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Identification of a Highly Conserved Module in E Proteins Required for in Vivo Helix-loop-helix Dimerization

1998; Elsevier BV; Volume: 273; Issue: 5 Linguagem: Inglês

10.1074/jbc.273.5.2866

ISSN

1083-351X

Autores

Adam N. Goldfarb, Kristine Lewandowska, Christopher A. Pennell,

Tópico(s)

Histone Deacetylase Inhibitors Research

Resumo

Basic helix-loop-helix (bHLH) transcription factors often function as heterodimeric complexes consisting of a tissue-specific factor such as SCL/tal or MyoD bound to a broadly expressed E protein. bHLH dimerization therefore appears to represent a key regulatory step in cell lineage determination and oncogenesis. Previous functional and structural studies have indicated that the well defined HLH domain is both necessary and sufficient for dimerization. Most of these studies, however, have employed in vitrosystems for analysis of HLH dimerization, and their implications for the requirements for in vivo dimerization remain unclear. Using multiple approaches, we have analyzed bHLH dimerization in intact, living cells and have identified a novel domain in E proteins, domain C, which is required for in vivo dimerization. Domain C, which lies just carboxyl-terminal to helix 2 of the HLH domain, represents the most highly conserved region within E proteins and appears to influence the in vivo conformation of the adjacent HLH domain. These results suggest that HLH dimerizationin vivo may represent a complex, regulated process that is distinct from HLH dimerization in vitro. Basic helix-loop-helix (bHLH) transcription factors often function as heterodimeric complexes consisting of a tissue-specific factor such as SCL/tal or MyoD bound to a broadly expressed E protein. bHLH dimerization therefore appears to represent a key regulatory step in cell lineage determination and oncogenesis. Previous functional and structural studies have indicated that the well defined HLH domain is both necessary and sufficient for dimerization. Most of these studies, however, have employed in vitrosystems for analysis of HLH dimerization, and their implications for the requirements for in vivo dimerization remain unclear. Using multiple approaches, we have analyzed bHLH dimerization in intact, living cells and have identified a novel domain in E proteins, domain C, which is required for in vivo dimerization. Domain C, which lies just carboxyl-terminal to helix 2 of the HLH domain, represents the most highly conserved region within E proteins and appears to influence the in vivo conformation of the adjacent HLH domain. These results suggest that HLH dimerizationin vivo may represent a complex, regulated process that is distinct from HLH dimerization in vitro. For several classes of transcription factors, such as members of the basic helix-loop-helix, leucine zipper, and nuclear receptor families, dimerization represents a key, obligatory step prior to DNA binding and transcriptional activation. This dimerization permits the mixing and matching of factors with different DNA half-site binding specificities and expands the repetoire of potential target sequences that may be recognized. Basic helix-loop-helix (bHLH) 1The abbreviations used are: HLH, helix-loop-helix; LacZ, β-galactosidase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; CMV, cytomegalovirus; GST, glutathione S-transferase; MBP, maltose-binding protein. factors in metazoan organisms often regulate the expression of target genes as heterodimeric complexes between tissue-specific factors, such as SCL/tal or MyoD, and broadly expressed E proteins (1Lassar A.B. Davis R.L. Wright W.E. Kadesch T. Murre C. Voronova A. Baltimore D. Weintraub H. Cell. 1991; 66: 305-315Abstract Full Text PDF PubMed Scopus (688) Google Scholar, 2Weintraub H. Davis R. Tapscott S. Thayer M. Krause M. Benezra R. Blackwell T.K. Turner D. Rupp R. Hollenberg S. Zhuang Y. Lassar A. Science. 1991; 251: 761-766Crossref PubMed Scopus (1237) Google Scholar, 3Jan Y.N. Jan L.Y. Cell. 1993; 75: 827-830Abstract Full Text PDF PubMed Scopus (392) Google Scholar). The composition of bHLH complexes is dictated in part by the dimerization specificities of the constituents; in particular, tissue-specific bHLH factors tend not to interact with one another but rather to bind universally to E protein partners (4Hsu H.-L. Wadman I. Baer R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3181-3185Crossref PubMed Scopus (105) Google Scholar, 5Goldfarb A.N. Lewandowska K. Blood. 1995; 85: 465-471Crossref PubMed Google Scholar). Likewise, the dominant negative HLH proteins of the Id family exert their inhibitory effect through preferential binding to E proteins (6Sun X.-H. Copeland N.G. Jenkins N.A. Baltimore D. Mol. Cell. Biol. 1991; 11: 5603-5611Crossref PubMed Scopus (532) Google Scholar, 7Jen Y. Weintraub H. Benezra R. Genes Dev. 1992; 6: 1466-1479Crossref PubMed Scopus (398) Google Scholar, 8Hara E. Hall M. Peters G. EMBO J. 1997; 16: 332-342Crossref PubMed Scopus (115) Google Scholar). Thus a progenitor cell during embryogenesis may contain an array of different cell lineage-specific bHLH factors all competing with one another, as well as with inhibitory HLH proteins, for dimerization with a common E protein partner. Such competition would allow the progenitor cell to make mutually exclusive, binary decisions with regard to lineage commitment, proliferation, and terminal differentiation (2Weintraub H. Davis R. Tapscott S. Thayer M. Krause M. Benezra R. Blackwell T.K. Turner D. Rupp R. Hollenberg S. Zhuang Y. Lassar A. Science. 1991; 251: 761-766Crossref PubMed Scopus (1237) Google Scholar). The structural basis for bHLH dimerization, according to x-ray crystallography, resides in the formation of a parallel, four-helix bundle in which dimerization contacts derive from conserved hydrophobic residues clustered within a shielded core (9D'Amare-Ferre A.R. Prendergast G.C. Ziff E.B. Burley S.K. Nature. 1993; 363: 38-45Crossref PubMed Scopus (599) Google Scholar, 10Ellenberger T. Fass D. Arnaud M. Harrison S.C. Genes Dev. 1994; 8: 970-980Crossref PubMed Scopus (356) Google Scholar, 11Ma P.C.M. Rould M.A. Weintraub H. Pabo C.O. Cell. 1994; 77: 451-459Abstract Full Text PDF PubMed Scopus (401) Google Scholar). However, the crystallographic structures have all been obtained with pre-formed, DNA-bound complexes and provide no information on the transition states that occur in the process of dimerization in solution. Furthermore, the data from the crystal structures provide no satisfactory explanation for dimerization specificities: all HLH factors possess similar hydrophobic dimerization contact residues, and yet tissue-specific bHLH factors show highly restricted dimerization specificities while E proteins demonstrate considerable promiscuity in dimerization. In vitro biochemical studies suggest that nonconserved hydrophilic residues may somehow contribute to HLH dimerization specificity (12Shirakata M. Friedman F.K. Paterson B.M. Genes Dev. 1993; 7: 2456-2470Crossref PubMed Scopus (49) Google Scholar). As an additional complication, in vitro dimerization of bHLH factors does not appear accurately to reflect the dimerization processin vivo. In most analyses, in vitro bHLH dimerization, whether with crude extracts or with purified proteins, is a highly inefficient process which requires subphysiologic temperatures and displays affinities in the micromolar range (13Sun X.-H. Baltimore D. Cell. 1991; 64: 459-470Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 14Fairman R. Beran-Steed R.K. Anthony-Cahill S.J. Lear J.D. Stafford W.F. DeGrado W.F. Benfield P.A. Brenner S.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10429-10433Crossref PubMed Scopus (96) Google Scholar, 15Maleki S.J. Royer C.A. Hurlburt B.K. Biochem. 1997; 36: 6762-6767Crossref PubMed Scopus (21) Google Scholar, 16Bishop P. Ghosh I. Jones C. Chmielewski J. J. Am. Chem. Soc. 1995; 117: 8283-8284Crossref Scopus (10) Google Scholar). Studies of in vivo dimerization portray a process that appears to be extremely efficient, with rapid heterodimerization occurring just prior to nuclear localization (17Goldfarb A.N. Lewandowska K. Exp. Cell Res. 1994; 214: 481-485Crossref PubMed Scopus (19) Google Scholar, 18Deed R.W. Armitage S. Norton J.D. J. Biol. Chem. 1996; 271: 23603-23606Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). To study factors influencing dimerization in vivo, we have exploited a number of established systems applicable to bHLH dimerization, including yeast two-hybrid, mammalian two hybrid, nuclear redirection assays, and coimmunoprecipitation (4Hsu H.-L. Wadman I. Baer R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3181-3185Crossref PubMed Scopus (105) Google Scholar, 17Goldfarb A.N. Lewandowska K. Exp. Cell Res. 1994; 214: 481-485Crossref PubMed Scopus (19) Google Scholar, 18Deed R.W. Armitage S. Norton J.D. J. Biol. Chem. 1996; 271: 23603-23606Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 19Staudinger J. Perry M. Elledge S.J. Olson E.N. J. Biol. Chem. 1993; 268: 4608-4611Abstract Full Text PDF PubMed Google Scholar, 20Finkel T. Duc J. Fearon E.R. Dang C.V. Tomaselli G.F. J. Biol. Chem. 1993; 268: 5-8Abstract Full Text PDF PubMed Google Scholar). We found that, contrary to findings in vitro, bHLH domains are not sufficient for dimerization in vivo. In particular, E proteins require an additional highly conserved domain, domain C, located carboxyl-terminal to helix 2. Tissue-specific bHLH factors, by contrast, require only the bHLH domain for heterodimerization with E proteins. Therefore, intrinsic structural differences exist between E proteins and tissue-specific bHLH factors, differences which may explain their different dimerization specificities. In particular, the role of domain C appears to be as an in vivo conformational determinant, maintaining the bHLH domain of E proteins in a “receptive” conformation for heterodimerization with tissue-specific bHLH proteins. Expression of LexA fusion proteins in yeast employed the vector pEG202, kindly provided by Dr. Roger Brent (Massachusetts General Hospital, Boston, MA) (21Zervos A.S. Gyuris J. Brent R. Cell. 1993; 72: 223-232Abstract Full Text PDF PubMed Scopus (666) Google Scholar). Expression of B42 activation domain fusion proteins in yeast employed the vector pJG45, also kindly provided by Dr. Roger Brent. PCR upon plasmid templates was used to generate DNA fragments encoding the following: the bHLH domain of SCL/tal (amino acids 186–242), the 20-kDa naturally occurring isoform of SCL/tal (amino acids 176–331) (22Aplan P.D. Begley C.G. Bertness V. Nussmeier M. Ezquerra A. Coligan J. Kirsch I.R. Mol. Cell. Biol. 1990; 10: 6426-6435Crossref PubMed Scopus (83) Google Scholar), the bHLH domain of MyoD (amino acids 108–163), the full-length coding region of MyoD, and the various truncation mutants of E2-2 (encoding amino acids 467–588, 484–541, 467–541, 484–588, 484–563, and 484–548). PCR fragments were cloned in-frame into pEG202 and pJG45 asEcoRI-XhoI fragments. pJG-Id1 and pJG-Id2, yeast expression plasmids encoding Id1 and Id2 as B42 fusion proteins, were previously isolated from a HeLa cDNA library in the vector pJG45 (library provided by Brent laboratory). For mammalian expression of the VP16 activation domain alone or fused to SCL/tal (amino acids 176–331), we used the plasmids pVP-HA1 and pVP16-TAL1, respectively, both generously provided by Dr. Richard Baer (University of Texas Southwestern Medical Center, Dallas, TX) (4Hsu H.-L. Wadman I. Baer R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3181-3185Crossref PubMed Scopus (105) Google Scholar). For mammalian expression of the GAL4 DNA-binding domain fusion proteins, our laboratory generated a parent vector pCMV-DB which contains the GAL4 DNA-binding domain downstream of the CMV immediate early promoter. The starting vector consisted of pCMV5 (23Andersson S. Davis D.L. Dahlback H. Jornvall H. Russell D.W. J. Biol. Chem. 1989; 264: 8222-8229Abstract Full Text PDF PubMed Google Scholar) from which the EcoRI site had been eliminated, yielding pCMV5 R-. AHindIII-PstI fragment encoding the GAL4 DNA-binding domain was released from the yeast expression vector pGBT9 (CLONTECH, Palo Alto, CA) and ligated into the corresponding sites in pCMV5 R-. E2-2 fragments (encoding amino acids 484–541 and 484–563) with EcoRI-XhoI ends were cloned into EcoRI-SalI sites of pCMV-DB, yielding plasmids with in-frame GAL4-E2-2 fusions, pCMV-DB-E2-2 bHLH, and pCMV-DB-E2-2 C. The G5E1bLUC reporter plasmid, with 5 GAL4-binding sites upstream of the E1b TATA sequence followed by the luciferase reporter gene, was generously provided by Dr. Richard Baer and has been described elsewhere (4Hsu H.-L. Wadman I. Baer R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3181-3185Crossref PubMed Scopus (105) Google Scholar). pEMSV-MyoD, kindly provided by the laboratory of Dr. Harold Weintraub (Fred Hutchinson Cancer Research Institute, Seattle WA), was used for mammalian expression of the full-length MyoD protein with its own activation domain. The mammalian expression plasmid for the nuclear localization-deficient mutant of SCL/tal, pCMB, has been previously described (24Goldfarb A.N. Goueli S. Mickelson D. Greenberg J.M. Blood. 1992; 80: 2858-2866Crossref PubMed Google Scholar). For bacterial expression of MBP and GST fusion proteins, SCL/tal (encoding amino acids 176–331) and E2-2 (encoding amino acids 484–541 or 484–563), were cloned asEcoRI-XhoI fragments into pMAL-c2 (New England Biolabs, Beverly, MA) and pGEX4T-1 (Pharmacia Biotech, Piscataway, NJ). The yeast two-hybrid system developed in the laboratory of Roger Brent was employed as we have previously described (21Zervos A.S. Gyuris J. Brent R. Cell. 1993; 72: 223-232Abstract Full Text PDF PubMed Scopus (666) Google Scholar, 25Goldfarb A.N. Lewandowska K. Shoham M. J. Biol. Chem. 1996; 271: 2683-2688Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The yeast strains (EGY48 and YPH499), mating protocols, library screening protocols, and β-galactosidase assays have all been described in an earlier publication (25Goldfarb A.N. Lewandowska K. Shoham M. J. Biol. Chem. 1996; 271: 2683-2688Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). For Western blot analysis of yeast expression of LexA fusion proteins, equivalent quantities of yeast grown to mid-log phase in CM-URA, -HIS, and -TRP media with 2% galactose, 1% raffinose were resuspended in SDS-PAGE loading buffer and boiled. Resultant Western blot membranes were probed with a rabbit polyclonal antibody to LexA, provided by Dr. Erica Golemis (Fox Chase Cancer Center, Philadelphia, PA). Error-prone PCR amplification of the bHLH encoding region of SCL/tal included 0.25 mm MnCl2 in a standard PCR reaction (with 2.5 mm MgCl2 and 200 μm dNTPs). After 30 cycles of mutagenic amplification, 0.1 μl out of a 100-μl reaction was subjected to a second 30 cycles of mutagenic amplification. Similarly, 0.1 μl of the latter reaction was subjected to a third round of 30 cycles of mutagenic amplification. Equal quantities of PCR products resulting from 30, 60, and 90 cycles of mutagenic amplification were pooled and cloned into the EcoRI-XhoI sites of pJG45. A library of 2 × 106 primary bacterial colonies was thereby generated. A plasmid preparation of this library was then transformed into the yeast strain YPH499, yielding 1 × 106 primary yeast colonies. K562 cells in mid-log phase were resuspended at a concentration of 1 × 106cells/ml in RPMI 1640 media with 5% fetal bovine serum. For each transfection, 1.6 × 106 cells were combined with 6 μg of DNA and 30 μl of DOTAP (Boehringer Mannheim, Indianapolis, IN) premixed in 400 μl of Hepes-buffered saline. The 6 μg of DNA consisted of 2 μg of GAL4 expression plasmid (pCMV-DB, pCMV-DB-E2-2 bHLH or pCMV-DB-E2-2 C), 2 μg of activation domain expression plasmid (pVPHA1, pVP16-TAL1, or pEMSV-MyoD), and 2 μg of the G5E1bLUC luciferase reporter plasmid. After overnight incubation, the cells were resuspended in fresh RPMI 1640 with 10% fetal bovine serum and cultured for an additional 24 h. To assay cells for luciferase activity, the Luciferase Assay System kit (Promega, Madison WI) was used, following the manufacturer's recommendations. In all cases, equivalent numbers of cells were harvested for luciferase assays. Light emission was measured on a Berthold Lumat LB 9501 luminometer (Berthold Systems Inc., Pittsburgh, PA). COS 7 cells were seeded on glass coverslips in RPMI 1640 with 5% fetal bovine serum at a density of 4 × 105 cells per 22-mm2 coverslip. Transfections were carried out overnight with 5 μg of plasmids and 25 μl of DOTAP (Boehringer Mannheim) per coverslip. The plasmids consisted of 2.5 μg of pCMB plus 2.5 μg of GAL4 expression plasmid (pCMV-DB, pCMV-DB-E2-2 bHLH, or pCMV-DB-E2-2 C). Cells were incubated in fresh RPMI 1640 with 10% fetal bovine serum for 72 h prior to fixation. The protocols for cell fixation and indirect immunofluorescent staining for SCL/tal have been previously described (17Goldfarb A.N. Lewandowska K. Exp. Cell Res. 1994; 214: 481-485Crossref PubMed Scopus (19) Google Scholar). The cells were visualized on an MRC-600 confocal laser scanning imaging system (Bio-Rad Molecular Bioscience Group, Hercules, CA). COS 7 cells grown to ∼60% confluency in 75-cm2 flasks were transfected with 5 μg of pCMV-SCL/tal, an expression vector for full-length SCL/tal protein (amino acids 1–331) (24Goldfarb A.N. Goueli S. Mickelson D. Greenberg J.M. Blood. 1992; 80: 2858-2866Crossref PubMed Google Scholar). In addition, the cells received 5 μg of either pCMV-DB-E2-2 bHLH or pCMV-DB-E2-2 C. Transfections were accomplished overnight in Dulbecco's modified Eagle's medium with 5% neonatal calf serum using 50 μg of DOTAP (Boehringer Mannheim) plus 10 μg of plasmid per flask. After supplying the cells with fresh media consisting of Dulbecco's modified Eagle's medium with 10% neonatal calf serum, the cells were incubated an additional 4 days prior to harvesting. For harvesting, cells were gently scraped in room temperature phosphate-buffered saline with 5 mm EDTA. Cell pellets were then resuspended in 300 μl of ice-cold NETN (20 mm Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40, 2 μg/ml leupeptin, 2 μg/ml pepstatin, 0.2% aprotinin, and 200 μmphenylmethylsulfonyl fluoride). After a 10-min incubation on ice with intermittent inversion, insoluble cellular debris was eliminated by pelleting. 50-μl portions of the extracts were saved for direct immunoblot analysis. To the remaining 250 μl of extracts, 0.5 μg of rabbit anti-GAL4 DNA-binding domain antibody (number sc-577, Santa Cruz Biotechnology, Santa Cruz, CA) was added followed by incubation on ice 45 min with intermittent inversion. A 10-μl packed volume of protein A-agarose beads, prewashed in phosphate-buffered saline with 1% bovine serum albumin, were then added to each tube, followed by rotation at 4 °C for 30 min. The protein A-agarose beads were then washed 4 times with ice-cold NETN and resuspended in 50 μl of SDS-PAGE loading buffer. Immunoprecipitates and crude cellular extracts were then subjected to SDS-PAGE on 12% gels followed by electrotransfer to nitrocellulose membranes. Immunoblots were probed with the BTL-73 mouse monoclonal anti-SCL/tal antibody that was kindly provided by Karen Pulford (Oxford, United Kingdom) (26Pulford K. Lecointe N. Leroy-Viard K. Jones M. Mathieu-Mahul D. Mason D.Y. Blood. 1995; 85: 675-684Crossref PubMed Google Scholar). BTL-73 was used as a one-half dilution of a tissue culture supernatant. In addition, crude extracts from COS cell transfectants were probed in parallel with the rabbit anti-GAL4 antibody at a 1/500 dilution (0.2 ng/ml). Western blots were otherwise carried out as described previously (24Goldfarb A.N. Goueli S. Mickelson D. Greenberg J.M. Blood. 1992; 80: 2858-2866Crossref PubMed Google Scholar). Production and purification of bacterially expressed GST and MBP fusion proteins followed previously described protocols (5Goldfarb A.N. Lewandowska K. Blood. 1995; 85: 465-471Crossref PubMed Google Scholar). Surface plasmon resonance studies employed a BIAcore biosensor device (Pharmacia Biosensor, Piscataway, NJ) and followed previously described guidelines (27Pauza M.E. Doumbia S.O. Pennell C.A. J. Immunol. 1997; 158: 3259-3269PubMed Google Scholar). In particular, GST-E2-2 fusion proteins (encoding E2-2 bHLH (amino acids 484–541) or E2-2 C (amino acids 484–563)) diluted in 10 mm sodium acetate (pH 4.3) were covalently linked to the carboxymethyl-dextran hydrogel matrix on a CM5 flow cell by the standard amine coupling procedure described by the manufacturer (Pharmacia Biosensor, Piscataway, NJ). For GST-E2-2 bHLH, 1800 resonance units were immobilized, and for GST-E2-2 C, 1700 resonance units were immobilized. The soluble analyte, MBP-SCL/tal176–331 was used at a concentration of 4 μm in protein interaction buffer (20 mmTris-HCl, pH 8, 100 mm NaCl, 2.5 mmMgCl2, 0.5 mm dithiothreitol, 100 μg/ml bovine serum albumin, and 0.005% p20 surfactant). Just prior to analysis, analyte preparations were centrifuged through Microsep® 50,000 Mr cut-off microconcentrators (Filtron Technology Corp., Northborough, MA) to remove aggregates. The analyte was then flowed over the immobilized ligands at a rate of 5 μl/min. The minimal bHLH domains of E2-2, SCL/tal, and MyoD have been well defined by alignment analysis of a broad range of bHLH proteins from a wide variety of organisms (28Atchley W.R. Fitch W.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5172-5176Crossref PubMed Scopus (503) Google Scholar). To assay interaction of minimal bHLH domains in the yeast two-hybrid system the amino acid sequences indicated in Fig. 1 were expressed in yeast as fusions with either the LexA DNA-binding domain or the B42 transcription activation domain. Interaction within yeast between coexpressed LexA fusions and B42 fusions was reflected by activation of a β-galactosidase reporter gene containing upstream LexA-binding sites. As shown in Fig. 2, LexA fusions with the minimal bHLH domains of MyoD and SCL/tal (LexA-MyoD bHLH and LexA-SCL/tal bHLH) displayed specific interaction with a larger fragment of E2-2 encompassing the carboxyl-terminal 121 amino acids (E2-2 467–588) fused to the B42 activation domain. Surprisingly, LexA-MyoD bHLH and LexA-SCL/tal bHLH manifested no interaction with the minimal bHLH domain of E2-2 (amino acids 484–541) fused to B42. As will be shown below, similar results were obtained with E2-2 as the LexA fusion component and were not attributable to poor expression of E2-2 bHLH fusion proteins in yeast. These data suggest that the structural requirements for dimerization in the yeast two-hybrid system differ for tissue-specific bHLH proteins as compared with E proteins, the former requiring only the minimal bHLH domain and the latter requiring additional sequence.Figure 2Relative quantitation of HLH interactions using the yeast two-hybrid system. LexA DNA-binding domain fusions were coexpressed in yeast with B42 activation domain fusions. Protein interaction was reflected by activation of the LexA operator β-galactosidase reporter plasmid pSH18-34. LexA fusions include: LexA only as a negative control, LexA fused to the minimal bHLH domain of MyoD (as depicted in Fig. 1), and LexA fused to the minimal bHLH domain of SCL/tal (as depicted in Fig. 1). B42 fusions include: B42 fused to E2-2 467–588 which includes the bHLH domain as well as flanking amino acids, and B42 fused to the minimal bHLH domain of E2-2 (as depicted in Fig. 1). Liquid β-galactosidase assays were performed on three separate occasions. Results shown are mean ± S.E.View Large Image Figure ViewerDownload (PPT) To identify additional sequence requirements for E2-2 heterodimerization, a number of E2-2 truncations were expressed in yeast as LexA fusion proteins (Fig. 3 A). These LexA-E2-2 truncations were analyzed for interaction with B42 fusions containing the following HLH proteins: Id1, Id2, SCL/tal, and MyoD. As shown in Fig. 3 B, domain C, a stretch of 22 amino acids carboxyl-terminal to the HLH domain (amino acids 541–563) is both sufficient and necessary for heterodimerization of the E2-2 bHLH domain with an array of HLH partners. Interestingly, domain A, an acidic region upstream of the bHLH domain, which has been implicated in selective heterodimerization of E12 with MyoD (29Shirakata M. Paterson B.M. EMBO J. 1995; 14: 1766-1772Crossref PubMed Scopus (25) Google Scholar), had no influence on the heterodimerization of E2-2 with the various HLH partners. To study further the role of domain C in the heterodimerization of E2-2, a number of control experiments were performed. First, Western blot analysis of LexA-E2-2 fusion expression in yeast demonstrated insufficient differences in expression levels of the various truncation mutants to account for the differences in interaction patterns (Fig. 4 A). For example, LexA-E2-2 467–541 (Fig. 4 A, lane 3) showed the same levels of expression as LexA-E2-2 484–563 (Fig. 4 A, lane 5), but only the latter LexA fusion demonstrated heterodimerization with the various HLH partners (Fig. 3 B). Second, we isolated two independent altered-specificity mutants of SCL/tal, SE1 and SS1, that exclusively recognized forms of E2-2 lacking domain C (Fig. 4 B). These mutants were isolated from a library of randomly mutated SCL/tal bHLH domains using a yeast two-hybrid screen for interaction with the minimal bHLH domain of E2-2 fused to LexA. The selectivity of SCL/tal mutants SE1 and SS1 for E2-2 lacking domain C rules out any trivial explanations for the role of domain C, i.e. nuclear localization or permitting DNA binding by LexA. In fact, these data indicate that domain C influences the conformation of the E2-2 bHLH domain such that the presence of domain C permits exclusive heterodimerization with wild type HLH partners and the absence of domain C permits exclusive heterodimerization with the SCL/tal mutants SE1 and SS1. To extend the findings in the yeast two-hybrid system, three independent assays were employed to analyze in vivoHLH dimerization in mammalian cells. In the mammalian two-hybrid system, K562 cells were transiently co-transfected with vectors expressing fusions with the GAL4 DNA-binding domain and with the VP16 activation domain. Also included in the transfection was the GAL5E1bLUC reporter plasmid which contains GAL4-binding sites upstream of the luciferase gene. As has been previously described, interaction of GAL4 fusions with VP16 fusions activates expression of the luciferase reporter gene (30Wadman I. Li J. Bash R.O. Forster A. Osada H. Rabbitts T.H. Baer R. EMBO J. 1994; 13: 4831-4839Crossref PubMed Scopus (219) Google Scholar). As shown in Fig. 5 A, a GAL4 fusion with the minimal E2-2 bHLH domain (E2-2 amino acids 484–541) showed no interaction above background with a VP16-SCL/tal fusion. However, inclusion of domain C in the GAL4-E2-2 fusion (E2-2 amino acids 484–563) permitted interaction with VP16-SCL/tal. In Fig. 5 B, GAL4 fusions were coexpressed with full-length MyoD. Because MyoD has its own potent activation domains, it was not necessary to express MyoD as a VP16 fusion. As with VP16-SCL/tal, MyoD failed to interact with a GAL4 fusion with the minimal E2-2 bHLH domain (E2-2 amino acids 484–541). As predicted, inclusion of domain C in the GAL4-E2-2 fusion (E2-2 amino acids 484–563) restored the in vivo interaction with MyoD. In the nuclear redirection assay, which has been previously described (17Goldfarb A.N. Lewandowska K. Exp. Cell Res. 1994; 214: 481-485Crossref PubMed Scopus (19) Google Scholar, 18Deed R.W. Armitage S. Norton J.D. J. Biol. Chem. 1996; 271: 23603-23606Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar), COS cells were transfected with an expression plasmid for a mutant SCL/tal which lacks a nuclear localization signal. In addition, the COS cells were cotransfected with expression plasmids for either E2-2 bHLH only or E2-2 bHLH with domain C, each fused to the GAL4 nuclear localization signal. Cells were then analyzed for subcellular localization of SCL/tal by indirect immunofluorescence with confocal microscopy. When coexpressed with only the GAL4 nuclear localization signal, the SCL/tal mutant showed predominantly cytoplasmic localization with perinuclear accumulation (Fig. 6 A). When coexpressed with E2-2 bHLH with domain C (E2-2 amino acids 484–563), the SCL/tal mutant showed efficient nuclear localization as a result of its heterodimerization with E2-2, a process referred to as nuclear redirection (Fig. 6 B). By contrast, no nuclear redirection of mutant SCL/tal was observed with coexpression of the minimal E2-2 bHLH domain (E2-2 amino acids 484–541), indicating an absence of heterodimerization (Fig. 6 C). In coimmunoprecipitation assays (Fig. 7), COS cells were cotransfected with expression vectors for full-length SCL/tal (pCMV-SCL/tal) and either GAL4-E2-2 bHLH (pCMV-DB-E2-2 bHLH) or GAL4-E2-2 C (pCMV-DB-E2-2 C). Transfectants were subjected to low stringency immunoprecipitation with rabbit anti-GAL4 antibodies. Immune complexes were then analyzed by immunoblot with the BTL-73 monoclonal antibody specific for SCL/tal (26Pulford K. Lecointe N. Leroy-Viard K. Jones M. Mathieu-Mahul D. Mason D.Y. Blood. 1995; 85: 675-684Crossref PubMed Google Scholar). As shown in Fig. 7, while no detectable SCL/tal protein could be detected in complex with GAL4-E2-2 bHLH (lane 1), the 42 kDa full-length isoform of SCL/tal could be detected in complex with GAL4-E2-2 C (lane 2). As shown in the immunoblots in the lower panels of Fig. 7, crude extracts from both COS cell transfectants contained similar quantities of SCL/tal and GAL4-E2-2 proteins. The doublet observed on immunoblotting for SCL/tal has been previously described (24Goldfarb A.N. Goueli S. Mickelson D. Greenberg J.M. Blood. 1992; 80: 2858-2866Crossref PubMed Google Scholar). Thus, three independent assay systems, mammalian two-hybrid, nuclear redirection, and coimmunoprecipitation, all confirm the requirement for domain C forin vivo heterodimerization of E2-2 in mammalian cells

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