The Leukemia-associated Protein Btg1 and the p53-regulated Protein Btg2 Interact with the Homeoprotein Hoxb9 and Enhance Its Transcriptional Activation
2000; Elsevier BV; Volume: 275; Issue: 1 Linguagem: Inglês
10.1074/jbc.275.1.147
ISSN1083-351X
AutoresDéborah Prévôt, Thibault Voeltzel, Anne‐Marie Birot, Anne‐Pierre Morel, Marie-Claude Rostan, Jean‐Pierre Magaud, Laura Corbo,
Tópico(s)Epigenetics and DNA Methylation
ResumoBTG1 and BTG2 belong to a family of functionally related genes involved in the control of the cell cycle. As part of an ongoing attempt to understand their biological functions, we used a yeast two-hybrid screening to look for possible functional partners of Btg1 and Btg2. Here we report the physical and functional association between these proteins and the homeodomain protein Hoxb9. We further show that Btg1 and Btg2 enhance Hoxb9-mediated transcription in transfected cells, and we report the formation of a Hoxb9·Btg2 complex on a Hoxb9-responsive target, and the fact that this interaction facilitates the binding of Hoxb9 to DNA. The transcriptional activity of the Hoxb9·Btg complex is essentially dependent on the activation domain of Hoxb9, located in the N-terminal portion of the protein. Our data indicate that Btg1 and Btg2 act as transcriptional cofactors of the Hoxb9 protein, and suggest that this interaction may mediate their antiproliferative function. BTG1 and BTG2 belong to a family of functionally related genes involved in the control of the cell cycle. As part of an ongoing attempt to understand their biological functions, we used a yeast two-hybrid screening to look for possible functional partners of Btg1 and Btg2. Here we report the physical and functional association between these proteins and the homeodomain protein Hoxb9. We further show that Btg1 and Btg2 enhance Hoxb9-mediated transcription in transfected cells, and we report the formation of a Hoxb9·Btg2 complex on a Hoxb9-responsive target, and the fact that this interaction facilitates the binding of Hoxb9 to DNA. The transcriptional activity of the Hoxb9·Btg complex is essentially dependent on the activation domain of Hoxb9, located in the N-terminal portion of the protein. Our data indicate that Btg1 and Btg2 act as transcriptional cofactors of the Hoxb9 protein, and suggest that this interaction may mediate their antiproliferative function. polymerase chain reaction polyacrylamide gel electrophoresis glutathione S-transferase thymidine kinase chloramphenicol acetyltransferase dithiothreitol enzyme-linked immunosorbent assay homeodomain binding site The BTG gene family, whose founding member isBTG1 (B-cell translocationgene 1) (1Rimokh R. Rouault J.P. Wahbi K. Gadoux M. Lafage M. Archimbaud E. Charrin C. Gentilhomme O. Germain D. Samarut J. Magaud Genes Chromosom. Cancer. 1991; 3: 24-36Crossref PubMed Scopus (81) Google Scholar, 2Rouault J.P. Rimokh R. Tessa C. Paranhos G. Ffrench M. Duret L. Garoccio M. Germain D. Samarut J. Magaud J.P. EMBO J. 1992; 11: 1663-1670Crossref PubMed Scopus (264) Google Scholar), is composed, in vertebrates, of at least seven distinct members: BTG1,BTG2/TIS21/PC3, BTG3/TOB5/ANA,TOB, TOB4, B9.10, andB9.15. The defining feature of this family is the presence of two short conserved domains (box A and box B) separated by a spacer sequence of 20–25 non-conserved amino acids (3Guehenneux F. Duret L. Callanan M.B. Bouhas R. Hayette S. Berthet C. Samarut C. Rimokh R. Birot A.M. Wang Q. Magaud J.P. Rouault J.P. Leukemia. 1997; 11: 370-375Crossref PubMed Scopus (106) Google Scholar). These proteins are found in organisms from nematodes to humans, and a large body of evidence suggests that Btg proteins may be mediators of multiple antiproliferative activities. Btg proteins are involved in cell growth control and differentiation in models such as T lymphocytes, 12-O-tetradecanoylphorbol-13-acetate-treated mouse fibroblasts, nerve growth factor-stimulated PC12 cells and neuron-cell birthday in the mouse embryo (4Fletcher B.S. Lim R.W. Varnum B.C. Kujubu D.A. Koski R.A. Herschman H.R. J. Biol. Chem. 1991; 266: 14511-14518Abstract Full Text PDF PubMed Google Scholar, 5Rouault J.P. Falette N. Guéhenneux F. Guillot C. Rimokh R. Wang Q. Berthet C. Moyret-Lalle C. Savatier P. Pain B. Shaw P. Berger R. Samarut J. Magaud J.P. Ozturk M. Samarut C. Puisieux A. Nat. Genet. 1996; 14: 482-486Crossref PubMed Scopus (343) Google Scholar, 6Bradbury A. Possenti R. Shooter E.M. Tirone F. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3353-3357Crossref PubMed Scopus (176) Google Scholar, 7Iacopetti P. Barsacchi G. Tirone F. Maffei L. Cremisi F. Mech. Dev. 1994; 47: 127-137Crossref PubMed Scopus (48) Google Scholar). Furthermore, BTG2expression is regulated by p53, and its inactivation in embryonic stem cells leads to the disruption of DNA damage-induced G2/M cell-cycle arrest (5Rouault J.P. Falette N. Guéhenneux F. Guillot C. Rimokh R. Wang Q. Berthet C. Moyret-Lalle C. Savatier P. Pain B. Shaw P. Berger R. Samarut J. Magaud J.P. Ozturk M. Samarut C. Puisieux A. Nat. Genet. 1996; 14: 482-486Crossref PubMed Scopus (343) Google Scholar). It had also been suggested that BTG2could be involved in programmed PC12 cell death (8Mesner P.W. Epting C.L. Hegarty J.L. Green S.H. J. Neurosci. 1995; 15: 7357-7366Crossref PubMed Google Scholar, 9Wang S. Dibenedetto A.J. Pittman R.N. Dev. Biol. 1997; 188: 322-336Crossref PubMed Scopus (40) Google Scholar). The functional specificity and selectivity of the different members of the Btg family could be achieved by the interaction with different cellular targets; Tob can associate with the ErbB2 growth factor receptor, which may modulate the signal elicited by epidermal growth factor (10Matsuda S. Kawamura-Tsuzuku J. Ohsugi M. Yoshida M. Emi M. Nakamura Y. Onda M. Yoshida Y. Nishiyama A. Yamamoto T. Oncogene. 1996; 12: 705-713PubMed Google Scholar); it has been shown that both Btg1 and Btg2 interact with a protein-arginineN-methyltransferase (Prmt1), and modulate its activity positively (11Lin W.J. Gary J.D. Yang M.C. Clarke S. Herschman H.R. J. Biol. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar); and we have demonstrated the physical interaction of both Btg1 and Btg2 with the mouse protein Caf1 (CCR4-associated factor 1) (12Rouault J.P. Prevot D. Berthet C. Birot A.M. Billaud M. Magaud J.P. Corbo L. J. Biol. Chem. 1998; 273: 22563-22569Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), the mouse homolog of the yeast yCAF/POP2 gene, which regulates the expression of a number of genes involved in cell-cycle regulation and progression. In this study, we report the characterization of another Btg-associated protein, the Hoxb9 homeotic protein, which is a member of a large family of transcription factors that share a similar DNA-binding motif known as the homeodomain, an evolutionarily conserved sequence of 60 amino acids. The Hox family of homeodomain-containing transcription factors regulates cell fates during the development of metazoans (13Gehring W.J. Affolter M. Burglin T. Annu. Rev. Biochem. 1994; 63: 487-526Crossref PubMed Scopus (839) Google Scholar). Despite their functional specificity in vivo, a large number of Hox proteins (up to 38 in mammals) appears to bind in vitro to a restricted set of very similar DNA sites with a similar degree of affinity (14Laughon A. Biochemistry. 1991; 30: 11357-11367Crossref PubMed Scopus (259) Google Scholar). There is considerable evidence that the diversity of the functions of Hoxin vivo depends not only on their DNA-binding properties, but also on transcriptional co-factors (15Kornberg T.B. J. Biol. Chem. 1993; 268: 26813-26816Abstract Full Text PDF PubMed Google Scholar, 16Mann R.S. Chan S.K. Trends Genet. 1996; 12: 258-262Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 17Mann R.S. Affolter M. Curr. Opin. Genet. Dev. 1998; 8: 423-429Crossref PubMed Scopus (323) Google Scholar). In the present study, we show that Btg1 and Btg2 interact with Hoxb9, and that the homeodomain is involved in these interactions. We also show that Btg1 and Btg2 can cooperate with Hoxb9 to activate transcription in transfected cells. The transcriptional activation domain of Hoxb9 was identified by deletion analysis, and was mapped to the N-terminal region of the protein. Our results indicate that Btg1 and Btg2 may function as cofactors for Hoxb9-mediated transcription. The Btg1 and Btg2 "bait" for the yeast two-hybrid system was based on the pPC62 yeast expression vector (18Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (476) Google Scholar). pPC62 was cut with SalI and XbaI, and ligated with SalI/XbaI BTG1 andBTG2 human cDNA, obtained by PCR,1 giving pPC62BTG1 and BTG2 encoding fusion proteins, consisting of the Gal4 DNA-binding domain fused to Btg1 and Btg2. The BTG1 deletion mutants, constructed as Gal4 fusions, which have already been described (12Rouault J.P. Prevot D. Berthet C. Birot A.M. Billaud M. Magaud J.P. Corbo L. J. Biol. Chem. 1998; 273: 22563-22569Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), are outlined in Fig. 1. The pPC86HOXB9/185–250 was generated cloning the fragment containing the Hoxb9 homeodomain (amino acid residues 180–246) obtained by PCR from the full-length coding sequence of mouseHOXB9 in frame with the Gal4 transactivaction domain of the pPC86 plasmid. The Hox2.5 plasmid, containing the full-length coding sequence of mouse HOXB9 gene, was kindly provided by F. Ruddle (Yale University, New Haven, CT). We used a yeast two-hybrid system with both LACZ and HIS3 selection (19Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5201) Google Scholar). The Y190 strain was transformed with pPC62BTG1. The Gal4Btg1-expressing yeast cells were subsequently transformed with the Gal4 transactivation-domain-tagged 14.5-day-old mouse embryo cDNA library (18Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (476) Google Scholar), following the protocol described (12Rouault J.P. Prevot D. Berthet C. Birot A.M. Billaud M. Magaud J.P. Corbo L. J. Biol. Chem. 1998; 273: 22563-22569Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). After 4–5 days at 30 °C on an Leu/Trp/His amino acid-depleted DOB medium, the transformants were tested for β-galactosidase activity using a yeast colony-filter assay. Positive (blue) colonies were grown in selective medium for 2 days to recover the prey plasmid. An Electromax DH10B bacterial strain was used to recover the expression plasmid from the selected transformed yeast (18Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (476) Google Scholar). To generate the bacterial expression vector for Hoxb9, its full-length coding sequence was inserted into the pGEX-expression vector (Amersham Pharmacia Biotech) in frame with the glutathioneS-transferase (GST) coding sequence. GST and GSTHoxb9 proteins were expressed in Escherichia coli DH5α, purified on glutathione-Sepharose beads (Amersham Pharmacia Biotech) and quantified using the Bradford method. Recombinant 6His vector for human Btg2 was constructed by inserting the cDNA into theBamHI site of pQE30 vector (Qiagen). This placed the Btg2 coding sequence in frame with six sequential histidine residues. Recombinant polyhistidine-tagged Btg2, expressed in M15 (pREP4), was purified from soluble lysates with a nickel chelation affinity column (Qiagen) according to the manufacturer's recommended protocol. SDS-PAGE and Coomassie staining were used to confirm the integrity of the full-length fusion proteins. The 6HisFlrg protein was provided by R. Rimokh (U453 INSERM, Lyon, France). The pHBS-TK-CAT plasmid was generated by inserting two copies of a SalI-SalI double-stranded oligonucleotide containing the Hoxb9-responsive element HBSII of the N-CAM gene promoter, into the SalI site of the plasmid pBLCAT2. HBSII sense strand consisted of 5′-gtcGACAAATTCCTGATTAAGGAACGTCGAC-3′. The core binding site is shown in bold letters. The pG4-TK-CAT reporter plasmid contains six GAL4 consensus elements upstream from the thymidine kinase (TK) promoter region, fused to the CAT gene. All the mammalian expression constructs used were derivatives of the SV40 promoter-driven expression vector pSG5 (Stratagene). The pSG5Flag plasmid, which was derived from pSG5 (12Rouault J.P. Prevot D. Berthet C. Birot A.M. Billaud M. Magaud J.P. Corbo L. J. Biol. Chem. 1998; 273: 22563-22569Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), contains the Flag peptide sequence (IBI Flag system; Eastman Kodak Co.). The full-length HOXB9 cDNA and the fragment coding for the region containing amino acids 79–250 and 185–250, obtained by PCR, were subcloned into theXhoI/SpeI sites of the pSG5Flag vector in order to generate pSG5FlagHOXB9, pSG5FlagHOXB9/79–250, and pSG5FlagHOXB9/180–246. pSG5FlagFOLL. was kindly provided by R. Rimokh (U453 INSERM, Lyon, France). The expression constructs producing Hoxc8 and Hoxd9 under SV40 promoter control (pTL1C8 and pSGH4C) were kindly provided by M. Volovitch (CNRS URA 1414, ENS, Paris, France). pSG5FlagBTG1, pSG5FlagBTG2, pGALBTG1, pGALBTG2, pGALBTG1/1–96, and pGAL4BTG1/1–38 have already been described (12Rouault J.P. Prevot D. Berthet C. Birot A.M. Billaud M. Magaud J.P. Corbo L. J. Biol. Chem. 1998; 273: 22563-22569Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The pGALHOXB9 vector was obtained by cloning the Hoxb9 coding region into Gal4polyII plasmid (20Green S. Issemann I. Sheer E. Nucleic Acids Res. 1988; 16: 369Crossref PubMed Scopus (543) Google Scholar), in frame with the yeast GAL4 DNA-binding domain coding sequence (amino acids 1–147), which contains GAL4 elements responsible for DNA binding, homodimerization, and nuclear localization. The cloned products were verified by DNA sequencing, and the correct expression of all the proteins was checked. The plasmids used for transfection were prepared by the alkaline lysis method, and purified by polyethylene glycol/LiCl. HeLa cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal calf serum, and seeded at 2.5 × 105 cells/well in six-well microtiter plates 8 h prior to transfection. The transfected DNA (1 μg total) included 0.5 μg of reporter plasmid, the amount of expression plasmids indicated in the figure legends, and 50 ng of the pCMV-LACZ control plasmid, in 1 ml of Opti-MEM (Life Technologies, Inc.) containing 5 μl of LipofectAMINE (Life Technologies, Inc.). The amount of SV40 promoter transfected was kept constant, where necessary, by the addition of pSG5 to the transfection mixture. The transfected cells were washed and collected 48 h after transfection. CAT-ELISA assays were performed using the Roche Molecular Biochemicals CAT-ELISA kit, following the manufacturer's instructions. The transfected cells were lysed in 150 μl of lysis buffer. The supernatants were assayed for Cat protein production and β-galactosidase activity. All transfection data were normalized by β-galactosidase assays, which were quantified by ONPG assay using a standard linear curve. Reporter activity was expressed as the ratio of -fold induction to the activity of the reporter vector alone. Each set of experiments was repeated at least three times, and similar results were obtained in each case. For the protein expression assays, 100 μl of a lysate of HeLa cells, transfected as described, were subjected to electrophoresis on a 12% SDS-polyacrylamide gel. The separated proteins were transferred to a polyvinylidene difluoride membrane (Millipore) by electroblotting. Equal amounts of protein were loaded into each lane, as measured by Bradford assay and confirmed by Ponceau Red staining of the transferred membrane. The M2 monoclonal antibody (Stratagene) was used to detect the FlagHoxb9 fusion protein. The membranes were then incubated with horseradish peroxidase-conjugated rabbit anti-mouse immunoglobulins. The proteins were visualized by means of the enhanced Amersham chemiluminescence kit, following the manufacturer's instructions. For in vitroprotein-protein interaction assays, 5 μg of GST, GSTHoxb9, or 6HisBtg2 purified proteins or induced whole bacterial extract were subjected to 10% SDS-PAGE and transferred to membrane. After denaturation/renaturation in 6–0.187 m guanidine-HCl in HB buffer (25 mm Hepes, pH 7.2, 5 mm NaCl, 5 mm MgCl2, 1 mm DTT), the blots were saturated at 4 °C in buffer H (20 mm Hepes, pH 7.7, 7.75 mm KCl, 0.1 mm EDTA, 2.5 mmMgCl2, 1 mm DTT, 0.05% Nonidet P-40, 1% milk), then incubated for 2 h at 4 °C with 50 μl of [35S]methionine-labeled in vitro-translated proteins, synthesized in a reticulocyte lysate-coupled transcription/translation system (Promega) in 3 ml of buffer H. After washing in buffer H for 1 h at 4 °C, the filters were dried and autoradiographed. For the DNA binding assays, purified bacterially produced GSTHoxb9 and 6His-fusion proteins were used. The oligonucleotide was end-labeled by filling in using the Klenow fragment of DNA polymerase and [α-32P]dCTP. EMSA were performed by incubating the bacterially produced proteins (2.5 μg) for 15 min at room temperature in 10 μl of buffer A (20 mm Hepes, pH 7.9, 20 mm KCl, 1 mm MgCl2, 0.5 mm DTT, 20% glycerol). We then added 5 × 104 cpm of 32P-labeled double-stranded templates together with the eventual cold competitor oligonucleotide or anti-GST antibody (TEBU, 200 ng) in 10 μl of buffer B (15 mm Tris-HCl, pH 7.9, 0.7 mm EDTA, 10 mm DTT, 150 mm KCl, 1 μg/μl bovine serum albumin), and the mixture was left at room temperature for 30 min. After incubation, the mixture was loaded onto a 4% (w/v) polyacrylamide gel (29:1 acrylamide/bisacrylamide ratio), 0.25× TBE, and run at room temperature at 10 V/cm for 3 h. The gels were dried, and the protein-DNA complexes were visualized by autoradiography. Double-stranded synthetic oligonucleotides containing the consensus binding site for Hoxb9 of promoter N-CAM and their mutated homologs were used (sense strands shown): HBSII, 5′-gtcGACAAATTCCTGATTAAGGAACGTCGAC-3′; mHBSII, 5′-gtcGACAAATTCCTGgTTcAGGAACGTCGAC-3′ (the core binding site is shown in bold letters). To study the role of Btg proteins in the control of cell growth, we focused on Btg1 and Btg2, which are closely related proteins sharing over 60% of amino acid identity, and tried to identify interacting proteins using the yeast two-hybrid screen (21Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4799) Google Scholar). A fusion protein made of the DNA-binding domain of Gal4 and the full-length Btg1 protein was used as a bait to screen a 14.5-day-old mouse embryo cDNA library cloned in pPC86, as already described (12Rouault J.P. Prevot D. Berthet C. Birot A.M. Billaud M. Magaud J.P. Corbo L. J. Biol. Chem. 1998; 273: 22563-22569Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Two out of clones producing β-galactosidase were further analyzed, and one of them was found to be identical to theCAF1 mouse gene (12Rouault J.P. Prevot D. Berthet C. Birot A.M. Billaud M. Magaud J.P. Corbo L. J. Biol. Chem. 1998; 273: 22563-22569Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The second one, which we named 4.2, was found to encode a protein with a sequence identical to that of the homeotic protein Hoxb9. Clone 4.2 begins at nucleotide 310 and at amino acid 78 according to Malicki (EMBL/GenBank™ accession number S66855) (22Malicki J. Bogarad L.D. Martin M.M. Ruddle F.H. McGinnis W. Mech. Dev. 1993; 42: 139-150Crossref PubMed Scopus (26) Google Scholar). The cDNA encompassing the entire mouse HOXB9 ORF was cloned by PCR from Hox2.5 plasmid kindly provided by F. Ruddle. To outline the region of Btg1 that mediates its interaction with Hoxb9, we made a series of Btg1 truncation constructs (Fig. 1). Using the yeast two-hybrid system, we found that the Btg1 region between residues 1 and 38 was sufficient for interaction with Hoxb9 (Fig. 1). When tested in two-hybrid system, the 4.2 clone product also interacted with the pPC62BTG2 expressed protein. Similarly, using a truncated fusion protein containing the homeodomain alone, we found that Hoxb9 homeodomain was sufficient for binding with Btg1 and Btg2. The results of these assays are summarized in Fig. 1. To confirm the interaction of Btg1 and Btg2 with Hoxb9 in vitro, we performed a Far-Western blot analysis. Purified GST, GSTHoxb9, and an induced bacterial extract containing GSTHoxb9 were subjected to SDS-PAGE, transferred from the gel to nitrocellulose, and probed with [35S]methionine-labeled Btg1 and Btg2 proteins. As shown in Fig. 2, specific hybridization was observed with GSTHoxb9, but not with control GST. In addition, incubation with [35S]methionine-labeled luciferase, done as a control, failed to show any interaction. These results point to a direct physical interaction of Hoxb9 with both Btg1 and Btg2. To test whether Btg proteins can interact with other homeodomain proteins, we did analogous experiments with Hoxd9, a paralog of Hoxb9 and Hoxc8, which is a member of a different paralog group. Under the conditions of our experiments, only Hoxc8 bound with Btg2 protein in vitro(Fig. 3).FIG. 3Btg interaction with other Hox proteins: Far Western analysis. GST, purified 6HisBtg2, and induced whole bacterial extract, shown as Coomassie staining, were subjected to 12% SDS-PAGE and transferred from the gel to nitrocellulose.35S-Labeled, in vitro translated, full-length Hoxc8 and Hoxd9 suspended in binding buffer were used as probes as in Fig. 2. Hoxc8 interacted specifically with Btg2 protein.View Large Image Figure ViewerDownload (PPT) Several in vitroand in vivo studies support the hypothesis that homeobox gene products modulate the expression of adhesion molecule genes (23Edelman G.M. Jones F.S. Philos. Trans. R. Soc. Lond. B Biol. Sci. 1995; 349: 305-312Crossref PubMed Scopus (55) Google Scholar, 24Goomer R.S. Holst B.D. Wood I.C. Jones F.S. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7985-7989Crossref PubMed Scopus (67) Google Scholar, 25Jones F.S. Prediger E.A. Bittner D.A. De Robertis E.M. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2086-2090Crossref PubMed Scopus (193) Google Scholar, 26Krushel L.A. Sporns O. Cunningham B.A. Crossin K.L. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4323-4327Crossref PubMed Scopus (57) Google Scholar, 27Wang Y. Jones F.S. Krushel L.A. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1892-1896Crossref PubMed Scopus (29) Google Scholar). The promoter for the neural cell adhesion molecule(N-CAM) contains a 47-bp region, including two potential homeodomain binding sites (HBSI and HBSII) (25Jones F.S. Prediger E.A. Bittner D.A. De Robertis E.M. Edelman G.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2086-2090Crossref PubMed Scopus (193) Google Scholar). These studies showed that HBSII is a target site for the binding and transcriptional control of the N-CAM promoter by the homeoprotein Hoxb9. To determine whether Hoxb9 and Btg proteins interact directly on DNA, we tested the influence of Btg on the in vitro binding of Hoxb9 to the HBSII sequence, using the full-length Hoxb9 protein as a GST-fusion in gel-mobility shift experiments, with a single HBSII site as a probe (Fig. 4 A). A retarded complex corresponding to GSTHoxb9·DNA was observed (Fig. 4 B, lane 4). This was specifically competed by unlabeled HBSII (lanes 6 and7), but not by the mutated HBSII oligonucleotide (lanes 8 and 9). No retarded complex was found when the GST protein was used (lane 1). The GSTHoxb9·DNA complex was supershifted by the anti-GST antibody (lane 5). When 6HisBtg2 was included in the Hoxb9·DNA-binding incubation mixture, an additional slow mobility complex was seen. This indicates that a DNA·Hoxb9·Btg2 complex was formed (lane 10). The complex exhibited the same DNA-binding specificity, as shown by the occurrence of specific competition with an excess of unlabeled oligonucleotide (lane 11). In contrast, no slow migrating retarded band was observed with the 6His-control protein, 6HisFlrg (lane 12). With 6HisBtg2 or 6HisFlrg alone, no shifted complexes were observed in our EMSA conditions (seelanes 2 and 3). Importantly, the addition of Btg2 enhanced Hoxb9 binding activity, as revealed by a stronger Hoxb9·DNA complex (Fig. 4 B, comparelanes 4 and 10), indicating that the Hoxb9·Btg interaction increased the affinity of Hoxb9 for HBSII sites. In order to study both the transcriptional properties of Hoxb9 and the possible functional interactions of Btg1 and Btg2 with Hoxb9, we performed transient-transfection assays in HeLa cells. We constructed a reporter plasmid, pHBS-TK-CAT, containing two copies of a Hoxb9 binding site (HBSII) upstream from the herpes simplex virus TK promoter, which directs the expression of the CATgene (Fig. 5 A). Transcriptional activity was measured indirectly as the amount of CAT protein production. As shown in Fig. 5 B, transfection of HeLa cells with the pHBS-TK-CAT reporter, together with increasing amounts of a construct expressing Hoxb9, activated transcription in a dose-dependent way. The control reporter construct pBLCAT2, lacking the HBSII sites, was not activated when cotransfected with the Hoxb9-expressing vector (Fig. 5 B, lane 2). The activity of Hoxb9 was enhanced linearly by cotransfection with increasing amounts of both the Btg1 and the Btg2 expression vectors (Fig. 5 C, lanes 3–6). No effect of Btg1 or Btg2 on reporter-gene activity was observed in the absence of Hoxb9 (lanes 8 and9). To ensure that this increase in transcriptional activity was not a result of additional Hoxb9 production, we monitored protein expression in whole cell extracts using Western blot analysis. As Fig. 5 D illustrates, Hoxb9 levels were not increased by Btg1 and Btg2 coexpression. We further looked at the possibility that a control protein could stimulate the transcriptional activation by Hoxb9. Co-transfection of pSG5FlagFOLL, which encodes an unrelated protein, with Hoxb9-expressing vector, showed no enhancement of the activation (Fig. 5 C, lane 7). Next we tested whether other Hox gene products could transactivate the pHBS-TK-CAT reporter alone or in combination with Btg1 and Btg2. When the reporter construct was cotransfected in HeLa cells with Hoxd9 and Hoxc8 alone, a very weak transactivation was observed. Since we could observe physical interaction in vitro between Hoxc8 and Btg2 (see Fig. 3), we tested if coexpression with Btg proteins could allow Hoxc8 to activate the reporter activity. Expression of Hoxc8 protein, as well as Hoxd9, in combination with Btg1 and Btg2 did not lead to an additional activation (data not shown). Taken together, these results indicate that the HBSII selectively mediate transcriptional activation by Hoxb9 and that its in vivo recognition and activation by a Hoxb9 is essential for the functional effect of the Btg proteins. Thus, it appears that Btg1 and Btg2 expression magnifies the Hoxb9 transcriptional response, which in turn suggests that the Btg proteins can act as effectors of the Hoxb9 signaling pathway. Regulation of the activity of transcription factors by other proteins can take place in a number of different ways, one of which involves synergistic effects in which the transcriptional activation caused by two or more transcription factors in combination is much greater than the sum of their individual activities. To test whether the potentiation of the Btg proteins for Hoxb9 activation is due to combinatorial synergism of two activation domains, we first identified the transcriptional activation domain of Hoxb9. For this purpose, we constructed a series of Hoxb9 deletion mutants (Fig. 6 A). The transcriptional activity of the N-terminal deletion mutants of Hoxb9, cloned under the control of the SV40 promoter, was examined using the pHBS-TK-CAT reporter plasmid in HeLa cells. As shown in Fig. 6 B, transfection of 0.3 μg of the pSGFlagHOXB9 plasmid led to a 13-fold increase in the activation of the pHBS-TK-CAT basal activity. Deletion of the 78 N-terminal amino acids notably decreased the transcriptional activity (Fig. 6 B, lane 3). These experiments indicate that the Hoxb9 N-terminal region, extending from amino acids 1 to 78, contains a functional domain that is necessary for transcriptional activation. Because Btg1 and Btg2 increased Hoxb9-dependent transcription, we next investigated whether the Btg proteins have an intrinsic transcriptional activation domain. We constructed vectors expressing Btg1, Btg2, and various portions of Btg1 cDNA (amino acids 1–38 and 1–96) fused to the Gal4 DNA-binding and dimerization domains of GAL4, downstream from the SV40 early promoter. These constructs were transfected in HeLa cells, together with the pG4-TK-CAT reporter plasmid containing six GAL4 consensus elements upstream from the TK promoter region fused to the CAT gene. No transactivation was detected for any Gal4-Btg1 fusion proteins, even though Gal4Vp16 construct exhibited potent activity (data not shown). This indicates that Btg1 and Btg2 lack an intrinsic transactivation domain. Consistent with this observation, neither Btg1 nor Btg2 was able to enhance Hoxb9/79–250-dependent transcription (Fig. 6 B, lanes 4 and 5), suggesting that transcriptional stimulation by Btg proteins requires the activation domain of Hoxb9. To further characterize the transcriptional properties of Hoxb9, and its functional interaction with the Btg proteins, we examined its activity when directed to a heterologous DNA binding element through the GAL4 DNA-binding domain. For this purpose, we constructed an expression plasmid that encoded a fusion protein containing the GAL4 DNA-binding domain fused to the full-length HOXB9 cDNA (pGALHOXB9). As reporter plasmid, we used the pG4-TK-CAT plasmid. The chimeric protein Gal4Hoxb9 was unable to activate transcription of pG4-TK-CAT, even in the presence of co-expressed Btg proteins (data not shown). Therefore, Btg proteins do not generically stimulate transcription through the minimal TK promoter, a
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