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• W2078806556 abstract "Enhancer II (ENII) of hepatitis B virus (HBV) is one of the essential cis-elements for the transcriptional regulation of HBV gene expression. Its function is highly liver-specific, suggesting that liver-enriched transcriptional factors play critical roles in regulating the activity of ENII. In this report, a novel hepatocyte transcription factor, which binds specifically to the B1 region (AACGACCGACCTTGAG) within the major functional unit (B unit) of ENII, has been cloned from a human liver cDNA library by yeast one-hybrid screening, and demonstrated to trans-activate ENII via the B1 region. We named this factor hB1F, for human B1-binding factor. Amino acid analysis revealed this factor structurally belongs to nuclear receptor superfamily. Based on the sequence similarities, hB1F is characterized to be a novel human homolog of the orphan receptor fushi tarazu factor I (FTZ-F1). Using reverse transcription-polymerase chain reaction, a splicing isoform of hB1F (hB1F-2) was identified, which has an extra 46 amino acid residues in the A/B region. Examination of the tissue distribution has revealed an abundant 5.2-kilobase transcript of hB1F is present specifically in human pancreas and liver. Interestingly, an additional transcript of 3.8 kilobases was found to be present in hepatoma cells HepG2. Fluorescent in situ hybridization has mapped the gene locus of hB1F to the region q31-32.1 of human chromosome 1. Altogether, this study provides the first report that a novel human homolog of FTZ-F1 binds and regulates ENII of HBV. The potential roles of this FTZ-F1 homolog in tissue-specific gene regulation, in embryonic development, as well as in liver carcinogenesis are discussed. Enhancer II (ENII) of hepatitis B virus (HBV) is one of the essential cis-elements for the transcriptional regulation of HBV gene expression. Its function is highly liver-specific, suggesting that liver-enriched transcriptional factors play critical roles in regulating the activity of ENII. In this report, a novel hepatocyte transcription factor, which binds specifically to the B1 region (AACGACCGACCTTGAG) within the major functional unit (B unit) of ENII, has been cloned from a human liver cDNA library by yeast one-hybrid screening, and demonstrated to trans-activate ENII via the B1 region. We named this factor hB1F, for human B1-binding factor. Amino acid analysis revealed this factor structurally belongs to nuclear receptor superfamily. Based on the sequence similarities, hB1F is characterized to be a novel human homolog of the orphan receptor fushi tarazu factor I (FTZ-F1). Using reverse transcription-polymerase chain reaction, a splicing isoform of hB1F (hB1F-2) was identified, which has an extra 46 amino acid residues in the A/B region. Examination of the tissue distribution has revealed an abundant 5.2-kilobase transcript of hB1F is present specifically in human pancreas and liver. Interestingly, an additional transcript of 3.8 kilobases was found to be present in hepatoma cells HepG2. Fluorescent in situ hybridization has mapped the gene locus of hB1F to the region q31-32.1 of human chromosome 1. Altogether, this study provides the first report that a novel human homolog of FTZ-F1 binds and regulates ENII of HBV. The potential roles of this FTZ-F1 homolog in tissue-specific gene regulation, in embryonic development, as well as in liver carcinogenesis are discussed. hepatitis B virus enhancer II enhancer I core promoter human B1-binding factor open reading frame GAL4 activation domain electrophoresis mobility shift assay chloramphenicol acetyltransferase fluorescent in situhybridization fushi tarazu factor 1 murine steroidogenic factor 1 murine embryonal long terminal repeat binding protein bovine adrenal AD4-binding protein human steroidogenic factor 1 zebrafish FTZ-F1 A and B Xenopus FTZ-F1-related A and B murine liver receptor homolog 1 rat fetoprotein transcription factor D. melanogaster FTZ-F1 mutant B1 activation domain cytomegalovirus polymerase chain reaction reverse transcriptase DNA-binding domain nucleotide base pair(s) kilobase pair(s) secreted placental alkaline phosphatase. Hepatitis B virus (HBV)1is the major cause of acute and chronic hepatitis, also closely associated with the development of hepatocellular carcinoma (1Raney A.K. McLachlan A. MaLachlan A. Molecular Biology of the Hepatitis B Virus. CRC Press, Boca Raton, FL1991: 1-37Google Scholar). HBV predominantly infects hepatocytes, which is a prominent characteristic of hepadnaviruses. The genome of HBV is a small, circular, partially double-stranded DNA of about 3.2 kb, which contains four partially overlapping open reading frames (ORF) encoding the surface antigens (preS/S), the core antigen/e antigen (preC/C), the polymerase (P) and the X protein (X), respectively (2Garnem D. Varmus H.E. Annu. Rev. Biochem. 1987; 56: 651-693Crossref PubMed Scopus (821) Google Scholar, 3Tiollais P. Pourcel C. Dejean A. Nature. 1985; 317: 489-495Crossref PubMed Scopus (977) Google Scholar). These HBV genes express specifically in liver, controlled by the combinatorial action of the promoters and enhancers. To date, four promoters (Sp1, Sp2, Cp, and Xp) (4Schaller H. Fisher M. Curr. Top. Microbiol. Immunol. 1991; 168: 21-39Crossref PubMed Scopus (79) Google Scholar) have been identified to be responsible for the transcription of the viral mRNAs, and they are regulated by two HBV enhancers. Enhancer I (ENI) functions in a relatively tissue independent manner (5Shaul Y. Rutter W.J. Laub O. EMBO J. 1985; 4: 427-430Crossref PubMed Scopus (165) Google Scholar, 6Tognoni A. Cattaneo R. Serfling E. Schaffner W. Nucleic Acids Res. 1985; 13: 7457-7472Crossref PubMed Scopus (57) Google Scholar), while enhancer II (ENII) shows strong hepatocyte specificity. Enhancer II of HBV is located within the X ORF, about 600 bp downstream of ENI (7Wang Y. Chen P. Wu X. Sun A.L. Wang H. Zhu Y.A. Li Z.P. J. Virol. 1990; 64: 3977-3981Crossref PubMed Google Scholar, 8Yee J.K. Science. 1989; 246: 658-661Crossref PubMed Scopus (132) Google Scholar, 9Yuh C.H. Ting L.P. J. Virol. 1993; 67: 142-149Crossref PubMed Google Scholar). In our previous study, ENII was mapped in a 148-bp region from nt 1627 to 1774 (HBV adr1 subtype), partially overlapped with the core promoter (Cp) (7Wang Y. Chen P. Wu X. Sun A.L. Wang H. Zhu Y.A. Li Z.P. J. Virol. 1990; 64: 3977-3981Crossref PubMed Google Scholar). According to the functional analyses, it can be divided into two parts, A and B, with part B as the basal functional unit, which could be further subdivided into three regions B1 (nt 1687–1704), B2 (nt 1705–1735), and B3 (nt 1736–1774) (Fig. 1) (7Wang Y. Chen P. Wu X. Sun A.L. Wang H. Zhu Y.A. Li Z.P. J. Virol. 1990; 64: 3977-3981Crossref PubMed Google Scholar, 10Wu X. Zhu L. Li Z.P. Koshy R. Wang Y. Virology. 1992; 191: 490-494Crossref PubMed Scopus (18) Google Scholar). ENII plays critical roles in the regulation of the liver-specific viral gene transcription. It is a major cis element regulating the liver-specific transcription initiated by Cp, which is critical for viral replication and morphogenesis (11Honigwachs J. Faktor O. Dikstein R. Shaul Y. Laub O. J. Virol. 1989; 63: 919-924Crossref PubMed Google Scholar). ENII also participates in the regulation of other promoters of HBV genome, and is able to activate heterologous promoters in a highly liver-specific manner (7Wang Y. Chen P. Wu X. Sun A.L. Wang H. Zhu Y.A. Li Z.P. J. Virol. 1990; 64: 3977-3981Crossref PubMed Google Scholar, 12Su H. Yee J.K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2708-2712Crossref PubMed Scopus (88) Google Scholar). To elucidate the regulatory mechanism of the liver-specific activity of ENII, a considerable amount of work has been focused on the study of trans-acting factors that interact with ENII. As shown in Fig. 1, many transcription factors have been identified to interact with different regions of ENII. Among these factors, a set of liver-enriched transcription factors such as HNF1, C/EBP, HNF3, and HNF4 are believed to be involved in the determination of hepatocyte specificity of ENII. Recently, we found the B1 region (nt 1688-AACGACCGACCTTGAG-nt 1703) was bound specifically by a novel hepatocyte nuclear factor (B1-binding factor, Fig. 1). 2Y. Xie, M. Li, Y. Wang, P. H. Hofschneider, and L. Weiss, submitted for publication. This binding activity is present in differentiated hepatoma cell line HepG2 but not in the nonhepatic cell line HeLa. Mutations in the B1 region not only aborted the specific binding by this factor, but also significantly reduced the activity of ENII in co-transfection analysis. In addition, the disruption of the specific binding was shown to cause a decrease in viral gene transcription initiated from the core promoter, resulting in a reduction of the 3.5-kb pregenomic RNA (13Xie, Y. H. (1997) Interaction of Cellular Transcription Factors with Enhancer II of Hepatitis B Virus. Ph.D. thesis, Shanghai Institute of Biochemistry, Chinese Academy of Sciences; Max-Plank-Institute of Biochemistry, GermanyGoogle Scholar, 14Xie Y.H. Wang Y. Hofschneider P.H. Weiss L. Molecular Biology of Hepatitis B Viruses Meeting. 1997; (, organized by C. Brechot and J. Summers, Institut Pasteur, Paris): O5Google Scholar). Therefore, this binding site has an important function in regulating the ENII activity, and consequently, it affects the global HBV gene expression. Our previous efforts to identify the B1-binding factor have suggested it not to be any members of known liver-enriched factors such as HNF1, HNF3, HNF4, or C/EBP (13Xie, Y. H. (1997) Interaction of Cellular Transcription Factors with Enhancer II of Hepatitis B Virus. Ph.D. thesis, Shanghai Institute of Biochemistry, Chinese Academy of Sciences; Max-Plank-Institute of Biochemistry, GermanyGoogle Scholar, 15Wang W.X. Li M. Wu X. Wang Y. Li Z.P. Res. Virol. 1998; 149: 99-108Crossref PubMed Scopus (36) Google Scholar), but a novel hepatocyte transcription factor uncharacterized before; it was thus considered of great interest to clone and characterized this factor. For this purpose, we employed a yeast one-hybrid screen (16Alexandre C. Grueneberg D.A. Gilman M.Z. Methods. 1993; 5: 147-155Crossref Scopus (20) Google Scholar, 17Li J.J Herskowitz I. Science. 1993; 262: 1870-1874Crossref PubMed Scopus (368) Google Scholar, 18Wang M.M. Reed R.R. Nature. 1993; 364: 121-126Crossref PubMed Scopus (371) Google Scholar) to clone the cDNA of the B1-binding factor. By screening GAL4 activation domain (AD) fused-cDNA library of human adult normal liver, we isolated a cDNA named hB1F (humanB1-binding factor) and investigated both its interaction with the B1 region in vitro and its trans-regulatory effects on ENII in vivo. hB1F was further characterized as a novel human homolog of FTZ-F1, an orphan nuclear receptor that was originally identified as a transcriptional factor involved in the regulation of fushi tarazu(ftz) expression in early Drosophila melanogasterembryos (19Lavorgna G. Ueda H. Clos J. Wu C. Science. 1991; 252: 848-851Crossref PubMed Scopus (247) Google Scholar). The two oligonucleotides, 5′-gatcAACGACCGACCTTGAG-3′ and 3′-TTGCTGGCTGGAACTCctag-5′, contained the B1 fragment in capital letters with extra nucleotides in lowercase letters added for cloning purpose, were chemically synthesized and annealed. Four tandem repeats of the B1 fragment were placed upstream of the E1b minimal promoter in HIS3 reporter plasmid (p6012), and the CYC1 minimal promoter in lacZ reporter plasmid (pCZII), respectively (CLONTECH). These two plasmids were linearized and sequentially integrated into Saccharomyces cerevisiae YM4271 (MAT a , ura3-52, his3-200, ade2-101, lys2-801, leu2-3, 112, trp1-903, tyr1-501) (CLONTECH) to obtain a reporter yeast strain YMB1-HB. The yeast was then transformed with human liver MATCHMAKER cDNA library (CLONTECH) and selected on histidine-deficient (His−) plates containing 3-aminotriazole (45 mm was shown optimal for suppressing HIS3 background growth). Large colonies (His+) were transferred onto Hybond N filters (Amersham Pharmacia Biotech) and further screened for β-galactosidase activity (20Breeden L. Nasmyth K. Cold Spring Harbor Symp. Quant. Biol. 1985; 50: 643-650Crossref PubMed Scopus (470) Google Scholar). After being placed in liquid nitrogen for 30 s, the filters were incubated in a buffer containing 0.8 mm6-bromo-4-chloro-3-indolyl-β-d-galactosidase at 30 °C. The positive interaction was determined by the appearance of blue colonies. The LacZ+ colonies were selected and plasmids were recovered (21Kaiser P. Auer B. BioTechniques. 1993; 14: 552PubMed Google Scholar). The candidate plasmids isolated from the positive clones were transformed into YMB1-HB to retest for His+phenotype and β-galactosidase activity. Those that could reproduce the positive phenotypes were called true positive clones and their cDNA inserts were sequenced and further characterized. EcoRI-digested 2.5-kb cDNA of the isolated positive clone (named pGAD-16) containing the complete hB1F coding sequence was inserted downstream of the T7 promoter of the pBS(+) (Stratagene), and used for in vitrotranslation in a typical 50-μl reaction volume of TNT coupled reticulocyte lysate systems (Promega), containing 25 μl of rabbit reticulocyte lysate, 1 μl of 1 mm amino acids, and 1 μg of DNA template. The BglII-NdeI fragment of pGAD-16 (nt 158–696) (see Fig. 3 B) containing the suspected DNA-binding domain of hB1F was made blunt-ended and in-frame fused with glutathione S-transferase by inserting into theSmaI digested pGEX-3X vector (Amersham Pharmacia Biotech). The glutathione S-transferase fusion protein was expressed in Eschericha coli. BL21 essentially as described by Smith and Johnson (22Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar), then was purified with glutathione-Sepharose 4B (Amersham Pharmacia Biotech). The nuclear extract of HepG2 cells and HeLa cells was prepared according to the method of Andreas and Faller (23Andreas N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2214) Google Scholar). The detailed procedure has been described previously (24Li M. Xie Y.H. Wu X. Kong Y.Y. Wang Y. Virology. 1995; 214: 371-378Crossref PubMed Scopus (62) Google Scholar). Briefly, cells were first suspended in 400 μl of buffer A (10 mm HEPES-KOH pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride), kept on ice for 15 min, and then homogenized in a Dounce homogenizer with a B pestle about 20–25 strokes. After centrifugation, the nuclear pellet was resuspended in 200 μl of buffer B (20 mm HEPES-KOH, pH 7.9, 420 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA, 1 mm dithiothreitol, 1 mmphenylmethylsulfonyl, fluoride, 25% glycerol), and kept on ice for 20 min. The supernatant was collected after centrifugation, immediately frozen in small aliquots in liquid nitrogen, and stored at −70 °C. Oligonucleotides used in EMSA were the following: B1, 5′-GATCAACGACCGACCTTGAG-3′ and 3′-TTGCTGGCTGGAACTCCTAG-5′; B1m (mutant B1, the mutated nucleotides are in lowercase letters), 5′-GATCAACtACaGAtCTcGAG-3′ and 3′-TTGaTGtCTaGAgCTCCTAG-5′. EMSA was performed as described previously (24Li M. Xie Y.H. Wu X. Kong Y.Y. Wang Y. Virology. 1995; 214: 371-378Crossref PubMed Scopus (62) Google Scholar). In the binding reaction,32P-end-labeled probe (0.1–1 ng, 10,000 cpm) was mixed with 1–2 μg of poly(dI-dC) (Amersham Pharmacia Biotech), 2–6 μg of nuclear extract, or 2 μl of in vitro translation product in a 20-μl reaction mixture containing 10 mmTris-HCl (pH 7.5), 90 mm NaCl, 0.15 mmMgCl2, 0.2 mm EDTA, 0.1 mmdithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 5% glycerol. After incubation at room temperature for 25 min, the mixture was resolved on a 5% nondenaturing polyacrylamide gel. In the competition assay, competitors were added to the reaction mixture and incubated at 0 °C for 10 min prior to the addition of the probe. The HBV genome fragment (nt 1634–1816) containing ENII and the core promoter (10Wu X. Zhu L. Li Z.P. Koshy R. Wang Y. Virology. 1992; 191: 490-494Crossref PubMed Scopus (18) Google Scholar) was amplified by PCR with [γ-32P]ATP-end-labeled 5′-primer (nt 1634-CCAGGTCTAGACCAAGGTC-nt 1651) and unlabeled 3′-primer (nt 1816-GGTGCTGGTTAACAGACCA-nt 1798), then used as a target DNA. The HBV sequence was derived from plasmid pADR-1, which contains an entire copy of the HBV genome of the subtype adr1 (25Gan R.B. Chu M.J. Shen L.P. Qian S.W. Li Z.P. Sci. Sin. 1987; 30: 507-521Google Scholar). For DNase I footprinting analysis, 2.5–7.5 μg of purified glutathioneS-transferase fusion protein was incubated with 5 μg of poly(dI-dC) and the probe in a 50-μl reaction mixture containing 10 mm Tris-HCl (pH 7.5), 90 mm NaCl, 0.15 mm MgCl2, 0.2 mm EDTA, 0.1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 5% glycerol for 25 min at room temperature. The DNase I digestion was then carried out as described in SureTrack footprinting kit (Amersham Pharmacia Biotech). To construct the CAT reporter plasmid pENII(B)/CpCAT, the ENII(B)/Cp fragment (nt 1686–1878) containing the basic functional unit of ENII (part B) and the Cp (10Wu X. Zhu L. Li Z.P. Koshy R. Wang Y. Virology. 1992; 191: 490-494Crossref PubMed Scopus (18) Google Scholar), was amplified by PCR and inserted into the XbaI and HindIII sites of pBS(+) (Stratagene). The CAT reporter gene was then cloned downstream of pENII(B)/Cp. Four point mutations of the B1 region (sequence was listed in an EMSA procedure) were introduced by PCR to construct mutant reporter plasmid pENII(B1m)/CpCAT. To construct the eukaryotic expression plasmid of hB1F, pCMV-hB1F, 2.5-kb hB1F cDNA from pGAD-16 released byEcoRI digestion was inserted downstream of CMV promoter in pCMV-poly vector (a gift from E. Lai). The antisense expression plasmid pAnti-hB1F, which transcribes hB1F in the antisense orientation, was constructed by inserting the hB1F cDNA in the reverse orientation downstream of CMV promoter in the same vector. HepG2 and HeLa cells were propagated in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 5% CO2 and 37 °C. DNAs were transfected into HepG2 or HeLa cells by the calcium phosphate precipitation method (26Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Cells were harvested 48 h after transfection and the protein extracts were prepared. CAT assay was performed according to the method of Gorman et al. (27Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar). In the co-transfection assay, different amounts of hB1F expression plasmid (pCMV-hB1F) and 1 μg of the reporter plasmid pENII(B)/CpCAT or pENII(B1m)/CpCAT were used; 1 μg of plasmid pCMV/SEAP (28Berger J. Hauber J. Hauber R. Geiger R. Cullen B.R. Gene (Amst.). 1988; 66: 1-10Crossref PubMed Scopus (583) Google Scholar), which expresses secreted placental alkaline phosphatase (SEAP), was included as internal control. The total amount of DNA plasmids used for each transfection experiment was 15 μg, and the plasmid pBS(+) (Stratagene) was used to make up the difference. Each transfection experiment was performed at least three times. Five primers were synthesized and used in the RT-PCR for amplification of the complete coding sequence of the hB1F in HepG2 cells: primer I, 5′-CCCCCAATCTCTTTTTGTTTTGAAAGC-3′ (nt 1591–1565 in hB1F); primer II, 5′-GAAAGCAGAGCTCCTAGGGGTTGTAAC-3′ (nt 1570–1544); primer III, 5′-GTAACTTATGCTCTTTTGGCATGCAAC-3′ (nt 1548–1522); primer IV, 5′-GAACTGCCTATAATTTCACTAAGAATGTC-3′ (nt 32–60); primer V, 5′-CTAAGAATGTCTTCTAATTCAGATACTGG-3′ (nt 50–78). 100 ng of poly(A)+-RNA from HepG2 or HeLa cells were reverse transcribed with 1 μl (200 units) of SUPERSCRIPT II RNase H− reverse transcriptase (Life Technologies, Inc.), using 2 pmol of primer I. The first round PCR was performed with primers I and IV, and the second round with primers II and V. To increase the specificity of the final PCR product, a third round PCR was performed with primers III and V. The resulting fragment was subcloned into pcDNA3 (Invitrogen) and subsequently sequenced. To examine the distribution of hB1F isoforms with difference within A/B regions, 1 μg of total RNA isolated separately from fetal liver, adult liver, HepG2 cells, and HeLa cells by TRIzol reagent (Life Technologies, Inc.) was reverse transcribed into cDNA with primer VI (5′-CTTGGATCACCTGAGACATGGCTTCTAGC-3′) (nt 521–493). Subsequently, first round PCR was performed using primer VI and primer IV, and second round using primer VII (5′-GTCTTTAAAGCACGGACTTACACCTATTG-3′) (nt 91–119) and primer VIII (5′-CTTCTAGCTTAAGTCCATTGGCTCGGATG-3′) (nt 500–472). The resulting PCR fragments were expected to include part of the A/B region and the C region, and were analyzed by electrophoresis on a 5% nondenaturing polyacrylamide gel. Multiple Tissue Northern blots containing 2 μg of poly(A)+ RNA per lane from multiple human adult tissues were purchased from CLONTECH. Blots were hybridized with random radiolabeled 2.5-kb hB1F cDNA probe (Prime-a-Gene labeling system, Promega) in ExpressHyb hybridization solution (CLONTECH) at 68 °C for 1 h. The filters were washed twice with wash solution I (0.3m NaCl, 0.03 m sodium citrate (pH 7.0), 0.05% SDS) for 20 min at room temperature, and twice with wash solution II (15 mm NaCl, 1.5 mm sodium citrate (pH 7.0), 0.1% SDS) for 30 min at 50 °C. The same filters were hybridized separately with a β-actin cDNA control probe. Twenty micrograms of total RNA isolated from HepG2 and HeLa cells by TRIzol reagent (Life Technologies, Inc.) were electrophoresed in 1.0% agarose-formaldehyde gels, blotted onto Hybond N+ nylon membranes (Amersham Pharmacia Biotech), and subjected to Northern blot hybridization as described above. A 2.5-kb hB1F cDNA probe was biotinylated with dATP using the Life Technologies, Inc. BioNick labeling kit. The FISH detection was performed in See DNA Biotech. Inc. (Canada), according to Heng and Tsui (29Heng H.H.Q. Tsui L.-C. Chromosoma. 1993; 102: 325-332Crossref PubMed Scopus (431) Google Scholar) and Heng et al.(30Heng H.H.Q. Squire J. Tsui L.-C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9509-9513Crossref PubMed Scopus (521) Google Scholar). The sequence of the hB1F cDNA determined in this study has been deposited in the GenBankTM data base under accession no. HSU80251. Our previous study has revealed that B1 region of HBV ENII is bound specifically by a novel liver-enriched nuclear factor (13Xie, Y. H. (1997) Interaction of Cellular Transcription Factors with Enhancer II of Hepatitis B Virus. Ph.D. thesis, Shanghai Institute of Biochemistry, Chinese Academy of Sciences; Max-Plank-Institute of Biochemistry, GermanyGoogle Scholar, 14Xie Y.H. Wang Y. Hofschneider P.H. Weiss L. Molecular Biology of Hepatitis B Viruses Meeting. 1997; (, organized by C. Brechot and J. Summers, Institut Pasteur, Paris): O5Google Scholar, 15Wang W.X. Li M. Wu X. Wang Y. Li Z.P. Res. Virol. 1998; 149: 99-108Crossref PubMed Scopus (36) Google Scholar). As shown in Fig. 2, using the 16-bp B1 fragment (AACGACCGACCTTGAG) as probe in EMSA, we detected a specific shift (band s) with the nuclear extract of the hepatoma HepG2 cell, but not with that of nonhepatic HeLa cell. When four mutations (AACtACaGAtCTcGAG) were introduced into the B1 fragment, the specific binding was no longer detected. Although HNF4 DNA binding consensus oligonucleotide in large excess amount seemed to be able to compete the B1 specific binding (Fig. 2), the possibility of HNF4 binding was ruled out since HNF4-specific antiserum failed to affect this specific band in a supershift assay (13Xie, Y. H. (1997) Interaction of Cellular Transcription Factors with Enhancer II of Hepatitis B Virus. Ph.D. thesis, Shanghai Institute of Biochemistry, Chinese Academy of Sciences; Max-Plank-Institute of Biochemistry, GermanyGoogle Scholar). As the interaction of the B1-binding factor with B1 fragment was of functional significance (13Xie, Y. H. (1997) Interaction of Cellular Transcription Factors with Enhancer II of Hepatitis B Virus. Ph.D. thesis, Shanghai Institute of Biochemistry, Chinese Academy of Sciences; Max-Plank-Institute of Biochemistry, GermanyGoogle Scholar, 14Xie Y.H. Wang Y. Hofschneider P.H. Weiss L. Molecular Biology of Hepatitis B Viruses Meeting. 1997; (, organized by C. Brechot and J. Summers, Institut Pasteur, Paris): O5Google Scholar), it was of great importance to identify this unknown factor. A yeast one-hybrid screen was employed to clone the B1- binding factor from human liver MATCHMAKER cDNA library (CLONTECH). In a total of approximately 4 × 106 transformants, 19 His+ colonies were selected, of which one colony showed strong β-galactosidase activity (LacZ+). The plasmid of this dual positive colony was recovered from the yeast, and named pGAD-16. When retransformed into the reporter yeast strain, pGAD-16 restored His and LacZ activity, indicating it was a true positive clone. Subsequent sequencing of the 2.5-kb cDNA insert of pGAD-16 revealed a 1.5-kb ORF. Surprisingly, the ORF is in an orientation reverse to the GAL4 AD (Fig. 3 A). This cDNA was probably transcribed by a cryptic promoter located around the ADH1 terminator region of the vector, 3M. Li and Y. Wang, unpublished data. and similar observations had been reported previously (31Chien C.-T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1225) Google Scholar). The complete sequence of the cDNA insert in pGAD-16 is shown in Fig. 3 B. The presence of an in-frame stop codon TAA upstream of the predicted initiation codon indicated that the cDNA insert contained the complete ORF encoding a protein of 495 amino acids. We named it hB1F for human B1-binding factor. The predicted molecular mass of 54 kDa for hB1F was verified by in vitro translation (data not shown). The amino acid sequence analysis showed that hB1F shared a common modular structure with the nuclear receptors (32Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Lumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6110) Google Scholar). It consists of subdomains A to E or F (Fig. 3 C), among which the C region (DNA-binding domain) and the E/F region (ligand-binding domain) are the two conserved domains of the nuclear receptor superfamily (33Evans R.M. Science. 1988; 240: 889-895Crossref PubMed Scopus (6341) Google Scholar, 34Tsai M.-J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2702) Google Scholar). The homology comparisons suggested hB1F to be a human homolog of orphan nuclear receptor FTZ-F1 (see below). Since the whole sequences including the DNA-binding domain and the activation domain have been encompassed in this factor, it is then not surprising that hB1F could activate the B1-driven HIS3 and lacZ reporter genes independent of GAL4 AD in the yeast screening. To confirm the binding specificity of the cloned hB1F for the B1 target sequence, in vitro translated hB1F was incubated with labeled B1 probe and analyzed by EMSA. As shown in Fig. 4 A, the formation of shifted complex (band s) could be efficiently competed by unlabeled B1 oligonucleotides, but not by B1m, or other nonspecific oligonucletides. Quite notably, the shifted complex (band s) migrated to a position similar to that of the complex found with the HepG2 nuclear extract (Fig. 2), suggesting that the binding fashion of the cloned hB1F to the B1 sequence was consistent with that of the B1-binding factor observed in the HepG2 nuclear extract. Since hB1F shares the common structure with nuclear receptors (Fig. 3,B and C), a partial hB1F cDNA (nt 158–696, encoding amino acids 35–215) containing the putative DNA-binding domain was fused with glutathione S-transferase and expressed in E. coli. The purified protein glutathioneS-transferase-hB1F(DBD) was able to bind to the B1 target region specifically (data not shown). To localize further the hB1F specific binding site(s) in HBV ENII and core promoter region, we performed a DNase I footprinting analysis, using the labeled fragment of HBV DNA (nt 1634–1816) as target. After incubation with the purified glutathione S-transferase-hB1F(DBD), one region of ENII (nt 1689-ACGACCGACCTTGAGGCA-nt 1706) was shown to be completely protected from DNase I digestion (Fig. 4 B). It agrees well with the B1 region of ENII (nt 1688-AACGACCGACCTTGAG-nt 1703). No other binding site of hB1F was found in ENII and Cp region. The above results provided strong evidence of the specific binding of hB1F to the B1 sequence. Together with the results obtained from a yeast one-hybrid screen, we have demonstrated that hB1F is the human cellular factor binding specifically to the B1 region of enhancer II of HBV. To investigate the functional importance of hB1F to the activity of enhancer II, we performed co-transfection analysis in nonhepatic cells (HeLa) which do not express hB1F protein (see the results of Northern blot analyses, Fig. 8 B). The CAT activity of the reporter plasmid pENII(B)/CpCAT is very low in HeLa cells due to the liver specificity of ENII (Fig. 5 A, lane 1) (24Li M. Xie Y.H. Wu X. Kong Y.Y. Wang Y. Virology. 1995; 214: 371-378Crossref PubMed Scopus (62) Google Scholar). When eukaryotic expression plasmid pCMV-hB1F, which contains hB1F cDNA under the control of CMV promoter was co-transfected, the CAT level of pENII(B)/CpCAT could be stimulated with the increasing amount of pCMV-hB1F (Fig. 5 A). However, if mutations were introduced into the B1 region, the activity of ENII could no longer be stimulated by hB1F (Fig. 5 B). These results indicated that hB1F activates ENII via the B1 site. The trans-activating effect" @default.
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• W2078806556 date "1998-11-01" @default.
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• W2078806556 title "Cloning and Characterization of a Novel Human Hepatocyte Transcription Factor, hB1F, Which Binds and Activates Enhancer II of Hepatitis B Virus" @default.
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• W2078806556 doi "https://doi.org/10.1074/jbc.273.44.29022" @default.
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