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Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1991 Apr;11(4):2273–2281. doi: 10.1128/mcb.11.4.2273

A conserved 28-base-pair element (HF-1) in the rat cardiac myosin light-chain-2 gene confers cardiac-specific and alpha-adrenergic-inducible expression in cultured neonatal rat myocardial cells.

H Zhu 1, A V Garcia 1, R S Ross 1, S M Evans 1, K R Chien 1
PMCID: PMC359928  PMID: 1848675

Abstract

To study the transcriptional regulatory mechanisms which mediate cardiac-specific and inducible expression during myocardial cell hypertrophy, we have extensively characterized the rat cardiac myosin light-chain-2 (MLC-2) gene as a model system. The MLC-2 gene encodes a relatively abundant contractile protein in slow skeletal and cardiac muscle and is upregulated during in vivo cardiac hypertrophy and alpha-adrenergic-mediated hypertrophy of neonatal rat myocardial cells. In transient expression assays employing a series of MLC-2-luciferase constructs, recent studies have identified a 250-bp fragment which is sufficient for both cardiac-specific and alpha-adrenergic-inducible expression. Within this 250-bp fragment lie three regions (HF-1, HF-2, and HF-3), each greater than 10 bp in length, which are conserved between the chicken and rat cardiac MLC-2 genes, suggesting their potential role in the regulated expression of this contractile protein gene. As assessed by substitution mutations within each of the conserved regions, the present study demonstrates that HF-1 and HF-2 are important in both cardiac-specific and inducible expression, while HF-3 has no detectable role in the regulated expression of the MLC-2 gene in transient expression assays. HF-1 sequences confer both cardiac-specific and inducible expression to a neutral promoter-luciferase construct but have no significant effect in the skeletal muscle or nonmuscle cell contexts. Thus, these studies have identified a new cardiac-specific regulatory element (HF-1) which plays a role in both cardiac-specific and inducible expression during myocardial cell hypertrophy.

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Selected References

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  1. Bergsma D. J., Grichnik J. M., Gossett L. M., Schwartz R. J. Delimitation and characterization of cis-acting DNA sequences required for the regulated expression and transcriptional control of the chicken skeletal alpha-actin gene. Mol Cell Biol. 1986 Jul;6(7):2462–2475. doi: 10.1128/mcb.6.7.2462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. 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]
  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. 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]
  7. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cherrington J. M., Mocarski E. S. Human cytomegalovirus ie1 transactivates the alpha promoter-enhancer via an 18-base-pair repeat element. J Virol. 1989 Mar;63(3):1435–1440. doi: 10.1128/jvi.63.3.1435-1440.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cribbs L. L., Shimizu N., Yockey C. E., Levin J. E., Jakovcic S., Zak R., Umeda P. K. Muscle-specific regulation of a transfected rabbit myosin heavy chain beta gene promoter. J Biol Chem. 1989 Jun 25;264(18):10672–10678. [PubMed] [Google Scholar]
  10. Dalla Libera L. A comparative study of atrial and ventricular myosin light subunits from different species. Comp Biochem Physiol B. 1986;83(4):751–755. doi: 10.1016/0305-0491(86)90140-9. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Devlin B. H., Wefald F. C., Kraus W. E., Bernard T. S., Williams R. S. Identification of a muscle-specific enhancer within the 5'-flanking region of the human myoglobin gene. J Biol Chem. 1989 Aug 15;264(23):13896–13901. [PubMed] [Google Scholar]
  14. Donoghue M., Ernst H., Wentworth B., Nadal-Ginard B., Rosenthal N. A muscle-specific enhancer is located at the 3' end of the myosin light-chain 1/3 gene locus. Genes Dev. 1988 Dec;2(12B):1779–1790. doi: 10.1101/gad.2.12b.1779. [DOI] [PubMed] [Google Scholar]
  15. Dunnmon P. M., Iwaki K., Henderson S. A., Sen A., Chien K. R. Phorbol esters induce immediate-early genes and activate cardiac gene transcription in neonatal rat myocardial cells. J Mol Cell Cardiol. 1990 Aug;22(8):901–910. doi: 10.1016/0022-2828(90)90121-h. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Gossett L. A., Kelvin D. J., Sternberg E. A., Olson E. N. A new myocyte-specific enhancer-binding factor that recognizes a conserved element associated with multiple muscle-specific genes. Mol Cell Biol. 1989 Nov;9(11):5022–5033. doi: 10.1128/mcb.9.11.5022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Gustafson T. A., Markham B. E., Morkin E. Effects of thyroid hormone on alpha-actin and myosin heavy chain gene expression in cardiac and skeletal muscles of the rat: measurement of mRNA content using synthetic oligonucleotide probes. Circ Res. 1986 Aug;59(2):194–201. doi: 10.1161/01.res.59.2.194. [DOI] [PubMed] [Google Scholar]
  19. Henderson S. A., Spencer M., Sen A., Kumar C., Siddiqui M. A., Chien K. R. Structure, organization, and expression of the rat cardiac myosin light chain-2 gene. Identification of a 250-base pair fragment which confers cardiac-specific expression. J Biol Chem. 1989 Oct 25;264(30):18142–18148. [PubMed] [Google Scholar]
  20. Henderson S. A., Xu Y. C., Chien K. R. Nucleotide sequence of full length cDNAs encoding rat cardiac myosin light chain-2. Nucleic Acids Res. 1988 May 25;16(10):4722–4722. doi: 10.1093/nar/16.10.4722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Horlick R. A., Benfield P. A. The upstream muscle-specific enhancer of the rat muscle creatine kinase gene is composed of multiple elements. Mol Cell Biol. 1989 Jun;9(6):2396–2413. doi: 10.1128/mcb.9.6.2396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Imagawa M., Chiu R., Karin M. Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell. 1987 Oct 23;51(2):251–260. doi: 10.1016/0092-8674(87)90152-8. [DOI] [PubMed] [Google Scholar]
  23. Iwaki K., Sukhatme V. P., Shubeita H. E., Chien K. R. Alpha- and beta-adrenergic stimulation induces distinct patterns of immediate early gene expression in neonatal rat myocardial cells. fos/jun expression is associated with sarcomere assembly; Egr-1 induction is primarily an alpha 1-mediated response. J Biol Chem. 1990 Aug 15;265(23):13809–13817. [PubMed] [Google Scholar]
  24. Izumo S., Nadal-Ginard B., Mahdavi V. Protooncogene induction and reprogramming of cardiac gene expression produced by pressure overload. Proc Natl Acad Sci U S A. 1988 Jan;85(2):339–343. doi: 10.1073/pnas.85.2.339. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Johnson J. E., Wold B. J., Hauschka S. D. Muscle creatine kinase sequence elements regulating skeletal and cardiac muscle expression in transgenic mice. Mol Cell Biol. 1989 Aug;9(8):3393–3399. doi: 10.1128/mcb.9.8.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Kumar C. C., Cribbs L., Delaney P., Chien K. R., Siddiqui M. A. Heart myosin light chain 2 gene. Nucleotide sequence of full length cDNA and expression in normal and hypertensive rat. J Biol Chem. 1986 Feb 25;261(6):2866–2872. [PubMed] [Google Scholar]
  28. 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]
  29. Lee H. R., Henderson S. A., Reynolds R., Dunnmon P., Yuan D., Chien K. R. Alpha 1-adrenergic stimulation of cardiac gene transcription in neonatal rat myocardial cells. Effects on myosin light chain-2 gene expression. J Biol Chem. 1988 May 25;263(15):7352–7358. [PubMed] [Google Scholar]
  30. Lin Z. Y., Dechesne C. A., Eldridge J., Paterson B. M. An avian muscle factor related to MyoD1 activates muscle-specific promoters in nonmuscle cells of different germ-layer origin and in BrdU-treated myoblasts. Genes Dev. 1989 Jul;3(7):986–996. doi: 10.1101/gad.3.7.986. [DOI] [PubMed] [Google Scholar]
  31. Long C. S., Ordahl C. P., Simpson P. C. Alpha 1-adrenergic receptor stimulation of sarcomeric actin isogene transcription in hypertrophy of cultured rat heart muscle cells. J Clin Invest. 1989 Mar;83(3):1078–1082. doi: 10.1172/JCI113951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mar J. H., Antin P. B., Cooper T. A., Ordahl C. P. Analysis of the upstream regions governing expression of the chicken cardiac troponin T gene in embryonic cardiac and skeletal muscle cells. J Cell Biol. 1988 Aug;107(2):573–585. doi: 10.1083/jcb.107.2.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Mar J. H., Ordahl C. P. M-CAT binding factor, a novel trans-acting factor governing muscle-specific transcription. Mol Cell Biol. 1990 Aug;10(8):4271–4283. doi: 10.1128/mcb.10.8.4271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Meidell R. S., Sen A., Henderson S. A., Slahetka M. F., Chien K. R. Alpha 1-adrenergic stimulation of rat myocardial cells increases protein synthesis. Am J Physiol. 1986 Nov;251(5 Pt 2):H1076–H1084. doi: 10.1152/ajpheart.1986.251.5.H1076. [DOI] [PubMed] [Google Scholar]
  35. Melton D. A., Krieg P. A., Rebagliati M. R., Maniatis T., Zinn K., Green M. R. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 1984 Sep 25;12(18):7035–7056. doi: 10.1093/nar/12.18.7035. [DOI] [PMC free article] [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. Minty A., Kedes L. Upstream regions of the human cardiac actin gene that modulate its transcription in muscle cells: presence of an evolutionarily conserved repeated motif. Mol Cell Biol. 1986 Jun;6(6):2125–2136. doi: 10.1128/mcb.6.6.2125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mohun T. J., Taylor M. V., Garrett N., Gurdon J. B. The CArG promoter sequence is necessary for muscle-specific transcription of the cardiac actin gene in Xenopus embryos. EMBO J. 1989 Apr;8(4):1153–1161. doi: 10.1002/j.1460-2075.1989.tb03486.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Morgan H. E., Gordon E. E., Kira Y., Chua H. L., Russo L. A., Peterson C. J., McDermott P. J., Watson P. A. Biochemical mechanisms of cardiac hypertrophy. Annu Rev Physiol. 1987;49:533–543. doi: 10.1146/annurev.ph.49.030187.002533. [DOI] [PubMed] [Google Scholar]
  40. Nagai R., Pritzl N., Low R. B., Stirewalt W. S., Zak R., Alpert N. R., Litten R. Z. Myosin isozyme synthesis and mRNA levels in pressure-overloaded rabbit hearts. Circ Res. 1987 May;60(5):692–699. doi: 10.1161/01.res.60.5.692. [DOI] [PubMed] [Google Scholar]
  41. Parmacek M. S., Leiden J. M. Structure and expression of the murine slow/cardiac troponin C gene. J Biol Chem. 1989 Aug 5;264(22):13217–13225. [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. Rosenthal N. Identification of regulatory elements of cloned genes with functional assays. Methods Enzymol. 1987;152:704–720. doi: 10.1016/0076-6879(87)52075-4. [DOI] [PubMed] [Google Scholar]
  45. Sassoon D. A., Garner I., Buckingham M. Transcripts of alpha-cardiac and alpha-skeletal actins are early markers for myogenesis in the mouse embryo. Development. 1988 Sep;104(1):155–164. doi: 10.1242/dev.104.1.155. [DOI] [PubMed] [Google Scholar]
  46. Schwartz K., de la Bastie D., Bouveret P., Oliviéro P., Alonso S., Buckingham M. Alpha-skeletal muscle actin mRNA's accumulate in hypertrophied adult rat hearts. Circ Res. 1986 Nov;59(5):551–555. doi: 10.1161/01.res.59.5.551. [DOI] [PubMed] [Google Scholar]
  47. Schäfer B. W., Blakely B. T., Darlington G. J., Blau H. M. Effect of cell history on response to helix-loop-helix family of myogenic regulators. Nature. 1990 Mar 29;344(6265):454–458. doi: 10.1038/344454a0. [DOI] [PubMed] [Google Scholar]
  48. Sen A., Dunnmon P., Henderson S. A., Gerard R. D., Chien K. R. Terminally differentiated neonatal rat myocardial cells proliferate and maintain specific differentiated functions following expression of SV40 large T antigen. J Biol Chem. 1988 Dec 15;263(35):19132–19136. [PubMed] [Google Scholar]
  49. Shubeita H. E., McDonough P. M., Harris A. N., Knowlton K. U., Glembotski C. C., Brown J. H., Chien K. R. Endothelin induction of inositol phospholipid hydrolysis, sarcomere assembly, and cardiac gene expression in ventricular myocytes. A paracrine mechanism for myocardial cell hypertrophy. J Biol Chem. 1990 Nov 25;265(33):20555–20562. [PubMed] [Google Scholar]
  50. Sills M. N., Xu Y. C., Baracchini E., Goodman R. H., Cooperman S. S., Mandel G., Chien K. R. Expression of diverse Na+ channel messenger RNAs in rat myocardium. Evidence for a cardiac-specific Na+ channel. J Clin Invest. 1989 Jul;84(1):331–336. doi: 10.1172/JCI114158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Simpson P. Norepinephrine-stimulated hypertrophy of cultured rat myocardial cells is an alpha 1 adrenergic response. J Clin Invest. 1983 Aug;72(2):732–738. doi: 10.1172/JCI111023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Starksen N. F., Simpson P. C., Bishopric N., Coughlin S. R., Lee W. M., Escobedo J. A., Williams L. T. Cardiac myocyte hypertrophy is associated with c-myc protooncogene expression. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8348–8350. doi: 10.1073/pnas.83.21.8348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. 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]
  54. Walsh K., Schimmel P. DNA-binding site for two skeletal actin promoter factors is important for expression in muscle cells. Mol Cell Biol. 1988 Apr;8(4):1800–1802. doi: 10.1128/mcb.8.4.1800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. 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]
  56. 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]
  57. Zeller R., Bloch K. D., Williams B. S., Arceci R. J., Seidman C. E. Localized expression of the atrial natriuretic factor gene during cardiac embryogenesis. Genes Dev. 1987 Sep;1(7):693–698. doi: 10.1101/gad.1.7.693. [DOI] [PubMed] [Google Scholar]
  58. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]

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