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. 1995 Jul;15(7):3813–3822. doi: 10.1128/mcb.15.7.3813

Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.

S M Hollenberg 1, R Sternglanz 1, P F Cheng 1, H Weintraub 1
PMCID: PMC230620  PMID: 7791788

Abstract

With modified two-hybrid technology, we have isolated a member of a new family of basic helix-loop-helix (bHLH) transcription factors. Thing1 (Th1) was identified in a screen of a mouse embryo cDNA library as a partner for the Drosophila E protein daughterless. RNA in situ hybridization and reverse transcriptase-PCR demonstrate a stage- and tissue-specific distribution for the expression of Th1. Although tissue specific, the expression pattern of Th1 is fairly complex. During development, Th1 mRNA is widely expressed in extraembryonic tissues, portions of the heart, autonomic ganglia, the gut, and pharyngeal arches. At embryonic day 7.5 (E7.5), extraembryonic derivatives show robust Th1 expression. By E8.5, expression in the embryonic heart becomes detectable. During the next 2 days of development, the signal also includes gut and pharyngeal arches. Predominant expression at E13.5 is in neural crest derivatives, especially the autonomic nervous system and adrenal medulla. Expression of Th1 persists in the adult, in which it is localized to the smooth muscle cells of the gut. In vitro, Th1 protein recognizes a set of DNA sites that are more degenerate than has been determined for other bHLH factors, indicating a reduced binding specificity. Transient transfection of NIH 3T3 cells with GAL4-Th1 fusions reveals a repression activity mediated by the Th1 bHLH domain. In combination, these properties define Th1 as a new bHLH protein with a unique set of properties.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Baer R. TAL1, TAL2 and LYL1: a family of basic helix-loop-helix proteins implicated in T cell acute leukaemia. Semin Cancer Biol. 1993 Dec;4(6):341–347. [PubMed] [Google Scholar]
  3. Bartel P., Chien C. T., Sternglanz R., Fields S. Elimination of false positives that arise in using the two-hybrid system. Biotechniques. 1993 Jun;14(6):920–924. [PubMed] [Google Scholar]
  4. Begley C. G., Visvader J., Green A. R., Aplan P. D., Metcalf D., Kirsch I. R., Gough N. M. Molecular cloning and chromosomal localization of the murine homolog of the human helix-loop-helix gene SCL. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):869–873. doi: 10.1073/pnas.88.3.869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Benezra R., Davis R. L., Lockshon D., Turner D. L., Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell. 1990 Apr 6;61(1):49–59. doi: 10.1016/0092-8674(90)90214-y. [DOI] [PubMed] [Google Scholar]
  6. Blackwell T. K., Huang J., Ma A., Kretzner L., Alt F. W., Eisenman R. N., Weintraub H. Binding of myc proteins to canonical and noncanonical DNA sequences. Mol Cell Biol. 1993 Sep;13(9):5216–5224. doi: 10.1128/mcb.13.9.5216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blackwell T. K., Weintraub H. Differences and similarities in DNA-binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science. 1990 Nov 23;250(4984):1104–1110. doi: 10.1126/science.2174572. [DOI] [PubMed] [Google Scholar]
  8. Braun T., Winter B., Bober E., Arnold H. H. Transcriptional activation domain of the muscle-specific gene-regulatory protein myf5. Nature. 1990 Aug 16;346(6285):663–665. doi: 10.1038/346663a0. [DOI] [PubMed] [Google Scholar]
  9. Brown L., Baer R. HEN1 encodes a 20-kilodalton phosphoprotein that binds an extended E-box motif as a homodimer. Mol Cell Biol. 1994 Feb;14(2):1245–1255. doi: 10.1128/mcb.14.2.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brown L., Espinosa R., 3rd, Le Beau M. M., Siciliano M. J., Baer R. HEN1 and HEN2: a subgroup of basic helix-loop-helix genes that are coexpressed in a human neuroblastoma. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8492–8496. doi: 10.1073/pnas.89.18.8492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Campos-Ortega J. A. Mechanisms of early neurogenesis in Drosophila melanogaster. J Neurobiol. 1993 Oct;24(10):1305–1327. doi: 10.1002/neu.480241005. [DOI] [PubMed] [Google Scholar]
  12. Campuzano S., Modolell J. Patterning of the Drosophila nervous system: the achaete-scute gene complex. Trends Genet. 1992 Jun;8(6):202–208. doi: 10.1016/0168-9525(92)90234-u. [DOI] [PubMed] [Google Scholar]
  13. Chen Q., Cheng J. T., Tasi L. H., Schneider N., Buchanan G., Carroll A., Crist W., Ozanne B., Siciliano M. J., Baer R. The tal gene undergoes chromosome translocation in T cell leukemia and potentially encodes a helix-loop-helix protein. EMBO J. 1990 Feb;9(2):415–424. doi: 10.1002/j.1460-2075.1990.tb08126.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Christy B. A., Sanders L. K., Lau L. F., Copeland N. G., Jenkins N. A., Nathans D. An Id-related helix-loop-helix protein encoded by a growth factor-inducible gene. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1815–1819. doi: 10.1073/pnas.88.5.1815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cowell I. G. Repression versus activation in the control of gene transcription. Trends Biochem Sci. 1994 Jan;19(1):38–42. doi: 10.1016/0968-0004(94)90172-4. [DOI] [PubMed] [Google Scholar]
  17. Dang C. V., Dolde C., Gillison M. L., Kato G. J. Discrimination between related DNA sites by a single amino acid residue of Myc-related basic-helix-loop-helix proteins. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):599–602. doi: 10.1073/pnas.89.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Edmondson D. G., Olson E. N. A gene with homology to the myc similarity region of MyoD1 is expressed during myogenesis and is sufficient to activate the muscle differentiation program. Genes Dev. 1989 May;3(5):628–640. doi: 10.1101/gad.3.5.628. [DOI] [PubMed] [Google Scholar]
  19. Ellenberger T., Fass D., Arnaud M., Harrison S. C. Crystal structure of transcription factor E47: E-box recognition by a basic region helix-loop-helix dimer. Genes Dev. 1994 Apr 15;8(8):970–980. doi: 10.1101/gad.8.8.970. [DOI] [PubMed] [Google Scholar]
  20. Ferré-D'Amaré A. R., Prendergast G. C., Ziff E. B., Burley S. K. Recognition by Max of its cognate DNA through a dimeric b/HLH/Z domain. Nature. 1993 May 6;363(6424):38–45. doi: 10.1038/363038a0. [DOI] [PubMed] [Google Scholar]
  21. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  22. Fields S., Sternglanz R. The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 1994 Aug;10(8):286–292. doi: 10.1016/0168-9525(90)90012-u. [DOI] [PubMed] [Google Scholar]
  23. Frohman M. A., Dush M. K., Martin G. R. Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8998–9002. doi: 10.1073/pnas.85.23.8998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Golemis E. A., Brent R. Fused protein domains inhibit DNA binding by LexA. Mol Cell Biol. 1992 Jul;12(7):3006–3014. doi: 10.1128/mcb.12.7.3006. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Green A. R., Lints T., Visvader J., Harvey R., Begley C. G. SCL is coexpressed with GATA-1 in hemopoietic cells but is also expressed in developing brain. Oncogene. 1992 Apr;7(4):653–660. [PubMed] [Google Scholar]
  26. Grueneberg D. A., Natesan S., Alexandre C., Gilman M. Z. Human and Drosophila homeodomain proteins that enhance the DNA-binding activity of serum response factor. Science. 1992 Aug 21;257(5073):1089–1095. doi: 10.1126/science.257.5073.1089. [DOI] [PubMed] [Google Scholar]
  27. Göbel V., Lipkowitz S., Kozak C. A., Kirsch I. R. NSCL-2: a basic domain helix-loop-helix gene expressed in early neurogenesis. Cell Growth Differ. 1992 Mar;3(3):143–148. [PubMed] [Google Scholar]
  28. Hill J., Donald K. A., Griffiths D. E., Donald G. DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res. 1991 Oct 25;19(20):5791–5791. doi: 10.1093/nar/19.20.5791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Hollenberg S. M., Cheng P. F., Weintraub H. Use of a conditional MyoD transcription factor in studies of MyoD trans-activation and muscle determination. Proc Natl Acad Sci U S A. 1993 Sep 1;90(17):8028–8032. doi: 10.1073/pnas.90.17.8028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Hopwood N. D., Pluck A., Gurdon J. B. A Xenopus mRNA related to Drosophila twist is expressed in response to induction in the mesoderm and the neural crest. Cell. 1989 Dec 1;59(5):893–903. doi: 10.1016/0092-8674(89)90612-0. [DOI] [PubMed] [Google Scholar]
  31. Hsu H. L., Huang L., Tsan J. T., Funk W., Wright W. E., Hu J. S., Kingston R. E., Baer R. Preferred sequences for DNA recognition by the TAL1 helix-loop-helix proteins. Mol Cell Biol. 1994 Feb;14(2):1256–1265. doi: 10.1128/mcb.14.2.1256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Hwang L. Y., Siegelman M., Davis L., Oppenheimer-Marks N., Baer R. Expression of the TAL1 proto-oncogene in cultured endothelial cells and blood vessels of the spleen. Oncogene. 1993 Nov;8(11):3043–3046. [PubMed] [Google Scholar]
  33. Ish-Horowicz D., Pinchin S. M. Pattern abnormalities induced by ectopic expression of the Drosophila gene hairy are associated with repression of ftz transcription. Cell. 1987 Nov 6;51(3):405–415. doi: 10.1016/0092-8674(87)90636-2. [DOI] [PubMed] [Google Scholar]
  34. Ishibashi M., Sasai Y., Nakanishi S., Kageyama R. Molecular characterization of HES-2, a mammalian helix-loop-helix factor structurally related to Drosophila hairy and Enhancer of split. Eur J Biochem. 1993 Aug 1;215(3):645–652. doi: 10.1111/j.1432-1033.1993.tb18075.x. [DOI] [PubMed] [Google Scholar]
  35. Kallianpur A. R., Jordan J. E., Brandt S. J. The SCL/TAL-1 gene is expressed in progenitors of both the hematopoietic and vascular systems during embryogenesis. Blood. 1994 Mar 1;83(5):1200–1208. [PubMed] [Google Scholar]
  36. Knust E., Bremer K. A., Vässin H., Ziemer A., Tepass U., Campos-Ortega J. A. The enhancer of split locus and neurogenesis in Drosophila melanogaster. Dev Biol. 1987 Jul;122(1):262–273. doi: 10.1016/0012-1606(87)90351-4. [DOI] [PubMed] [Google Scholar]
  37. Lillie J. W., Green M. R. Transcription activation by the adenovirus E1a protein. Nature. 1989 Mar 2;338(6210):39–44. doi: 10.1038/338039a0. [DOI] [PubMed] [Google Scholar]
  38. Ma P. C., Rould M. A., Weintraub H., Pabo C. O. Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation. Cell. 1994 May 6;77(3):451–459. doi: 10.1016/0092-8674(94)90159-7. [DOI] [PubMed] [Google Scholar]
  39. Murre C., McCaw P. S., Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 1989 Mar 10;56(5):777–783. doi: 10.1016/0092-8674(89)90682-x. [DOI] [PubMed] [Google Scholar]
  40. Patanjali S. R., Parimoo S., Weissman S. M. Construction of a uniform-abundance (normalized) cDNA library. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1943–1947. doi: 10.1073/pnas.88.5.1943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Quertermous E. E., Hidai H., Blanar M. A., Quertermous T. Cloning and characterization of a basic helix-loop-helix protein expressed in early mesoderm and the developing somites. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7066–7070. doi: 10.1073/pnas.91.15.7066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Quong M. W., Massari M. E., Zwart R., Murre C. A new transcriptional-activation motif restricted to a class of helix-loop-helix proteins is functionally conserved in both yeast and mammalian cells. Mol Cell Biol. 1993 Feb;13(2):792–800. doi: 10.1128/mcb.13.2.792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Roberts V. J., Steenbergen R., Murre C. Localization of E2A mRNA expression in developing and adult rat tissues. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7583–7587. doi: 10.1073/pnas.90.16.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Rupp R. A., Weintraub H. Ubiquitous MyoD transcription at the midblastula transition precedes induction-dependent MyoD expression in presumptive mesoderm of X. laevis. Cell. 1991 Jun 14;65(6):927–937. doi: 10.1016/0092-8674(91)90545-a. [DOI] [PubMed] [Google Scholar]
  45. Sadowski I., Ptashne M. A vector for expressing GAL4(1-147) fusions in mammalian cells. Nucleic Acids Res. 1989 Sep 25;17(18):7539–7539. doi: 10.1093/nar/17.18.7539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  47. Silver P. A., Brent R., Ptashne M. DNA binding is not sufficient for nuclear localization of regulatory proteins in Saccharomyces cerevisiae. Mol Cell Biol. 1986 Dec;6(12):4763–4766. doi: 10.1128/mcb.6.12.4763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Staudinger J., Perry M., Elledge S. J., Olson E. N. Interactions among vertebrate helix-loop-helix proteins in yeast using the two-hybrid system. J Biol Chem. 1993 Mar 5;268(7):4608–4611. [PubMed] [Google Scholar]
  49. Sun X. H., Baltimore D. An inhibitory domain of E12 transcription factor prevents DNA binding in E12 homodimers but not in E12 heterodimers. Cell. 1991 Jan 25;64(2):459–470. doi: 10.1016/0092-8674(91)90653-g. [DOI] [PubMed] [Google Scholar]
  50. Sun X. H., Copeland N. G., Jenkins N. A., Baltimore D. Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins. Mol Cell Biol. 1991 Nov;11(11):5603–5611. doi: 10.1128/mcb.11.11.5603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Thayer M. J., Weintraub H. A cellular factor stimulates the DNA-binding activity of MyoD and E47. Proc Natl Acad Sci U S A. 1993 Jul 15;90(14):6483–6487. doi: 10.1073/pnas.90.14.6483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Van Antwerp M. E., Chen D. G., Chang C., Prochownik E. V. A point mutation in the MyoD basic domain imparts c-Myc-like properties. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9010–9014. doi: 10.1073/pnas.89.19.9010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Visvader J., Begley C. G. Helix-loop-helix genes translocated in lymphoid leukemia. Trends Biochem Sci. 1991 Sep;16(9):330–333. doi: 10.1016/0968-0004(91)90137-k. [DOI] [PubMed] [Google Scholar]
  54. Vojtek A. B., Hollenberg S. M., Cooper J. A. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. doi: 10.1016/0092-8674(93)90307-c. [DOI] [PubMed] [Google Scholar]
  55. Wang Z., Brown D. D. A gene expression screen. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11505–11509. doi: 10.1073/pnas.88.24.11505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Weintraub H., Davis R., Lockshon D., Lassar A. MyoD binds cooperatively to two sites in a target enhancer sequence: occupancy of two sites is required for activation. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5623–5627. doi: 10.1073/pnas.87.15.5623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Weintraub H. The MyoD family and myogenesis: redundancy, networks, and thresholds. Cell. 1993 Dec 31;75(7):1241–1244. doi: 10.1016/0092-8674(93)90610-3. [DOI] [PubMed] [Google Scholar]
  58. Wolf C., Thisse C., Stoetzel C., Thisse B., Gerlinger P., Perrin-Schmitt F. The M-twist gene of Mus is expressed in subsets of mesodermal cells and is closely related to the Xenopus X-twi and the Drosophila twist genes. Dev Biol. 1991 Feb;143(2):363–373. doi: 10.1016/0012-1606(91)90086-i. [DOI] [PubMed] [Google Scholar]
  59. Wright W. E., Binder M., Funk W. Cyclic amplification and selection of targets (CASTing) for the myogenin consensus binding site. Mol Cell Biol. 1991 Aug;11(8):4104–4110. doi: 10.1128/mcb.11.8.4104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wright W. E., Sassoon D. A., Lin V. K. Myogenin, a factor regulating myogenesis, has a domain homologous to MyoD. Cell. 1989 Feb 24;56(4):607–617. doi: 10.1016/0092-8674(89)90583-7. [DOI] [PubMed] [Google Scholar]
  61. Xia Y., Brown L., Yang C. Y., Tsan J. T., Siciliano M. J., Espinosa R., 3rd, Le Beau M. M., Baer R. J. TAL2, a helix-loop-helix gene activated by the (7;9)(q34;q32) translocation in human T-cell leukemia. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11416–11420. doi: 10.1073/pnas.88.24.11416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Young D., Riggs M., Field J., Vojtek A., Broek D., Wigler M. The adenylyl cyclase gene from Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1989 Oct;86(20):7989–7993. doi: 10.1073/pnas.86.20.7989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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