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The Hematopoietic Transcription Factor SCL Binds the p44 Subunit of TFIIH

1999; Elsevier BV; Volume: 274; Issue: 3 Linguagem: Inglês

10.1074/jbc.274.3.1388

ISSN

1083-351X

Autores

Xianfeng Zhao, Peter D. Aplan,

Tópico(s)

T-cell and Retrovirus Studies

Resumo

SCL is a basic domain helix-loop-helix (bHLH) oncoprotein that is involved in T-cell acute lymphoblastic leukemia as well as in normal hematopoiesis. Although it is believed that SCL functions as a tissue-specific transcription factor, no molecular mechanism has thus far been identified for this putative function. In this report, we show that SCL interacts with p44, a subunit of the basal transcription factor TFIIH. The minimal region of SCL that interacts with p44 was mapped to a 101-amino acid sequence that includes, but is not limited to, the bHLH region; the SCL-binding site of p44 is located in the carboxyl-terminal half of p44. This interaction was confirmed by glutathione S-transferase fusion protein pull-down assays and a co-immunoprecipitation assay. As analyzed with a yeast two-hybrid system, p44 interacts specifically with SCL, but not with the other class A or B bHLH proteins tested. E2A did not compete with p44 for SCL binding, as demonstrated by anin vitro binding assay. These findings document a previously unsuspected interaction between SCL and a subunit of the basal transcription factor TFIIH, suggesting a potential means by which SCL might modulate transcription. SCL is a basic domain helix-loop-helix (bHLH) oncoprotein that is involved in T-cell acute lymphoblastic leukemia as well as in normal hematopoiesis. Although it is believed that SCL functions as a tissue-specific transcription factor, no molecular mechanism has thus far been identified for this putative function. In this report, we show that SCL interacts with p44, a subunit of the basal transcription factor TFIIH. The minimal region of SCL that interacts with p44 was mapped to a 101-amino acid sequence that includes, but is not limited to, the bHLH region; the SCL-binding site of p44 is located in the carboxyl-terminal half of p44. This interaction was confirmed by glutathione S-transferase fusion protein pull-down assays and a co-immunoprecipitation assay. As analyzed with a yeast two-hybrid system, p44 interacts specifically with SCL, but not with the other class A or B bHLH proteins tested. E2A did not compete with p44 for SCL binding, as demonstrated by anin vitro binding assay. These findings document a previously unsuspected interaction between SCL and a subunit of the basal transcription factor TFIIH, suggesting a potential means by which SCL might modulate transcription. Since its isolation from the multipotential DU528 stemcell leukemia cell line, which carries a t(1;14)(p32;q11) chromosome translocation (1Begley C.G. Aplan P.D. Denning S.M. Haynes B.F. Waldmann T.A. Kirsch I.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 10128-10132Crossref PubMed Scopus (289) Google Scholar, 2Chen Q. Cheng J.-T. Tsai L.-H. Schneider N. Buchanan G. Carroll A. Crist W. Ozanne B. Siciliano M.J. Baer R. EMBO J. 1990; 9: 415-424Crossref PubMed Scopus (275) Google Scholar, 3Finger L.R. Kagan J. Christopher G. Kurtzberg J. Hershfield M.S. Nowell P.C. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5039-5043Crossref PubMed Scopus (191) Google Scholar), the SCL(TCL5 or tal-1) gene has been studied in the context of both normal and abnormal hematopoiesis. The observation that the SCL locus is frequently disrupted in T-cell acute lymphoblastic leukemia cells (4Aplan P.D. Lombardi D.P. Ginsberg A.M. Cossman J. Bertness V.L. Kirsch I.R. Science. 1990; 250: 1426-1429Crossref PubMed Scopus (236) Google Scholar, 5Brown L. Cheng J.-T. Chen Q. Siciliano M.J. Crist W. Buchanan G. Baer R. EMBO J. 1990; 9: 3343-3351Crossref PubMed Scopus (288) Google Scholar, 6Bernard O. Lecointe N. Jonveaux P. Suoyri M. Mauchauffe M. Berger R. Larsen C.J. Mathieu-Mahul D. Oncogene. 1991; 6: 1477-1488PubMed Google Scholar), resulting in aberrantSCL expression, has led to speculation that the gene product of SCL is an oncoprotein. Indeed, recent transgenic mouse models have confirmed that unscheduled SCL expression leads to aggressive T-cell malignancies (7Larson R.C. Lavenir I. Larson T.A. Baer R. Warren A.J. Wadman I. Nottage K. Rabbitts T.H. EMBO J. 1996; 15: 1021-1027Crossref PubMed Scopus (186) Google Scholar, 8Aplan P.D. Jones C.A. Chervinsky D.S. Zhao X.-F. Ellsworth M. Wu C. McGuire E.A. Gross K.W EMBO J. 1997; 9: 2408-2419Crossref Scopus (125) Google Scholar). Moreover, despite its initial identification in leukemic cells, targeted disruption of theSCL locus has demonstrated that SCL expression is absolutely required for normal hematopoietic development (9Robb L. Lyons I. Li R. Hartley L. Köntgen F. Harvey R.P. Metcalf D. Begley C.G Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7075-7079Crossref PubMed Scopus (479) Google Scholar, 10Shivdasani R.A. Mayer E.L. Orkin S.H Nature. 1995; 373: 432-434Crossref PubMed Scopus (775) Google Scholar, 11Robb L. Elwood N.J. Elefanty A.G. Kontgen F. Li R. Barnett L.D. Begley C.G. EMBO J. 1996; 15: 4123-4129Crossref PubMed Scopus (284) Google Scholar, 12Porcher C. Swat W. Rockwell K. Fujiwara Y. Alt F.W. Orkin S.H. Cell. 1996; 86: 47-57Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar).SCL belongs to the basic domain helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic domain helix-loop-helix; SD, synthetic dextrose; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PCR, polymerase chain reaction; Tp44, truncated p44; GAD, GAL4 transcription activation domain; GBD, GAL4 DNA-binding domain. 1The abbreviations used are: bHLH, basic domain helix-loop-helix; SD, synthetic dextrose; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PCR, polymerase chain reaction; Tp44, truncated p44; GAD, GAL4 transcription activation domain; GBD, GAL4 DNA-binding domain. family of proteins (13Murre C. McCaw P.S. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1837) Google Scholar). Three principal classes of bHLH proteins have been identified (14Murre C. McCaw P.S. Vaessin H. Caudy M. Jan L.Y. Jan Y.N. Cabrera C.V. Buskin J.N. Hauschica S.D. Lassar A.B. Weintraub H. Baltimore D. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1294) Google Scholar). Class A bHLH proteins include the E proteins E2A/ITF1 (immunoglobulin transcriptionfactor 1), E2-2/ITF2, and HEB (an E protein related to E2A/ITF1 and E2-2/ITF2); these proteins are expressed ubiquitously. In contrast, the expression of class B bHLH proteins, such as MyoD and SCL, is restricted to specific organs or tissues. The myogenic (MyoD, myogenin, myf5) bHLH proteins have been studied extensively (15Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (924) Google Scholar) and serve as a useful paradigm for the actions of tissue-restricted bHLH proteins. MyoD forms a heterodimer with the ubiquitously expressed E2A proteins (16Lassar 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 (684) Google Scholar), binds specific DNA sequence at the regulatory regions of genes coding for muscle-specific proteins (such as muscle creatine kinase), and activates transcription of these genes (17Jaynes J.B. Johnson J.E. Buskin J.N. Gartside C.L. Hauschka S.D. Mol. Cell. Biol. 1988; 8: 62-70Crossref PubMed Scopus (170) Google Scholar). Although unproven, it is thought that SCL activates transcription of genes required for normal hematopoietic development in an analogous fashion (18Shivadasani R.A. Orkin S.H. Blood. 1996; 87: 4025-4039Crossref PubMed Google Scholar).Several forms of the SCL protein have been found in mammalian cells (19Elwood N.J. Green A.R. Melder A. Begley C.G. Nicola N. Leukemia. 1994; 8: 106-114PubMed Google Scholar); the full-length form of SCL has a molecular mass variously reported to be between 42 and 49 kDa, whereas an amino-terminally truncated form of 22–26 kDa is produced by translation of alternatively spliced transcripts (20Aplan P.D. Begley C.G. Bertness V. Nussmeier M. Ezquerra A. Colligan J. Kirsch I.R. Mol. Cell. Biol. 1990; 10: 6426-6435Crossref PubMed Scopus (83) Google Scholar) in both normal and leukemic cells. Although a transcription activation domain has been mapped to its amino terminus (21Wadman I.A. Hsu H.-L. Cobb M.H. Baer R. Oncogene. 1994; 9: 3713-3716PubMed Google Scholar), unscheduled synthesis of an SCL protein that lacks the transactivation domain leads to T-cell leukemia in transgenic mice, indicating that this transactivation domain is not required for leukemogenesis (8Aplan P.D. Jones C.A. Chervinsky D.S. Zhao X.-F. Ellsworth M. Wu C. McGuire E.A. Gross K.W EMBO J. 1997; 9: 2408-2419Crossref Scopus (125) Google Scholar). Similar to many other bHLH proteins, SCL has been shown to bind several E proteins, including E2-2/ITF2 (22Goldfarb A.N. Lewandowska K. Shoham M. J. Biol. Chem. 1996; 271: 2683-2688Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), E12, and E47 (two alternatively spliced forms of E2A/ITF1) (23Hsu H.-L. Cheng J.-T. Chen Q. Baer R. Mol. Cell. Biol. 1991; 11: 3037-3042Crossref PubMed Scopus (133) Google Scholar). When bound to E12, the SCL·E12 complex preferentially binds to a CAGATG nucleotide sequence in vitro (24Hsu H.-L. Huang L. Tsan T. Funk W. Wright W.E. Hu J.-S. Kingston R.E. Baer R. Mol. Cell. Biol. 1994; 14: 1256-1265Crossref PubMed Scopus (147) Google Scholar). Although SCL is suspected to function as a sequence-specific transcription factor, no target genes for SCL have thus far been convincingly identified, and any mechanism by which SCL may control transcription remains unknown.TFIIH is a multicomponent basal transcription factor complex that is also known to function in certain DNA repair pathways (25Svejstrup J.Q. Vichi P. Egly J.-M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar). Nine subunits have been identified within the TFIIH holoenzyme complex; various enzymatic activities, including DNA repair, helicase (26Schaeffer L. Roy R. Humbert S. Moncollin V. Vermeulen W. Hoejimakers J.H.J. Chambon P. Egly J.-M. Science. 1993; 260: 58-63Crossref PubMed Scopus (664) Google Scholar), and cyclin-dependent kinase (27Adamczewski J.P. Rossignol M. Tassan J.-P. Nigg E.A. Moncollin V. Egly J.-M. EMBO J. 1996; 15: 1877-1884Crossref PubMed Scopus (114) Google Scholar) activities, have been identified. The p62, p52, p44, and p34 subunits are thought to constitute the “core” of the TFIIH transcription machinery (28Marinoni J.C. Roy R. Vermeulen W. Miniou P. Lutz Y. Weeda G. Seroz T. Gomez D.M. Hoeijmakers J.H. Egly J.-M. EMBO J. 1997; 16: 1093-1102Crossref PubMed Scopus (60) Google Scholar). Although the p44 and p34 subunits have no defined enzymatic activity, their zinc finger structures suggest that they may be DNA-binding proteins (29Humbert S. van Vuuren H. Lutz Y. Hoeijmakers J.H. Egly J.-M. Moncollin V. EMBO J. 1994; 13: 2393-2398Crossref PubMed Scopus (100) Google Scholar) that might mediate interactions with soluble transcription factors.Here we report the identification of an unexpected interaction between SCL and the p44 subunit of TFIIH. This observation provides a link between SCL and the basal transcription machinery, suggesting that SCL may exert its suspected transcription regulatory effects through an interaction with TFIIH.DISCUSSIONUsing a yeast two-hybrid system, we have isolated several SCL-binding proteins, both expected and unexpected, from three different human cDNA libraries. As expected, the most prevalent SCL-binding partners recovered from these screens were E proteins, including E2A/ITF1, E2-2/ITF2, and HEB (14Murre C. McCaw P.S. Vaessin H. Caudy M. Jan L.Y. Jan Y.N. Cabrera C.V. Buskin J.N. Hauschica S.D. Lassar A.B. Weintraub H. Baltimore D. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1294) Google Scholar). We also used the yeast two-hybrid system to demonstrate previously unreported interactions between SCL and NHLH1 as well as a homodimeric interaction of SCL with itself. A human form of the DRG protein was also isolated from a human thymus cDNA library; this was not surprising given that mouse DRG has been isolated from a mouse erythroleukemia cell line (MEL) (35Mahajan M.A. Park S.T. Sun X.-H. Oncogene. 1996; 12: 2343-2350PubMed Google Scholar) using the 22–26-kDa form of SCL as the bait protein in a yeast two-hybrid screen.However, we were surprised by the isolation of the p44 subunit of TFIIH from the human thymus cDNA library. Although interactions between bHLH proteins (e.g. c-Myc and MyoD) and basal transcription machinery have been documented (41Maheswaran S. Lee H. Sonenshein G.E. Mol. Cell. Biol. 1994; 14: 1147-1152Crossref PubMed Scopus (70) Google Scholar, 42Heller H. Bengal E. Nucleic Acids Res. 1998; 26: 2112-2120Crossref PubMed Scopus (23) Google Scholar), those studies involved the interaction with TBP, the first basal transcription factor that is recruited to an activated promoter (43Kornberg R.D. Trends Biochem. Sci. 1996; 21: 325-326Abstract Full Text PDF PubMed Scopus (32) Google Scholar). The interaction between a bHLH protein and TFIIH, which is recruited to the transcription initiation complex relatively late and functions in promoter clearance and elongation of transcription (25Svejstrup J.Q. Vichi P. Egly J.-M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar), has not been previously reported.Several functional domains have been mapped to portions of the SCL protein; the amino-terminal region is proline-rich and has been shown to contain a transcription activation domain (21Wadman I.A. Hsu H.-L. Cobb M.H. Baer R. Oncogene. 1994; 9: 3713-3716PubMed Google Scholar), whereas the bHLH domain is involved in both DNA binding and dimerization with other bHLH proteins (44Tapscott S.J. Weintraub H. J. Clin. Invest. 1991; 87: 1133-1138Crossref PubMed Scopus (106) Google Scholar). The p44-binding region of SCL was mapped to a region that includes the bHLH domain as well as amino acid residues immediately N- and C-terminal to the bHLH domain. Although the bHLH domain was included in the region essential for binding, it is possible that the bHLH domain may only serve as an arm to link the N- and C-terminal flanking regions together to form a binding domain for p44. Support for this possibility is found in the observation that only SCL, among several bHLH proteins tested, was able to bind p44 in the yeast two-hybrid system, indicating that the bHLH structure alone is not sufficient for this interaction. For example, LYL1 (a bHLH protein named by virtue of its involvement in a chromosomal translocation associated with lymphoblasticleukemia) has a bHLH region that is quite similar (49/55 identical amino acids and four conservative substitutions) to SCL (1Begley C.G. Aplan P.D. Denning S.M. Haynes B.F. Waldmann T.A. Kirsch I.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 10128-10132Crossref PubMed Scopus (289) Google Scholar). Despite this, an interaction between LYL1 and p44 was not detected with this system, suggesting that the interaction between SCL and p44 requires amino acids proximal and distal to the bHLH region. Our results also show that the C-terminal polyglycine tract is important for the interaction; this is not surprising because glycine-rich domains have been shown to mediate other protein/protein interactions (45Cartegni L. Maconi M. Morandi E. Cobianchi F. Riva S. Biamonti G. J. Mol. Biol. 1996; 259: 337-348Crossref PubMed Scopus (157) Google Scholar). However, the SCL C-terminal glycine tract itself is not sufficient for the SCL/p44 interaction (see Fig. 4). The SCL-binding region of p44 was mapped to the C-terminal region of p44. This region is composed of 166 amino acids (from Lys-229 to Val-395), containing two putative functional motifs (29Humbert S. van Vuuren H. Lutz Y. Hoeijmakers J.H. Egly J.-M. Moncollin V. EMBO J. 1994; 13: 2393-2398Crossref PubMed Scopus (100) Google Scholar). One of these motifs, the TFIIIA-like zinc finger motif, was found to interact with DNA; the second motif, whose function is unknown, is conserved in both p44 and Ssl1, the yeast homologue of human p44. Although the SCL-binding domain of p44 has not yet been clearly defined, the above two motifs are potential targets for future studies.It is not clear how the interaction between SCL and p44 might function in a biological context. However, some insight may be gained through the study of previously reported interactions between nonessential transcription factors and elements of the basal transcription complex. Several proteins in addition to those of the basal transcription complex have been shown to interact with TFIIH subunits. These proteins include p53, which has been shown to interact in vitro with the XPB subunit of TFIIH (46Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (632) Google Scholar); this interaction is thought to modulate either the nucleotide excision repair activity of TFIIH (47Wang X.W. Yhe H. Schaeffer L. Roy R. Moncollin V. Egly J.-M. Wang Z. Friedberg E.C. Evans M.K. Taffe B.G. Bohr V.A. Weeda G. Hoeijmaker J.H.J. Forrester K. Harris C.C. Nat. Genet. 1995; 10: 188-195Crossref PubMed Scopus (514) Google Scholar) or p53-mediated apoptosis (48Wang X.W. Vermeulen W. Coursen J.D. Gibson M. Lupold S.E Forrester K. Xu G. Elmore L. Yeh H. Hoeijmaker J.H.J. Harris C.C. Genes Dev. 1996; 10: 1219-1232Crossref PubMed Scopus (309) Google Scholar). Additionally, the transcription activation domain of VP16 has been shown to bind the p62 subunit of TFIIH (49Xiao H. Pearson A. Coulombe B. Truant R. Zhang S. Regier J.L. Triezenberg S.J. Reinberg D. Flores O. Ingles C.J. Greenblatt J. Mol. Cell. Biol. 1994; 14: 7013-7024Crossref PubMed Scopus (327) Google Scholar), resulting in activation of transcription from an adenovirus type 2 major late promoter. With regard to interactions between bHLH proteins and basal transcription factors, the bHLH transcription factor c-Myc has been shown to bind the basal transcription factor TBP (50Hateboer G. Timmers H.T. Rustgi A.K. Billaud M. van't Veer L.J. Bernards R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8489-8493Crossref PubMed Scopus (129) Google Scholar). Therefore, one hypothesis regarding the functional relevance of an SCL/p44 interaction speculates that SCL, itself or as a heterodimer with an E protein, interacts with p44, thus modulating the activity of TFIIH and eventually the transcription elongation.In summary, we have demonstrated a specific interaction, both in vivo and in vitro, between the bHLH protein SCL and the p44 subunit of the basal transcription factor TFIIH. Although this interaction was mapped to a region of SCL that encompassed, but was not limited to, the bHLH domain, other bHLH proteins did not demonstrate binding to p44. This novel interaction between a bHLH protein and TFIIH suggests a potential mechanism by which the tissue-restricted bHLH protein SCL might function during hematopoietic and vascular development. Since its isolation from the multipotential DU528 stemcell leukemia cell line, which carries a t(1;14)(p32;q11) chromosome translocation (1Begley C.G. Aplan P.D. Denning S.M. Haynes B.F. Waldmann T.A. Kirsch I.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 10128-10132Crossref PubMed Scopus (289) Google Scholar, 2Chen Q. Cheng J.-T. Tsai L.-H. Schneider N. Buchanan G. Carroll A. Crist W. Ozanne B. Siciliano M.J. Baer R. EMBO J. 1990; 9: 415-424Crossref PubMed Scopus (275) Google Scholar, 3Finger L.R. Kagan J. Christopher G. Kurtzberg J. Hershfield M.S. Nowell P.C. Croce C.M. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5039-5043Crossref PubMed Scopus (191) Google Scholar), the SCL(TCL5 or tal-1) gene has been studied in the context of both normal and abnormal hematopoiesis. The observation that the SCL locus is frequently disrupted in T-cell acute lymphoblastic leukemia cells (4Aplan P.D. Lombardi D.P. Ginsberg A.M. Cossman J. Bertness V.L. Kirsch I.R. Science. 1990; 250: 1426-1429Crossref PubMed Scopus (236) Google Scholar, 5Brown L. Cheng J.-T. Chen Q. Siciliano M.J. Crist W. Buchanan G. Baer R. EMBO J. 1990; 9: 3343-3351Crossref PubMed Scopus (288) Google Scholar, 6Bernard O. Lecointe N. Jonveaux P. Suoyri M. Mauchauffe M. Berger R. Larsen C.J. Mathieu-Mahul D. Oncogene. 1991; 6: 1477-1488PubMed Google Scholar), resulting in aberrantSCL expression, has led to speculation that the gene product of SCL is an oncoprotein. Indeed, recent transgenic mouse models have confirmed that unscheduled SCL expression leads to aggressive T-cell malignancies (7Larson R.C. Lavenir I. Larson T.A. Baer R. Warren A.J. Wadman I. Nottage K. Rabbitts T.H. EMBO J. 1996; 15: 1021-1027Crossref PubMed Scopus (186) Google Scholar, 8Aplan P.D. Jones C.A. Chervinsky D.S. Zhao X.-F. Ellsworth M. Wu C. McGuire E.A. Gross K.W EMBO J. 1997; 9: 2408-2419Crossref Scopus (125) Google Scholar). Moreover, despite its initial identification in leukemic cells, targeted disruption of theSCL locus has demonstrated that SCL expression is absolutely required for normal hematopoietic development (9Robb L. Lyons I. Li R. Hartley L. Köntgen F. Harvey R.P. Metcalf D. Begley C.G Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7075-7079Crossref PubMed Scopus (479) Google Scholar, 10Shivdasani R.A. Mayer E.L. Orkin S.H Nature. 1995; 373: 432-434Crossref PubMed Scopus (775) Google Scholar, 11Robb L. Elwood N.J. Elefanty A.G. Kontgen F. Li R. Barnett L.D. Begley C.G. EMBO J. 1996; 15: 4123-4129Crossref PubMed Scopus (284) Google Scholar, 12Porcher C. Swat W. Rockwell K. Fujiwara Y. Alt F.W. Orkin S.H. Cell. 1996; 86: 47-57Abstract Full Text Full Text PDF PubMed Scopus (608) Google Scholar). SCL belongs to the basic domain helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic domain helix-loop-helix; SD, synthetic dextrose; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PCR, polymerase chain reaction; Tp44, truncated p44; GAD, GAL4 transcription activation domain; GBD, GAL4 DNA-binding domain. 1The abbreviations used are: bHLH, basic domain helix-loop-helix; SD, synthetic dextrose; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; PCR, polymerase chain reaction; Tp44, truncated p44; GAD, GAL4 transcription activation domain; GBD, GAL4 DNA-binding domain. family of proteins (13Murre C. McCaw P.S. Baltimore D. Cell. 1989; 56: 777-783Abstract Full Text PDF PubMed Scopus (1837) Google Scholar). Three principal classes of bHLH proteins have been identified (14Murre C. McCaw P.S. Vaessin H. Caudy M. Jan L.Y. Jan Y.N. Cabrera C.V. Buskin J.N. Hauschica S.D. Lassar A.B. Weintraub H. Baltimore D. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1294) Google Scholar). Class A bHLH proteins include the E proteins E2A/ITF1 (immunoglobulin transcriptionfactor 1), E2-2/ITF2, and HEB (an E protein related to E2A/ITF1 and E2-2/ITF2); these proteins are expressed ubiquitously. In contrast, the expression of class B bHLH proteins, such as MyoD and SCL, is restricted to specific organs or tissues. The myogenic (MyoD, myogenin, myf5) bHLH proteins have been studied extensively (15Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (924) Google Scholar) and serve as a useful paradigm for the actions of tissue-restricted bHLH proteins. MyoD forms a heterodimer with the ubiquitously expressed E2A proteins (16Lassar 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 (684) Google Scholar), binds specific DNA sequence at the regulatory regions of genes coding for muscle-specific proteins (such as muscle creatine kinase), and activates transcription of these genes (17Jaynes J.B. Johnson J.E. Buskin J.N. Gartside C.L. Hauschka S.D. Mol. Cell. Biol. 1988; 8: 62-70Crossref PubMed Scopus (170) Google Scholar). Although unproven, it is thought that SCL activates transcription of genes required for normal hematopoietic development in an analogous fashion (18Shivadasani R.A. Orkin S.H. Blood. 1996; 87: 4025-4039Crossref PubMed Google Scholar). Several forms of the SCL protein have been found in mammalian cells (19Elwood N.J. Green A.R. Melder A. Begley C.G. Nicola N. Leukemia. 1994; 8: 106-114PubMed Google Scholar); the full-length form of SCL has a molecular mass variously reported to be between 42 and 49 kDa, whereas an amino-terminally truncated form of 22–26 kDa is produced by translation of alternatively spliced transcripts (20Aplan P.D. Begley C.G. Bertness V. Nussmeier M. Ezquerra A. Colligan J. Kirsch I.R. Mol. Cell. Biol. 1990; 10: 6426-6435Crossref PubMed Scopus (83) Google Scholar) in both normal and leukemic cells. Although a transcription activation domain has been mapped to its amino terminus (21Wadman I.A. Hsu H.-L. Cobb M.H. Baer R. Oncogene. 1994; 9: 3713-3716PubMed Google Scholar), unscheduled synthesis of an SCL protein that lacks the transactivation domain leads to T-cell leukemia in transgenic mice, indicating that this transactivation domain is not required for leukemogenesis (8Aplan P.D. Jones C.A. Chervinsky D.S. Zhao X.-F. Ellsworth M. Wu C. McGuire E.A. Gross K.W EMBO J. 1997; 9: 2408-2419Crossref Scopus (125) Google Scholar). Similar to many other bHLH proteins, SCL has been shown to bind several E proteins, including E2-2/ITF2 (22Goldfarb A.N. Lewandowska K. Shoham M. J. Biol. Chem. 1996; 271: 2683-2688Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), E12, and E47 (two alternatively spliced forms of E2A/ITF1) (23Hsu H.-L. Cheng J.-T. Chen Q. Baer R. Mol. Cell. Biol. 1991; 11: 3037-3042Crossref PubMed Scopus (133) Google Scholar). When bound to E12, the SCL·E12 complex preferentially binds to a CAGATG nucleotide sequence in vitro (24Hsu H.-L. Huang L. Tsan T. Funk W. Wright W.E. Hu J.-S. Kingston R.E. Baer R. Mol. Cell. Biol. 1994; 14: 1256-1265Crossref PubMed Scopus (147) Google Scholar). Although SCL is suspected to function as a sequence-specific transcription factor, no target genes for SCL have thus far been convincingly identified, and any mechanism by which SCL may control transcription remains unknown. TFIIH is a multicomponent basal transcription factor complex that is also known to function in certain DNA repair pathways (25Svejstrup J.Q. Vichi P. Egly J.-M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar). Nine subunits have been identified within the TFIIH holoenzyme complex; various enzymatic activities, including DNA repair, helicase (26Schaeffer L. Roy R. Humbert S. Moncollin V. Vermeulen W. Hoejimakers J.H.J. Chambon P. Egly J.-M. Science. 1993; 260: 58-63Crossref PubMed Scopus (664) Google Scholar), and cyclin-dependent kinase (27Adamczewski J.P. Rossignol M. Tassan J.-P. Nigg E.A. Moncollin V. Egly J.-M. EMBO J. 1996; 15: 1877-1884Crossref PubMed Scopus (114) Google Scholar) activities, have been identified. The p62, p52, p44, and p34 subunits are thought to constitute the “core” of the TFIIH transcription machinery (28Marinoni J.C. Roy R. Vermeulen W. Miniou P. Lutz Y. Weeda G. Seroz T. Gomez D.M. Hoeijmakers J.H. Egly J.-M. EMBO J. 1997; 16: 1093-1102Crossref PubMed Scopus (60) Google Scholar). Although the p44 and p34 subunits have no defined enzymatic activity, their zinc finger structures suggest that they may be DNA-binding proteins (29Humbert S. van Vuuren H. Lutz Y. Hoeijmakers J.H. Egly J.-M. Moncollin V. EMBO J. 1994; 13: 2393-2398Crossref PubMed Scopus (100) Google Scholar) that might mediate interactions with soluble transcription factors. Here we report the identification of an unexpected interaction between SCL and the p44 subunit of TFIIH. This observation provides a link between SCL and the basal transcription machinery, suggesting that SCL may exert its suspected transcription regulatory effects through an interaction with TFIIH. DISCUSSIONUsing a yeast two-hybrid system, we have isolated several SCL-binding proteins, both expected and unexpected, from three different human cDNA libraries. As expected, the most prevalent SCL-binding partners recovered from these screens were E proteins, including E2A/ITF1, E2-2/ITF2, and HEB (14Murre C. McCaw P.S. Vaessin H. Caudy M. Jan L.Y. Jan Y.N. Cabrera C.V. Buskin J.N. Hauschica S.D. Lassar A.B. Weintraub H. Baltimore D. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1294) Google Scholar). We also used the yeast two-hybrid system to demonstrate previously unreported interactions between SCL and NHLH1 as well as a homodimeric interaction of SCL with itself. A human form of the DRG protein was also isolated from a human thymus cDNA library; this was not surprising given that mouse DRG has been isolated from a mouse erythroleukemia cell line (MEL) (35Mahajan M.A. Park S.T. Sun X.-H. Oncogene. 1996; 12: 2343-2350PubMed Google Scholar) using the 22–26-kDa form of SCL as the bait protein in a yeast two-hybrid screen.However, we were surprised by the isolation of the p44 subunit of TFIIH from the human thymus cDNA library. Although interactions between bHLH proteins (e.g. c-Myc and MyoD) and basal transcription machinery have been documented (41Maheswaran S. Lee H. Sonenshein G.E. Mol. Cell. Biol. 1994; 14: 1147-1152Crossref PubMed Scopus (70) Google Scholar, 42Heller H. Bengal E. Nucleic Acids Res. 1998; 26: 2112-2120Crossref PubMed Scopus (23) Google Scholar), those studies involved the interaction with TBP, the first basal transcription factor that is recruited to an activated promoter (43Kornberg R.D. Trends Biochem. Sci. 1996; 21: 325-326Abstract Full Text PDF PubMed Scopus (32) Google Scholar). The interaction between a bHLH protein and TFIIH, which is recruited to the transcription initiation complex relatively late and functions in promoter clearance and elongation of transcription (25Svejstrup J.Q. Vichi P. Egly J.-M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar), has not been previously reported.Several functional domains have been mapped to portions of the SCL protein; the amino-terminal region is proline-rich and has been shown to contain a transcription activation domain (21Wadman I.A. Hsu H.-L. Cobb M.H. Baer R. Oncogene. 1994; 9: 3713-3716PubMed Google Scholar), whereas the bHLH domain is involved in both DNA binding and dimerization with other bHLH proteins (44Tapscott S.J. Weintraub H. J. Clin. Invest. 1991; 87: 1133-1138Crossref PubMed Scopus (106) Google Scholar). The p44-binding region of SCL was mapped to a region that includes the bHLH domain as well as amino acid residues immediately N- and C-terminal to the bHLH domain. Although the bHLH domain was included in the region essential for binding, it is possible that the bHLH domain may only serve as an arm to link the N- and C-terminal flanking regions together to form a binding domain for p44. Support for this possibility is found in the observation that only SCL, among several bHLH proteins tested, was able to bind p44 in the yeast two-hybrid system, indicating that the bHLH structure alone is not sufficient for this interaction. For example, LYL1 (a bHLH protein named by virtue of its involvement in a chromosomal translocation associated with lymphoblasticleukemia) has a bHLH region that is quite similar (49/55 identical amino acids and four conservative substitutions) to SCL (1Begley C.G. Aplan P.D. Denning S.M. Haynes B.F. Waldmann T.A. Kirsch I.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 10128-10132Crossref PubMed Scopus (289) Google Scholar). Despite this, an interaction between LYL1 and p44 was not detected with this system, suggesting that the interaction between SCL and p44 requires amino acids proximal and distal to the bHLH region. Our results also show that the C-terminal polyglycine tract is important for the interaction; this is not surprising because glycine-rich domains have been shown to mediate other protein/protein interactions (45Cartegni L. Maconi M. Morandi E. Cobianchi F. Riva S. Biamonti G. J. Mol. Biol. 1996; 259: 337-348Crossref PubMed Scopus (157) Google Scholar). However, the SCL C-terminal glycine tract itself is not sufficient for the SCL/p44 interaction (see Fig. 4). The SCL-binding region of p44 was mapped to the C-terminal region of p44. This region is composed of 166 amino acids (from Lys-229 to Val-395), containing two putative functional motifs (29Humbert S. van Vuuren H. Lutz Y. Hoeijmakers J.H. Egly J.-M. Moncollin V. EMBO J. 1994; 13: 2393-2398Crossref PubMed Scopus (100) Google Scholar). One of these motifs, the TFIIIA-like zinc finger motif, was found to interact with DNA; the second motif, whose function is unknown, is conserved in both p44 and Ssl1, the yeast homologue of human p44. Although the SCL-binding domain of p44 has not yet been clearly defined, the above two motifs are potential targets for future studies.It is not clear how the interaction between SCL and p44 might function in a biological context. However, some insight may be gained through the study of previously reported interactions between nonessential transcription factors and elements of the basal transcription complex. Several proteins in addition to those of the basal transcription complex have been shown to interact with TFIIH subunits. These proteins include p53, which has been shown to interact in vitro with the XPB subunit of TFIIH (46Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (632) Google Scholar); this interaction is thought to modulate either the nucleotide excision repair activity of TFIIH (47Wang X.W. Yhe H. Schaeffer L. Roy R. Moncollin V. Egly J.-M. Wang Z. Friedberg E.C. Evans M.K. Taffe B.G. Bohr V.A. Weeda G. Hoeijmaker J.H.J. Forrester K. Harris C.C. Nat. Genet. 1995; 10: 188-195Crossref PubMed Scopus (514) Google Scholar) or p53-mediated apoptosis (48Wang X.W. Vermeulen W. Coursen J.D. Gibson M. Lupold S.E Forrester K. Xu G. Elmore L. Yeh H. Hoeijmaker J.H.J. Harris C.C. Genes Dev. 1996; 10: 1219-1232Crossref PubMed Scopus (309) Google Scholar). Additionally, the transcription activation domain of VP16 has been shown to bind the p62 subunit of TFIIH (49Xiao H. Pearson A. Coulombe B. Truant R. Zhang S. Regier J.L. Triezenberg S.J. Reinberg D. Flores O. Ingles C.J. Greenblatt J. Mol. Cell. Biol. 1994; 14: 7013-7024Crossref PubMed Scopus (327) Google Scholar), resulting in activation of transcription from an adenovirus type 2 major late promoter. With regard to interactions between bHLH proteins and basal transcription factors, the bHLH transcription factor c-Myc has been shown to bind the basal transcription factor TBP (50Hateboer G. Timmers H.T. Rustgi A.K. Billaud M. van't Veer L.J. Bernards R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8489-8493Crossref PubMed Scopus (129) Google Scholar). Therefore, one hypothesis regarding the functional relevance of an SCL/p44 interaction speculates that SCL, itself or as a heterodimer with an E protein, interacts with p44, thus modulating the activity of TFIIH and eventually the transcription elongation.In summary, we have demonstrated a specific interaction, both in vivo and in vitro, between the bHLH protein SCL and the p44 subunit of the basal transcription factor TFIIH. Although this interaction was mapped to a region of SCL that encompassed, but was not limited to, the bHLH domain, other bHLH proteins did not demonstrate binding to p44. This novel interaction between a bHLH protein and TFIIH suggests a potential mechanism by which the tissue-restricted bHLH protein SCL might function during hematopoietic and vascular development. Using a yeast two-hybrid system, we have isolated several SCL-binding proteins, both expected and unexpected, from three different human cDNA libraries. As expected, the most prevalent SCL-binding partners recovered from these screens were E proteins, including E2A/ITF1, E2-2/ITF2, and HEB (14Murre C. McCaw P.S. Vaessin H. Caudy M. Jan L.Y. Jan Y.N. Cabrera C.V. Buskin J.N. Hauschica S.D. Lassar A.B. Weintraub H. Baltimore D. Cell. 1989; 58: 537-544Abstract Full Text PDF PubMed Scopus (1294) Google Scholar). We also used the yeast two-hybrid system to demonstrate previously unreported interactions between SCL and NHLH1 as well as a homodimeric interaction of SCL with itself. A human form of the DRG protein was also isolated from a human thymus cDNA library; this was not surprising given that mouse DRG has been isolated from a mouse erythroleukemia cell line (MEL) (35Mahajan M.A. Park S.T. Sun X.-H. Oncogene. 1996; 12: 2343-2350PubMed Google Scholar) using the 22–26-kDa form of SCL as the bait protein in a yeast two-hybrid screen. However, we were surprised by the isolation of the p44 subunit of TFIIH from the human thymus cDNA library. Although interactions between bHLH proteins (e.g. c-Myc and MyoD) and basal transcription machinery have been documented (41Maheswaran S. Lee H. Sonenshein G.E. Mol. Cell. Biol. 1994; 14: 1147-1152Crossref PubMed Scopus (70) Google Scholar, 42Heller H. Bengal E. Nucleic Acids Res. 1998; 26: 2112-2120Crossref PubMed Scopus (23) Google Scholar), those studies involved the interaction with TBP, the first basal transcription factor that is recruited to an activated promoter (43Kornberg R.D. Trends Biochem. Sci. 1996; 21: 325-326Abstract Full Text PDF PubMed Scopus (32) Google Scholar). The interaction between a bHLH protein and TFIIH, which is recruited to the transcription initiation complex relatively late and functions in promoter clearance and elongation of transcription (25Svejstrup J.Q. Vichi P. Egly J.-M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar), has not been previously reported. Several functional domains have been mapped to portions of the SCL protein; the amino-terminal region is proline-rich and has been shown to contain a transcription activation domain (21Wadman I.A. Hsu H.-L. Cobb M.H. Baer R. Oncogene. 1994; 9: 3713-3716PubMed Google Scholar), whereas the bHLH domain is involved in both DNA binding and dimerization with other bHLH proteins (44Tapscott S.J. Weintraub H. J. Clin. Invest. 1991; 87: 1133-1138Crossref PubMed Scopus (106) Google Scholar). The p44-binding region of SCL was mapped to a region that includes the bHLH domain as well as amino acid residues immediately N- and C-terminal to the bHLH domain. Although the bHLH domain was included in the region essential for binding, it is possible that the bHLH domain may only serve as an arm to link the N- and C-terminal flanking regions together to form a binding domain for p44. Support for this possibility is found in the observation that only SCL, among several bHLH proteins tested, was able to bind p44 in the yeast two-hybrid system, indicating that the bHLH structure alone is not sufficient for this interaction. For example, LYL1 (a bHLH protein named by virtue of its involvement in a chromosomal translocation associated with lymphoblasticleukemia) has a bHLH region that is quite similar (49/55 identical amino acids and four conservative substitutions) to SCL (1Begley C.G. Aplan P.D. Denning S.M. Haynes B.F. Waldmann T.A. Kirsch I.R. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 10128-10132Crossref PubMed Scopus (289) Google Scholar). Despite this, an interaction between LYL1 and p44 was not detected with this system, suggesting that the interaction between SCL and p44 requires amino acids proximal and distal to the bHLH region. Our results also show that the C-terminal polyglycine tract is important for the interaction; this is not surprising because glycine-rich domains have been shown to mediate other protein/protein interactions (45Cartegni L. Maconi M. Morandi E. Cobianchi F. Riva S. Biamonti G. J. Mol. Biol. 1996; 259: 337-348Crossref PubMed Scopus (157) Google Scholar). However, the SCL C-terminal glycine tract itself is not sufficient for the SCL/p44 interaction (see Fig. 4). The SCL-binding region of p44 was mapped to the C-terminal region of p44. This region is composed of 166 amino acids (from Lys-229 to Val-395), containing two putative functional motifs (29Humbert S. van Vuuren H. Lutz Y. Hoeijmakers J.H. Egly J.-M. Moncollin V. EMBO J. 1994; 13: 2393-2398Crossref PubMed Scopus (100) Google Scholar). One of these motifs, the TFIIIA-like zinc finger motif, was found to interact with DNA; the second motif, whose function is unknown, is conserved in both p44 and Ssl1, the yeast homologue of human p44. Although the SCL-binding domain of p44 has not yet been clearly defined, the above two motifs are potential targets for future studies. It is not clear how the interaction between SCL and p44 might function in a biological context. However, some insight may be gained through the study of previously reported interactions between nonessential transcription factors and elements of the basal transcription complex. Several proteins in addition to those of the basal transcription complex have been shown to interact with TFIIH subunits. These proteins include p53, which has been shown to interact in vitro with the XPB subunit of TFIIH (46Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (632) Google Scholar); this interaction is thought to modulate either the nucleotide excision repair activity of TFIIH (47Wang X.W. Yhe H. Schaeffer L. Roy R. Moncollin V. Egly J.-M. Wang Z. Friedberg E.C. Evans M.K. Taffe B.G. Bohr V.A. Weeda G. Hoeijmaker J.H.J. Forrester K. Harris C.C. Nat. Genet. 1995; 10: 188-195Crossref PubMed Scopus (514) Google Scholar) or p53-mediated apoptosis (48Wang X.W. Vermeulen W. Coursen J.D. Gibson M. Lupold S.E Forrester K. Xu G. Elmore L. Yeh H. Hoeijmaker J.H.J. Harris C.C. Genes Dev. 1996; 10: 1219-1232Crossref PubMed Scopus (309) Google Scholar). Additionally, the transcription activation domain of VP16 has been shown to bind the p62 subunit of TFIIH (49Xiao H. Pearson A. Coulombe B. Truant R. Zhang S. Regier J.L. Triezenberg S.J. Reinberg D. Flores O. Ingles C.J. Greenblatt J. Mol. Cell. Biol. 1994; 14: 7013-7024Crossref PubMed Scopus (327) Google Scholar), resulting in activation of transcription from an adenovirus type 2 major late promoter. With regard to interactions between bHLH proteins and basal transcription factors, the bHLH transcription factor c-Myc has been shown to bind the basal transcription factor TBP (50Hateboer G. Timmers H.T. Rustgi A.K. Billaud M. van't Veer L.J. Bernards R. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8489-8493Crossref PubMed Scopus (129) Google Scholar). Therefore, one hypothesis regarding the functional relevance of an SCL/p44 interaction speculates that SCL, itself or as a heterodimer with an E protein, interacts with p44, thus modulating the activity of TFIIH and eventually the transcription elongation. In summary, we have demonstrated a specific interaction, both in vivo and in vitro, between the bHLH protein SCL and the p44 subunit of the basal transcription factor TFIIH. Although this interaction was mapped to a region of SCL that encompassed, but was not limited to, the bHLH domain, other bHLH proteins did not demonstrate binding to p44. This novel interaction between a bHLH protein and TFIIH suggests a potential mechanism by which the tissue-restricted bHLH protein SCL might function during hematopoietic and vascular development. We thank David S. Chervinsky for excellent technical assistance, Ellen Greco for art work, and the Roswell Park Cancer Institute Biopolymer Facility for oligonucleotide synthesis. We are also grateful to Dr. Ilan Kirsch for the NHLH1 cDNA, Dr. Adam Goldfarb for the E2-5 cDNA, and Dr. Karen Pulford for BTL73 monoclonal antibodies.

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