Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1991 Oct;10(10):2957–2964. doi: 10.1002/j.1460-2075.1991.tb07846.x

Different mechanisms regulate muscle-specific AChR gamma- and epsilon-subunit gene expression.

M Numberger 1, I Dürr 1, W Kues 1, M Koenen 1, V Witzemann 1
PMCID: PMC453010  PMID: 1655408

Abstract

Five different subunits, alpha, beta, gamma, delta and epsilon, constitute the acetylcholine receptors from mammalian skeletal muscle. Their corresponding mRNA levels are regulated differentially. In particular, mRNAs encoding the gamma- and epsilon-subunits, which specify two AChR isoforms, show a reciprocal behaviour during synapse formation and maturation. We have isolated 5' flanking sequences of the gamma- and epsilon-subunit genes that confer muscle-specific expression upon transient transfection of primary cultures of rat muscle cells. The gamma-subunit gene fragment contains two adjacent CANNTG sequence motifs that are essential for muscle-specific transcriptional activity suggesting transactivation by helix-loop-helix proteins. The epsilon-subunit gene fragment carries only a single CANNTG consensus motif which is not required for expression in transfected muscle cells. This sequence motif is, however, necessary to repress transcriptional activity in non-muscle cells and thus may control the muscle-specific expression of the epsilon-subunit gene. The results suggest that CANNTG motifs together with their 3' and 5' flanking nucleotides provide binding sites for both activating as well as repressing trans-acting factors. These elements could thus contribute to the muscle-specific expression of AChR subunit genes.

Full text

PDF
2961

Images in this article

2958Image
on p.2958
2959Image
on p.2959
2960Image
on p.2960
2961Image
on p.2961

Selected References

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

  1. 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]
  2. Baldwin T. J., Burden S. J. Muscle-specific gene expression controlled by a regulatory element lacking a MyoD1-binding site. Nature. 1989 Oct 26;341(6244):716–720. doi: 10.1038/341716a0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bouvagnet P. F., Strehler E. E., White G. E., Strehler-Page M. A., Nadal-Ginard B., Mahdavi V. Multiple positive and negative 5' regulatory elements control the cell-type-specific expression of the embryonic skeletal myosin heavy-chain gene. Mol Cell Biol. 1987 Dec;7(12):4377–4389. doi: 10.1128/mcb.7.12.4377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Braun T., Tannich E., Buschhausen-Denker G., Arnold H. H. Promoter upstream elements of the chicken cardiac myosin light-chain 2-A gene interact with trans-acting regulatory factors for muscle-specific transcription. Mol Cell Biol. 1989 Jun;9(6):2513–2525. doi: 10.1128/mcb.9.6.2513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Brenner H. R., Witzemann V., Sakmann B. Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses. Nature. 1990 Apr 5;344(6266):544–547. doi: 10.1038/344544a0. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Daubas P., Klarsfeld A., Garner I., Pinset C., Cox R., Buckingham M. Functional activity of the two promoters of the myosin alkali light chain gene in primary muscle cell cultures: comparison with other muscle gene promoters and other culture systems. Nucleic Acids Res. 1988 Feb 25;16(4):1251–1271. doi: 10.1093/nar/16.4.1251. [DOI] [PMC free article] [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. 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]
  15. Eftimie R., Brenner H. R., Buonanno A. Myogenin and MyoD join a family of skeletal muscle genes regulated by electrical activity. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1349–1353. doi: 10.1073/pnas.88.4.1349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gardner P. D., Heinemann S., Patrick J. Transcriptional regulation of nicotinic acetylcholine receptor genes: identification of control elements of a gamma-subunit gene. Brain Res. 1987 Dec;427(1):69–76. doi: 10.1016/0169-328x(87)90046-5. [DOI] [PubMed] [Google Scholar]
  17. Goldman D., Brenner H. R., Heinemann S. Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity. Neuron. 1988 Jun;1(4):329–333. doi: 10.1016/0896-6273(88)90081-5. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Gu Y., Hall Z. W. Immunological evidence for a change in subunits of the acetylcholine receptor in developing and denervated rat muscle. Neuron. 1988 Apr;1(2):117–125. doi: 10.1016/0896-6273(88)90195-x. [DOI] [PubMed] [Google Scholar]
  20. Herbomel P., Bourachot B., Yaniv M. Two distinct enhancers with different cell specificities coexist in the regulatory region of polyoma. Cell. 1984 Dec;39(3 Pt 2):653–662. doi: 10.1016/0092-8674(84)90472-0. [DOI] [PubMed] [Google Scholar]
  21. Herlitze S., Koenen M. A general and rapid mutagenesis method using polymerase chain reaction. Gene. 1990 Jul 2;91(1):143–147. doi: 10.1016/0378-1119(90)90177-s. [DOI] [PubMed] [Google Scholar]
  22. Imler J. L., Lemaire C., Wasylyk C., Wasylyk B. Negative regulation contributes to tissue specificity of the immunoglobulin heavy-chain enhancer. Mol Cell Biol. 1987 Jul;7(7):2558–2567. doi: 10.1128/mcb.7.7.2558. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Klarsfeld A., Daubas P., Bourachot B., Changeux J. P. A 5'-flanking region of the chicken acetylcholine receptor alpha-subunit gene confers tissue specificity and developmental control of expression in transfected cells. Mol Cell Biol. 1987 Feb;7(2):951–955. doi: 10.1128/mcb.7.2.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Luckow B., Schütz G. CAT constructions with multiple unique restriction sites for the functional analysis of eukaryotic promoters and regulatory elements. Nucleic Acids Res. 1987 Jul 10;15(13):5490–5490. doi: 10.1093/nar/15.13.5490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mishina M., Takai T., Imoto K., Noda M., Takahashi T., Numa S., Methfessel C., Sakmann B. Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature. 1986 May 22;321(6068):406–411. doi: 10.1038/321406a0. [DOI] [PubMed] [Google Scholar]
  27. Pierce J. W., Gifford A. M., Baltimore D. Silencing of the expression of the immunoglobulin kappa gene in non-B cells. Mol Cell Biol. 1991 Mar;11(3):1431–1437. doi: 10.1128/mcb.11.3.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  30. Schuetze S. M., Role L. W. Developmental regulation of nicotinic acetylcholine receptors. Annu Rev Neurosci. 1987;10:403–457. doi: 10.1146/annurev.ne.10.030187.002155. [DOI] [PubMed] [Google Scholar]
  31. Shen R. A., Goswami S. K., Mascareno E., Kumar A., Siddiqui M. A. Tissue-specific transcription of the cardiac myosin light-chain 2 gene is regulated by an upstream repressor element. Mol Cell Biol. 1991 Mar;11(3):1676–1685. doi: 10.1128/mcb.11.3.1676. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Tsay H. J., Neville C. M., Schmidt J. Protein synthesis is required for the denervation-triggered activation of acetylcholine receptor genes. FEBS Lett. 1990 Nov 12;274(1-2):69–72. doi: 10.1016/0014-5793(90)81331-h. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Wang X. M., Tsay H. J., Schmidt J. Expression of the acetylcholine receptor delta-subunit gene in differentiating chick muscle cells is activated by an element that contains two 16 bp copies of a segment of the alpha-subunit enhancer. EMBO J. 1990 Mar;9(3):783–790. doi: 10.1002/j.1460-2075.1990.tb08174.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Weintraub H., Tapscott S. J., Davis R. L., Thayer M. J., Adam M. A., Lassar A. B., Miller A. D. Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5434–5438. doi: 10.1073/pnas.86.14.5434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Witzemann V., Barg B., Criado M., Stein E., Sakmann B. Developmental regulation of five subunit specific mRNAs encoding acetylcholine receptor subtypes in rat muscle. FEBS Lett. 1989 Jan 2;242(2):419–424. doi: 10.1016/0014-5793(89)80514-9. [DOI] [PubMed] [Google Scholar]
  40. Witzemann V., Barg B., Nishikawa Y., Sakmann B., Numa S. Differential regulation of muscle acetylcholine receptor gamma- and epsilon-subunit mRNAs. FEBS Lett. 1987 Oct 19;223(1):104–112. doi: 10.1016/0014-5793(87)80518-5. [DOI] [PubMed] [Google Scholar]
  41. Witzemann V., Brenner H. R., Sakmann B. Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses. J Cell Biol. 1991 Jul;114(1):125–141. doi: 10.1083/jcb.114.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Witzemann V., Sakmann B. Differential regulation of MyoD and myogenin mRNA levels by nerve induced muscle activity. FEBS Lett. 1991 May 6;282(2):259–264. doi: 10.1016/0014-5793(91)80490-t. [DOI] [PubMed] [Google Scholar]
  43. Witzemann V., Stein E., Barg B., Konno T., Koenen M., Kues W., Criado M., Hofmann M., Sakmann B. Primary structure and functional expression of the alpha-, beta-, gamma-, delta- and epsilon-subunits of the acetylcholine receptor from rat muscle. Eur J Biochem. 1990 Dec 12;194(2):437–448. doi: 10.1111/j.1432-1033.1990.tb15637.x. [DOI] [PubMed] [Google Scholar]
  44. Yutzey K. E., Rhodes S. J., Konieczny S. F. Differential trans activation associated with the muscle regulatory factors MyoD1, myogenin, and MRF4. Mol Cell Biol. 1990 Aug;10(8):3934–3944. doi: 10.1128/mcb.10.8.3934. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES