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• W2017019224 abstract "The hepatitis B virus X protein interacts with the basic-region, leucine zipper protein (bZip) domain of cAMP response element-binding protein increasing its affinity for the cAMP response element site in vitro and its transcriptional efficacyin vivo (Williams, J. S., and Andrisani, O. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3819–3823). Here we examine pX interactions with bZip transcription factors ATF3, gadd153/Chop10, ICER IIγ, and NF-IL6. We demonstrate direct interactions in vitro between pX and the bZip proteins tested. In contrast MyoD and Gal41–147 fail to interact with pX. We also demonstrate by the mammalian two-hybrid assay the direct interaction of pX with cAMP response element- binding protein, ICER IIγ, ATF3, and NF-IL6 in hepatocytes. In addition, pX increases the DNA binding potential of bZip proteins for their cognate DNA-binding site in vitro. In transient transfections in hepatocytes (AML12 cell line), pX increases the transcriptional efficacy of the bZip transcription factors. NF-IL6-mediated transcriptional activation is enhanced 3-fold by pX. Most interestingly, pX augments the repression mediated by bZip repressors ATF3 and ICER IIγ, by 6- and 7-fold, respectively, demonstrating for the first time the involvement of pX in gene repression. We conclude that pX is an enhancer of the DNA binding potential of bZip transcription factors, thereby increasing the transactivation or repression efficacy of bZip-responsive genes. The hepatitis B virus X protein interacts with the basic-region, leucine zipper protein (bZip) domain of cAMP response element-binding protein increasing its affinity for the cAMP response element site in vitro and its transcriptional efficacyin vivo (Williams, J. S., and Andrisani, O. M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3819–3823). Here we examine pX interactions with bZip transcription factors ATF3, gadd153/Chop10, ICER IIγ, and NF-IL6. We demonstrate direct interactions in vitro between pX and the bZip proteins tested. In contrast MyoD and Gal41–147 fail to interact with pX. We also demonstrate by the mammalian two-hybrid assay the direct interaction of pX with cAMP response element- binding protein, ICER IIγ, ATF3, and NF-IL6 in hepatocytes. In addition, pX increases the DNA binding potential of bZip proteins for their cognate DNA-binding site in vitro. In transient transfections in hepatocytes (AML12 cell line), pX increases the transcriptional efficacy of the bZip transcription factors. NF-IL6-mediated transcriptional activation is enhanced 3-fold by pX. Most interestingly, pX augments the repression mediated by bZip repressors ATF3 and ICER IIγ, by 6- and 7-fold, respectively, demonstrating for the first time the involvement of pX in gene repression. We conclude that pX is an enhancer of the DNA binding potential of bZip transcription factors, thereby increasing the transactivation or repression efficacy of bZip-responsive genes. The hepatitis B virus genome encodes a 16.5-kDa protein, termed X antigen (2Tiollais P. Pourcell C. DeJean A. Nature. 1985; 317: 489-495Crossref PubMed Scopus (977) Google Scholar), expressed during viral infection (3Kay A. Mandart E. Trepo C. Galibert F. EMBO J. 1985; 4: 1287-1292Crossref PubMed Scopus (72) Google Scholar, 4Moriarty A.M. Alexander H. Lerner R.A. Thornton G.B. Science. 1985; 227: 429-433Crossref PubMed Scopus (123) Google Scholar, 5Siddiqui A. Jameel S. Mapoles J. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 2513-2517Crossref PubMed Scopus (109) Google Scholar) and required for the viral life cycle (6Chen H.-S. Kaneko S. Girones R. Anderson R.W. Hornbuckle W.E. Tennant B.C. Cote P.J. Gerin J.L. Purcell R.H. Miller R.H. J. Virol. 1993; 67: 1218-1226Crossref PubMed Google Scholar, 7Zoulim F. Saputelli J. Seeger C. J. Virol. 1994; 68: 2026-2030Crossref PubMed Google Scholar). Expression of pX in liver of transgenic animals has been shown in some cases to induce liver cancer (8Kim C. Koike K. Saito I. Miyamura T. Jay G. Nature. 1991; 351: 317-351Crossref PubMed Scopus (1056) Google Scholar), implicating pX in the development of hepatocarcinogenesis in humans chronically infected with HBV. 1The abbreviations used are: HBV, hepatitis B virus; bZip, basic-region, leucine zipper proteins; HBV pX, hepatitis B virus X protein; GST, glutathione S-transferase; CRE, cAMP-response element; CREB, CRE-binding protein; ICER, inducible cAMP early repressor; HIV-LTR, human immunodeficiency virus-long terminal repeat; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; IL, interleukin; CAT, chloramphenicol acetyltransferase; RSV, Rous sarcoma virus. However, the mechanism of hepatocarcinogenesis remains unknown. Many studies have examined the role of pX in cellular signaling, but its mechanism of action remains obscure. HBV X is a multifunctional protein; it interacts in vitro and in vivo with the tumor suppressor p53 protein (9Feitelson M.A. Zhu M. Duan L.-X. London W.T. Oncogene. 1993; 8: 1109-1117PubMed Google Scholar, 10Wang X.W. Forresier K. Yeh H. 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, 11Truant R. Antunovic J. Greenblatt J. Prives C. Cromlish J.A. J. Virol. 1995; 69: 1851-1859Crossref PubMed Google Scholar), with the putative DNA repair protein XAP-1 (12Lee T.-H. Elledge S.J. Butel J.S. J. Virol. 1995; 69: 1107-1114Crossref PubMed Google Scholar), and acts as a promiscuous transactivator (13Rosner M.T. J. Med. Virol. 1992; 36: 101-117Crossref PubMed Scopus (192) Google Scholar). pX transactivates cis-acting elements, such as those present within the HBV (14Spandau D. Lee C.-H. J. Virol. 1988; 62: 427-434Crossref PubMed Google Scholar, 15Colgrove R. Simon G. Ganem D. J. Virol. 1989; 63: 4019-4026Crossref PubMed Google Scholar) and SV40 enhancers (16Twu J.S. Robinson W.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2046-2050Crossref PubMed Scopus (107) Google Scholar, 17Luber B. Burgelt E. Fromental C. Kanno M. Koch W. Virology. 1991; 184: 808-813Crossref PubMed Scopus (13) Google Scholar), and the HIV-LTR (18Seto E. Yen T. Peterlin B. Ou J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8286-8290Crossref PubMed Scopus (150) Google Scholar, 19Twu J.S. Chu K. Robinson W. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5168-5172Crossref PubMed Scopus (92) Google Scholar, 20Siddiqui A. Gaynor R. Srinivasan A. Mapoles J. Farr R.W. Virology. 1989; 169: 479-484Crossref PubMed Scopus (107) Google Scholar, 21Levrero M. Balsano C. Natoli G. Avantaggiati M.L. Elfassi E. J. Virol. 1990; 64: 3082-3086Crossref PubMed Google Scholar). X-responsive elements include the NF-κB (18Seto E. Yen T. Peterlin B. Ou J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8286-8290Crossref PubMed Scopus (150) Google Scholar, 20Siddiqui A. Gaynor R. Srinivasan A. Mapoles J. Farr R.W. Virology. 1989; 169: 479-484Crossref PubMed Scopus (107) Google Scholar, 21Levrero M. Balsano C. Natoli G. Avantaggiati M.L. Elfassi E. J. Virol. 1990; 64: 3082-3086Crossref PubMed Google Scholar, 22Twu J.-S. Wu J.Y. Robinson W.S. Virology. 1990; 177: 406-410Crossref PubMed Scopus (21) Google Scholar, 23Lucito R. Schneider R.J. J. Virol. 1992; 66: 983-991Crossref PubMed Google Scholar), AP-1 (24Seto E. Mitchell P.J. Yen T.S.B. Nature. 1990; 344: 72-74Crossref PubMed Scopus (213) Google Scholar, 25Twu J.-S. Lai M.-Y. Chen D.-S. Robinson W.S. Virology. 1993; 192: 346-350Crossref PubMed Scopus (110) Google Scholar, 26Kekule A.S. Lauer U. Weiss L. Luber B. Hofschneider P.H. Nature. 1993; 361: 742-745Crossref PubMed Scopus (336) Google Scholar, 27Benn J. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10350-10354Crossref PubMed Scopus (404) Google Scholar, 28Natoli G. Avantaggiati M.L. Chirillo P. Costanzo A. Artini M. Balsano C. Levrero M. Mol. Cell. Biol. 1994; 14: 989-998Crossref PubMed Scopus (125) Google Scholar), AP-2 (24Seto E. Mitchell P.J. Yen T.S.B. Nature. 1990; 344: 72-74Crossref PubMed Scopus (213) Google Scholar), and CRE (1Williams J.S. Andrisani O.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3819-3823Crossref PubMed Scopus (147) Google Scholar, 29Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (379) Google Scholar) sites. Interestingly, pX does not appear to bind double-stranded DNA. These multiple X-responsive, cis-acting elements suggest that the mechanism of pX action is pleiotropic. Studies to date support pX transactivation by a dual mechanism, i.e. both the activation of cytoplasmic signaling pathways and direct interactions with cellular transcription factors and the transcriptional machinery. Regarding this dual mechanism, pX activates in vivo the Ras, Raf, mitogen-activated protein kinase, and JNK signaling pathways, leading to transactivation of AP-1 and NF-κB sites (27Benn J. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10350-10354Crossref PubMed Scopus (404) Google Scholar, 30Doria M. Klein N. Lucito R. Schneider R.J. EMBO J. 1995; 14: 4747-4757Crossref PubMed Scopus (275) Google Scholar, 31Benn J. Su F. Doria M. Schneider R.J. J. Virol. 1996; 70: 4978-4985Crossref PubMed Google Scholar). pX interacts with several members of the basal transcriptional apparatus, such as the RPB5 subunit of eukaryotic RNA polymerases (32Cheong J.-H. Yi M.-K. Lin Y. Murakami S. EMBO J. 1995; 14: 143-150Crossref PubMed Scopus (241) Google Scholar), TBP (33Qadri I. Maguire H.F. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1003-1007Crossref PubMed Scopus (185) Google Scholar), and with components of TFIIH (34Qadri I. Conaway J.W. Conaway R.C. Schaack J. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10578-10583Crossref PubMed Scopus (131) Google Scholar). Furthermore, it has been proposed that pX is a general viral transactivator, acting by influencing the coactivator process (35Haviv I. Vaizel D. Shaul Y. Mol. Cell. Biol. 1995; 15: 1079-1085Crossref PubMed Google Scholar). Direct interactions between pX and cellular transcription factors have been uniquely demonstrated for the CREB/ATF family of proteins (1Williams J.S. Andrisani O.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3819-3823Crossref PubMed Scopus (147) Google Scholar, 29Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (379) Google Scholar). CREB, which mediates the transcriptional induction of the cAMP-transduction pathway (36Andrisani O. Zhu Z. Pot D. Dixon J.E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2181-2185Crossref PubMed Scopus (28) Google Scholar, 37Gonzalez G.A. Montiminy M. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2064) Google Scholar), is also the interaction target of the HTLVI Tax oncoprotein (38Adya N. Giam C.-Z. J. Virol. 1995; 69: 1834-1841Crossref PubMed Google Scholar, 39Bantignies F. Rousset R. Desbois C. Jalinot P. Mol. Cell. Biol. 1996; 16: 2174-2182Crossref PubMed Scopus (53) Google Scholar). Our studies demonstrated that the interaction of pX with CREB increases by 1 order of magnitude its affinity for the CRE site, thereby increasing its in vivotranscriptional efficacy by 13-fold. In contrast to the CREB/Tax interactions (40Wagner S. Green M.R. Science. 1993; 262: 395-399Crossref PubMed Scopus (290) Google Scholar), pX does not enhance the dimerization rate of CREB; it seems to target the basic, DNA-binding region of CREB (1Williams J.S. Andrisani O.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3819-3823Crossref PubMed Scopus (147) Google Scholar). In this study we investigate the potential interaction of pX with other bZip transcription factors normally expressed in the hepatocyte. Our hypothesis is that pX interacts not only with CREB but also with other bZip proteins, by recognizing common structural features shared by bZip proteins (41Baranger A.M. Palmer C.R. Hamm M.K. Giebler H.A. Brauweller A. Nyborg J.K. Schepartz A. Nature. 1995; 376: 606-608Crossref PubMed Scopus (162) Google Scholar). To test this hypothesis, we selected for analysis bZip proteins that play a role in hepatocyte physiology such as, NF-IL6 or C/EBPβ (42Akira S. Ishiki H. Sugita T. Tanabe O. Kinoshita S. Hishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1212) Google Scholar), gadd153/Chop10 (43Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (986) Google Scholar), ATF3 (44Hai T. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (760) Google Scholar), and ICER IIγ (45Molina C.A. Foulkes N.S. Lalli E. Sassone-Corsi P. Cell. 1993; 75: 875-886Abstract Full Text PDF PubMed Scopus (527) Google Scholar, 80Servillo G. Penna L. Foulkes N.S. Magni M.V. Fazia M.A.D. Sassone-Corsi P. Oncogene. 1997; 14: 1601-1606Crossref PubMed Scopus (55) Google Scholar) which may play such a role. NF-IL6, a member of the C/EBP leucine zipper family (42Akira S. Ishiki H. Sugita T. Tanabe O. Kinoshita S. Hishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1212) Google Scholar), is selected in this study, since it is expressed in liver (46Descombes P. Chojkier M. Lichtsteiner S. Falvey E. Schibler U. EMBO J. 1990; 4: 1541-1551Google Scholar) where it is implicated as the master regulator of the acute phase response genes (42Akira S. Ishiki H. Sugita T. Tanabe O. Kinoshita S. Hishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1212) Google Scholar). Expression of NF-IL6 is induced by IL-6 (42Akira S. Ishiki H. Sugita T. Tanabe O. Kinoshita S. Hishio Y. Nakajima T. Hirano T. Kishimoto T. EMBO J. 1990; 9: 1897-1906Crossref PubMed Scopus (1212) Google Scholar, 47Poli V. Mancini F.P. Cortese R. Cell. 1990; 63: 643-653Abstract Full Text PDF PubMed Scopus (459) Google Scholar), IL-1 (48Cao Z. Umek R. McKnight S. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1350) Google Scholar), and other inflammatory mediators. NF-IL6 forms heterodimers with other bZip proteins in the liver (43Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (986) Google Scholar), including gadd153/Chop10 (43Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (986) Google Scholar) which is induced by cellular stress conditions (49Carlson S.G. Fawcett T.W. Bartlett J.D. Bernier M. Holbrook N.J. Mol. Cell. Biol. 1993; 13: 4736-4744Crossref PubMed Scopus (189) Google Scholar, 50Price B. Caldewood S. Cancer Res. 1992; 52: 3814-3817PubMed Google Scholar), acute phase (inflammatory) response (51Alam T. An M.R. Papaconstantinou J. J. Biol. Chem. 1992; 267: 5021-5024Abstract Full Text PDF PubMed Google Scholar), and exposure to toxins (52Chen Q. Yu K. Holbrook N.J. Stevens J.L. J. Biol. Chem. 1992; 267: 8207-8212Abstract Full Text PDF PubMed Google Scholar, 53Luethy J.D. Holbrook N.J. Cancer Res. 1992; 52: 5-10PubMed Google Scholar). While gadd153/Chop10 has strong sequence similarity to C/EBP-like proteins within the bZip region (43Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (986) Google Scholar), it contains substitutions of three conserved residues in the basic region critical for DNA binding. Therefore, heterodimers of gadd153/Chop10 and C/EBP-like proteins are unable to bind to their cognate DNA binding site (43Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (986) Google Scholar). Accordingly, it has been proposed that gadd153/Chop10 functions as a stress-inducible transcriptional inhibitor (43Ron D. Habener J.F. Genes Dev. 1992; 6: 439-453Crossref PubMed Scopus (986) Google Scholar). However, recent studies indicate that in certain cases gadd153/Chop10 may function as a direct transcriptional activator (54Ubeda M. Wang X.-Z. Zinszner H. Wu I. Habener J.F. Ron D. Mol. Cell. Biol. 1996; 16: 1479-1489Crossref PubMed Google Scholar). ATF3, is also of interest in this study since it is a member of the CREB/ATF family of transcription factors, sharing sequence similarity with the bZip domain of CREB and binding to the CRE site (44Hai T. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (760) Google Scholar). However, while CREB is a positive regulator of transcription, ATF3 is a transcriptional repressor (56Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 269: 15819-15826Abstract Full Text PDF PubMed Google Scholar). Recent studies indicate that ATF3 is induced by a variety of physiological stress conditions, such as mechanically injured and toxin-injured liver (55Chen B.P.C. Wolfgang C.D. Hai T. Mol. Cell. Biol. 1996; 16: 1157-1168Crossref PubMed Scopus (259) Google Scholar, 57Liang G. Wolfgang C.D. Chen B.P.C. Chen T.-H. Hai T. J. Biol. Chem. 1996; 271: 1695-1701Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). ATF3 and gadd153/Chop10 form non-DNA-binding heterodimers (55Chen B.P.C. Wolfgang C.D. Hai T. Mol. Cell. Biol. 1996; 16: 1157-1168Crossref PubMed Scopus (259) Google Scholar). Interestingly, during carbon tetrachloride injury of liver, gadd153/Chop10 and ATF3mRNAs are inversely induced but in an overlapping manner; gadd153/Chop10 mRNA, high in normal liver, decreases upon CCl4 exposure; ATF3 mRNA, low in normal liver, increases upon CCl4 exposure (55Chen B.P.C. Wolfgang C.D. Hai T. Mol. Cell. Biol. 1996; 16: 1157-1168Crossref PubMed Scopus (259) Google Scholar). ICER IIγ (inducible cAMP earlyrepressor) (45Molina C.A. Foulkes N.S. Lalli E. Sassone-Corsi P. Cell. 1993; 75: 875-886Abstract Full Text PDF PubMed Scopus (527) Google Scholar), a member of the cAMP-responsive element modulator subfamily of bZip proteins, is of interest since it is an inducible repressor, displaying high degree of amino acid sequence identity with the bZip of CREB and is devoid of a transactivation domain. Thus, ICER IIγ provides an ideal model system to demonstrate the effect of pX in repression of bZip transcription. Recent studies demonstrated ICER IIγ expression in liver regeneration (80Servillo G. Penna L. Foulkes N.S. Magni M.V. Fazia M.A.D. Sassone-Corsi P. Oncogene. 1997; 14: 1601-1606Crossref PubMed Scopus (55) Google Scholar). In this study, we employ in vitro and in vivo(cellular) assays to examine the interaction of pX with the aforementioned bZip transcription factors. Transcription factors ICER IIγ (45Molina C.A. Foulkes N.S. Lalli E. Sassone-Corsi P. Cell. 1993; 75: 875-886Abstract Full Text PDF PubMed Scopus (527) Google Scholar), the bZip domain of NF-IL6, amino acid residues 259–335, and CREB327 (59Andrisani O.M. Dixon J.E. J. Biol. Chem. 1991; 266: 21444-21450Abstract Full Text PDF PubMed Google Scholar) were cloned in T7-7vector. ATF3 (55Chen B.P.C. Wolfgang C.D. Hai T. Mol. Cell. Biol. 1996; 16: 1157-1168Crossref PubMed Scopus (259) Google Scholar) and gadd153/Chop10 (55Chen B.P.C. Wolfgang C.D. Hai T. Mol. Cell. Biol. 1996; 16: 1157-1168Crossref PubMed Scopus (259) Google Scholar) were in a derivative of pTM1 (60Moss B. Elroy-Stein O. Alexander W.A. Fuerst T.R. Nature. 1990; 348: 91-92Crossref PubMed Scopus (450) Google Scholar) vector. 35S-Labeled proteins were synthesized by the TnT (Promega) in vitrotranscription/translation system. Plasmids GST-X1–154 and GST-X49–154 were constructed by polymerase chain reaction of pX DNA fragments corresponding either to amino acid residues 1–154 or 49–154 cloned into the EcoRI-HindIII sites of plasmid pGEX-KG (61Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar). GST-X fusion proteins were expressed inEscherichia coli and purified on glutathione-Sepharose 4B resin (Pharmacia Biotech Inc.), as described previously (62Williams J.S. Dixon J.E. Andrisani O.M. DNA Cell Biol. 1993; 12: 183-190Crossref PubMed Scopus (19) Google Scholar). Bacterial extract obtained from 1 liter of bacterial culture was bound to 500 μl of resin for 30 min and washed (62Williams J.S. Dixon J.E. Andrisani O.M. DNA Cell Biol. 1993; 12: 183-190Crossref PubMed Scopus (19) Google Scholar). Protein concentration of GST and GST-X1–154 bound to the resin was estimated by Coomassie Blue staining of SDS-PAGE and by comparison to known amounts of bovine serum albumin run on the same gel. In vitroprotein-protein interaction assays were carried out as follows: 10 μg of GST and 2 μg of GST-X1–154 proteins immobilized onto 20 μl of glutathione-Sepharose 4B resin were incubated with 4 μl of TnT lysate for 3 h at 4 °C, in buffer containing 25 mm Hepes, pH 7.5, 100 mm KCl, 5 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 40 μg/ml bovine serum albumin, 0.1% Triton X-100, and a protease inhibitor mixture. After incubation, the beads were washed six times in the above buffer. Analysis of the bound protein was by SDS-PAGE and fluorography. DNA protein binding assays were carried out as described previously (1Williams J.S. Andrisani O.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3819-3823Crossref PubMed Scopus (147) Google Scholar). The somatostatin CRE (62Williams J.S. Dixon J.E. Andrisani O.M. DNA Cell Biol. 1993; 12: 183-190Crossref PubMed Scopus (19) Google Scholar, 63Andrisani O.M. Pot D.A. Zhu Z. Dixon J.E. Mol. Cell. Biol. 1989; 8: 1947-1956Crossref Scopus (43) Google Scholar) was used as the oligonucleotide probe for CREB, ICER IIγ, ATF3, and gadd153/Chop10. The nucleotide sequence spanning positions −165 to −173 of the HIV-LTR (58Tesmer V.M. Rajadhyaksha A. Babin J. Bina M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7298-7302Crossref PubMed Scopus (91) Google Scholar) was used as the DNA-binding probe for NF-IL6. The MyoD/E47 E-box DNA-binding site (64Murre C. McCaw P. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1863) Google Scholar) was kindly provided by Dr. S. Konieczny. Proteins for ICER IIγ, ATF3, gadd153/Chop10, ATF3/gadd153/Chop10, and MyoD/E47 were obtained by in vitro translation (TnT, Promega); 1–3 μl of in vitro translation mixture was used for each reaction. Bacterially produced CREB327 (70Colbran J.L. Fiol C.J. Roach P. Dixon J.E. Andrisani O.M. Corbin J.D. Biochem. Cell Biol. 1993; 70: 1277-1282Crossref Scopus (40) Google Scholar) and bZip NF-IL6, 15 ng each, were employed in the binding reactions. Recombinant GST-X49–154 was added to binding reactions; control lanes contained equal amounts of GST protein. The reaction mixtures were analyzed by native acrylamide gel electrophoresis, as described (62Williams J.S. Dixon J.E. Andrisani O.M. DNA Cell Biol. 1993; 12: 183-190Crossref PubMed Scopus (19) Google Scholar,63Andrisani O.M. Pot D.A. Zhu Z. Dixon J.E. Mol. Cell. Biol. 1989; 8: 1947-1956Crossref Scopus (43) Google Scholar). AML12 cells were kindly provided by Dr. N. Fausto. AML12 cells were propagated in Dulbecco's modified Eagle's medium/Ham's F-12, supplemented with 10% fetal calf serum, a mixture of insulin, transferrin, and selenium (Life Technologies, Inc.), 0.1 μm dexamethasone, and gentamicin, 50 μg/ml (65Wu J.C. Merlino G. Fausto N. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 674-678Crossref PubMed Scopus (250) Google Scholar). In functional assays, AML12 cells were transfected by the calcium phosphate coprecipitation method, using the Life Technologies, Inc. transfection kit. Briefly, 10–20% subconfluent cultures were transfected with 5 μg of CAT reporter plasmid and the indicated amount of expressor plasmid. Cells were harvested 48 h after transfection. CAT activity was determined as described (66Fiol C.J. Williams J.S. Chou C.-H. Wang Q.M. Roach P.J. Andrisani O.M. J. Biol. Chem. 1994; 269: 32187-32193Abstract Full Text PDF PubMed Google Scholar) using equal amounts of protein extract per assay. Protein concentration of cellular extracts was determined by the Bio-Rad protein assay. Each experiment was repeated a minimum of three times. The CAT reporter vector pC15ΔXE, kindly provided by Dr. M. Bina, contains only the NF-IL6-binding site I, at position −158 to −178 (58Tesmer V.M. Rajadhyaksha A. Babin J. Bina M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7298-7302Crossref PubMed Scopus (91) Google Scholar). The mammalian two-hybrid assay was carried out using the 5 × Gal4-E1b TATAA-luciferase reporter plasmid (67Sun P. Enslen H. Myung P.S. Maurer R.A. Genes Dev. 1994; 8: 2527-2539Crossref PubMed Scopus (649) Google Scholar). The RSV-CREB-VP16 and RSV-Gal4 vectors were kindly provided by Dr. R. Gaynor (68Yin M. Paulssen E. Seeler J. Gaynor R.B. J. Virol. 1995; 69: 3420-3432Crossref PubMed Google Scholar). The CMV4-ICER IIγ (45Molina C.A. Foulkes N.S. Lalli E. Sassone-Corsi P. Cell. 1993; 75: 875-886Abstract Full Text PDF PubMed Scopus (527) Google Scholar) was kindly provided by Dr. C. Molina. The ATF3, gadd153/Chop10, ICER IIγ, and bZip of NF-IL6 (amino acid residues 259–335) were cloned by polymerase chain reaction amplification of the respective fragments into the RSV-VP16 vector (68Yin M. Paulssen E. Seeler J. Gaynor R.B. J. Virol. 1995; 69: 3420-3432Crossref PubMed Google Scholar), at the NcoI site, resulting in the construction of bZip-VP16 fusion proteins, as described by Yin et al.(68Yin M. Paulssen E. Seeler J. Gaynor R.B. J. Virol. 1995; 69: 3420-3432Crossref PubMed Google Scholar). Similarly, RSV-X-Gal4 constructs were prepared by inserting the coding region of pX at the NcoI site, resulting in fusion proteins with the Gal4 DNA-binding domain (amino acids 1–147) at its C terminus. 5 μg of reporter plasmid, 5 × Gal4-E1b TATAA-luciferase, were cotransfected with 5–10 μg of each of the expressor plasmids. Transfections were performed in duplicates (60-mm plates) and repeated a minimum of three times. Interactions between pX and CREB were demonstrated by enhanced CREB binding to the CRE site in the presence of pX (1Williams J.S. Andrisani O.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3819-3823Crossref PubMed Scopus (147) Google Scholar, 29Maguire H.F. Hoeffler J.P. Siddiqui A. Science. 1991; 252: 842-844Crossref PubMed Scopus (379) Google Scholar) and by altered methylation interference assays (1Williams J.S. Andrisani O.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3819-3823Crossref PubMed Scopus (147) Google Scholar). In the present study we employed the CREB/pX-interacting proteins as the model system for developing an assay to detect direct protein-protein interactions. For this analysis the full-length X protein was produced as a fusion with glutathione S-transferase (61Guan K. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1641) Google Scholar). The GST-X1–154fusion protein is selectively retained by glutathione-Sepharose 4B resin and thus the complex provides a suitable affinity resin. Recombinant CREB327 (69Zhu Z. Andrisani O.M. Pot D.A. Dixon J.E. J. Biol. Chem. 1989; 264: 6550-6556Abstract Full Text PDF PubMed Google Scholar, 70Colbran J.L. Fiol C.J. Roach P. Dixon J.E. Andrisani O.M. Corbin J.D. Biochem. Cell Biol. 1993; 70: 1277-1282Crossref Scopus (40) Google Scholar), 32P-radiolabeled (66Fiol C.J. Williams J.S. Chou C.-H. Wang Q.M. Roach P.J. Andrisani O.M. J. Biol. Chem. 1994; 269: 32187-32193Abstract Full Text PDF PubMed Google Scholar), was employed to establish the in vitro conditions for detecting specific binding of CREB327 to the resin-immobilized GST-X1–154. We observe selective binding of 32P-CREB327 to GST-X1–154 but no binding to the control GST-resin (Fig.1 A). Additional control experiments include the following: first, the demonstration that under the conditions detecting CREB/pX interactions (Fig. 1 A), transcription factors of a different class, namely Gal41–147 (71Ma J. Ptashne M. Cell. 1987; 48: 847-853Abstract Full Text PDF PubMed Scopus (604) Google Scholar) and MyoD (64Murre C. McCaw P. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1863) Google Scholar), did not display detectable binding to GST-X1–154 (Fig. 1, B andC); and second, the demonstration that the interaction of CREB327 with GST-X1–154 involved the bZip domain of CREB327. For this analysis, CREB327and the N-terminal portion of CREB327, amino acid residues 1–198, were synthesized in vitro (Fig.2 A). In comparative analysis, the N-terminal region of CREB327 did not interact with GST-X1–154 (Fig. 2 B), whereas35S-CREB327 displayed specific binding to GST-X1–154 (Fig. 2 C). The results confirm our earlier observations that the bZip of CREB is the interacting target of pX and show that under the established conditions (Fig. 1) we detect specific binding of CREB327 to pX in vitro.Figure 2In vitro translated35S-labeled CREB variants. A, full-length CREB327 synthesized in vitro via the T7-CREB327 template (58Tesmer V.M. Rajadhyaksha A. Babin J. Bina M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7298-7302Crossref PubMed Scopus (91) Google Scholar); CREB1–198 contains the N-terminal 198 amino acid residues, synthesized in vitro from T7-CREB327template digested with KpnI; the resulting truncated CREB peptide, lacks the bZip domain. 4 μl of in vitrotranslated CREB327 or CREB1–198 proteins (TnT, Promega), analyzed by 12% SDS-PAGE and autoradiography. B, protein-protein interaction assay with35S-CREB1–198. Input (INPUT) is 4 μl of 35S-CREB1–198 loaded onto GSF- or GST-X1–154 resin, respectively.35S-CREB1–198 bound to GST or GST-X1–154 resin, respectively. Analysis is by 12% SDS-PAGE and autoradiography. C, protein-protein interaction assay with 35S-CREB327, as described in Fig.1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We employed the in vitro protein-protein interaction assay described in Fig. 1 A to examine the interaction of bZip transcription factors ATF3, gadd153/Chop10, NF-IL6, and ICER IIγ with pX. The binding reactions with bZip proteins were carried out exactly as described for CREB327 (Fig.1 A and Fig. 2 C). Fig.3 A shows the binding of the bZip of NF-IL6 to the immobilized GST-X1–154 is much higher th" @default.
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• W2017019224 title "The Hepatitis B Virus X Protein Enhances the DNA Binding Potential and Transcription Efficacy of bZip Transcription Factors" @default.
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