Hepatitis B Viral Transactivator HBx Alleviates p53-mediated Repression of α-Fetoprotein Gene Expression
2000; Elsevier BV; Volume: 275; Issue: 36 Linguagem: Inglês
10.1074/jbc.m004449200
ISSN1083-351X
AutoresStacey K. Ogden, Kathleen C. Lee, Michelle Barton,
Tópico(s)Cell death mechanisms and regulation
ResumoChronic infection with hepatitis B virus (HBV) is associated with development of hepatocellular carcinoma (HCC). The exact mechanism by which chronic infection with HBV contributes to onset of HCC is unknown. However, previous studies have implicated the HBV transactivator protein, HBx, in progression of HCC through its ability to bind the human tumor suppressor protein, p53. In this study, we have examined the ability of HBx to modify p53 regulation of the HCC tumor marker gene, α-fetoprotein (AFP). By utilizing in vitro chromatin assembly of DNA templates prior to transcription analysis, we have demonstrated that HBx functionally disrupts p53-mediated repression of AFP transcription through protein-protein interaction. HBx modification of p53 gene regulation is both tissue-specific and dependent upon the p53 binding element. Our data suggest that the mechanism by which HBx alleviates p53 repression of AFP transcription is through an association with DNA-bound p53, resulting in a loss of p53 interaction with liver-specific transcriptional co-repressors. Chronic infection with hepatitis B virus (HBV) is associated with development of hepatocellular carcinoma (HCC). The exact mechanism by which chronic infection with HBV contributes to onset of HCC is unknown. However, previous studies have implicated the HBV transactivator protein, HBx, in progression of HCC through its ability to bind the human tumor suppressor protein, p53. In this study, we have examined the ability of HBx to modify p53 regulation of the HCC tumor marker gene, α-fetoprotein (AFP). By utilizing in vitro chromatin assembly of DNA templates prior to transcription analysis, we have demonstrated that HBx functionally disrupts p53-mediated repression of AFP transcription through protein-protein interaction. HBx modification of p53 gene regulation is both tissue-specific and dependent upon the p53 binding element. Our data suggest that the mechanism by which HBx alleviates p53 repression of AFP transcription is through an association with DNA-bound p53, resulting in a loss of p53 interaction with liver-specific transcriptional co-repressors. hepatitis B virus hepatocellular carcinoma HBV transactivator X protein human immunodeficiency virus α-fetoprotein polyacrylamide gel electrophoresis electrophoretic mobility shift assay general transcription factors 2-[bis(2-hydroxyethyl)amino]-2- (hydroxymethyl)propane-1,3-diol Chronic infection with hepatitis B virus (HBV)1 is a predominant risk factor associated with development of hepatocellular carcinoma (HCC). Multiple lines of evidence support the relationship between chronic HBV infection and HCC; geographic correlation exists between global distribution of HCC and the prevalence of HBV carrier states; a high incidence of HBV markers in blood and tissue samples is detected in HCC patients; 30% of all virally induced human tumors involve HBV infection (1Kew M.C. Clin. Lab. Med. 1996; 16: 395-405Abstract Full Text PDF PubMed Google Scholar, 2Hildt E. Hofschneider P.H. Recent Results Cancer Res. 1998; 154: 315-329Crossref PubMed Scopus (78) Google Scholar). Based on epidemiological studies involving chronic HBV infection, it is estimated that the relative risk of developing HCC may be between 100- and 200-fold higher for HBV carriers than for non-carriers (2Hildt E. Hofschneider P.H. Recent Results Cancer Res. 1998; 154: 315-329Crossref PubMed Scopus (78) Google Scholar, 3Feitelson M.A. Duan L.X. Am. J. Pathol. 1997; 150: 1141-1157PubMed Google Scholar). The most likely scenario for HBVs role in HCC predisposition is by modification of host gene regulation (4Andrisani O.M. Barnabas S. Intl. J. Cancer. 1999; 15: 373-379Google Scholar, 5Yoo Y.D. Ueda H. Park K. Flanders K.C. Lee Y.I. Jay G. Kim S.J. J. Clin. Invest. 1996; 97: 388-395Crossref PubMed Scopus (154) Google Scholar, 6Hsu T.Y. Monroy T. Etiemble J. Louise A. Trepo C. Tiollais P. Buendia M.A. Cell. 1988; 55: 627-635Abstract Full Text PDF PubMed Scopus (162) Google Scholar). Integration of viral DNA into the host genome can mediate host gene deregulation by a variety of mechanisms: integration of viral promoters can activate and/or mutate neighboring host genes (6Hsu T.Y. Monroy T. Etiemble J. Louise A. Trepo C. Tiollais P. Buendia M.A. Cell. 1988; 55: 627-635Abstract Full Text PDF PubMed Scopus (162) Google Scholar); integration of viral DNA encoding the HBV transactivator X protein (HBx) enhances HBx expression and subsequent interaction with cellular genes and regulatory proteins (2Hildt E. Hofschneider P.H. Recent Results Cancer Res. 1998; 154: 315-329Crossref PubMed Scopus (78) Google Scholar, 7Stuver S.O. Semin. Cancer Biol. 1998; 8: 299-306Crossref PubMed Scopus (51) Google Scholar, 8Buendia M.A. Semin. Cancer Biol. 1992; 3 (20): 309PubMed Google Scholar). Although HBx has not been reported to bind double-stranded DNA, it can activate transcription of both viral and cellular genes through interaction with a variety of host DNA-binding proteins (reviewed in Ref. 4Andrisani O.M. Barnabas S. Intl. J. Cancer. 1999; 15: 373-379Google Scholar). HBx association with cellular transcriptional activators and general transcription factors such as C/EBPα, TBP, and TFIIH enhances gene activation. In contrast, HBx binding to human p53 protein antagonizes p53-mediated transcriptional activation (9Wang X.W. Forrester K. Yeh J. Feitelson M.A. Gu J.-R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (637) Google Scholar, 10Truant R. Antunovic J. Greenblat J. Prives C. Cromlish J. J. Virol. 1995; 69: 1851-1859Crossref PubMed Google Scholar), and p53-mediated apoptosis (11Wang X.W. Gibson M.K. Vermeulen W. Yeh H. Forrester K. Sturzbecker H.W. et al.Cancer Res. 1995; 55: 6012-6016PubMed Google Scholar, 12Elmore L.W. Hancock A.R. Chang S.F. Wang X.W. Chang S. Callahan C.P. Geller D.A. Will H. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14707-14712Crossref PubMed Scopus (307) Google Scholar). p53 is a classical tumor suppressor with a diverse range of functions in transcriptional activation, cell cycle arrest, DNA damage repair, apoptosis (13Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar, 14Levine A. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar) and, as recently demonstrated, transcriptional repression by sequence-specific DNA binding (15Lee K.C. Crowe A.J. Barton M.C. Mol. Cell. Biol. 1999; 19: 1279-1288Crossref PubMed Scopus (152) Google Scholar, 16Thornborrow E.C. Manfredi J.J. J. Biol. Chem. 1999; 274: 33747-33756Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). While p53 mutation or inactivation is detected in over 60% of human cancers, p53 mutations are rare in the early stages of HCC (17Feitelson M.A. Zhuy M. Duan L.X. London W.T. Oncogene. 1993; 8: 1109-1117PubMed Google Scholar). However, p53-HBx interaction may disrupt p53 function, leading to genomic instability and accumulation of p53 mutations. Multiple studies support HBx-mediated p53 dysfunction, but the exact mechanism by which HBx inactivates p53 remains unclear. Strong support for HBx disruption of p53 function during development of HCC was demonstrated by a transgenic mouse study in which mice expressing HBx developed liver tumors with 80–90% penetrance within 4 months of birth (18Kim C. Koike K. Saito I. Miyamura T. Jay G. Nature. 1991; 351: 317-320Crossref PubMed Scopus (1056) Google Scholar, 19Ueda H. Ullrich S.J. Gangemi J.D. Kappel C.A. Ngo L. Feitlson M.A. Jay G. Nature Gen. 1995; 9: 41-47Crossref PubMed Scopus (328) Google Scholar). In these studies, HBx protein and murine p53 protein were found sequestered in the cytoplasm. These studies suggested that HBx-p53 interaction prevented entry into the nucleus. However, more recent studies demonstrated nuclear localization of HBx (reviewed in Ref. 20Murakami S. Intervirology. 1999; 42: 81-99Crossref PubMed Scopus (140) Google Scholar), where it could potentially disrupt the ability of nuclear p53 to bind DNA (9Wang X.W. Forrester K. Yeh J. Feitelson M.A. Gu J.-R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (637) Google Scholar), communicate with general transcription factors, or form stable tetramers (10Truant R. Antunovic J. Greenblat J. Prives C. Cromlish J. J. Virol. 1995; 69: 1851-1859Crossref PubMed Google Scholar). We have shown recently that p53 represses transcription of the α-fetoprotein (AFP) gene by sequence-specific DNA binding (15Lee K.C. Crowe A.J. Barton M.C. Mol. Cell. Biol. 1999; 19: 1279-1288Crossref PubMed Scopus (152) Google Scholar). This repression is both tissue- and developmental-specific, and contributes to the developmental regulation pattern of AFP expression. AFP is secreted by the visceral endoderm of the yolk sac, and is the predominant fetal serum protein synthesized by the developing liver (21Godbout R. Ingram R. Tilghman S.M. Mol. Cell. Biol. 1986; 6: 477-487Crossref PubMed Scopus (115) Google Scholar, 22Crandall B.F. Crit. Rev. Clin. Lab. Sci. 1981; 15: 127-185Crossref PubMed Scopus (96) Google Scholar). At birth, AFP expression is silenced: its mRNA level is down-regulated approximately 10,000-fold. This nearly undetectable expression is maintained throughout adult life, except in cases of liver regeneration and/or HCC where AFP expression is reactivated in 75–80% of hepatocarcinomas (23Belayew A. Tilghman S.M. Mol. Cell. Biol. 1982; 2: 1427-1435Crossref PubMed Scopus (99) Google Scholar, 24Camper S.A. Godbout R. Tilghman S.M. Prog. Nucleic Acids Res. Mol Biol. 1989; 36: 131-143Crossref PubMed Scopus (21) Google Scholar). In these studies we have utilized regulation of AFP expression in a cell-free assay system as a marker for HBx-mediated disruption of p53 function. By employing fractionated Xenopus egg extracts to assemble AFP templates into chromatin prior to in vitrotranscription analysis, we have examined regulated AFP expression devoid of effects mediated by nonspecific transcriptional squelching. Our results demonstrate that HBx can destroy the ability of p53 to regulate AFP in a tissue-specific manner. Additionally, the data demonstrate that the mechanism by which HBx alleviates p53-mediated repression of AFP transcription is by physically interacting with p53, blocking interaction with hepatic-specific proteins that may act in transcription repression. The AFP/lacZ template contains 3.8 kilobases of the AFP 5′-flanking sequence, including the previously defined distal and proximal promoter elements and enhancer I, fused to the β-galactosidase coding region (25Spear B.T. Longley T. Moulder S. Wang S.L. Peterson M.L. DNA Cell Biol. 1995; 14: 635-642Crossref PubMed Scopus (21) Google Scholar, 26Spear B.T. Mol. Cell. Biol. 1994; 14: 6497-6505Crossref PubMed Google Scholar). The AFP/DelA template was prepared from the AFPmut5 plasmid previously described (15Lee K.C. Crowe A.J. Barton M.C. Mol. Cell. Biol. 1999; 19: 1279-1288Crossref PubMed Scopus (152) Google Scholar) by three-step polymerase chain reaction (27Nelson R. Long G. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar). The 10-base pair deletion within the p53-binding site, located at −853 relative to the AFP transcriptional start site, was created through polymerase chain reaction amplification using the AFP/mut5 template and the following primers: A, 5′-CCTCCATTTTATGAGTACACTATA-3′; B, 5′-GTGTCTTAAGCGTTGCTAAGG-3′; C, 5′-CGAGGGGAAAATAGGTGGTTGCGCG-3′; D, 5′-CCTTAGCAACGCTTAAGACAC-3′. A and B primers were used in the 5′ amplification step, with C and D primers used in the 3′ amplification step. Primers A and D were used for final amplification. The polymerase chain reaction product containing the mutated p53-binding site was subcloned into the TA vector, pCR2.1 (Invitrogen), and recovered byBamHI and HindIII restriction digest and gel isolation. The fragment was subcloned into an AFP(−1.0)/lacZ template lacking enhancer I (25Spear B.T. Longley T. Moulder S. Wang S.L. Peterson M.L. DNA Cell Biol. 1995; 14: 635-642Crossref PubMed Scopus (21) Google Scholar, 26Spear B.T. Mol. Cell. Biol. 1994; 14: 6497-6505Crossref PubMed Google Scholar). The −3.8 to −1-kilobase enhancer I region was subsequently cloned as a BamHI fragment into this intermediate plasmid to yield AFP/DelA. The β-globin template contains the chick β-globin upstream promoter, structural gene, and enhancer. Its construction has been previously described (28Emerson B.M. Nickol J.M. Fong T.C. Cell. 1989; 57: 1189-1200Abstract Full Text PDF PubMed Scopus (45) Google Scholar). Recombinant histidine-tagged p53Δ30 protein (29Hupp T.R. Meek D.W. Midgley C.A. Lane D.P. Cell. 1992; 71: 875-886Abstract Full Text PDF PubMed Scopus (866) Google Scholar), lacking 30 amino acids from the C terminus of the protein, was prepared from the pET23bp53Δ30 plasmid as described previously for HNF-3α protein residing in inclusion bodies (30Zaret K.S. Stevens K. Proc. Exp. Purif. 1995; 6: 821-825Crossref PubMed Scopus (24) Google Scholar), or as described below for the soluble p53 protein fraction. Briefly, 500-ml cultures were grown at 37 °C to anA 600 of 0.4. Protein production was induced by addition of isopropyl-1-thio-β-d-galactopyranoside to a 1 mm final concentration. Cultures were grown for an additional 2 h, then collected, pelleted, and resuspended in 20 ml of 1 × binding buffer (5 mm imidazole, 0.5m NaCl, 20 mm Tris-HCl, pH 7.9). Cells were sonicated on ice for 40 s. The crude protein extract was collected by centrifugation at 24,000 × g for 10 min at 4 °C. Soluble p53 was purified from the crude fraction by affinity chromatography over a Ni2+-NTA agarose column (Qiagen). Bound protein was washed and eluted as described (30Zaret K.S. Stevens K. Proc. Exp. Purif. 1995; 6: 821-825Crossref PubMed Scopus (24) Google Scholar). Purified p53 was dialyzed against dialysis buffer (20 mm Tris-HCl, pH 8.0, 0.5 mm EDTA, pH 8.0, 100 mm KCl, 20% glycerol, 1.0 mm phenylmethylsulfonyl fluoride, 0.5 mmdithiothreitol). Recombinant histidine-tagged HBx was prepared from the pRSETc::X plasmid as described previously (31Haviv I. Vaizel D. Shaul Y. EMBO J. 1996; 15: 3413-3420Crossref PubMed Scopus (87) Google Scholar). HepG2 whole cell extracts were prepared as described previously, (32Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar, 33Crowe A.J. Sang L. Lee K.C. Spear B.T. Barton M.C. J. Biol. Chem. 1999; 274: 25113-25120Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). HeLa cell nuclear extracts were prepared exactly as described in Ref. 34Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Janssen K. Current Protocols in Molecular Biology. 1st Ed. John Wiley & Sons, Inc., Boston1987: 12.1.1-12.1.9Google Scholar. Xenopus egg extracts were prepared exactly as described previously (35Crowe A.J. Barton M.C. Methods. 1999; 17: 173-187Crossref PubMed Scopus (11) Google Scholar, 36Barton M.C. Emerson B.M. Methods Enzymol. 1996; 274: 299-312Crossref PubMed Scopus (9) Google Scholar). High speed supernatant soluble fractions used for chromatin assembly had protein concentrations ranging from 50 to 100 μg/μl. In vitro transcription analysis of templates as chromatin-free (naked) and chromatin-assembled DNA was performed as described previously (33Crowe A.J. Sang L. Lee K.C. Spear B.T. Barton M.C. J. Biol. Chem. 1999; 274: 25113-25120Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), with minor modifications. For naked DNA transcriptions, recombinant p53 and HBx proteins were added to 500 ng of supercoiled DNA templates in transcription reaction mixture and allowed to bind for 5 min at room temperature prior to addition of transcribing extract. RNA products were purified and analyzed by primer extension. Solid-phase DNA templates for chromatin transcriptions were prepared as described (33Crowe A.J. Sang L. Lee K.C. Spear B.T. Barton M.C. J. Biol. Chem. 1999; 274: 25113-25120Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 35Crowe A.J. Barton M.C. Methods. 1999; 17: 173-187Crossref PubMed Scopus (11) Google Scholar). Briefly, AFP DNA was digested with EcoRI and ClaI, then biotinylated with Biotin-21 dUTP and Biotin-14 dATP and Klenow fragment DNA polymerase (Life Technologies, Inc.) prior to coupling to streptavidin-coated, paramagnetic beads (Dynal). In chromatin transcription reactions, p53 and HBx were added to 500 ng of solid-phase DNA templates during a 20-min preincubation in HepG2 or HeLa cellular extracts prior to chromatin assembly. After 1 h chromatin assembly in fractionated Xenopus egg extract, solid-phase DNA templates were washed three times in modified nuclear dialysis buffer (mNDB) (20 mm Hepes, pH 7.9, 50 mm KCl, 0.2 mm EDTA, 10% glycerol, 1 mm dithiothreitol) plus 0.01% Nonidet P-40, then transcribed in HeLa extract and analyzed as above. Immunoprecipitations were performed in HepG2 whole cell extract or HeLa nuclear extract diluted to a total protein concentration of 8 μg/μl in mNDB. Recombinant p53 (approximately 600 ng) and HBx proteins (approximately 300 ng) were added to 25 μl of cellular extract and incubated at 4 °C for 20 min. Anti-p53 antibody (Santa Cruz pAB 240) was added to the reaction mixture and bound for 1 h in the presence of 1% Nonidet P-40 with gentle rocking at 4 °C. Immunocomplexes were collected with Protein A/Protein G+ agarose beads (Santa Cruz) equilibrated in mNDB plus 1% Nonidet P-40. Immunocomplexes were washed three times with IP wash buffer (100 mm Tris, 1 m NaCl, 0.3% SDS), then resuspended in sample buffer (0.06 m Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 0.001% bromphenol blue). Additional wash buffers used included: AC wash buffer 1 (100 mm Tris, pH 8.0, 1 m NaCl); AC wash buffer 2 (100 mm Tris, pH 8.0, 100 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 0.3% SDS); AC wash buffer 3 (10 mm Tris, pH 8.0, 0.1% SDS); and mNDB wash buffer (mNDB + 1% Nonidet P-40). Samples were analyzed by SDS-PAGE and Western blot. Western blots were performed as described previously (37Crowe A.J. Hayman M.J. Oncogene. 1993; 8: 181-189PubMed Google Scholar), with minor modifications. Membranes were probed with the appropriate antibody (anti-p53 pAB 240, Santa Cruz, and anti-HBx 11/121/52, gift of Dr. Claus H. Schroder, Virus-Host Interactions, German Cancer Center (38Su Q. Schroder C.H. Hofmann W.J. Otto G. Pichlmayr R. Bannasch P. Hepatology. 1998; 27: 1109-1120Crossref PubMed Scopus (193) Google Scholar)). Binding was visualized by ECL Western blot Analysis System (Amersham Pharmacia Biotech). EMSA was performed using the double-stranded p53 regulatory element from the AFP distal promoter (bases −862 through −830) 5′-GATCCTTAGCAAACATGTCTGGACCTCTAGAC as described previously (15Lee K.C. Crowe A.J. Barton M.C. Mol. Cell. Biol. 1999; 19: 1279-1288Crossref PubMed Scopus (152) Google Scholar), with protein-DNA binding carried out for 30 min at 30 °C. Protein binding assays contained 7 μg of HepG2 or HeLa cell extract and approximately 1 μg of purified p53 protein and 1 μg of purified HBx protein, except as indicated. Solid-phase DNA oligomers were generated by annealing 5′-biotinylated p53 regulatory element (5′ Bio-GATCCTTAGCAAACATGTCTGGACCTCTAGAC) (Life Technologies, Inc.) to complementary strand prior to coupling to streptavidin-coated paramagnetic beads (Dynal). Control reactions to assess protein binding specificity were performed in parallel with AFP site −1007 (5′ Bio-GATCCAATATCCTCTTCAG) solid-phase DNA oligomers prepared in the same way. Approximately 200 ng of p53 regulatory element or −1007 solid-phase oligos were washed in 1 × phosphate-buffered saline, 1% bovine serum albumin prior to incubation with 1 μg of p53 in the presence or absence of HBx (1 μg) and/or 70 μg (total protein) of HepG2 or HeLa cell extract. Binding reactions proceeded for 30 min at 22 °C. DNA-bound protein complexes were collected by magnetic concentration and washed two times in 1 × phosphate-buffered saline, 1% Nonidet P-40 and once in wash buffer (100 mm NaCl, 50 mm Bis-Tris HCl, 10 mm MgCl2, 1 mm dithiothreitol, pH 6.0) prior to elution. DNA-associated proteins were eluted for 10 min at 37 °C in urea elution buffer (5 m urea, 10 mm Tris, pH 8.0, 100 mmNaH2P04, 1% β-mercaptoethanol). Eluted proteins were analyzed by gel electrophoresis and silver stain or Western blot. Image analysis was performed by use of ImageQuant 5.0 software (Molecular Dynamics). In order to examine the regulatory consequences of HBx transactivator expression on a hepatic-expressed cellular gene, we performed in vitro transcription analysis of AFP DNA templates in the presence of HBx and p53 proteins. Transcription extracts isolated from the human hepatoma cell line HepG2, which actively expresses AFP but does not carry integrated HBV DNA, were used for in vitrotranscription (33Crowe A.J. Sang L. Lee K.C. Spear B.T. Barton M.C. J. Biol. Chem. 1999; 274: 25113-25120Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). We utilized a constitutively activated p53 protein harboring a C-terminal truncation to examine the ability of p53 to regulate AFP transcription independently of post-translational modifications within the protein C terminus, which activate p53 for DNA binding (13Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar). Addition of recombinant p53 protein resulted in 2–3-fold repression of AFP transcribed as naked (chromatin-free) DNA in HepG2 extract (Fig. 1 A, lanes 2 and 3, compared with lane 1). Addition of recombinant HBx protein to p53-repressed transcription reactions alleviated p53-mediated repression (Fig. 1 A, compare lanes 3and 4). The level of AFP transcription detected upon addition of HBx was derepressed 4-fold to a level slightly higher than transcription in the absence of p53 (Fig. 1 A, comparelanes 1 and 4). Addition of HBx without p53 resulted in modest activation (less than 2-fold) of AFP transcription (Fig. 1 A, compare lanes 1 and 5), suggesting that the observed activation of AFP is dependent primarily upon HBx effects on p53, rather than HBx association with hepatoma-enriched transcriptional activators.Figure 3HBx alleviates p53-mediated repression of chromatin assembled AFP DNA. Immobilized AFP templates were incubated with nuclear extract buffer (lanes 1 and2) or HepG2 whole cell extract (20 μl: approximately 200 μg of total protein; lanes 3–6) prior to 1 h chromatin assembly in fractionated Xenopus egg extract. Reactions were supplemented with recombinant p53 protein (370 ng,lane 4; 1.8 μg, lane 5) or recombinant p53 (1.8 μg) plus recombinant HBx (470 ng, lane 6). Chromatin-assembled templates were washed in nuclear extract buffer andin vitro transcribed in HeLa nuclear extract. AFP primer extension products are indicated.View Large Image Figure ViewerDownload (PPT) AFP is expressed in the fetus by endoderm-derived cells of the yolk sac, liver, and gut (39Tilghman S.M. Belayew A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 5254-5257Crossref PubMed Scopus (286) Google Scholar, 40Tilghman S.M. Oxf. Surv. Eukaryotic Genes. 1985; 2: 160-206PubMed Google Scholar). We have previously shown by cell culture transfection studies that p53 repression of AFP is tissue-specific (15Lee K.C. Crowe A.J. Barton M.C. Mol. Cell. Biol. 1999; 19: 1279-1288Crossref PubMed Scopus (152) Google Scholar). To determine if the observed effects of HBx on p53-regulated AFP expression were also tissue-specific, we performed in vitrotranscription analysis in cervical cancer-derived HeLa cell extract. In contrast to the observed repression of AFP transcription following addition of p53 to HepG2 reactions, p53 introduction to HeLa transcription reactions resulted in modest activation of AFP expression (less than 2-fold) (Fig. 1 B, compare lane 1 withlanes 2 and 3). HBx addition to transcription reactions in the presence of p53 slightly augmented this activation to a level of 3-fold over basal expression (Fig. 1 B,lanes 4 and 5). This is in sharp contrast to results observed in the hepatoma extract where HBx addition strongly reversed p53 effects on AFP transcription. Addition of low concentrations of HBx protein, in the absence of p53, activated AFP expression approximately 2-fold (Fig. 1 B, compare lane 1 with lanes 6 and 7). This level of HBx-mediated activation, in the absence of p53, is comparable to that observed in hepatoma extracts, again supporting the hypothesis that HBx-mediated activation of AFP is due primarily to a modification of p53 regulation. Interestingly, increasing concentrations of HBx protein in the absence of p53 did not activate AFP transcription, but diminished transcription to basal levels (Fig.1 B, compare lanes 1 and 8). This result could be due to apparent squelching by HBx through self-oligomerization or transcription factor binding. HBx has not previously been demonstrated to squelch transcription; however, because of its ability to associate with multiple transcriptional activators and general transcription factors (GTFs) (reviewed in Ref. 4Andrisani O.M. Barnabas S. Intl. J. Cancer. 1999; 15: 373-379Google Scholar), addition of high concentrations of HBx in the absence of p53 may promote nonfunctional HBx-protein interactions. To determine if derepression of p53-regulated AFP transcription was due to a direct interaction between HBx and p53 that occurs only in a hepatoma extract, we performed immunoprecipitations with anti-p53 antibody (pAB 240, Santa Cruz) in both HepG2 and HeLa transcription extracts. p53 and HBx proteins incubated in HepG2 extract formed a complex, as shown by immunoprecipitation and Western blot analysis with anti-p53 (pAB 240) and anti-HBx (11/121/52) antibodies (Fig. 1 C, lane 3). Additionally, p53 and HBx interacted in both HeLa extract and extract buffer (Fig. 1 C, lanes 6 and 8), demonstrating that p53 and HBx proteins form a stable complex in the absence of DNA or additional proteins present in hepatoma cell extracts. Taken together, these data demonstrate that the tissue-specific effects of HBx on p53-regulated AFP transcription are not due to an inability of the proteins to form a stable complex in a non-hepatic cell extract, but rather are likely due to tissue specificity of p53 transcriptional repression. The ability of p53 to repress in vitrotranscription of AFP templates could be explained by a number of mechanisms. Multiple regions of p53 protein interact with and bind a wide range of proteins mediating, in part, the pleiotropic functions of the tumor suppressor (13Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2294) Google Scholar, 14Levine A. Cell. 1997; 88: 323-331Abstract Full Text Full Text PDF PubMed Scopus (6759) Google Scholar). p53 can interact with GTFs including TFIID subunits TBP, TAF31, and TAF70, and TFIIH subunits p62, XPB, and XPD. The ability of p53 to squelch transcription through interactions with GTFs, particularly TPB, is well documented (41Seto E. Usheva A. Zambetti G.P. Momand J. Horikoski N. Weinmann R. Levine A.J. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 12028-12032Crossref PubMed Scopus (464) Google Scholar, 42Ragimov N. Krauskopf A. Navot N. Rotter V. Oren M. Aloni Y. Oncogene. 1993; 8: 1183-1193PubMed Google Scholar, 43Horikoshi N. Usheva A. Chen J. Levine A.J. Weinmann R. Shenk T. Mol. Cell. Biol. 1995; 15: 227-234Crossref PubMed Scopus (162) Google Scholar). If p53-mediated AFP repression was due in part to p53 squelching of TBP in the hepatoma extract, the apparent derepression upon HBx addition could be due to HBx disruption of p53-TBP or p53-GTF interactions. To determine if HBx could reverse p53-mediated transcriptional squelching, in vitro transcription analysis was performed using chick β-globin DNA as template. β-Globin DNA has no p53-binding site and is not directly regulated by p53 (data not shown). In the presence of high levels of p53 protein, apparent transcriptional repression can be attributed, most likely, to p53-mediated squelching of basal transcription factors. As demonstrated in Fig.2, addition of increasing amounts of p53 to β-globin in vitro transcription reactions resulted in greater than 5-fold squelching of transcription (compare lane 1 with lanes 2–4). Addition of HBx resulted in very little (1.5-fold) reactivation of squelched transcription (Fig. 2, compare lanes 5 and 6 with lane 4). Addition of HBx in the absence of p53 resulted in approximately 2-fold repression of transcription, potentially due to HBx-mediated squelching, as discussed above (Fig. 2, compare lanes 1 and7). Because HBx reactivation of p53-squelched β-globin transcription was much lower than the reactivation of p53-repressed AFPin vitro transcription, we suspected that HBx alleviation of p53-mediated AFP repression was not due simply to reversal of p53 squelching. This inability of HBx to activate transcription from a promoter lacking a p53-binding site suggested that HBx must be targeted to DNA by promoter-bound p53 in order to render its activating effects. In order to demonstrate conclusively that HBx-mediated derepression of AFP expression in our in vitro transcription system was not an effect on squelching, we utilized in vitro chromatin assembly of AFP DNA templates prior to transcription analysis. Solid-phase AFP DNA templates were prepared by coupling biotinylated AFP DNA to paramagnetic beads as described previously (44Crowe A.J. Barton M.C. Methods Enzmol. 1999; 304: 63-76Crossref PubMed Scopus (7) Google Scholar). Chromatin assembly was achieved by incubating the solid-phase DNA templates in fractionated Xenopus egg extracts (33Crowe A.J. Sang L. Lee K.C. Spear B.T. Barton M.C. J. Biol. Ch
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