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• W1994539200 abstract "We demonstrate that human activating transcription factor 4 (hATF4), a member of the activating transcription factor/cAMP-responsive element-binding protein (ATF/CREB) family of transcription factors, is a potent transcriptional activator in both mammalian cells and yeast. The N-terminal 113 amino acids of hATF4 activate transcription efficiently, and unexpectedly, the C-terminal bZip DNA binding domain of hATF4 also activates transcription, albeit weakly. Our results indicate that hATF4 interacts with several general transcription factors: TATA-binding protein, TFIIB, and the RAP30 subunit of TFIIF. In addition, hATF4 interacts with the coactivator CREB-binding protein (CBP) at four regions: 1) the KIX domain, 2) a region that contains the third zinc finger and the E1A-interacting domain, 3) a C-terminal region that contains the p160/SRC-1-interacting domain, and 4) the recently identified histone acetyltransferase domain. Interestingly, both the N-terminal and C-terminal regions of hATF4 interact with the above general transcription factors and CBP, providing a mechanistic explanation for their ability to activate transcription. Consistent with its role as a coactivator, CBP potentiates the ability of hATF4 to activate transcription. The potential significance of the interaction between hATF4 and multiple factors is discussed. We demonstrate that human activating transcription factor 4 (hATF4), a member of the activating transcription factor/cAMP-responsive element-binding protein (ATF/CREB) family of transcription factors, is a potent transcriptional activator in both mammalian cells and yeast. The N-terminal 113 amino acids of hATF4 activate transcription efficiently, and unexpectedly, the C-terminal bZip DNA binding domain of hATF4 also activates transcription, albeit weakly. Our results indicate that hATF4 interacts with several general transcription factors: TATA-binding protein, TFIIB, and the RAP30 subunit of TFIIF. In addition, hATF4 interacts with the coactivator CREB-binding protein (CBP) at four regions: 1) the KIX domain, 2) a region that contains the third zinc finger and the E1A-interacting domain, 3) a C-terminal region that contains the p160/SRC-1-interacting domain, and 4) the recently identified histone acetyltransferase domain. Interestingly, both the N-terminal and C-terminal regions of hATF4 interact with the above general transcription factors and CBP, providing a mechanistic explanation for their ability to activate transcription. Consistent with its role as a coactivator, CBP potentiates the ability of hATF4 to activate transcription. The potential significance of the interaction between hATF4 and multiple factors is discussed. Transcription of protein-coding genes is a major regulatory step for gene expression in eukaryotes. Intensive studies in this area have revealed that transcriptional activators exert their effects by contacting general transcription factors (GTFs) 1The abbreviations used are: GTF, general transcription factor; ATF, activating transcription factor; hATF, human ATF; bZip, basic region/leucine zipper; CAT, chloramphenicol acetyltransferase; CRE, cAMP-responsive element; CREB, CRE-binding protein; CBP, CREB-binding protein; CRE-BP1, cAMP-responsive element-binding protein 1; HAT, histone acetyltransferase; GST, glutathione S-transferase; RAP, RNAP (RNA polymerase) II-associating protein; TBP, TATA-binding protein; TF, transcription factor; HTLV, human T-cell leukemia virus. 1The abbreviations used are: GTF, general transcription factor; ATF, activating transcription factor; hATF, human ATF; bZip, basic region/leucine zipper; CAT, chloramphenicol acetyltransferase; CRE, cAMP-responsive element; CREB, CRE-binding protein; CBP, CREB-binding protein; CRE-BP1, cAMP-responsive element-binding protein 1; HAT, histone acetyltransferase; GST, glutathione S-transferase; RAP, RNAP (RNA polymerase) II-associating protein; TBP, TATA-binding protein; TF, transcription factor; HTLV, human T-cell leukemia virus. directly or indirectly (for reviews, see Refs. 1Goodrich J.A. 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Nature. 1993; 365: 855-859Crossref PubMed Scopus (1763) Google Scholar), a transcription factor with the basic region/leucine zipper (bZip) DNA binding domain. Strikingly and unexpectedly, CBP/p300 proteins function as coactivators for not only CREB (7Kwok R.P.S. Lundblad J.R. Chrivia J.C. Richards J.P. Bächinger H.P. Brennan R.G. Roberts S.G.E. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1280) Google Scholar, 8Arias J. Alberts A.S. Brindle P. Claret F.X. Smeal T. Karin M. Feramisco J. Montminy M. Nature. 1994; 370: 226-229Crossref PubMed Scopus (680) Google Scholar, 9Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (921) Google Scholar, 10Arany Z. Sellers W.R. Livingston D.M. Eckner R. Cell. 1994; 77: 799-800Abstract Full Text PDF PubMed Scopus (370) Google Scholar) but also a variety of other sequence-specific transcription factors, such as c-Jun (8Arias J. 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Nature. 1996; 383: 99-103Crossref PubMed Scopus (844) Google Scholar), c-Myb (17Dai 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, 18Oelgeschläger M. Janknecht R. Krieg J. Schreek S. Lüscher B. EMBO J. 1996; 15: 2771-2780Crossref PubMed Scopus (193) Google Scholar), YY1 (19Lee J.-S. Galvin K.M. See R.H. Eckner R. Livingston D. Moran E. Shi Y. Genes Dev. 1995; 9: 1188-1198Crossref PubMed Scopus (280) Google Scholar, 20Lee J.-S. Zhang X. Shi Y. J. Biol. Chem. 1996; 271: 17666-17674Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), Sap-1a (21Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar), MyoD (22Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 23Sartorelli V. Huang J. Hamamori Y. Kedes L. Mol. Cell. Biol. 1997; 17: 1010-1026Crossref PubMed Scopus (321) Google Scholar), Stat2 (24Bhattacharya S. Eckner R. Grossman S. Oldread E. Arany Z. D'Andrea A. Livingston D.M. Nature. 1996; 383: 344-347Crossref PubMed Scopus (419) Google Scholar), SREBP (25Oliner J.D. Andresen J.M. Hansen S.K. Zhou S. Tjian R. Genes Dev. 1996; 10: 2903-2911Crossref PubMed Scopus (140) Google Scholar), and NF-κB (26Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D.H. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar). These observations provide an explanation for the ability of some of these transcription factors to inhibit each other. For example, nuclear receptors have been demonstrated to inhibit the activity of c-Jun by competing with c-Jun for CBP (14Kamei 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 (1918) Google Scholar). Significantly, in addition to interacting with sequence-specific transcription factors, CBP/p300 also interacts with other proteins such as the S6 kinase pp90 rsk (27Nakajima T. Fukamizu A. Takahashi J. Gage F.H. Fisher T. Blenis J. Montminy M.R. Cell. 1996; 86: 465-474Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar), nuclear receptor coactivator p160/SRC-1 (14Kamei 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 (1918) Google Scholar, 15Smith C.L. Oñate S.A. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8884-8888Crossref PubMed Scopus (367) Google Scholar), several GTFs (see below), and viral proteins E1A (9Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (921) Google Scholar, 28Arany Z. Newsome D. Oldread E. Livingston D.M. Eckner R. Nature. 1995; 374: 81-84Crossref PubMed Scopus (490) Google Scholar, 29Lundblad 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), SV40 T antigen (30Avantaggiati M.L. Carbone M. Graessmann A. Nakatani Y. Howard B. Levine A.S. EMBO J. 1996; 15: 2236-2248Crossref PubMed Scopus (136) Google Scholar, 31Eckner R. Ludlow J.W. Lill N.L. Oldread E. Arany Z. Modjtahedi N. DeCaprio J.A. Livingston D.M. Morgan J.A. Mol. Cell. Biol. 1996; 16: 3454-3464Crossref PubMed Scopus (225) Google Scholar), and HTLV1 Tax (32Kwok R.P.S. Laurance M.E. Lundblad J.R. Goldman P.S. Shih H.-M. Connor L.M. Marriott S.J. Goodman R.H. Nature. 1996; 380: 642-646Crossref PubMed Scopus (308) Google Scholar). Therefore, CBP/p300 functions as a target for a variety of proteins involved in different regulatory circuits, allowing cells to coordinate or integrate signals from different pathways. Consequently, it has been suggested that it be named an “integrator” (14Kamei 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 (1918) Google Scholar) or “co-integrator” (16Chakravarti 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 (844) Google Scholar). Although the mechanisms by which CBP/p300 functions as a coactivator remain to be determined, the current understanding suggests several possibilities. One possibility is that it acts as an adapter between the sequence-specific transcriptional activators and GTFs such as TBP (22Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 33Abraham S.E. Lobo S. Yaciuk P. Wang H.G. Moran E. Oncogene. 1993; 8: 1639-1647PubMed Google Scholar), TFIIB (7Kwok R.P.S. Lundblad J.R. Chrivia J.C. Richards J.P. Bächinger H.P. Brennan R.G. Roberts S.G.E. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1280) Google Scholar), and RNA polymerase II (34Kee B. Arias J. Montminy M.R. J. Biol. Chem. 1996; 271: 2373-2375Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Another possibility is that CBP/p300 enhances the acetylation of histones; this presumably destabilizes the nucleosome and facilitates access of DNA by regulatory factors (for reviews see Refs. 35Brownell J.E. Allis C.D. Curr. Opin. Genet. Dev. 1996; 6: 176-184Crossref PubMed Scopus (464) Google Scholar and 36Wolffe A.P. Pruss D. Cell. 1996; 84: 817-819Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). Recent studies indicate that CBP/p300 can enhance acetylation of histones by at least two mechanisms; it has an intrinsic histone acetyltransferase (HAT) activity (37Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2377) Google Scholar, 38Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1526) Google Scholar), and it recruits P/CAF, which also has HAT activity (39Yang X.-J. Ogryzko V.V. Nishikawa J.-I. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1311) Google Scholar). In this report, we demonstrate that human ATF4 (hATF4), a member of the ATF/CRE family of proteins, also interacts with CBP/p300. Binding sites for this family of proteins are present in many cellular and viral promoters (for a review see Ref. 40Sassone-Corsi P. Annu. Rev. Cell Dev. Biol. 1995; 11: 355-377Crossref PubMed Scopus (336) Google Scholar), suggesting an involvement of this family of proteins in the regulation of many genes. A partial cDNA clone of hATF4 was originally isolated by its ability to bind to the consensus ATF/CRE site (41Hai T. Liu F. Coukos W.J. Green M.R. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (754) Google Scholar). The full-length clone was later isolated and named as TAXREB67 (42Tsujimoto A. Nyunoya H. Morita T. Sato T. Shimotohno K. J. Virol. 1991; 65: 1420-1426Crossref PubMed Google Scholar) or CREB2 (43Karpinski B.A. Morle G.D. Huggenvik J. Uhler M.D. Leiden J.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4820-4824Crossref PubMed Scopus (208) Google Scholar). However, the term “CREB2” was also used to refer to CRE-BP1/ATF2 (44Benbrook D.M. Jones N.C. Oncogene. 1990; 5: 295-302PubMed Google Scholar) and an alternatively spliced form of CREB that lacks 14 amino acids (45Yoshimura T. Fujisawa J.I. Yoshida M. EMBO J. 1990; 9: 2537-2542Crossref PubMed Scopus (151) Google Scholar, 46Willems L. Kettmann R. Chen G. Portetelle D. Burny A. Derse D. DNA Seq. 1991; 1: 415-417Crossref PubMed Scopus (4) Google Scholar). Therefore, to avoid confusion, we will refer to the human clone as hATF4 in the rest of this report. Three mouse cDNAs, mATF4 (47Mielnicki L.M. Pruitt S.C. Nucleic Acids Res. 1991; 19: 6332Crossref PubMed Scopus (24) Google Scholar), mTR67 (48Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Crossref PubMed Scopus (479) Google Scholar), and C/ATF (49Vallejo M. Ron D. Miller C.P. Habener J.F. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4679-4683Crossref PubMed Scopus (208) Google Scholar), share over 90% identity to hATF4 (Fig. 1 A). Therefore, ATF4 represents a group of conserved proteins. Interestingly, all ATF4 proteins have a second zipper in addition to the C-terminal leucine zipper (Fig. 1 A). It is not clear, however, whether this second zipper is functionally important. Although all ATF4 proteins are bZip proteins, the only similarity they share with CREB (50Hoeffler J.P. Meyer T.E. Yun Y. Jameson J.L. Habener J.F. Science. 1988; 242: 1430-1433Crossref PubMed Scopus (524) Google Scholar, 51Gonzalez G.A. Yamamoto K.K. Fischer W.H. Karr D. Menzel P. Biggs III, W. Vale W.W. Montminy M.R. Nature. 1989; 337: 749-752Crossref PubMed Scopus (648) Google Scholar) and other ATF proteins is the conserved residues in the basic region and the conserved leucine residues in the leucine zipper (for reviews see Refs. 40Sassone-Corsi P. Annu. Rev. Cell Dev. Biol. 1995; 11: 355-377Crossref PubMed Scopus (336) Google Scholar and 52Hurst H.C. Protein Profile. 1994; 1: 123-168PubMed Google Scholar). In the rest of the bZip domain and outside of the bZip domain, ATF4 proteins are completely different from CREB and other ATF proteins. Therefore, the names of various ATF proteins reflect more the history of discovery rather than the similarities between them. Thus far, the physiological functions of ATF4 proteins have not been defined. Tissue distribution of ATF4 mRNA demonstrated that it is present in most tissues or cell lines examined, revealing little clues about its physiological functions. In this context, it is interesting to note that aplysia CREB2/ATF4 (ApCREB2/ATF4), which has been demonstrated to repress long term facilitation (53Bartsch D. Ghirardi M. Skehel P.A. Karl K.A. Herder S.P. Chen M. Bailey C.H. Kandel E.R. Cell. 1995; 83: 979-992Abstract Full Text PDF PubMed Scopus (492) Google Scholar), is 50% identical to hATF4 in the bZip domain and has a second zipper (Fig.1 B). However, it is not clear whether the mammalian ATF4 plays a similar role. In this report, we show that hATF4 is a strong activator in both mammalian cells and yeast. It interacts with several GTFs and with the coactivator CBP at multiple domains including the recently identified HAT domain. N-terminal and C-terminal deletion constructs of hATF4 were generated by ExoIII deletion. GAL4 fusions were generated by placing GAL4 DNA binding domain at either the N or C terminus of various hATF4 fragments, and the resulting fusions are indicated as GAL4-hATF4 or hATF4-GAL4, respectively. To express the proteins, various DNA fragments were cloned into appropriate expression vectors: pCG (from W. Herr) for mammalian cells, pTM1 (from B. Moss) for reticulocyte translation, pGBT-9 (from S. Fields) for yeast expression, and pET-His (54Chen B.P.C. Hai T. Gene ( Amst. ). 1994; 139: 73-75Crossref PubMed Scopus (71) Google Scholar) for expression in Escherichia coli. Yeast strain Y190 (from S. J. Elledge), which lacks the endogenous functional GAL4 protein is MATa, leu2–3,112, ura3–52, trp1–901, his3-Δ200, ade2–101, gal4Δgal80Δ URA3 GAL-lacZ, LYS GAL-HIS3, cyhr (55Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5220) Google Scholar). Yeast transformation and the β-galactosidase assay were carried out according to standard protocols (56Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Plainview, NY1990Google Scholar, 57Guthrie C. Fink G.R. Methods in Enzymology: Guide to Yeast Genetics and Molecular Biology.194. Academic Press, New York1991Google Scholar). Cell culture, calcium phosphate transfection, and CAT assays were performed as described previously (58Chen B.P.C. Liang G. Whelan J. Hai T. J. Biol. Chem. 1994; 269: 15819-15826Abstract Full Text PDF PubMed Google Scholar). For transfection, unless otherwise indicated, 1.5 μg of reporter DNA and 0.5 μg of effector DNA expressing the indicated proteins were used for each 60-mm plate of cells. Appropriate amounts of pGEM3 carrier DNA were included to make a total 7 μg of DNA for each transfection. hATF4 tagged with six contiguous histidines was expressed in E. coli BL21 (DE3/LysS) (from F. W. Studier) according to Studier et al. (59Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5998) Google Scholar) and purified at 4 °C as follows. Cell pellets were resuspended in ice-cold 1 m bZip buffer (1 mNaCl, 50 mm sodium phosphate, pH 7.8, 5% glycerol, 2 mm β-mercaptoethanol) containing various protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 0.1m sodium metabisulfite, 2 μg/ml aprotinin, and 2 μg/ml leupeptin). The high concentration of NaCl appears to increase the solubility of hATF4. Tween 20 was added to a final concentration of 0.1%, and the cell suspension was lysed by sonication on ice. After centrifugation at 15,000 × g at 4 °C for 15 min, the supernatant was separated from the cell debris and mixed with Ni2+ nitrilotriacetic acid-agarose beads (Qiagen) that had been preequilibrated in 1 m bZip buffer. After gentle rocking for 1 h, the beads were packed into column and washed with two column volumes of 1 m bZip buffer, two column volumes of 0.3 m bZip buffer (same as 1 m bZip except NaCl was 0.3 m), and five column volumes of 0.3m bZip buffer containing 25 mm imidazole. Bound proteins were eluted with 0.3 m bZip buffer containing 0.5m imidazole and dialyzed against buffer D (20 mm Hepes, pH 7.9, 0.1 m NaCl, 20% glycerol, 0.2 mm EDTA, and 0.5 mm dithiothreitol) containing 0.5 mm phenylmethylsulfonyl fluoride and 0.1m sodium metabisulfite. Protein concentrations were determined by Bio-Rad assay, and bovine serum albumin was added, if necessary, to make the final protein concentration 1 mg/ml. In vitro transcription was carried out using primer extension as described previously (60Hai T. Horikoshi M. Roeder R.G. Green M.R. Cell. 1988; 54: 1043-1051Abstract Full Text PDF PubMed Scopus (102) Google Scholar). The CAT primer 5′-GCCATTGGGATATATCAACGG-3′ is complementary to the region from +29 to +49 of the CAT mRNA. Nuclear extracts were made from HeLa cells according to Dignam et al. (61Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9150) Google Scholar). pTM1 derivatives encoding the indicated proteins were transcribed by T7 polymerase and translated in the presence of [35S]methionine using the TNT reticulocyte lysate system (Promega) according to the manufacturer's instructions. E. colicells expressing the indicated GST fusion proteins were collected and resuspended in ice-cold 1 m TED buffer (1 mKCl, 50 mm Tris-HCl, pH 8, 2 mm EDTA, 1 mm dithiothreitol) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 0.1 m sodium metabisulfite, 2 μg/ml aprotinin, and 2 μg/ml leupeptin). Triton X-100 was added to 1%, and the cell suspension was sonicated on ice. After centrifugation at 15,000 × g for 15 min at 4 °C, the supernatant was separated from the cell debris. Aliquots were quick-frozen in liquid nitrogen and stored at −80 °C. Each aliquot (0.25–1 ml, depending on the levels of expression) of the frozen supernatant was thawed and incubated at 4 °C for 30 min with 10 μl of glutathione-conjugated agarose beads (Sigma) that had been prewashed in 1 m TED buffer, and the unbound proteins were removed by washes in GST binding buffer (100 mm KCl, 40 mm Hepes, pH 7.5, 5 mm MgCl2, 0.2 mm EDTA, 1 mm dithiothreitol, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 0.1 msodium metabisulfite, 2 μg/ml aprotinin, and 2 μg/ml leupeptin). The resulting glutathione-agarose beads bound with the fusion protein were used in the binding assay immediately. The beads were incubated with the indicated 35S-labeled proteins at 4 °C in 0.5 ml of GST binding buffer for 1 h to allow binding and washed with GST binding buffer for three times (4 °C, 5 min each time). The bound proteins were eluted by boiling the beads with 20 μl of SDS-PAGE loading buffer and analyzed by electrophoresis. The GST fusion proteins were visualized by Coomassie Blue stain, and the labeled proteins were visualized by autoradiography. To examine the function of hATF4, we transfected HeLa cells with pCG-hATF4, which expresses hATF4, and a CAT reporter driven by tandem ATF sites. As shown in Fig.2 A, overexpression of hATF4 activated the reporter more than 150-fold. This activity can be transferred to a heterologous DNA binding domain, the GAL4 DNA binding domain. A plasmid expressing the hATF4-GAL4 fusion protein activated the CAT reporter driven by tandem GAL4 sites. The -fold activation ranged from approximately 60 (Fig. 4) to 700 (Fig.3), depending on the amount of DNA used (10 and 500 ng, respectively). This activity was comparable with that of GAL4-VP16 (data not shown), a well characterized, strong viral activator. Although the DNA binding domain of hATF4 is at its C terminus, the orientation of fusion does not significantly affect the activity (data not shown). Therefore, fusion proteins with either orientation were used in this study. In the rest of this report, “GAL4-hATF4” refers to fusion proteins with a GAL4 DNA binding domain at the N terminus, and “hATF4-GAL4” refers to fusion proteins with a GAL4 DNA binding domain at the C terminus.Figure 4The N-terminal 113 amino acids of hATF4 function as a potent activator. The CAT reporter driven by tandem GAL4 sites was transfected into HeLa cells with 10 ng of pCG or pCG derivatives, producing the indicated hATF4-GAL4 fusion proteins. The fold of activation by hATF4 is calculated by defining the reporter activity in the presence of pCG as 1. The activity of hATF4-(1–339) is then defined as 100%. This construct lacks the last 12 amino acids of hATF4 (to remove the termination codon) and has an activity comparable with that of the full-length hATF4 with GAL4 fused at its N terminus (data not shown). The result is the average of five experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Both the N- and the C-terminal regions of hATF4 contain a transcriptional activation domain. A, the N-terminal region is important for the function of hATF4. The CAT reporter driven by three tandem ATF sites was transfected into HeLa cells with pCG vector or pCG derivatives producing the indicated hATF4 proteins. The fold of activation by each derivative is calculated by defining the reporter activity in the presence of pCG as 1. The relative activity is calculated by defining the activity of the full-length hATF4 as 100%. The result is the average of five experiments. B, the bZip region of hATF4 has transcriptional activity when fused to the GAL4 DNA binding domain. The CAT reporter driven by five tandem GAL4 sites was transfected into HeLa cells with pCG vector or pCG derivatives, producing the indicated fusion proteins. The fold of activation and the relative activity are calculated as inpanel A. The result is the average of five experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because the CAT reporters used above were driven by artificial promoters, we asked whether ATF4 can activate transcription from naturally occurring promoters. We examined the following promoters containing functional ATF or ATF-like sites: adenovirus E4, E-selectin, proenkephalin (ENK), HTLV-1, somatostatin (SOM), and tyrosine aminotransferase (TAT) promoters (Fig. 2). As shown in Fig. 2 A, hATF4 activated all of these promoters, ranging from a few to 50-fold. The fold of activation was not as high as that observed on the artificial promoter, possibly because the natural promoters contain binding sites for other transcription factors and have higher basal transcriptional activity, leading to less activation; alternatively, the activation may be lower because hATF4 does not interact well with other factors on the promoters and consequently does not activate the promoter efficiently. To test whether the ability of hATF4 to activate transcription can be recapitulated in vitro, we examined its activity by anin vitro transcription assay. As shown in Fig.2 B, hATF4 activated transcription from a promoter containing tandem ATF sites in a dose-dependent manner. However, when hATF4 was added at a high concentration, the activity of the promoter started to decrease (Fig. 2 B, last lane), presumably because hATF4 sequestered factors/co-factors away from the promoter at high concentrations, a phenomenon commonly referred to as “squelching” (62Gill G. Ptashne M. Nature. 1988; 334: 721-724Crossref PubMed Scopus (495) Google Scholar). Because many mammalian transcription factors can function in yeast, we examined whether hATF4 also activates transcription in yeast. We took a GAL4 fusion approach to avoid potential interference from the endogenous yeast ATF-related proteins (63Nojima H. Leem S.-H. Araki H. Sakai A. Nakashima N. Kanaoka Y. Ono Y. Nucl. Acids Res. 1994; 22: 5279-5288Crossref PubMed Scopus (58) Google Scholar, 64Vincent A.C. Struhl K. Mol. Cell. Biol. 1992; 12: 5394-5405Crossref PubMed Scopus (70) Google Scholar, 65Nehlin J.O. Carlberg M. Ronne H. Nucleic Acids Res. 1992; 20: 5271-5278Crossref PubMed Scopus (90) Google Scholar, 66Jones R.H. Jones N.C. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2176-2180Crossref PubMed Scopus (40) Google Scholar). As shown in Fig.2 C, the full-length (FL) GAL4-hATF4 activated a β-galactosidase reporter driven by tandem GAL4 sites. The activation by GAL4-hATF4 was comparable with that by the full-length GAL4, a well characterized yeast activator, indicating that hATF4 can function as a potent activator in yeast. In summary, we demonstrate that hATF4 can activate transcription in a mammalian system both in vivoand in vitro; in addition, it functions as a potent transcriptional activator in yeast. To examine whether hATF4 contains discrete transcriptional activation domains, we made a series of N- and C-terminal deletions and examined their activities by transfection. Fig. 3 A shows the results of the N-terminal deletions. Deletion of the first N-terminal 22 amino acids (ΔN22) abolished 80% of the activity. Experiments in yeast using GAL4 fusion proteins showed a comparable result (Fig.2 C), indicating that the first 22 amino acids are import" @default.
• W1994539200 created "2016-06-24" @default.
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• W1994539200 date "1997-09-01" @default.
• W1994539200 modified "2023-09-30" @default.
• W1994539200 title "Characterization of Human Activating Transcription Factor 4, a Transcriptional Activator That Interacts with Multiple Domains of cAMP-responsive Element-binding Protein (CREB)-binding Protein (CBP)" @default.
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• W1994539200 doi "https://doi.org/10.1074/jbc.272.38.24088" @default.
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