-cycle-arrested phenotype of an HCF-1 temperature-sensitive mutant. However, neither the mechanisms involved nor specific cellular transcriptional targets have been identified. As the protein is essential for cell viability and proliferation, a genetic system was developed to specifically sequester the nuclear factor in the cell cytoplasm in a regulated manner. This approach exhibits no significant cell toxicity yet clearly demonstrates the requirement of available nuclear HCF-1 for herpes simplex virus immediate early gene expression during productive infection. Additionally, cellular transcriptional events were identified that contribute to understanding the functions ascribed to the protein and implicate the protein in events that impact the regulation of critical cellular processes. HCF-1, 1The abbreviations used are: HCF-1, host cell factor-1; HSV, herpes simplex virus; IE, immediate early; VP16, virion protein 16; αTIF, α-trans-induction-factor; ts, temperature-sensitive; HA, hemagglutinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pfu, plaque-forming unit. 1The abbreviations used are: HCF-1, host cell factor-1; HSV, herpes simplex virus; IE, immediate early; VP16, virion protein 16; αTIF, α-trans-induction-factor; ts, temperature-sensitive; HA, hemagglutinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; pfu, plaque-forming unit. a cellular transcriptional coactivator, was originally identified by its requirement for the stable assembly of the herpes simplex virus (HSV) immediate early (IE) enhanceosome (1Wilson A.C. LaMarco K. Peterson M.G. Herr W. Cell. 1993; 74: 115-125Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 2Kristie T.M. Sharp P.A. J. Biol. Chem. 1993; 268: 6525-6534Abstract Full Text PDF PubMed Google Scholar, 3Kristie 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). In vitro, the multiprotein IE regulatory complex is nucleated by the recognition of the 5′ sequences in the IE enhancer core element by Oct-1, a POU-homeo-domain protein (4Roizman B. Sears A.E. Fields B. Knipe D.M. Howley P.M. Fundamental Virology. Lippincott-Raven, Philadelphia1996: 1043-1107Google Scholar, 5Wilson 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, 6Vogel J.L. Kristie T.M. Creighton T.E. The Encyclopedia of Molecular Medicine. 1. John Wiley and Sons, Inc., New York2001: 732-735Google Scholar, 7Wysocka J. Herr W. Trends Biochem. Sci. 2003; 28: 294-304Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar). The viral encoded IE transactivator protein, VP16 (αTIF), recognizes the 3′ sequences of the core and cooperatively interacts via specific recognition of the Oct-1 homeosubdomain (8Kristie T.M. Sharp P.A. Genes Dev. 1990; 4: 2383-2396Crossref PubMed Scopus (156) Google Scholar, 9Liu Y. Gong W. Huang C.C. Herr W. Cheng X. Genes Dev. 1999; 13: 1692-1703Crossref PubMed Scopus (47) Google Scholar, 10Lai J.S. Cleary M.A. Herr W. Genes Dev. 1992; 6: 2058-2065Crossref PubMed Scopus (116) Google Scholar, 11Pomerantz J.L. Kristie T.M. Sharp P.A. Genes Dev. 1992; 6: 2047-2057Crossref PubMed Scopus (97) Google Scholar). HCF-1 interacts with VP16 with high affinity, resulting in a stable enhanceosome assembly. HCF-1 is a ubiquitously expressed family of polypeptides derived from a single 230-kDa precursor protein by site-specific proteolytic processing (1Wilson A.C. LaMarco K. Peterson M.G. Herr W. Cell. 1993; 74: 115-125Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 3Kristie 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, 12Vogel J.L. Kristie T.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 9425-9430Crossref PubMed Scopus (26) Google Scholar). With a single notable exception, the protein is localized predominantly in the nucleus and has been described as a transcriptional coactivator for VP16 (13Luciano R.L. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13403-13408Crossref PubMed Scopus (36) Google Scholar) and for cellular transcription factors such as GA-binding protein (GABP) (14Vogel J.L. Kristie T.M. EMBO J. 2000; 19: 683-690Crossref PubMed Scopus (72) Google Scholar), KROX20 (15Luciano R.L. Wilson A.C. J. Biol. Chem. 2003; 278: 51116-51124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), E2F4 (15Luciano R.L. Wilson A.C. J. Biol. Chem. 2003; 278: 51116-51124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and LZIP (13Luciano R.L. Wilson A.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13403-13408Crossref PubMed Scopus (36) Google Scholar, 16Lu R. Yang P. O'Hare P. Misra V. Mol. Cell. Biol. 1997; 17: 5117-5126Crossref PubMed Scopus (147) Google Scholar). In addition, multiple roles have been proposed for HCF-1 in basic cellular processes such as cell-cycle progression (17Scarr R.B. Smith M.R. Beddall M. Sharp P.A. Mol. Cell. Biol. 2000; 20: 3568-3575Crossref PubMed Scopus (7) Google Scholar, 18Julien E. Herr W. EMBO J. 2003; 22: 2360-2369Crossref PubMed Scopus (97) Google Scholar, 19Goto 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), positive and negative transcriptional regulation (20Scarr R.B. Sharp P.A. Oncogene. 2002; 21: 5245-5254Crossref PubMed Scopus (44) Google Scholar, 21Wysocka J. Myers M.P. Laherty C.D. Eisenman R.N. Herr W. Genes Dev. 2003; 17: 896-911Crossref PubMed Scopus (315) Google Scholar), chromatin remodeling (21Wysocka J. Myers M.P. Laherty C.D. Eisenman R.N. Herr W. Genes Dev. 2003; 17: 896-911Crossref PubMed Scopus (315) Google Scholar), and mRNA processing (22Ajuh P. Chusainow J. Ryder U. Lamond A.I. EMBO J. 2002; 21: 6590-6602Crossref PubMed Scopus (19) Google Scholar). These functions have been based primarily upon identified HCF-1-protein interactions and upon the pleiotropic phenotypes of cells containing a temperature-sensitive mutation in the amino terminal kelch domain of the protein. The HCF-1 kelch domain consists of reiterations of four antiparallel β-sheets connected via linker segments (23Mahajan S.S. Wilson A.C. Mol. Cell. Biol. 2000; 20: 919-928Crossref PubMed Scopus (25) Google Scholar, 24Adams J. Kelso R. Cooley L. Trends Cell Biol. 2000; 10: 17-24Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). Interactions of the domain with transcriptional activators (VP16, LZIP, Zhangfei, Krox20, and E2F4 (5Wilson 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, 15Luciano R.L. Wilson A.C. J. Biol. Chem. 2003; 278: 51116-51124Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 23Mahajan S.S. Wilson A.C. Mol. Cell. Biol. 2000; 20: 919-928Crossref PubMed Scopus (25) Google Scholar, 25Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar, 26Lu R. Yang P. Padmakumar S. Misra V. J. Virol. 1998; 72: 6291-6297Crossref PubMed Google Scholar, 27Lu R. Misra V. Nucleic Acids Res. 2000; 28: 2446-2454Crossref PubMed Google Scholar, 28Wilson A.C. Freiman R.N. Goto H. Nishimoto T. Herr W. Mol. Cell. Biol. 1997; 17: 6139-6146Crossref PubMed Scopus (90) Google Scholar)), coactivators (peroxisome proliferator-activated receptor γ coactivator (29Lin J. Puigserver P. Donovan J. Tarr P. Spiegelman B.M. J. Biol. Chem. 2002; 277: 1645-1648Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar)), and chromatin modification components (Set1/Ash2 HMT (21Wysocka J. Myers M.P. Laherty C.D. Eisenman R.N. Herr W. Genes Dev. 2003; 17: 896-911Crossref PubMed Scopus (315) Google Scholar)) have suggested that HCF-1 may be involved in global transcriptional regulation. In addition, cells containing the HCF-1 temperature-sensitive mutation in this domain arrest in G0-G1 cell-cycle stage after prolonged incubation at the nonpermissive temperature (19Goto 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). However, other than the HSV IE genes, the regulatory targets have not been identified. While various genetic systems could be utilized to address these issues, HCF-1 appears to be essential for cell viability, general transcription, and cell-cycle progression. Approaches such as the generation of dominant negative mutants, ts mutants, or RNA interference-mediated depletion have inherent problems for the analysis of essential proteins including the resulting cell toxicity, the time frame for phenotypic effects to become evident due to protein turnover rates, and the difficulties in determining the primary effects of the mutation or depletion. Therefore, a system was developed to inducibly alter the specific localization of HCF-1 by sequestering the protein in the cell cytoplasm. This approach is reflective of the specific cytoplasmic sequestering of HCF-1 in sensory neurons, the single exception to the nuclear localization pattern of the protein in most cell types (30Kristie T.M. Vogel J.L. Sears A.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1229-1233Crossref PubMed Scopus (98) Google Scholar). This system produces a regulated shift in the nuclear-cytoplasmic ratio of HCF-1, exhibits low toxicity, and allows for the delineation of events that are critically dependent upon the levels of nuclear HCF-1. Using this approach, cytoplasmic sequestering of HCF-1 inhibited HSV IE gene expression, indicating the potential of the system for analysis of essential protein functions. In addition, transcriptional regulatory targets were identified that may account for the pleiotropic effects of the protein and also defines HCF-1 as a critical transcriptional component with impact on many basic regulatory processes. Plasmids—pHA-actin was constructed by insertion of an HA epitope tag and start codon (ATG) into the NheI-BglII sites of pEGFP-actin (Clontech). pHA-TIF-Act and pHA-TIFEH were constructed by insertion of the coding sequences for the αTIF-HCF-1 interaction domain (AKLDSYSSFTTSPSEAVMREHAYSRARTKNNYGSTIEGLLD) or mutant domain (AKLDSYSSFTTSPSEAVMRAAAYSRARTKNNYGSTIEGLLD) between the HA tag and the actin coding sequences in pHA-actin. The coding regions from pHA-actin, pHA-TIF-Act, and pHA-TIFEH-Act were subsequently ligated into pMEP4 (Invitrogen) to generate the respective metallothionein promoter controlled expression vectors. Cell Culture and Virus—Cell lines expressing the actin fusion proteins under the control of a metallothionein promoter were generated by transfection of HeLa cells with pMEP4 plasmids (Invitrogen) expressing either the HA-TIF EHAY motif-actin fusion protein (T-Act), HA-TIF EHAY mutant motif-actin fusion protein (TEH-Act), or HA-actin fusion protein (Act). Hygromycin B-resistant colonies were pooled and used for all experiments. HSV-1 (F) viral stocks were produced and titered by infection of Vero cells according to standard procedures. Immunofluorescence—Cells were plated on glass coverslips and 4 μm CdSO4 was added for various defined time periods. The cells were fixed in 4% paraformaldehyde and permeabilized with 1% Triton X-100. The expressed fusion proteins and HCF-1 were detected with anti-HA monoclonal antibody (mHA.11, Covance) and anti-HCF-1 antibody (Ab2131 (3Kristie 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)), respectively, followed by the appropriate Alexafluor secondary antibody (Molecular Probes). Actin was detected using Texas Red-X phalloidin (Molecular Probes). Confocal images were obtained using a confocal laser scanning microscope (Leica TCS-SP2) equipped with a 63× PL Fluotar objective, a 488 nm argon-ion laser, and a 568 nm krypton-ion laser. The images from 488 and 568 nm channels were collected independently and merged using the accompanying software. For quantitation of the HCF-1 fluorescence, polygons were drawn around the entire cell and the nucleus using the accompanying Leica software and the fluorescence values of each were obtained. The HCF-1 fluorescence in the cell cytoplasm was determined by subtracting the nuclear fluorescence values from those of the entire cell. Immunoblot Analysis—Cells were lysed in buffer A (50 mm Tris, pH 8.0, 150 mm NaCl, 1.0% Nonidet P-40, 0.5% deoxycholate) and protein concentrations were determined by Bio-Rad assay (Bio-Rad). Equivalent amounts of protein were resolved in Tris-glycine gels, transferred to Immobilon-P (Millipore), and probed with antibodies to ICP4 (HIA021, Virusys Corp.), HA (mHA.11, Covance), ELK1 (sc-355, Santa Cruz Biotechnology), MCM2 (sc-9839, Santa Cruz Biotechnology), c/EBPβ (sc-150, Santa Cruz Biotechnology), ARF6 (sc-7971, Santa Cruz Biotechnology), CASP9 (sc-17784, Santa Cruz Biotechnology), or GAPDH (TRK5G4-6C5, Research Diagnostic Inc.). The blots were developed using the appropriate horseradish peroxidase-conjugated secondary antibodies and Pierce Super Signal West Pico (Pierce) chemiluminescent reagent. Protein bands were quantitated using a Kodak ID v6.3.1 imaging system (Kodak Scientific) and normalized to the levels of endogenous GAPDH. RNA and cDNA Preparation—Cells were harvested 32 h post-addition of CdSO4. Total RNA was isolated using TRIzol reagent (Invitrogen) followed by purification with an RNeasy midi-column (Qiagen) as directed by the manufacturer. RNA integrity and concentrations were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Massy, France) and a Ultrospec 3000 spectrophotometer (Amersham Biosciences), respectively. Fluorescently labeled first strand cDNAs were prepared by direct incorporation of fluorescent nucleotide analogs during a reverse transcription reaction consisting of either 20 (Cy3 labeling) or 40 μg (Cy5 labeling) of total RNA, 2 μg of oligo(dT)20 primers, 0.25 mm each of dATP, dCTP, and dGTP, 0.025 mm dTTP, 10 mm dithiothreitol, 300 units of reverse transcriptase (Superscript II, Stratagene), and 2 nmol of either Cy3- or Cy5-dUTP (Amersham Biosciences). The cDNA probes were concentrated by filtering through Vivaspin 0.5-ml concentrators (Vivascience). For each microarray hybridization, the appropriate Cy3- and Cy5-labeled cDNA pools to be compared were mixed. Microarray Hybridization and Data Analysis—Microarray slides representing 14,000 human genes (NIAID Microarray Facility) were incubated for 1 h at 42 °C with prehybridization solution (1% bovine serum albumin, 0.1% SDS, 5× SSC), washed two times in double-distilled H2O, washed once in isopropyl alcohol, and dried by centrifugation at 600 rpm for 3 min. Each microarray slide received 66 μl of hybridization solution containing the appropriate Cy3- and Cy5-labeled cDNA pools, 1 μg of poly(d(A))40-60, 10 μg of Cot-1 DNA, 4 μg of yeast tRNA, and 33 μl of hybridization solution (10× SSC, 50% formamide, 0.2% SDS). The mixtures were applied by capillary action under a coverslip (LifterSlip, Erie Scientific Co., Portsmouth, NH) placed over the microarray, and the assembly was incubated in a humidified hybridization chamber for 16 h at 42 °C. Microarray slides were washed twice in 1× SSC, 0.05% SDS for 2 min and twice in 0.1× SSC for 2 min and dried by centrifugation at 600 rpm for 3 min. The slides were immediately scanned and analyzed with a scanner/software package (Axon GenePix 4000B/GenePix Pro 4.0; Axon Instruments, Inc., Foster City, CA). For each microarray set, four replicate hybridizations were done including a dye-bias hybridization control. Microarray Analysis—The average signal intensity and local background measurements for each spot on the array were analyzed using custom mAdb software (developed by Center for Information Technology/BioInformatics & Molecular Analysis Section in collaboration with NCI/Center for Cancer Research & NIAID and available at madb.niaid.nih.gov/index.shtml). The local background was subtracted from the mean intensity value of each spot on the array. Spots were considered negative and eliminated from further analysis if the values for both channels were less than a threshold value defined as one S.D. above the background. The two channels were normalized with respect to the median values for the remaining set of spots. The Cy5/Cy3 fluorescence ratios were calculated from the normalized values and reflect the differential value. Three sets of microarray analyses were performed: T-Act versus TEH-Act, T-Act versus Act, and Act versus MEP (control). To ensure reproducibility in the cDNA preparation and hybridization steps, all experimental data were collected from the hybridization of four independent cDNA preparations from each RNA preparation. Luciferase Reporter Constructs and Assays—Luciferase reporter constructs contained the promoter domains for ELK1 (-500 to +34, NT_011568), c/EBPβ (-500 to +66, NT_011362), Rous sarcoma virus long terminal repeat (-303 to +23), and ubiquitin C (-333 to +877) in pGL3-Basic (Promega). pLG3FOR2 (gift of C. J. Ciudad, University of Barcelona) contained the Sp1 promoter (-217 to +44)-luciferase reporter (31Nicolas M. Noe V. Ciudad C.J. Biochem. J. 2003; 371: 265-275Crossref PubMed Scopus (58) Google Scholar). For transfections, duplicate plates seeded with 4 × 104 tsBN67 cells per 1.9-cm2 well (19Goto 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) were incubated at 31 and 40 °C for 24 h prior to transfection with DNA mixes containing various amounts of the promoter-luciferase reporter (25, 50, 100, 200, and 400 ng). The dishes were incubated at the appropriate temperatures for 24 h prior to the measurement of luciferase activity (dual luciferase assay system, Promega). In all cases, the firefly luciferase activity was normalized to the activity of a cotransfected Renilla control (pRL-TK), and the results of duplicate transfections were averaged. Development of a Cytoplasmic Sequestering System—In contrast to the predominantly nuclear localization of HCF-1 in most cell types, the protein is uniquely sequestered in the cytoplasm of sensory neurons. This localization has been hypothesized to regulate the transcriptional functions of HCF-1 and to be a major controlling determinant in the establishment of and reactivation from the latent state for herpes simplex virus (30Kristie T.M. Vogel J.L. Sears A.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1229-1233Crossref PubMed Scopus (98) Google Scholar). The phenomenon provided a concept for the development of an inducible cytoplasmic sequestering system for the analysis of the cellular functions of HCF-1. The sequestering system is dependent upon the inducible expression of an actin fusion protein containing a small, high affinity HCF-1 interaction motif derived from VP16 (7Wysocka J. Herr W. Trends Biochem. Sci. 2003; 28: 294-304Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 25Freiman R.N. Herr W. Genes Dev. 1997; 11: 3122-3127Crossref PubMed Scopus (116) Google Scholar, 26Lu R. Yang P. Padmakumar S. Misra V. J. Virol. 1998; 72: 6291-6297Crossref PubMed Google Scholar). As illustrated in Fig. 1, incorporation of the expressed actin fusion protein into cytoplasmic actin filaments would act as a high affinity anchor by binding HCF-1 and preventing the normal nuclear transport of the protein. In this manner, the nuclear to cytoplasmic ratio of the endogenous HCF-1 could be shifted in a regulated manner to allow modulation of HCF-1 localization without total ablation of the protein. Theoretically, this system would promote cell survival while affecting functions that are critically dependent upon the available nuclear pool of HCF-1. In developing this system, the high affinity HCF-1 interaction motif from VP16 or a control containing mutations in the critical EHAY core of the motif were fused to actin under the control of an inducible metallothionein promoter (Fig. 2A). Stable cell lines containing the transgenes were selected and analyzed by Western blot for the expression of the fusion proteins in the absence and presence of the inducer (CdSO4). As shown in Fig. 2B, both the wild-type fusion protein (T-Act) and the fusion protein containing the EHAY core mutations (TEHAct) were not detected in the absence of CdSO4 and were expressed at equivalent levels in the presence of the inducer. Furthermore, both fusion proteins exhibited immunofluorescent patterns indicating that they had been incorporated into cellular actin filaments (Fig. 2C). Cytoplasmic Sequestering of HCF-1—The effect of the expression of the actin fusion proteins upon the localization of endogenous HCF-1 was analyzed by quantitative immunofluorescent labeling (Fig. 3, top). T-Act, TEH-Act, and control cells containing the vector alone (MEP) were fixed at various times after induction with CdSO4. The cells were costained with anti-HA sera to detect the expressed actin fusion protein and anti-HCF-1 sera to determine the localization of HCF-1. As shown in Fig. 3(top), cells expressing the T-Act fusion protein exhibited a clear visual increase in the level of cytoplasmic HCF-1 over time as compared with control MEP (Fig. 3, right panels) or TEH-Act cells (data not shown). Quantitative assessment of the cytoplasmic/nuclear ratio of HCF-1 in each cell line confirmed the visual interpretation and demonstrated that the cytoplasmic/nuclear ratio of HCF-1 increased in T-Act cells to nearly 1.4, while MEP and TEH-Act cells maintained a stable ratio ranging from 0.3 to 0.5 (Fig. 3, bottom right). Sequestering of HCF-1 Inhibits the Expression of HSV IE Genes and Viral Replication—Induced T-Act cells exhibited a significant shift in the cytoplasmic to nuclear ratio of HCF-1. As in vitro data and studies using the ts-HCF-1 mutant have supported the model that HCF-1 is required for the regulated transcription of the HSV IE genes, T-Act, TEH-Act, and control cells expressing the HA-tagged actin (Act) were induced for 24 h and infected with HSV at either 0.01 or 0.1 pfu/cell. At 2 h post-infection, the expression of the viral IE protein ICP4 was determined by Western blot analysis of cell lysates. As shown in Fig. 4A (left panel), the level of ICP4 expression was significantly reduced in induced T-Act cells relative to those in TEHAct and Act cells (60% reduction, Fig. 4B). The levels of a second IE protein, ICP0, were similarly affected (data not shown). In contrast to this, infection of the T-Act cells at 0.1 pfu/cell resulted in expression of nearly wild-type levels of both IE proteins (Fig. 4A, right panel, ICP0 data not shown). The contrasting impacts of HCF-1 cytoplasmic sequestering on viral IE gene expression at 0.01 and 0.1 pfu/cell were likely due to the introduction of high levels of wild-type VP16 protein into the cells by the infecting viral particles (4Roizman B. Sears A.E. Fields B. Knipe D.M. Howley P.M. Fundamental Virology. Lippincott-Raven, Philadelphia1996: 1043-1107Google Scholar) that would be expected to compete for the sequestered HCF-1 protein (see “Discussion”). The overall effect of sequestering HCF-1 on viral replication was also assessed. T-Act, TEH-Act, and Act cells were induced and infected with HSV at 0.01 or 0.1 pfu/cell. The resulting progeny virus was harvested at 17 h post-infection. As shown in Fig. 4C, infection of T-Act cells at 0.01 pfu/cell resulted in a significant decrease in the resulting viral yield relative to the TEH-Act and Act control lines. Surprisingly, a decrease in the viral yield was also observed after infection of T-Act cells at 0.1 pfu/cell. The less significant but reproducible decrease in viral yield at 0.1 pfu/cell in T-Act cells suggests that HCF-1 may also play additional roles later in the HSV viral lytic cycle (see “Discussion”). Cellular Regulatory Targets of HCF-1—While the role of HCF-1 in HSV IE gene expression has been well studied, little is known concerning the cellular HCF-1 functions or targets. The effect of sequestering of HCF-1 on HSV IE gene expression and viral replication demonstrated that this system could provide an approach to the analysis of HCF-1-dependent cellular transcriptional functions including those events that might account for the various phenotypic functions ascribed to the protein. Therefore, to identify targets that might be critically dependent upon HCF-1 nuclear function, human oligonucleotide microarrays were hybridized with mRNA isolated from the HCF-1 sequestering and control cell lines in three independent microarray sets: T-Act versus Act, T-Act versus TEH-Act, and Act versus MEP. The resulting microarray raw data were subjected to a series of successive criteria to ensure the validity of the final values and the selected gene list. As listed in Table I, the oligonucleotide microarrays represented 14,000 known and predicted human open reading frames. Those genes that were detected in an array set and that were represented in at least three of the four replicate array hybridizations were selected for further analysis. Genes identified in the T-Act versus Act array set and the control Act versus MEP set were then filtered for those with differential values ≤0.3 or ≥3.0 in at least one replicate hybridization. As the TEH-Act mutant control is not a null mutant and does have a limited sequestering phenotype (refer to Fig. 3), this array set was filtered less stringently for those genes with differential values ≤0.5 or ≥1.5 in at least two replicate hybridizations. From each of these array sets, genes represented in the filtered Act versus MEP array set were deleted to eliminate any impact of the overexpression of actin or fluctuations in the actin cytoskeleton. For those genes remaining in the T-Act versus Act and T-Act versus TEH-Act array sets, the values of the replicate hybridizations were averaged, and the gene list and values were compared between each independent array set. Only those genes that were represented in both the T-Act versus Act and T-Act versus TEH-Act arrays and whose values were consistent in both array sets were selected. Finally, the average of the replicate hybridizations of the T-Act versus Act and T-Act versus TEH-Act for each gene was filtered for a differential of ≤0.5 or ≥1.5. The final 188 selected genes are listed in Tables II, III, and IV.Table IMicroarray data analysis chartT-Act vs. ActT-Act vs. TEH-ActAct vs. MepRepresented on arrays14,00014,00014,000Detected in microarray hybridization845191578588Filtered for 75% representation in replicate sets699578167422Applied filters:≤0.3 or ≥3.0389276≤0.5 or ≥1.5500Deleted genes represented in Act vs. Mep3674470Compared average values from T-Act vs. Act to T-Act vs. TEH-Act1) Genes represented in both sets of arrays2) Array values consistent in both independent array sets3) Average of both array sets was ≤0.5 or ≥1.5Selected188 Open table in a new tab Table IIGenes affected by cytoplasmic sequestering of HCF-1UniGeneGeneCategory 2FunctionT-Act vs. TEH-ActT-Act vs. ActAverageHCF-1ApoptosisHs.1048KITLGUp-regulates Bcl-2 and Bcl-XL, antiapoptotic0.220.190.21+Hs.122552GTSE1DNA damage responseControls p53-dependent apoptosis after DNA damage0.480.460.47+Hs.80019PDCD6T cell receptor-, Fas-, GR-induced apoptosis1.511.661.58−Hs.100641CASP9DNA damage responseInitiator caspase, UV-dependent1.651.681.66−Cell cycleHs.121028ASPMMitotic spindle function0.180.180.18+Hs.23348SKP2DNA damage responseG1-S, SCF ubiquitin ligase-reg c-Myc, p21, DNA repair0.320.390.35+Hs.152983HUS1DNA damage responseS-phase cell cycle checkpoint, DNA damage response0.430.480.46+Hs.82906CDC20Coactivator of anaphase-promoting complex1.601.501.55−Hs.423615CDC34Degrades CDK for DNA replication initiation1.531.721.62−Hs.256697HINT1TranscriptionAdenosine monophosphoramidase binds Cdk71.591.851.72−ChromatinHs.143042HIST1H3DHistone H3, nucleosome formation1.661." @default.
• W1980316914 created "2016-06-24" @default.
• W1980316914 creator A5076006283 @default.
• W1980316914 creator A5087749389 @default.
• W1980316914 date "2004-08-01" @default.
• W1980316914 modified "2023-09-29" @default.
• W1980316914 title "A Protein Sequestering System Reveals Control of Cellular Programs by the Transcriptional Coactivator HCF-1" @default.
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