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. 1991 Jul;11(7):3633–3641. doi: 10.1128/mcb.11.7.3633

Inefficient homooligomerization contributes to the dependence of myogenin on E2A products for efficient DNA binding.

T Chakraborty 1, T J Brennan 1, L Li 1, D Edmondson 1, E N Olson 1
PMCID: PMC361113  PMID: 1646392

Abstract

Myogenin is a muscle-specific transcription factor that can activate myogenesis; it belongs to a family of transcription factors that share homology within a basic region and an adjacent helix-loop-helix (HLH) motif. Although myogenin alone binds DNA inefficiently, in the presence of the widely expressed HLH proteins E12 and E47 (encoded by the E2A gene), it forms heterooligomers that bind with high affinity to a DNA sequence known as a kappa E-2 site. In contrast, E47 and to a lesser extent E12 are both able to bind the kappa E-2 site relatively efficiently as homooligomers. To define the relative contributions of the basic regions of myogenin and E12 to DNA binding and muscle-specific gene activation, we created chimeras of the two proteins by swapping their basic regions. We showed that myogenin's weak affinity for the kappa E-2 site is attributable to inefficient homooligomerization and that the myogenin basic domain alone can mediate high-affinity DNA binding when placed in E12. Within a heterooligomeric complex, two basic regions were required to form a high-affinity DNA-binding domain. Basic-domain mutants of myogenin or E2A gene products that cannot bind DNA retained the ability to oligomerize and could abolish DNA binding of the wild-type proteins in vitro. These myogenin and E2A mutants also acted as trans-dominant inhibitors of muscle-specific gene activation in vivo. These findings support the notion that muscle-specific gene activation requires oligomerization between myogenin and E2A gene products and that E2A gene products play an important role in myogenesis by enhancing the DNA-binding activity of myogenin, as well as other myogenic HLH proteins.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alonso M. C., Cabrera C. V. The achaete-scute gene complex of Drosophila melanogaster comprises four homologous genes. EMBO J. 1988 Aug;7(8):2585–2591. doi: 10.1002/j.1460-2075.1988.tb03108.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beckmann H., Su L. K., Kadesch T. TFE3: a helix-loop-helix protein that activates transcription through the immunoglobulin enhancer muE3 motif. Genes Dev. 1990 Feb;4(2):167–179. doi: 10.1101/gad.4.2.167. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Braun T., Bober E., Buschhausen-Denker G., Kohtz S., Grzeschik K. H., Arnold H. H., Kotz S. Differential expression of myogenic determination genes in muscle cells: possible autoactivation by the Myf gene products. EMBO J. 1989 Dec 1;8(12):3617–3625. doi: 10.1002/j.1460-2075.1989.tb08535.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Braun T., Bober E., Winter B., Rosenthal N., Arnold H. H. Myf-6, a new member of the human gene family of myogenic determination factors: evidence for a gene cluster on chromosome 12. EMBO J. 1990 Mar;9(3):821–831. doi: 10.1002/j.1460-2075.1990.tb08179.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Braun T., Buschhausen-Denker G., Bober E., Tannich E., Arnold H. H. A novel human muscle factor related to but distinct from MyoD1 induces myogenic conversion in 10T1/2 fibroblasts. EMBO J. 1989 Mar;8(3):701–709. doi: 10.1002/j.1460-2075.1989.tb03429.x. [DOI] [PMC free article] [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. Brennan T. J., Edmondson D. G., Olson E. N. Aberrant regulation of MyoD1 contributes to the partially defective myogenic phenotype of BC3H1 cells. J Cell Biol. 1990 Apr;110(4):929–937. doi: 10.1083/jcb.110.4.929. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Brennan T. J., Olson E. N. Myogenin resides in the nucleus and acquires high affinity for a conserved enhancer element on heterodimerization. Genes Dev. 1990 Apr;4(4):582–595. doi: 10.1101/gad.4.4.582. [DOI] [PubMed] [Google Scholar]
  11. Buskin J. N., Hauschka S. D. Identification of a myocyte nuclear factor that binds to the muscle-specific enhancer of the mouse muscle creatine kinase gene. Mol Cell Biol. 1989 Jun;9(6):2627–2640. doi: 10.1128/mcb.9.6.2627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Carr C. S., Sharp P. A. A helix-loop-helix protein related to the immunoglobulin E box-binding proteins. Mol Cell Biol. 1990 Aug;10(8):4384–4388. doi: 10.1128/mcb.10.8.4384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Caudy M., Vässin H., Brand M., Tuma R., Jan L. Y., Jan Y. N. daughterless, a Drosophila gene essential for both neurogenesis and sex determination, has sequence similarities to myc and the achaete-scute complex. Cell. 1988 Dec 23;55(6):1061–1067. doi: 10.1016/0092-8674(88)90250-4. [DOI] [PubMed] [Google Scholar]
  14. Chakraborty T., Brennan T., Olson E. Differential trans-activation of a muscle-specific enhancer by myogenic helix-loop-helix proteins is separable from DNA binding. J Biol Chem. 1991 Feb 15;266(5):2878–2882. [PubMed] [Google Scholar]
  15. Davis R. L., Cheng P. F., Lassar A. B., Weintraub H. The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation. Cell. 1990 Mar 9;60(5):733–746. doi: 10.1016/0092-8674(90)90088-v. [DOI] [PubMed] [Google Scholar]
  16. Davis R. L., Weintraub H., Lassar A. B. Expression of a single transfected cDNA converts fibroblasts to myoblasts. Cell. 1987 Dec 24;51(6):987–1000. doi: 10.1016/0092-8674(87)90585-x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Ellis H. M., Spann D. R., Posakony J. W. extramacrochaetae, a negative regulator of sensory organ development in Drosophila, defines a new class of helix-loop-helix proteins. Cell. 1990 Apr 6;61(1):27–38. doi: 10.1016/0092-8674(90)90212-w. [DOI] [PubMed] [Google Scholar]
  19. French B. A., Chow K. L., Olson E. N., Schwartz R. J. Heterodimers of myogenic helix-loop-helix regulatory factors and E12 bind a complex element governing myogenic induction of the avian cardiac alpha-actin promoter. Mol Cell Biol. 1991 May;11(5):2439–2450. doi: 10.1128/mcb.11.5.2439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Garrell J., Modolell J. The Drosophila extramacrochaetae locus, an antagonist of proneural genes that, like these genes, encodes a helix-loop-helix protein. Cell. 1990 Apr 6;61(1):39–48. doi: 10.1016/0092-8674(90)90213-x. [DOI] [PubMed] [Google Scholar]
  21. Gregor P. D., Sawadogo M., Roeder R. G. The adenovirus major late transcription factor USF is a member of the helix-loop-helix group of regulatory proteins and binds to DNA as a dimer. Genes Dev. 1990 Oct;4(10):1730–1740. doi: 10.1101/gad.4.10.1730. [DOI] [PubMed] [Google Scholar]
  22. Henthorn P., Kiledjian M., Kadesch T. Two distinct transcription factors that bind the immunoglobulin enhancer microE5/kappa 2 motif. Science. 1990 Jan 26;247(4941):467–470. doi: 10.1126/science.2105528. [DOI] [PubMed] [Google Scholar]
  23. Hu Y. F., Lüscher B., Admon A., Mermod N., Tjian R. Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity. Genes Dev. 1990 Oct;4(10):1741–1752. doi: 10.1101/gad.4.10.1741. [DOI] [PubMed] [Google Scholar]
  24. Jaynes J. B., Johnson J. E., Buskin J. N., Gartside C. L., Hauschka S. D. The muscle creatine kinase gene is regulated by multiple upstream elements, including a muscle-specific enhancer. Mol Cell Biol. 1988 Jan;8(1):62–70. doi: 10.1128/mcb.8.1.62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lassar A. B., Buskin J. N., Lockshon D., Davis R. L., Apone S., Hauschka S. D., Weintraub H. MyoD is a sequence-specific DNA binding protein requiring a region of myc homology to bind to the muscle creatine kinase enhancer. Cell. 1989 Sep 8;58(5):823–831. doi: 10.1016/0092-8674(89)90935-5. [DOI] [PubMed] [Google Scholar]
  27. Lin H., Yutzey K. E., Konieczny S. F. Muscle-specific expression of the troponin I gene requires interactions between helix-loop-helix muscle regulatory factors and ubiquitous transcription factors. Mol Cell Biol. 1991 Jan;11(1):267–280. doi: 10.1128/mcb.11.1.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Miner J. H., Wold B. Herculin, a fourth member of the MyoD family of myogenic regulatory genes. Proc Natl Acad Sci U S A. 1990 Feb;87(3):1089–1093. doi: 10.1073/pnas.87.3.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Murre C., McCaw P. S., Vaessin H., Caudy M., Jan L. Y., Jan Y. N., Cabrera C. V., Buskin J. N., Hauschka S. D., Lassar A. B. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989 Aug 11;58(3):537–544. doi: 10.1016/0092-8674(89)90434-0. [DOI] [PubMed] [Google Scholar]
  31. Murre C., Voronova A., Baltimore D. B-cell- and myocyte-specific E2-box-binding factors contain E12/E47-like subunits. Mol Cell Biol. 1991 Feb;11(2):1156–1160. doi: 10.1128/mcb.11.2.1156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nelson C., Shen L. P., Meister A., Fodor E., Rutter W. J. Pan: a transcriptional regulator that binds chymotrypsin, insulin, and AP-4 enhancer motifs. Genes Dev. 1990 Jun;4(6):1035–1043. doi: 10.1101/gad.4.6.1035. [DOI] [PubMed] [Google Scholar]
  33. Olson E. N. MyoD family: a paradigm for development? Genes Dev. 1990 Sep;4(9):1454–1461. doi: 10.1101/gad.4.9.1454. [DOI] [PubMed] [Google Scholar]
  34. Piette J., Bessereau J. L., Huchet M., Changeux J. P. Two adjacent MyoD1-binding sites regulate expression of the acetylcholine receptor alpha-subunit gene. Nature. 1990 May 24;345(6273):353–355. doi: 10.1038/345353a0. [DOI] [PubMed] [Google Scholar]
  35. Pinney D. F., Pearson-White S. H., Konieczny S. F., Latham K. E., Emerson C. P., Jr Myogenic lineage determination and differentiation: evidence for a regulatory gene pathway. Cell. 1988 Jun 3;53(5):781–793. doi: 10.1016/0092-8674(88)90095-5. [DOI] [PubMed] [Google Scholar]
  36. Prendergast G. C., Ziff E. B. Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region. Science. 1991 Jan 11;251(4990):186–189. doi: 10.1126/science.1987636. [DOI] [PubMed] [Google Scholar]
  37. Rhodes S. J., Konieczny S. F. Identification of MRF4: a new member of the muscle regulatory factor gene family. Genes Dev. 1989 Dec;3(12B):2050–2061. doi: 10.1101/gad.3.12b.2050. [DOI] [PubMed] [Google Scholar]
  38. Rosenthal N., Berglund E. B., Wentworth B. M., Donoghue M., Winter B., Bober E., Braun T., Arnold H. H. A highly conserved enhancer downstream of the human MLC1/3 locus is a target for multiple myogenic determination factors. Nucleic Acids Res. 1990 Nov 11;18(21):6239–6246. doi: 10.1093/nar/18.21.6239. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sartorelli V., Webster K. A., Kedes L. Muscle-specific expression of the cardiac alpha-actin gene requires MyoD1, CArG-box binding factor, and Sp1. Genes Dev. 1990 Oct;4(10):1811–1822. doi: 10.1101/gad.4.10.1811. [DOI] [PubMed] [Google Scholar]
  40. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  41. Sternberg E. A., Spizz G., Perry W. M., Vizard D., Weil T., Olson E. N. Identification of upstream and intragenic regulatory elements that confer cell-type-restricted and differentiation-specific expression on the muscle creatine kinase gene. Mol Cell Biol. 1988 Jul;8(7):2896–2909. doi: 10.1128/mcb.8.7.2896. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Tapscott S. J., Davis R. L., Thayer M. J., Cheng P. F., Weintraub H., Lassar A. B. MyoD1: a nuclear phosphoprotein requiring a Myc homology region to convert fibroblasts to myoblasts. Science. 1988 Oct 21;242(4877):405–411. doi: 10.1126/science.3175662. [DOI] [PubMed] [Google Scholar]
  44. Thayer M. J., Tapscott S. J., Davis R. L., Wright W. E., Lassar A. B., Weintraub H. Positive autoregulation of the myogenic determination gene MyoD1. Cell. 1989 Jul 28;58(2):241–248. doi: 10.1016/0092-8674(89)90838-6. [DOI] [PubMed] [Google Scholar]
  45. Thisse B., Stoetzel C., Gorostiza-Thisse C., Perrin-Schmitt F. Sequence of the twist gene and nuclear localization of its protein in endomesodermal cells of early Drosophila embryos. EMBO J. 1988 Jul;7(7):2175–2183. doi: 10.1002/j.1460-2075.1988.tb03056.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Voronova A., Baltimore D. Mutations that disrupt DNA binding and dimer formation in the E47 helix-loop-helix protein map to distinct domains. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4722–4726. doi: 10.1073/pnas.87.12.4722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Walker M. D., Park C. W., Rosen A., Aronheim A. A cDNA from a mouse pancreatic beta cell encoding a putative transcription factor of the insulin gene. Nucleic Acids Res. 1990 Mar 11;18(5):1159–1166. doi: 10.1093/nar/18.5.1159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. Wentworth B. M., Donoghue M., Engert J. C., Berglund E. B., Rosenthal N. Paired MyoD-binding sites regulate myosin light chain gene expression. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1242–1246. doi: 10.1073/pnas.88.4.1242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]

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