A431 cells have an amplification of the epidermal growth factor (EGF) receptor gene, the cellular homolog of the v-erb B oncogene, and overproduce an aberrant 2.9-kilobase RNA that encodes a portion of the EGF receptor. A cDNA (pE15) for the aberrant RNA was cloned, sequenced, and used to analyze genomic DNA blots from A431 and normal cells. These data indicate that the aberrant RNA is created by a gene rearrangement within chromosome 7, resulting in a fusion of the 5' portion of the EGF receptor gene to an unidentified region of genomic DNA. The unidentified sequences are amplified to about the same degree (20- to 30-fold) as the EGF receptor sequences. In situ hybridization to chromosomes from normal cells and A431 cells show that both the EGF receptor gene and the unidentified DNA are localized to the p14-p12 region of chromosome 7. By using cDNA fragments to probe DNA blots from mouse-A431 somatic cell hybrids, the rearranged receptor gene was shown to be associated with translocation chromosome M4.
The transcription factor ATF-2 (also called CRE-BP1), whose DNA-binding domain consists of a basic amino acid cluster and a leucine zipper (b-ZIP) region, binds to the cAMP response element as a homodimer or as a heterodimer with c-Jun. The amino-terminal region of ATF-2 containing the transcriptional activation domain is phosphorylated by stress-activated kinases, which leads to activation of ATF-2. We report here that CBP, which was originally identified as a co-activator of CREB, directly binds to the b-ZIP region of ATF-2 via a Cys/His-rich region termed C/H2, and potentiates trans-activation by ATF-2. The b-ZIP region of ATF-2 was previously shown to interact with the amino-terminal region intramolecularly and to inhibit trans-activating capacity. The binding of CBP to the b-ZIP region abrogates this intramolecular interaction. The adenovirus 13S E1A protein which binds to the b-ZIP region of ATF-2 also inhibited this intramolecular interaction, suggesting that both CBP and 13S E1A share a similar function as positive regulators of ATF-2. We found that the b-ZIP regions of c-Jun and CREB also interact with the C/H2 domain of CBP, suggesting that CBP acts as a regulator for a group of b-ZIP-containing proteins. These results shed light on a novel aspect of CBP function as a regulator for a group of b-ZIP-containing proteins. The transcription factor ATF-2 (also called CRE-BP1), whose DNA-binding domain consists of a basic amino acid cluster and a leucine zipper (b-ZIP) region, binds to the cAMP response element as a homodimer or as a heterodimer with c-Jun. The amino-terminal region of ATF-2 containing the transcriptional activation domain is phosphorylated by stress-activated kinases, which leads to activation of ATF-2. We report here that CBP, which was originally identified as a co-activator of CREB, directly binds to the b-ZIP region of ATF-2 via a Cys/His-rich region termed C/H2, and potentiates trans-activation by ATF-2. The b-ZIP region of ATF-2 was previously shown to interact with the amino-terminal region intramolecularly and to inhibit trans-activating capacity. The binding of CBP to the b-ZIP region abrogates this intramolecular interaction. The adenovirus 13S E1A protein which binds to the b-ZIP region of ATF-2 also inhibited this intramolecular interaction, suggesting that both CBP and 13S E1A share a similar function as positive regulators of ATF-2. We found that the b-ZIP regions of c-Jun and CREB also interact with the C/H2 domain of CBP, suggesting that CBP acts as a regulator for a group of b-ZIP-containing proteins. These results shed light on a novel aspect of CBP function as a regulator for a group of b-ZIP-containing proteins. cAMP response element histone acetyltransferase cAMP-dependent protein kinase stress-activated kinase phosphate-buffered saline glutathioneS-transferase polyacrylamide gel electrophoresis chloramphenicol acetyltransferase. So far, a number of transcription factors of the ATF/CREB family have been identified. All members of this family contain a DNA-binding domain consisting of a cluster of basic amino acids and a leucine zipper region, the so-called b-ZIP. They form homodimers or heterodimers through the leucine zipper and bind to the cAMP response element (CRE)1 (1Hai T. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Crossref PubMed Scopus (1117) Google Scholar). Among many of the transcription factors of the ATF/CREB family, three factors, ATF-2 (also called CRE-BP1), ATF-a, and CRE-BPa, form a subgroup (2Maekawa T. Sakura H. Kanei-Ishii C. Sudo T. Yoshimura T. Fujisawa J. Yoshida M. Ishii S. EMBO J. 1989; 8: 2023-2028Crossref PubMed Scopus (292) Google Scholar, 3Hai T. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (760) Google Scholar, 4Gaire M. Chatton B. Kedinger C. Nucleic Acids Res. 1990; 18: 3461-3473Crossref Scopus (75) Google Scholar, 5Nomura N. Zu Y.-L. Maekawa T. Tabata S. Akiyama T. Ishii S. J. Biol. Chem. 1993; 268: 4259-4266Abstract Full Text PDF PubMed Google Scholar). A common characteristic of this group of factors is the presence of a transcriptional activation domain containing the metal finger structure located in the amino-terminal region (5Nomura N. Zu Y.-L. Maekawa T. Tabata S. Akiyama T. Ishii S. J. Biol. Chem. 1993; 268: 4259-4266Abstract Full Text PDF PubMed Google Scholar, 6Matsuda S. Maekawa T. Ishii S. J. Biol. Chem. 1991; 266: 18188-18193Abstract Full Text PDF PubMed Google Scholar). These factors are capable of forming homodimers or heterodimers with c-Jun and bind to CRE (1Hai T. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Crossref PubMed Scopus (1117) Google Scholar, 5Nomura N. Zu Y.-L. Maekawa T. Tabata S. Akiyama T. Ishii S. J. Biol. Chem. 1993; 268: 4259-4266Abstract Full Text PDF PubMed Google Scholar, 6Matsuda S. Maekawa T. Ishii S. J. Biol. Chem. 1991; 266: 18188-18193Abstract Full Text PDF PubMed Google Scholar). Among these three factors, ATF-2 has been more extensively studied and shown to be ubiquitously expressed with the highest level of expression being observed in the brain (7Takeda J. Maekawa T. Sudo T. Seino Y. Imura H. Saito N. Tanaka C. Ishii S. Oncogene. 1991; 6: 1009-1014PubMed Google Scholar). The mutant mice generated by gene targeting exhibited decreased postnatal viability and growth with a defect in endochondrial ossification and a decreased number of cerebellar Purkinje cells (8Reimond A.M. Grusby M.J. Kosaras B. Fries J.W.U. Mori R. Maniwa S. Clauss I.M. Collins T. Sidman R.L. Glimcher M.J. Glimcher L.H. Nature. 1996; 379: 262-265Crossref PubMed Scopus (243) Google Scholar). The stress-activated protein kinases (SAPK) such as Jun amino-terminal kinase and p38 phosphorylate this group of factors at sites close to the amino-terminal transcriptional activation domain, and stimulate their trans-activating capacity (9Gupta S. Campbell D. Dérijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1339) Google Scholar, 10Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (476) Google Scholar, 11van Dam H. Wilhelm D. Herr I. Steffen A. Herrlich P. Angel P. EMBO J. 1995; 14: 1798-1811Crossref PubMed Scopus (571) Google Scholar). Since a group of factors of the ATF/CREB family including CREB are activated via direct phosphorylation by cAMP-dependent protein kinase (PKA) (12Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2063) Google Scholar), these two groups of factors, CREB and ATF-2, are linked to the distinct signaling cascades involving the PKA and SAPK pathways. The adenovirus 13S E1A activates CRE-dependent transcription, and this transcriptional activation is mediated by ATF-2 (13Liu F. Green M.R. Cell. 1990; 67: 1217-1224Abstract Full Text PDF Scopus (266) Google Scholar, 14Maekawa T. Matsuda S. Fujisawa J.-I. Yoshida M. Ishii S. Oncogene. 1991; 6: 627-632PubMed Google Scholar). E1A binds to the b-ZIP region of ATF-2 (15Liu F. Gree M.R. Nature. 1994; 368: 520-525Crossref PubMed Scopus (225) Google Scholar). Recently, it was reported that the b-ZIP region of ATF-2 interacts with the amino-terminal region intramolecularly (16Li X.-Y. Green M.R. Genes Dev. 1996; 10: 517-527Crossref PubMed Scopus (107) Google Scholar), and this interaction appears to inhibit thetrans-activating capacity of ATF-2. However, the co-activator that binds to the amino-terminal activation domain remains unidentified, and the mechanism of transcriptional activation by ATF-2 needs to be clarified. The transcriptional co-activator CBP was originally identified as a protein that binds to the PKA-phosphorylated form of CREB (17Chrivia J.C. Kwok R.P.S. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1770) Google Scholar). CBP also binds to multiple components of the basal transcriptional machinery, including TFIIB (18Kwok R.P.S. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G.R. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1283) Google Scholar) and the RNA polymerase II holoenzyme complex (19Kee B.L. Arias J. Montminy M.R. J. Biol. Chem. 1996; 271: 2373-2375Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), suggesting that CBP serves as a molecular bridge between CREB and the basal transcriptional machinery. In addition to CREB, many other transcription factors including c-Jun (20Arias J. Alberts A.S. Brindle P. Claret F.X. Smeal T. Karin M. Feramisco J. Montminy M. Nature. 1994; 370: 226-229Crossref PubMed Scopus (681) Google Scholar), c-Fos (21Bannister A.J. Kouzarides T. EMBO J. 1995; 14: 4758-4762Crossref PubMed Scopus (319) Google Scholar), c-Myb (22Dai P. Akimaru H. Tanaka Y. Hou D.-X. Yasukawa T. Kanei-Ishii C. Takahashi T. Ishii S. Genes Dev. 1996; 10: 528-540Crossref PubMed Scopus (303) Google Scholar), nuclear hormone receptors (23Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.-C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1928) Google Scholar, 24Chakravarti D. LaMorte V.J. Nelson M.C. Nakajima T. Schulman I.G. Juguilon H. Montminy M. Evans R.M. Nature. 1996; 383: 99-103Crossref PubMed Scopus (851) Google Scholar), Stat2 (25Bhattacharya S. Eckner R. Grossman S. Oldread E. Arany Z. D'Andrea A. Livingston D.M. Nature. 1996; 383: 344-347Crossref PubMed Scopus (423) Google Scholar), and MyoD (26Eckner R. Yao T.-P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (320) Google Scholar) were recently demonstrated to bind to CBP (for review, see Ref. 27Giles R.H. Peters D.J.M. Breuning M.H. Trends Genet. 1998; 14: 178-183Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). CBP contributes to the transcriptional activation mediated by each of these factors. Although multiple transcription factors bind to CBP, there is a striking difference in the role of CBP depending on the transcriptional activator. For instance, CBP binds to the transcriptional activation domains of CREB and c-Myb (17Chrivia J.C. Kwok R.P.S. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1770) Google Scholar, 22Dai P. Akimaru H. Tanaka Y. Hou D.-X. Yasukawa T. Kanei-Ishii C. Takahashi T. Ishii S. Genes Dev. 1996; 10: 528-540Crossref PubMed Scopus (303) Google Scholar). In the case of nuclear hormone receptors, however, other co-activators bind to the transcriptional activation domain, and CBP binds to a different domain, indicating that CBP functions as a integrator for nuclear hormone receptors (23Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.-C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1928) Google Scholar, 24Chakravarti D. LaMorte V.J. Nelson M.C. Nakajima T. Schulman I.G. Juguilon H. Montminy M. Evans R.M. Nature. 1996; 383: 99-103Crossref PubMed Scopus (851) Google Scholar). The amount of CBP in mammalian cells appears to be limiting, as a 50% reduction in the amount of CBP causes abnormal pattern formation in human (known as Rubinstein-Taybi syndrome) (28Petrij F. Giles R.H. Dauwerse H.G. Saris J.J. Hennekam R.C.M. Masuno M. Tommerup N. van Ommen G.B. Goodman R.H. Peters D.J.M. Breuning M.H. Nature. 1995; 376: 348-351Crossref PubMed Scopus (1029) Google Scholar) and mouse (29Tanaka Y. Naruse I. Maekawa T. Masuya H. Shiroishi T. Ishii S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10215-10220Crossref PubMed Scopus (261) Google Scholar). Recent genetic analyses using Drosophila CBP mutants indicated that CBP is required for transcriptional activation by Cubitus interruptus (Ci) and Dorsal (Dl), which are homologs of mammalian factors glioblastoma (GLI) and NF-κB, respectively (30Akimaru H. Chen Y. Dai P. Hou D.-X. Nonaka M. Smolik S.M. Armstromg S. Goodman R. Ishii S. Nature. 1997; 386: 735-738Crossref PubMed Scopus (238) Google Scholar, 31Akimaru H. Hou D.-X. Ishii S. Nat. Genet. 1997; 17: 211-214Crossref PubMed Scopus (97) Google Scholar). These results suggest that the decreased expression level of target genes of these transcription factors such as Bmp and Twist lead to the deficiency in pattern formation. In addition to the finding that CBP itself has histone acetyltransferase (HAT) activity (32Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar, 33Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1535) Google Scholar), CBP forms a complex with multiple HATs such as P/CAF, ACTR, and SRC-1 (34Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar, 35Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1270) Google Scholar, 36Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.-J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1070) Google Scholar), suggesting that the CBP complex contributes to transcriptional activation by disrupting the repressive chromatin structure. The cbp gene family contains at least one other member, p300, that was originally identified through its ability to bind to the adenovirus E1A protein (37Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCapiro J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (927) Google Scholar), and E1A binds to both CBP and p300 (38Lundblad J.R. Kwok R.P.S. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (531) Google Scholar, 39Arany Z. Newsome D. Oldread E. Livingston D.M. Eckner R. Nature. 1995; 374: 81-84Crossref PubMed Scopus (492) Google Scholar). Binding of E1A to CBP inhibits transcriptional activation mediated by CBP. Since E1A and HAT P/CAF bind to the same region of CBP, the mechanism of inhibition of CBP activity by E1A was postulated to be due to blocking of P/CAF binding to CBP (34Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar). To investigate the possibility that CBP is also involved in transcriptional activation by ATF-2, we have examined for a direct interaction between CBP and ATF-2. Our results indicate that CBP functions as a regulator of ATF-2 by binding to its b-ZIP region. To express various forms of GST-CBP fusion proteins in Escherichia coli, the plasmids pGEX-KIX, pGEX-Bromo, pGEX-C/H2, and pGEX-C/H3 were made by inserting the appropriate fragment encoding the 265- (amino acids 454–718), 104- (amino acids 1087–1190), 437- (amino acids 1190–1626), and 257-amino acid (amino acids 1621–1877) regions of mouse CBP, respectively, into the appropriate site of the pGEX vector (see Ref. 40Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5047) Google Scholar; Amersham Pharmacia Biotech). The plasmid to express the GST fusion protein containing the amino-terminal 253 amino acids of ATF-2 was constructed using the pGEX-2TK vector. The modified pSP65 vector pSPUTK (Stratagene) was used for in vitro transcription/translation of various forms of ATF-2. A series of mutants of ATF-2 was described previously (6Matsuda S. Maekawa T. Ishii S. J. Biol. Chem. 1991; 266: 18188-18193Abstract Full Text PDF PubMed Google Scholar). The plasmid to express CREB, c-Jun, c-Fos, or E1A byin vitro transcription/translation system was also made using pGEM (Promega), pBluescript, and the pcDNA3 vector (Invitrogen), respectively. The plasmids to express Gal4-CBPC/H2 in which the C/H2 domain of CBP (amino acids 1182–1500) was fused to the DNA-binding domain of Gal4 (amino acids 1–147) were constructed by the polymerase chain reaction-based method using the cytomegalovirus promoter-containing vector, pSTCX556 (41Severne Y. Wieland S. Schaffner W. Rusconi S. EMBO J. 1988; 7: 2503-2508Crossref PubMed Scopus (134) Google Scholar). The plasmid encoding the VP16 fusion protein containing the carboxyl-terminal region of ATF-2 (amino acids 291–505), CREB (amino acids 191–341), or c-Jun (amino acids 201–334) joined to the VP16 activation domain was constructed similarly. The plasmids to express CBP and E1A were described previously (22Dai P. Akimaru H. Tanaka Y. Hou D.-X. Yasukawa T. Kanei-Ishii C. Takahashi T. Ishii S. Genes Dev. 1996; 10: 528-540Crossref PubMed Scopus (303) Google Scholar). The GST pull-down assay using GST-CBP and in vitro translated ATF-2, CREB, c-Jun, or c-Fos was essentially performed as described (22Dai P. Akimaru H. Tanaka Y. Hou D.-X. Yasukawa T. Kanei-Ishii C. Takahashi T. Ishii S. Genes Dev. 1996; 10: 528-540Crossref PubMed Scopus (303) Google Scholar). The expression of the GST fusion protein or GST alone in E. coliand preparation of the bacterial lysates containing these proteins were done as described (42Nomura T. Sakai N. Sarai A. Sudo T. Kanei-Ishii C. Ramsay R.G. Favier D. Gonda T.J. Ishii S. J. Biol. Chem. 1993; 268: 21914-21923Abstract Full Text PDF PubMed Google Scholar). Samples of bacterial lysate containing 20 μg of GST or GST-CBP were rocked for 2–3 h at 4 °C with 100 μl of glutathione-Sepharose beads (Amersham Pharmacia Biotech). The beads were washed with 1 ml of PBS 5 times and then with 1 ml of binding buffer (20 mm Hepes, pH 7.7, 75 mm KCl, 0.1 mm EDTA, 2.5 mm MgCl2, 1% skim milk, 1 mm dithiothreitol, 0.05% Nonidet P-40). Various forms of ATF-2, CREB, c-Jun, or c-Fos were synthesized with [35S]methionine using an in vitrotranscription/translation kit according to the procedures described by the supplier (Promega). Then, a sample from the reaction was mixed with 750 μl of binding buffer and the GST or GST-CBP affinity resin. After rocking at 4 °C overnight, the resin was washed with 1 ml of binding buffer 5 times and mixed with SDS-sample buffer, and the bound proteins were released by boiling. The proteins were analyzed by SDS-PAGE followed by autoradiography. In the experiments to examine the effects of phosphorylation by PKA, 25 μl of lysate containing the in vitro translated CREB was mixed with 175 μl of the kinase buffer to give final concentrations of 20 mm Tris-HCl, pH 7.5, 100 mm NaCl, 12 mm MgCl2, and 2.5 mm ATP, and the mixture was incubated with or without 100 units of the catalytic subunit of bovine PKA (Sigma). The potato acid phosphatase treatment of the in vitro translated CREB was performed as described (22Dai P. Akimaru H. Tanaka Y. Hou D.-X. Yasukawa T. Kanei-Ishii C. Takahashi T. Ishii S. Genes Dev. 1996; 10: 528-540Crossref PubMed Scopus (303) Google Scholar) except for the use of 1 milliunit of phosphatase. To examine the intramolecular interaction of ATF-2, the GST pull-down assay using the GST fusion protein containing the amino-terminal portion of ATF-2 and the in vitro translated carboxyl-terminal region of ATF-2 was done similarly, except for the use of a low stringency binding buffer (10 mm Hepes, pH 8.0, 50 mm KCl, 2.5 mm MgCl2, 50 mm ZnCl2, 0.025% Nonidet P-40, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 100 μg/ml bovine serum albumin). To investigate whether the SAPK-phosphorylated activation domain of ATF-2 binds to CBP, GST pull-down assays were performed as follows. HepG2 cells were transfected with 5 μg of the ATF-2 expression plasmid, and the transfected cells were treated with sorbitol (0.5m) or PBS for 30 min before the preparation of cell lysates. Three days after transfection, cell lysates were prepared using the lysis buffer (50 mm Hepes, pH 7.5, 250 mm NaCl, 0.2 mm EDTA, 50 mm NaF, 0.5% Nonidet P-40, 2 mm Na3VO4, 25 mm β-glycerophosphate, and 0.1 nm okadaic acid) containing the protease inhibitor mixture (Boehringer Mannheim) and mixed with the resin containing 20 μg of the GST-CBP fusion protein containing various parts of CBP. The bound proteins were eluted by SDS sample buffer and analyzed by Western blotting using the anti-ATF-2 antibody and chemiluminescent detection reagents (New England Biolabs). To investigate thein vivo interaction between the C/H2 domain of CBP and the b-ZIP region of ATF-2, two-hybrid assays were done using HepG2 cells essentially as described (43Kanei-Ishii C. Tanikawa J. Nakai A. Morimoto R.I. Ishii S. Science. 1997; 277: 246-248Crossref PubMed Scopus (55) Google Scholar). A mixture containing 1 μg of the luciferase reporter plasmid containing three copies of the Gal4-binding site linked to the TK promoter, 3 μg of the Gal4-CBPC/H2 expression plasmid, 3 μg of VP16-ATF-2, or VP16 expression plasmid, and 0.5 μg of the internal control plasmid pRL-TK (Promega), in which the sea-pansy luciferase gene is linked to the TK promoter, was transfected into HepG2 cells by using the CaPO4 method. Luciferase assays were performed using the dual-luciferase assay system (Promega). For co-immunoprecipitation of CBP and ATF-2, a mixture of 5 μg of the CBP expression plasmid, pcDNA3-CBP, and 5 μg of the plasmid to express wild type ATF-2 was transfected by using the CaPO4 method into 293T cells which corresponds to the adenovirus type 5-transformed human embryonic kidney 293 cells containing the SV40 large T antigen (44DuBridge R.B. Tang P. Hsia H.C. Phaik-Mooi L. Miller J.H. Calos M.P. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 156-160Crossref PubMed Scopus (754) Google Scholar). Two days after transfection, cells were lysed by rocking in the lysis buffer (50 mm Hepes, pH 7.5, 250 mm NaCl, 0.2 mm EDTA, 10 mm NaF, 0.5% Nonidet P-40) at 4 °C for 1 h and centrifuged at 13,000 rpm for 20 min. The supernatant was mixed with 1.5 volume of the lysis buffer lacking NaCl to decrease the salt concentration to 100 mm. Lysates were immunoprecipitated using anti-CBP antibodies NT (Upstate Biotechnology Inc.) or anti-β-galactosidase, and the immune complexes were separated on 10% SDS gels and analyzed by Western blotting using anti-ATF-2 antibodies (7Takeda J. Maekawa T. Sudo T. Seino Y. Imura H. Saito N. Tanaka C. Ishii S. Oncogene. 1991; 6: 1009-1014PubMed Google Scholar) and ECL detection reagents (Amersham Pharmacia Biotech). For co-immunoprecipitation of Gal4-CBPC/H2 and VP16-CREBDBD or VP16-c-JunDBD, a mixture of 5 μg of the Gal4-CBPC/H2 expression plasmid and 5 μg of the plasmid to express VP16-CREBDBD or VP16-c-JunDBD was transfected into 293 cells. Immunoprecipitation was performed similarly using anti-VP16 antibody V-20 (Santa Cruz), and the immune complexes were separated on 10% SDS gels and analyzed by Western blotting using anti-Gal4 antibody DBD (Santa Cruz) and chemiluminescent detection reagents (New England Biolabs). The CAT co-transfection assay was done essentially as described (14Maekawa T. Matsuda S. Fujisawa J.-I. Yoshida M. Ishii S. Oncogene. 1991; 6: 627-632PubMed Google Scholar). A mixture of 4 μg of the reporter plasmid DNA pMFcolCAT6MBS-I, in which the CAT gene is linked to the mouse α2(I)-collagen promoter and six tandem repeats of the Myb-binding site MBS-I, 5 μg of effector plasmid DNA to express c-Myb-ATF-2 fusion protein consisting of the DNA-binding domain of c-Myb and full-length ATF-2, and 1 μg of the internal control plasmid pact-β-gal was transfected into Chinese hamster ovary cells (CHO-K1). The plasmid to express c-MybDBD-ATF-2-Gla4, in which the carboxyl-terminal 512 amino acids of c-MybDBA-ATF-2 containing the b-ZIP region was replaced by the DNA-binding domain of Gal4 (amino acids 1–147), was constructed using the polymerase chain reaction-based method and also used for co-transfection assays as the effector plasmid. To examine the effect of E1A and CBP, 5 μg of the E1A13S expression plasmid and 6 μg of the CBP expression plasmid were added. The total amount of DNA was adjusted to 21 μg by adding the control plasmid DNA lacking the cDNA to be expressed. Forty hours after transfection, cell lysates were prepared, and CAT assays were done. The amounts of lysates used for the CAT assay were normalized with the β-galactosidase activity expressed from the internal control plasmid pact-β-gal. For UV stimulation, cells were washed with PBS 8 h before preparation of cell lysates and irradiated (45 J/m2) for 15 s. All co-transfection experiments were repeated at least two times, and the difference between each set of experiments was no more than 20%. Typical results are shown in Fig. 4. To examine whether CBP directly binds to ATF-2, we first used the GST pull-down assay. The full-length form of mouse CBP synthesized using an in vitrotranscription/translation system bound to the GST fusion protein containing full-length ATF-2 (data not shown). To narrow down the specific region in CBP responsible for interaction with ATF-2, a series of GST fusion proteins containing various parts of CBP were made and used for the GST pull-down assays (Fig. 1, A and B). The35S-ATF-2 protein was synthesized using an in vitro transcription/translation system and mixed with various GST-CBP resins. Approximately 51% of input ATF-2 bound to the GST fusion protein containing the Cys/His-rich region of CBP, termed the C/H2 domain, whereas other GST fusion proteins containing other regions of CBP and GST alone as a control did not interact with ATF-2 (less than 2% of input) (Fig. 1 C). To examine further which region of ATF-2 interacts with CBP, we used deletion mutants of ATF-2 for the binding assay (Fig. 2, A and B). We previously identified the amino-terminal region between amino acids 19 and 50, which contains a metal finger, as a transcriptional activation domain (6Matsuda S. Maekawa T. Ishii S. J. Biol. Chem. 1991; 266: 18188-18193Abstract Full Text PDF PubMed Google Scholar). The NT50 mutant lacking this activation domain still retained almost full capacity to bind to CBP. About 27 and 30% of the input wild type ATF-2 protein and NT50 bound to the GST-CBP resin, respectively. The two amino-truncated mutants, NT253 and NT341, lacking the amino-terminal 253 and 341 amino acids, respectively, also bound to CBP as efficiently as the wild type. These results indicate that CBP binds to the carboxyl-terminal 164-amino acid region containing the b-ZIP region. The two mutants lacking the cluster of basic amino acids of the b-ZIP region, ΔBR and NT253ΔBR, failed to interact with CBP. In addition, the two mutants in which the third and fourth leucine in the b-ZIP region were changed to valine, L34V and NT253L34V, did not bind to CBP. The CT91 mutant lacking the carboxyl-terminal 91 amino acid region bound to the GST-CBP resin, but its binding efficiency was significantly lower than that of wild type. These results indicate that CBP binds to the b-ZIP region of ATF-2 (amino acids 338–407) and that the region downstream from the b-ZIP region enhances the interaction with CBP. To confirm thein vivo interaction between ATF-2 and CBP in mammalian cells, co-immunoprecipitation was performed (Fig. 3 A). The two plasmids to express ATF-2 and CBP were transfected into 293T cells, and the cell lysates were immunoprecipitated with anti-CBP antibody or control antibody against anti-β-galactosidase. ATF-2 was co-immunoprecipitated with anti-CBP antibody but not with the anti-β-galactosidase antibody. To confirm further the in vivo interaction between ATF-2 and CBP, we performed in vivo two-hybrid assays in mammalian cells (Fig. 3 B). Two chimeric proteins were created by fusing the CBP fragment containing the C/H2 region in frame to the DNA-binding domain of Gal4 and by fusing the carboxyl-terminal region of ATF-2 containing the b-ZIP to the transcriptional activation domain of VP16. Transcriptional activation was then examined in HepG2 cells transfected with a combination of these constructs. The basal activity is the luciferase activity obtained by a combination of Gal4 DNA-binding domain and VP-16. The VP16 protein fused to the carboxyl-terminal region of ATF-2 stimulated Gal4-CBP activity 7.3-fold, whereas VP16 alone stimulated only 2.2-fold. Furthermore, VP16-ATF-2 enhanced the Gal4 activity only 1.7-fold. These results indicated that the C/H2 domain of CBP interact with the carboxyl-terminal region of ATF-2 containing the b-ZIP structure. To investigate the role of CBP in transcriptional activation by ATF-2, we performed some CAT co-transfection experiments using Chinese hamster ovary cells (Fig. 4). Since ATF-2 was expressed in all of the cell lines examined, it was difficult to analyze the transcriptional activation resulting from the exogenous ATF-2 expressed from the transfected DNA. Therefore, we used the fusion protein consisting of the c-myb gene product (c-Myb) and ATF-2 (14Maekawa T. Matsuda S. Fujisawa J.-I. Yoshida M. Ishii S. Oncogene. 1991; 6: 627-632PubMed Google Scholar). c-Myb is a sequence-specific DNA-binding protein, which is predominantly expressed in immature hematopoietic cells but not in many other cells. Therefore, the transcriptional activation by the fusion protein consisting of the DNA-binding domain of c-Myb and full-length ATF-2 (MybDBD-ATF-2) can be analyzed without any interference from the endogenous protein. The plasmid pMFcolCAT6MBS-I, in which the CAT gene is linked to the mouse α2(I)-collagen promoter and six tandem repeats of the Myb-binding site MBS-I, was used as a reporter. Under the conditions used, CAT expression from this reporter plasmid was activated 3.5-fold by the MybDBD-ATF-2 fusion protein (comparelanes 1 and 4). Co-transfection of the CBP expression plasmid with the MybDBD-ATF-2 expression plasmids enhanced the level of CAT activity about 2.7-fold (cf. lanes 4 and 5). The E1A13S product also potentiated by 2.6-fold the trans-activation by MybDBD-ATF-2 as reported previously (cf. lanes 4 and 6), but additional enhancement of the trans-activating capacity of Myb
At least two different types of proteins, NF-kappa B/KBF1 and HIV-EP1/PRDII-BF1/MBP-1, which are members of a family of rel oncoproteins and metal-finger proteins, respectively, bind to the human immunodeficiency virus type (HIV-1) enhancer. As a new member of a HIV-EP1 family that is expressed at a high level in T cells, we have isolated cDNA clones of HIV-EP2 by cross-hybridization with HIV-EP1 cDNA. HIV-EP2 protein consists of 1,833 amino acids and has a molecular weight of 211,000. HIV-EP2 protein is highly homologous with HIV-EP1/PRDII-BF1/MBP-1 in three regions. These three regions contain the potential nuclear localization signal followed by a Ser/Thr-rich region, the DNA-binding domain consisting of a metal-finger structure, and a cluster of acidic amino acids. The DNA-binding property of HIV-EP2 was similar to that of HIV-EP1. Northern blot analysis of HIV-EP2 mRNA indicated relatively high expression in the T cell line Molt-4 and in some tumor cell lines. Furthermore, like HIV-EP1, expression of HIV-EP2 mRNA was greatly induced by mitogen and phorbol ester treatment of Jurkat T cells, suggesting that HIV-EP2 acts in HIV production from latently infected T cells.
Abstract Increasing evidence indicates that parental diet affects the metabolism and health of offspring. It is reported that paternal low-protein diet (pLPD) induces glucose intolerance and the expression of genes involved in cholesterol biosynthesis in mouse offspring liver. The aim of the present study was to determine the effect of a pLPD on gene expression in offspring white adipose tissue (WAT), another important tissue for the regulation of metabolism. RNA-seq analysis indicated that pLPD up- and down-regulated 54 and 274 genes, respectively, in offspring WAT. The mRNA expression of many genes involved in lipogenesis was down-regulated by pLPD feeding, which may contribute to metabolic disorder. The expression of carbohydrate response element-binding protein β (ChREBP-β), an important lipogenic transcription factor, was also significantly lower in the WAT of pLPD offspring, which may have mediated the down-regulation of the lipogenic genes. By contrast, the LPD did not affect the expression of lipogenic genes in the WAT of the male progenitor, but increased the expression of lipid oxidation genes, suggesting that a LPD may reduce lipogenesis using different mechanisms in parents and offspring. These findings add to our understanding of how paternal diet can regulate metabolism in their offspring.
Assisted reproductive technologies, including in vitro fertilization (IVF), are now frequently used, and increasing evidence indicates that IVF causes gene expression changes in children and adolescents that increase the risk of metabolic diseases. Although such gene expression changes are thought to be due to IVF-induced epigenetic changes, the mechanism remains elusive. We tested whether the transcription factor ATF7, – which mediates stress-induced changes in histone H3K9 tri- and di-methylation, typical marks of epigenetic silencing – is involved in the IVF-induced gene expression changes. IVF up- and down-regulated the expression of 688 and 204 genes, respectively, in the liver of 3-week-old wild-type (WT) mice, whereas 87% and 68% of these were not changed, respectively, by IVF in ATF7-deficient (Atf7—/—) mice. The genes, which are involved in metabolism, such as pyrimidine and purine metabolism, were up-regulated in WT mice but not in Atf7—/— mice. Of the genes whose expression was up-regulated by IVF in WT mice, 37% were also up-regulated by a loss of ATF7. These results indicate that ATF7 is a key factor in establishing the memory of IVF effects on metabolic pathways.
