Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1997 Jan 15;25(2):423–430. doi: 10.1093/nar/25.2.423

Inhibition of muscle-specific gene expression by Id3: requirement of the C-terminal region of the protein for stable expression and function.

B Chen 1, B H Han 1, X H Sun 1, R W Lim 1
PMCID: PMC146444  PMID: 9016574

Abstract

We have examined the role of an Id-like protein, Id3 (also known as HLH462), in the regulation of muscle-specific gene expression. Id proteins are believed to block expression of muscle-specific genes by preventing the dimerization between ubiquitous bHLH proteins (E proteins) and myogenic bHLH proteins such as MyoD. Consistent with its putative role as an inhibitor of differentiation, Id3 mRNA was detected in proliferating skeletal muscle cells, was further induced by basic fibroblast growth factor (bFGF) and was down-regulated in differentiated muscle cultures. Overexpression of Id3 efficiently inhibited the MyoD-mediated activation of the muscle-specific creatine kinase (MCK) reporter gene. Deletion analysis indicated that the C-terminal 15 amino acids of Id3 are critical for the full inhibitory activity while deleting up to 42 residues from the C-terminus of the related protein, Id2, did not affect its ability to inhibit the MCK reporter gene. Chimeric protein containing the N-terminal region of Id3 and the C-terminus of Id2 was also non-functional in transfected cells. In contrast, wild-type Id3, the C-terminal mutants, and the Id3/Id2 chimera could all interact with the E-protein E47in vitro. Additional studies indicated that truncation of the Id3 C-terminus might have adversely affected the expression level of the mutant proteins but the Id3/Id2 chimera was stably expressed. Taken together, our results revealed a more complex requirement for the expression and proper function of the Id family proteins than was hitherto expected.

Full Text

The Full Text of this article is available as a PDF (250.5 KB).

Selected References

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

  1. Arber S., Halder G., Caroni P. Muscle LIM protein, a novel essential regulator of myogenesis, promotes myogenic differentiation. Cell. 1994 Oct 21;79(2):221–231. doi: 10.1016/0092-8674(94)90192-9. [DOI] [PubMed] [Google Scholar]
  2. Bain G., Gruenwald S., Murre C. E2A and E2-2 are subunits of B-cell-specific E2-box DNA-binding proteins. Mol Cell Biol. 1993 Jun;13(6):3522–3529. doi: 10.1128/mcb.13.6.3522. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barone M. V., Pepperkok R., Peverali F. A., Philipson L. Id proteins control growth induction in mammalian cells. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4985–4988. doi: 10.1073/pnas.91.11.4985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Biggs J., Murphy E. V., Israel M. A. A human Id-like helix-loop-helix protein expressed during early development. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1512–1516. doi: 10.1073/pnas.89.4.1512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blanar M. A., Rutter W. J. Interaction cloning: identification of a helix-loop-helix zipper protein that interacts with c-Fos. Science. 1992 May 15;256(5059):1014–1018. doi: 10.1126/science.1589769. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Clegg C. H., Linkhart T. A., Olwin B. B., Hauschka S. D. Growth factor control of skeletal muscle differentiation: commitment to terminal differentiation occurs in G1 phase and is repressed by fibroblast growth factor. J Cell Biol. 1987 Aug;105(2):949–956. doi: 10.1083/jcb.105.2.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. 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]
  14. Deed R. W., Bianchi S. M., Atherton G. T., Johnston D., Santibanez-Koref M., Murphy J. J., Norton J. D. An immediate early human gene encodes an Id-like helix-loop-helix protein and is regulated by protein kinase C activation in diverse cell types. Oncogene. 1993 Mar;8(3):599–607. [PubMed] [Google Scholar]
  15. Desprez P. Y., Hara E., Bissell M. J., Campisi J. Suppression of mammary epithelial cell differentiation by the helix-loop-helix protein Id-1. Mol Cell Biol. 1995 Jun;15(6):3398–3404. doi: 10.1128/mcb.15.6.3398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dias P., Dilling M., Houghton P. The molecular basis of skeletal muscle differentiation. Semin Diagn Pathol. 1994 Feb;11(1):3–14. [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. Edmondson D. G., Olson E. N. Helix-loop-helix proteins as regulators of muscle-specific transcription. J Biol Chem. 1993 Jan 15;268(2):755–758. [PubMed] [Google Scholar]
  19. Ellmeier W., Aguzzi A., Kleiner E., Kurzbauer R., Weith A. Mutually exclusive expression of a helix-loop-helix gene and N-myc in human neuroblastomas and in normal development. EMBO J. 1992 Jul;11(7):2563–2571. doi: 10.1002/j.1460-2075.1992.tb05321.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  21. Hanks S. K., Quinn A. M. Protein kinase catalytic domain sequence database: identification of conserved features of primary structure and classification of family members. Methods Enzymol. 1991;200:38–62. doi: 10.1016/0076-6879(91)00126-h. [DOI] [PubMed] [Google Scholar]
  22. Hara E., Uzman J. A., Dimri G. P., Nehlin J. O., Testori A., Campisi J. The helix-loop-helix protein Id-1 and a retinoblastoma protein binding mutant of SV40 T antigen synergize to reactivate DNA synthesis in senescent human fibroblasts. Dev Genet. 1996;18(2):161–172. doi: 10.1002/(SICI)1520-6408(1996)18:2<161::AID-DVG9>3.0.CO;2-7. [DOI] [PubMed] [Google Scholar]
  23. Hara E., Yamaguchi T., Nojima H., Ide T., Campisi J., Okayama H., Oda K. Id-related genes encoding helix-loop-helix proteins are required for G1 progression and are repressed in senescent human fibroblasts. J Biol Chem. 1994 Jan 21;269(3):2139–2145. [PubMed] [Google Scholar]
  24. Hardy S., Kong Y., Konieczny S. F. Fibroblast growth factor inhibits MRF4 activity independently of the phosphorylation status of a conserved threonine residue within the DNA-binding domain. Mol Cell Biol. 1993 Oct;13(10):5943–5956. doi: 10.1128/mcb.13.10.5943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hu J. S., Olson E. N., Kingston R. E. HEB, a helix-loop-helix protein related to E2A and ITF2 that can modulate the DNA-binding ability of myogenic regulatory factors. Mol Cell Biol. 1992 Mar;12(3):1031–1042. doi: 10.1128/mcb.12.3.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Iavarone A., Garg P., Lasorella A., Hsu J., Israel M. A. The helix-loop-helix protein Id-2 enhances cell proliferation and binds to the retinoblastoma protein. Genes Dev. 1994 Jun 1;8(11):1270–1284. doi: 10.1101/gad.8.11.1270. [DOI] [PubMed] [Google Scholar]
  27. Jen Y., Weintraub H., Benezra R. Overexpression of Id protein inhibits the muscle differentiation program: in vivo association of Id with E2A proteins. Genes Dev. 1992 Aug;6(8):1466–1479. doi: 10.1101/gad.6.8.1466. [DOI] [PubMed] [Google Scholar]
  28. Kaushal S., Schneider J. W., Nadal-Ginard B., Mahdavi V. Activation of the myogenic lineage by MEF2A, a factor that induces and cooperates with MyoD. Science. 1994 Nov 18;266(5188):1236–1240. doi: 10.1126/science.7973707. [DOI] [PubMed] [Google Scholar]
  29. Kreider B. L., Benezra R., Rovera G., Kadesch T. Inhibition of myeloid differentiation by the helix-loop-helix protein Id. Science. 1992 Mar 27;255(5052):1700–1702. doi: 10.1126/science.1372755. [DOI] [PubMed] [Google Scholar]
  30. Kurabayashi M., Jeyaseelan R., Kedes L. Two distinct cDNA sequences encoding the human helix-loop-helix protein Id2. Gene. 1993 Nov 15;133(2):305–306. doi: 10.1016/0378-1119(93)90658-p. [DOI] [PubMed] [Google Scholar]
  31. Lasorella A., Iavarone A., Israel M. A. Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins. Mol Cell Biol. 1996 Jun;16(6):2570–2578. doi: 10.1128/mcb.16.6.2570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lassar A. B., Davis R. L., Wright W. E., Kadesch T., Murre C., Voronova A., Baltimore D., Weintraub H. Functional activity of myogenic HLH proteins requires hetero-oligomerization with E12/E47-like proteins in vivo. Cell. 1991 Jul 26;66(2):305–315. doi: 10.1016/0092-8674(91)90620-e. [DOI] [PubMed] [Google Scholar]
  33. Li L., Zhou J., James G., Heller-Harrison R., Czech M. P., Olson E. N. FGF inactivates myogenic helix-loop-helix proteins through phosphorylation of a conserved protein kinase C site in their DNA-binding domains. Cell. 1992 Dec 24;71(7):1181–1194. doi: 10.1016/s0092-8674(05)80066-2. [DOI] [PubMed] [Google Scholar]
  34. Lim R. W., Zhu C. Y., Stringer B. Differential regulation of primary response gene expression in skeletal muscle cells through multiple signal transduction pathways. Biochim Biophys Acta. 1995 Apr 6;1266(1):91–100. doi: 10.1016/0167-4889(94)00226-5. [DOI] [PubMed] [Google Scholar]
  35. Martin J. F., Li L., Olson E. N. Repression of myogenin function by TGF-beta 1 is targeted at the basic helix-loop-helix motif and is independent of E2A products. J Biol Chem. 1992 Jun 5;267(16):10956–10960. [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Olson E. N., Perry M., Schulz R. A. Regulation of muscle differentiation by the MEF2 family of MADS box transcription factors. Dev Biol. 1995 Nov;172(1):2–14. doi: 10.1006/dbio.1995.0002. [DOI] [PubMed] [Google Scholar]
  39. Pesce S., Benezra R. The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity. Mol Cell Biol. 1993 Dec;13(12):7874–7880. doi: 10.1128/mcb.13.12.7874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Peverali F. A., Ramqvist T., Saffrich R., Pepperkok R., Barone M. V., Philipson L. Regulation of G1 progression by E2A and Id helix-loop-helix proteins. EMBO J. 1994 Sep 15;13(18):4291–4301. doi: 10.1002/j.1460-2075.1994.tb06749.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]
  42. Riechmann V., van Crüchten I., Sablitzky F. The expression pattern of Id4, a novel dominant negative helix-loop-helix protein, is distinct from Id1, Id2 and Id3. Nucleic Acids Res. 1994 Mar 11;22(5):749–755. doi: 10.1093/nar/22.5.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Shoji W., Yamamoto T., Obinata M. The helix-loop-helix protein Id inhibits differentiation of murine erythroleukemia cells. J Biol Chem. 1994 Feb 18;269(7):5078–5084. [PubMed] [Google Scholar]
  44. Springhorn J. P., Singh K., Kelly R. A., Smith T. W. Posttranscriptional regulation of Id1 activity in cardiac muscle. Alternative splicing of novel Id1 transcript permits homodimerization. J Biol Chem. 1994 Feb 18;269(7):5132–5136. [PubMed] [Google Scholar]
  45. Sun X. H. Constitutive expression of the Id1 gene impairs mouse B cell development. Cell. 1994 Dec 2;79(5):893–900. doi: 10.1016/0092-8674(94)90078-7. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Therrien M., Drouin J. Cell-specific helix-loop-helix factor required for pituitary expression of the pro-opiomelanocortin gene. Mol Cell Biol. 1993 Apr;13(4):2342–2353. doi: 10.1128/mcb.13.4.2342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Weintraub H., Davis R., Tapscott S., Thayer M., Krause M., Benezra R., Blackwell T. K., Turner D., Rupp R., Hollenberg S. The myoD gene family: nodal point during specification of the muscle cell lineage. Science. 1991 Feb 15;251(4995):761–766. doi: 10.1126/science.1846704. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Yuan W., Condorelli G., Caruso M., Felsani A., Giordano A. Human p300 protein is a coactivator for the transcription factor MyoD. J Biol Chem. 1996 Apr 12;271(15):9009–9013. doi: 10.1074/jbc.271.15.9009. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES