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Absence of Mutation at the 5′-Upstream Promoter Region of the TPM4 Gene From Cardiac Mutant Axolotl (Ambystoma mexicanum)

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Abstract

Tropomyosins are a family of actin-binding proteins that show cell-specific diversity by a combination of multiple genes and alternative RNA splicing. Of the 4 different tropomyosin genes, TPM4 plays a pivotal role in myofibrillogenesis as well as cardiac contractility in amphibians. In this study, we amplified and sequenced the upstream regulatory region of the TPM4 gene from both normal and mutant axolotl hearts. To identify the cis-elements that are essential for the expression of the TPM4, we created various deletion mutants of the TPM4 promoter DNA, inserted the deleted segments into PGL3 vector, and performed promoter–reporter assay using luciferase as the reporter gene. Comparison of sequences of the promoter region of the TPM4 gene from normal and mutant axolotl revealed no mutations in the promoter sequence of the mutant TPM4 gene. CArG box elements that are generally involved in controlling the expression of several other muscle-specific gene promoters were not found in the upstream regulatory region of the TPM4 gene. In deletion experiments, loss of activity of the reporter gene was noted upon deletion which was then restored upon further deletion suggesting the presence of both positive and negative cis-elements in the upstream regulatory region of the TPM4 gene. We believe that this is the first axolotl promoter that has ever been cloned and studied with clear evidence that it functions in mammalian cell lines. Although striated muscle-specific cis-acting elements are absent from the promoter region of TPM4 gene, our results suggest the presence of positive and negative cis-elements in the promoter region, which in conjunction with positive and negative trans-elements may be involved in regulating the expression of TPM4 gene in a tissue-specific manner.

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References

  1. Martson, S. B., & Redwood, C. S. (2003). Modulation of thin filament activation by breakdown or isoform switiching of thin filament proteins physiological and pathological implications. Circulation Research, 93, 1170–1178.

    Article  Google Scholar 

  2. Perry, S. V. (2001). Vertebrate tropomyosin: distribution, properties and function. Journal of Muscle Research and Cell Motility, 22, 5–49.

    Article  PubMed  CAS  Google Scholar 

  3. Lees-Miller, J. P., & Helfman, D. M. (1991). The molecular basis for tropomyosin diversity. BioEssays, 13, 429–437.

    Article  PubMed  CAS  Google Scholar 

  4. Nadal-Ginard, B. (1990). Muscle cell differentiation, alternative splicing. Current Opinion in Cell Biology, 6, 96–104.

    Google Scholar 

  5. Wieczorek, D. F., Smith, C. W. J., & Nadal-Ginard, B. (1988). The rat alpha-tropomyosin gene generates a minimum of six different mRNAs coding for striated, smooth, and nonmuscle isoforms by alternative splicing. Molecular and Cellular Biology, 8, 679–694.

    PubMed  CAS  Google Scholar 

  6. Gunning, P. (2008). Introduction and historical perspective. Advances in Experimental Medicine and Biology, 644, 1–5.

    Article  PubMed  CAS  Google Scholar 

  7. Hardy, S., Theze, N., Lepetit, D., Allo, M., & Thiebaud, P. (1995). The Xenopus laevis TM4 gene encodes non-muscle and cardiac tropomyosin isoforms through alternative splicing. Gene, 156, 265–270.

    Article  PubMed  CAS  Google Scholar 

  8. Fleenor, D. E., Hickman, K. H., Lindquester, G. J., & Devlin, R. B. (1992). Avian cardiac tropomyosin gene produces tissue-specific isoforms through alternative RNA splicing. Journal of Muscle Research and Cell Motility, 13(1), 55–63.

    Article  PubMed  CAS  Google Scholar 

  9. Forry-Schaudies, S., Gruber, C. E., & Hughes, S. H. (1990). Chicken tropomyosin and a low molecular weight non-muscle tropomyosin are related by alternative splicing. Cell Growth and Differentiation, 10, 473–481.

    Google Scholar 

  10. Zajdel, R. W., Denz, C. R., Lee, S., Dube, S., Ehler, E., Perriard, E., et al. (2003). Identification, characterization, and expression of a novel alpha-tropomyosin isoform in cardiac tissues in developing chicken. Journal of Cellular Biochemistry, 89(3), 427–439.

    Article  PubMed  CAS  Google Scholar 

  11. Spinner, B. J., Zajdel, R. W., McLean, M. D., Denz, C. R., Dube, S., Mehta, S., et al. (2002). Characterization of a TM-4 type tropomyosin that is essential for myofibrillogenesis and contractile activity in embryonic hearts of the Mexican axolotl. Journal of Cellular Biochemistry, 85(4), 747–761.

    Article  PubMed  CAS  Google Scholar 

  12. Lemanski, L. F. (1973). Morphology of developing heart in cardiac lethal mutant Mexican axolotls, Ambystoma mexicanum. Developmental Biology, 33(2), 312–333.

    Article  PubMed  CAS  Google Scholar 

  13. Lemanski, L. F., Nakatsugawa, M., Bhatia, R., Erginel-Unaltuna, N., Spinner, B. J., & Dube, D. K. (1996). A specific synthetic RNA promotes cardiac myofibrillogenesis in the Mexican axolotl. Biochemical and Biophysical Research Communications, 229(3), 974–981.

    Article  PubMed  CAS  Google Scholar 

  14. Zhang, C., Jia, P., Huang, X., Sferrazza, G. F., Athauda, G., Achary, M. P., et al. (2009). Myofibril-inducing RNA (MIR) is essential for tropomyosin expression and myofibrillogenesis in axolotl hearts. Journal of Biomedical Science, 16, 81.

    Article  PubMed  CAS  Google Scholar 

  15. Luque, E. A., Spinner, B. J., Dube, S., Dube, D. K., & Lemanski, L. F. (1997). Differential expression of a novel isoform of alpha-tropomyosin in cardiac and skeletal muscle of the Mexican axolotl (Ambystoma mexicanum). Gene, 185(2), 175–180.

    Article  PubMed  CAS  Google Scholar 

  16. Zajdel, R. W., Sanger, J. M., Denz, C. R., Lee, S., Dube, S., Poiesz, B. J., et al. (2002). A novel striated tropomyosin incorporated into organized myofibrils of cardiomyocytes in cell and organ culture. FEBS Letters, 520(1–3), 35–39.

    Article  PubMed  CAS  Google Scholar 

  17. Ruiz-Opazo, N., & Nadal-Ginard, B. (1987). Alpha-tropomyosin gene organization. Alternative splicing of duplicated isotype-specific exons accounts for the production of smooth and striated muscle isoforms. Journal of Biological Chemistry, 262(10), 4755–4765.

    PubMed  CAS  Google Scholar 

  18. Taylor, M. V. (1991). A family of muscle gene promoter element (CArG) binding activities in Xenopus embryos: CArG/SRE discrimination and distribution during myogenesis. Nucleic Acids Research, 19(10), 2669–2675.

    Article  PubMed  CAS  Google Scholar 

  19. Mohun, T. J., Taylor, M. V., Garrett, N., & Gurdon, J. B. (1989). The CArG promoter sequence is necessary for muscle-specific transcription of the cardiac actin gene in Xenopus embryos. EMBO Journal, 8(4), 1153–1161.

    PubMed  CAS  Google Scholar 

  20. Chen, C. Y., & Schwartz, R. J. (1996). Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription. Molecular and Cellular Biology, 16(11), 6372–6384.

    PubMed  CAS  Google Scholar 

  21. Lee, T. C., Chow, K. L., Fang, P., & Schwartz, R. J. (1991). Activation of skeletal alpha-actin gene transcription: the cooperative formation of serum response factor- binding complexes over positive cis-acting promoter serum response elements displaces a negative-acting nuclear factor enriched in replicating myoblasts and nonmyogenic cells. Molecular and Cellular Biology, 11(10), 5090–5100.

    PubMed  CAS  Google Scholar 

  22. Nakamura, M., Nishida, W., Mori, S., Hiwada, K., Hayashi, K., & Sobue, K. (2001). Transcriptional activation of beta-tropomyosin mediated by serum response factor and a novel Barx homologue, Barx1b, in smooth muscle cells. Journal of Biological Chemistry, 276(21), 18313–18320.

    PubMed  CAS  Google Scholar 

  23. Bhatia, R., Gaur, A., Lemanski, L. F., & Dube, D. K. (1998). Cloning and sequencing of the cDNA for an RNA-binding protein from the Mexican axolotl: binding affinity of the in vitro synthesized protein. Biochem Biophys Acta, 1398(3), 265–274.

    PubMed  CAS  Google Scholar 

  24. Narshi, A., Denz, C. R., Nakatsugawa, M., Zajdel, R. W., Dube, S., Poiesz, B. J., et al. (2005). Cardiac myofibril formation is not affected by modification of both N- and C-termini of sarcomeric tropomyosin. Cardiovascular Toxicology, 5(1), 1–8.

    Article  PubMed  CAS  Google Scholar 

  25. Li, S., Czubryt, M. P., McAnally, J., Bassel-Duby, R., Richardson, J. A., Wiebel, F. F., et al. (2005). Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice. Proceedings of the National Academy of Sciences, 102(4), 1082–1087.

    Article  CAS  Google Scholar 

  26. Latinkic, B. V., Cooper, B., Smith, S., Kotecha, S., Towers, N., Sparrow, D., et al. (2004). Transcriptional regulation of the cardiac-specific MLC2 gene during Xenopus embryonic development. Development, 131(3), 669–679.

    Article  PubMed  CAS  Google Scholar 

  27. Qasba, P., Lin, E., Zhou, M. D., Kumar, A., & Siddiqui, M. A. (1992). A single transcription factor binds to two divergent sequence elements with a common function in cardiac myosin light chain-2 promoter. Molecular and Cellular Biology, 12(3), 1107–1116.

    PubMed  CAS  Google Scholar 

  28. Zhou, M. D., Goswami, S. K., Martin, M. E., & Siddiqui, M. A. (1993). A new serum-responsive, cardiac tissue-specific transcription factor that recognizes the MEF-2 site in the myosin light chain-2 promoter. Molecular and Cellular Biology, 13(2), 1222–1231.

    PubMed  CAS  Google Scholar 

  29. Shen, R. A., Goswami, S. K., Mascareno, E., Kumar, A., & Siddiqui, M. A. (1991). Tissue-specific transcription of the cardiac myosin light-chain 2 gene is regulated by an upstream repressor element. Molecular and Cellular Biology, 11(3), 1676–1685.

    PubMed  CAS  Google Scholar 

  30. Gaillard, C., Thézé, N., Hardy, S., Allo, M. R., Ferrasson, E., & Thiébaud, P. (1998). Alpha- tropomyosin gene expression in Xenopus laevis: differential promoter usage during development and controlled expression by myogenic factors. Development Genes and Evolution, 207(7), 435–445.

    Article  PubMed  CAS  Google Scholar 

  31. Wang, D., Chang, P. S., Wang, Z., Sutherland, L., Richardson, J. A., Small, E., et al. (2001). Activation of cardiac gene expression by myocardin, a transcriptional cofactor for serum response factor. Cell, 105(7), 851–862.

    Article  PubMed  CAS  Google Scholar 

  32. Yoshida, T., Sinha, S., Dandré, F., Wamhoff, B. R., Hoofnagle, M. H., Kremer, B. E., et al. (2003). Myocardin is a key regulator of CArG-dependent transcription of multiple smooth muscle marker genes. Circulation Research, 92(8), 856–864.

    Article  PubMed  CAS  Google Scholar 

  33. Ueyama, T., Kasahara, H., Ishiwata, T., Nie, Q., & Izumo, S. (2003). Myocardin expression is regulated by Nkx2.5, and its function is required for cardiomyogenesis. Molecular and Cellular Biology, 23(24), 9222–9232.

    Article  PubMed  CAS  Google Scholar 

  34. Qasba, P., Danishefsky, K., Gadot, M., & Siddiqui, M. A. (1992). Functional analysis of a CArG-like promoter element in cardiac myosin light chain 2 gene. Molecular Biology of the Cell, 38(5–6), 561–569.

    CAS  Google Scholar 

  35. Ren, Y., & Liao, W. S. (2001). Transcription factor AP-2 functions as a repressor that contributes to the liver-specific expression of serum amyloid A1 gene. Journal of Biological Chemistry, 276(21), 17770–17778.

    Article  PubMed  CAS  Google Scholar 

  36. Kageyama, R., & Pastan, I. (1989). Molecular cloning and characterization of a human DNA binding factor that represses transcription. Cell, 59(5), 815–825.

    Article  PubMed  CAS  Google Scholar 

  37. Pasquet, S., Naye, F., Faucheux, C., Bronchain, C., Cheseneau, A., Thiebaud, P., et al. (2006). Transcription enhancer Factor-1-dependent expression of the α-tropomyosin gene in the three muscle cell types. Journal of Biological Chemistry, 281(45), 34406–34420.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

The work was supported by grants from American Heart Association (NY Affiliate # 951038T) and from the Department of Medicine to DKD and NIH S6- GM073621 and AHA Great Southeast Affiliate (09GRNT2400138) to XH.

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Authors and Affiliations

  1. Department of Medicine, SUNY Upstate Medical University, 750 East Adams Street, Syracuse, NY, 13210, USA

    Christopher R. Denz, Syamalima Dube, Anish Thomas, Bernard J. Poiesz & Dipak K. Dube

  2. Department of Biomedical Science, Florida Atlantic University, Boca Raton, FL, 33431, USA

    Chi Zhang, Pingping Jia, Jianfeng Du & Xupei Huang

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  1. Christopher R. Denz
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  2. Chi Zhang
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Correspondence to Dipak K. Dube.

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Denz, C.R., Zhang, C., Jia, P. et al. Absence of Mutation at the 5′-Upstream Promoter Region of the TPM4 Gene From Cardiac Mutant Axolotl (Ambystoma mexicanum). Cardiovasc Toxicol 11, 235–243 (2011). https://doi.org/10.1007/s12012-011-9117-z

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  • DOI: https://doi.org/10.1007/s12012-011-9117-z

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