Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Sep;13(9):5133–5140. doi: 10.1128/mcb.13.9.5133

An E box mediates activation and repression of the acetylcholine receptor delta-subunit gene during myogenesis.

A M Simon 1, S J Burden 1
PMCID: PMC360201  PMID: 8355673

Abstract

The genes encoding the skeletal muscle acetylcholine receptor (AChR) are induced during muscle development and are regulated subsequently by innervation. Because both the initiation and the subsequent regulation of AChR expression are controlled by transcriptional mechanisms, an understanding of the steps that regulate AChR expression following innervation is likely to require knowledge of the pathway that activates AChR genes during myogenesis. Thus, we sought to identify the cis-acting sequences that regulate expression of the AChR delta-subunit gene during muscle differentiation. We transfected muscle and nonmuscle cell lines with gene fusions between 5'-flanking DNA from the AChR delta-subunit gene and the human growth hormone gene, and we show here that 148 bp of 5'-flanking DNA from the AChR delta-subunit gene contains two regulatory elements that control muscle-specific gene expression. One element is an E box, which is important both for activation of the delta-subunit gene in myotubes and for its repression in myoblasts and nonmuscle cells. Mutation of this E box, which prevents binding of MyoD-E2A and myogenin-E2A heterodimers, decreases expression in myotubes and increases expression in myoblasts and nonmuscle cells. An E-box binding activity, which does not contain MyoD, myogenin, or E2A proteins, is present in muscle and nonmuscle cells and may be responsible for repressing the delta-subunit gene in myoblasts and nonmuscle cells. An enhancer, which lacks E boxes, is also required for expression of the delta-subunit gene but does not confer muscle-specific expression.

Full text

PDF
5133

Images in this article

5136Image
on p.5136
5137Image
on p.5137
5138Image
on p.5138

Selected References

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

  1. Akerblom I. E., Slater E. P., Beato M., Baxter J. D., Mellon P. L. Negative regulation by glucocorticoids through interference with a cAMP responsive enhancer. Science. 1988 Jul 15;241(4863):350–353. doi: 10.1126/science.2838908. [DOI] [PubMed] [Google Scholar]
  2. Baldwin T. J., Burden S. J. Isolation and characterization of the mouse acetylcholine receptor delta subunit gene: identification of a 148-bp cis-acting region that confers myotube-specific expression. J Cell Biol. 1988 Dec;107(6 Pt 1):2271–2279. doi: 10.1083/jcb.107.6.2271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bassel-Duby R., Hernandez M. D., Gonzalez M. A., Krueger J. K., Williams R. S. A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter. Mol Cell Biol. 1992 Nov;12(11):5024–5032. doi: 10.1128/mcb.12.11.5024. [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. Berkowitz L. A., Riabowol K. T., Gilman M. Z. Multiple sequence elements of a single functional class are required for cyclic AMP responsiveness of the mouse c-fos promoter. Mol Cell Biol. 1989 Oct;9(10):4272–4281. doi: 10.1128/mcb.9.10.4272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blackwell T. K., Kretzner L., Blackwood E. M., Eisenman R. N., Weintraub H. Sequence-specific DNA binding by the c-Myc protein. Science. 1990 Nov 23;250(4984):1149–1151. doi: 10.1126/science.2251503. [DOI] [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. 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]
  9. Brenner H. R., Herczeg A., Slater C. R. Synapse-specific expression of acetylcholine receptor genes and their products at original synaptic sites in rat soleus muscle fibres regenerating in the absence of innervation. Development. 1992 Sep;116(1):41–53. doi: 10.1242/dev.116.1.41. [DOI] [PubMed] [Google Scholar]
  10. Buonanno A., Merlie J. P. Transcriptional regulation of nicotinic acetylcholine receptor genes during muscle development. J Biol Chem. 1986 Sep 5;261(25):11452–11455. [PubMed] [Google Scholar]
  11. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dutton E. K., Simon A. M., Burden S. J. Electrical activity-dependent regulation of the acetylcholine receptor delta-subunit gene, MyoD, and myogenin in primary myotubes. Proc Natl Acad Sci U S A. 1993 Mar 1;90(5):2040–2044. doi: 10.1073/pnas.90.5.2040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gilman M. Z., Wilson R. N., Weinberg R. A. Multiple protein-binding sites in the 5'-flanking region regulate c-fos expression. Mol Cell Biol. 1986 Dec;6(12):4305–4316. doi: 10.1128/mcb.6.12.4305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gilmour B. P., Fanger G. R., Newton C., Evans S. M., Gardner P. D. Multiple binding sites for myogenic regulatory factors are required for expression of the acetylcholine receptor gamma-subunit gene. J Biol Chem. 1991 Oct 25;266(30):19871–19874. [PubMed] [Google Scholar]
  15. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Jaynes J. B., O'Farrell P. H. Activation and repression of transcription by homoeodomain-containing proteins that bind a common site. Nature. 1988 Dec 22;336(6201):744–749. doi: 10.1038/336744a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Jo S. A., Burden S. J. Synaptic basal lamina contains a signal for synapse-specific transcription. Development. 1992 Jul;115(3):673–680. doi: 10.1242/dev.115.3.673. [DOI] [PubMed] [Google Scholar]
  20. Kato G. J., Lee W. M., Chen L. L., Dang C. V. Max: functional domains and interaction with c-Myc. Genes Dev. 1992 Jan;6(1):81–92. doi: 10.1101/gad.6.1.81. [DOI] [PubMed] [Google Scholar]
  21. Klarsfeld A., Bessereau J. L., Salmon A. M., Triller A., Babinet C., Changeux J. P. An acetylcholine receptor alpha-subunit promoter conferring preferential synaptic expression in muscle of transgenic mice. EMBO J. 1991 Mar;10(3):625–632. doi: 10.1002/j.1460-2075.1991.tb07990.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lamb P., McKnight S. L. Diversity and specificity in transcriptional regulation: the benefits of heterotypic dimerization. Trends Biochem Sci. 1991 Nov;16(11):417–422. doi: 10.1016/0968-0004(91)90167-t. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Laufer R., Changeux J. P. Activity-dependent regulation of gene expression in muscle and neuronal cells. Mol Neurobiol. 1989 Spring-Summer;3(1-2):1–53. doi: 10.1007/BF02935587. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Merlie J. P., Kornhauser J. M. Neural regulation of gene expression by an acetylcholine receptor promoter in muscle of transgenic mice. Neuron. 1989 Apr;2(4):1295–1300. doi: 10.1016/0896-6273(89)90067-6. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Numberger M., Dürr I., Kues W., Koenen M., Witzemann V. Different mechanisms regulate muscle-specific AChR gamma- and epsilon-subunit gene expression. EMBO J. 1991 Oct;10(10):2957–2964. doi: 10.1002/j.1460-2075.1991.tb07846.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ohkuma Y., Horikoshi M., Roeder R. G., Desplan C. Binding site-dependent direct activation and repression of in vitro transcription by Drosophila homeodomain proteins. Cell. 1990 May 4;61(3):475–484. doi: 10.1016/0092-8674(90)90529-n. [DOI] [PubMed] [Google Scholar]
  30. Oro A. E., Hollenberg S. M., Evans R. M. Transcriptional inhibition by a glucocorticoid receptor-beta-galactosidase fusion protein. Cell. 1988 Dec 23;55(6):1109–1114. doi: 10.1016/0092-8674(88)90255-3. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Roy A. L., Meisterernst M., Pognonec P., Roeder R. G. Cooperative interaction of an initiator-binding transcription initiation factor and the helix-loop-helix activator USF. Nature. 1991 Nov 21;354(6350):245–248. doi: 10.1038/354245a0. [DOI] [PubMed] [Google Scholar]
  34. Ruezinsky D., Beckmann H., Kadesch T. Modulation of the IgH enhancer's cell type specificity through a genetic switch. Genes Dev. 1991 Jan;5(1):29–37. doi: 10.1101/gad.5.1.29. [DOI] [PubMed] [Google Scholar]
  35. Sanes J. R., Johnson Y. R., Kotzbauer P. T., Mudd J., Hanley T., Martinou J. C., Merlie J. P. Selective expression of an acetylcholine receptor-lacZ transgene in synaptic nuclei of adult muscle fibers. Development. 1991 Dec;113(4):1181–1191. doi: 10.1242/dev.113.4.1181. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Selden R. F., Howie K. B., Rowe M. E., Goodman H. M., Moore D. D. Human growth hormone as a reporter gene in regulation studies employing transient gene expression. Mol Cell Biol. 1986 Sep;6(9):3173–3179. doi: 10.1128/mcb.6.9.3173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Simon A. M., Hoppe P., Burden S. J. Spatial restriction of AChR gene expression to subsynaptic nuclei. Development. 1992 Mar;114(3):545–553. doi: 10.1242/dev.114.3.545. [DOI] [PubMed] [Google Scholar]
  39. Smale S. T., Baltimore D. The "initiator" as a transcription control element. Cell. 1989 Apr 7;57(1):103–113. doi: 10.1016/0092-8674(89)90176-1. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. 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]
  42. Taylor J. W., Ott J., Eckstein F. The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA. Nucleic Acids Res. 1985 Dec 20;13(24):8765–8785. doi: 10.1093/nar/13.24.8765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tsay H. J., Schmidt J. Skeletal muscle denervation activates acetylcholine receptor genes. J Cell Biol. 1989 Apr;108(4):1523–1526. doi: 10.1083/jcb.108.4.1523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES