Abstract
To understand how cardiac gene expression is regulated, the identification and characterization of cis-regulatory elements and their trans-acting factors by gel mobility shift assay (GMSA) or gel retardation assay are essential and common steps. In addition to providing a general protocol for GMSA, this chapter describes some applications of this assay to characterize cardiac-specific and ubiquitous trans-acting factors bound to regulatory elements [novel TCTG(G/C) direct repeat and A/T-rich region] of the rat cardiac troponin T promoter. In GMSA, the specificity of the binding of trans-acting factor to labeled DNA probe should be verified by the addition of unlabeled probe in the reaction mixture. The migratory property of DNA-protein complexes formed by protein extracts prepared from different tissues can be compared to determine the tissue specificity of trans-acting factors. GMSA, coupled with specific antibody to trans-acting factor (antibody supershift assay), is used to identify proteins present in the DNA-protein complex. The gel-shift competition assay with an unlabeled probe containing a slightly different sequence is a powerful technique used to assess the sequence specificity and relative binding affinity of a DNA-protein interaction. GMSA with SDS-PAGE fractionated proteins allows for the determination of the apparent molecular mass of bound trans-acting factor.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wang, Q., Reiter, R. S., Huang, Q.-Q., Jin, J.-P., and Lin, J. J.-C. (2001) Comparative studies on the expression patterns of three troponin T genes during mouse development. Anat. Rec. 263, 72–84.
Wang, Q., Sigmund, C. D., and Lin, J. J.-C. (2000) Identification of cis elements in the cardiac troponin T gene conferring specific expression in cardiac muscle of transgenic mice. Circ. Res. 86, 478–484.
Wang, G., Yeh, H.-L, and Lin, J. J.-C. (1994) Characterization of cis-regulating elements and trans-activating factors of the rat cardiac troponin T gene. J. Biol. Chem. 269, 30595–30603.
Wang, Q., Lin, J. L.-C., and Lin, J. J.-C. (2002) A novel TCTG(G/C) direct repeat and an A/T-rich HMG2-binding site control the expression of the rat cardiac troponin T gene. J. Mol. Cell. Cardiol. 34, 1667–1679.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.
Chen, C. Y. and Schwartz, R. J. (1996) Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac α-actin gene transcription. Mol. Cell. Biol. 16, 6372–6384.
Navankasattusas, S., Zhu, H., Garcia, A. V., Evans, S. M., and Chien, K. R. (1992) A ubiquitous factor (HF-1a) and a distinct muscle factor (HF-1b/MEF-2) form an E-box-independent pathway for cardiac muscle gene expression. Mol. Cell. Biol. 12, 1469–1479.
Zou, Y. and Chien, K. R. (1995) EFIa/YB-1 is a component of cardiac HF-1A binding activity and positively regulates transcription of the myosin light-chain 2v gene. Mol. Cell. Biol. 15, 2972–2982.
Mar, J. H. and Ordahl, C. P. (1990) M-CAT binding factor, a novel trans-acting factor governing muscle-specific transcription. Mol. Cell. Biol. 10, 4271–4283.
Abmayr, S. M. and Workman, J. L. (1998) Preparation of nuclear and cytoplasmic extracts from mammalian cells, in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., et al., eds.), John Wiley & Sons, New York, pp. 12.11.11–12.11.19.
Buratowski, S. and Chodosh, L. A. (1998) Mobility shift DNA-binding assay using gel electrophoresis. in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., et al., eds.), John Wiley & Sons, New York, pp. 12.12.11–12.12.11.
Zhu, H., Nguyen, V. T., Brown, A. B., et al. (1993) A novel, tissue-restricted zinc finger protein (HF-1b) binds to the cardiac regulatory element (HF-1b/MEF-2) within the rat myosin light chain-2 gene. Mol. Cell. Biol. 13, 4432–4444.
Navankasattusas, S., Sawadogo, M., van Bilsen, M., Dang, C. V., and Chien, K. R. (1994) The basic helix-loop-helix protein upstream stimulating factor regulates the cardiac ventricular myosin light-chain 2 gene via independent cis regulatory elements. Mol. Cell. Biol. 14, 7331–7339.
Yu, Y.-T., Breitbart, R. E., Smoot, L. B., Lee, Y., Mahdavi, V., and Nadal-Ginard, B. (1992) Human myocyte-specific enhancer factor 2 comprises a group of tissue-restricted MADS box transcription factors. Genes Dev. 6, 1783–1798.
Lee, K. J., Hickey, R., Zhu, H., and Chien, K. R. (1994) Positive regulatory elements (HF-1a and HF-1b) and a novel negative regulatory element (HF-3) mediate ventricular muscle-specific expression of myosin light-chain 2 luciferase fusion genes in transgenic mice. Mol. Cell. Biol. 14, 1220–1229.
Ossipow, V., Laemmli, U. K., and Schibier, U. (1993) A simple method to renature DNA-binding proteins separated by SDS-polyacrylamide gel electrophoresis. Nucleic Acids Res. 21, 6040–6041.
Cooper, T. A. and Ordahl, C. P. (1984) A single cardiac troponin T gene regulated by different programs in cardiac and skeletal muscle development. Science 226, 979–982.
Mar, J. H., Antin, P. B., Cooper, T. A., and Ordahl, C. P. (1988) Analysis of the upstream regions governing expression of the chicken cardiac troponin T gene in embryonic cardiac and skeletal muscle cells. J. Cell Biol. 107, 573–585.
Saggin, L., Gorza, L., Ausoni, S., and Schiaffino, S. (1990) Cardiac troponin T in developing, regenerating and denervated rat skeletal muscle. Development 110, 547–554.
Swiderski, R. E. and Solursh, M. (1990) Precocious appearance of cardiac troponin T pre-mRNAs during early avian embryonic skeletal muscle development in ovo. Dev. Biol. 140, 73–82.
Sutherland, C. J., Elsom, V. L., Gordon, M. L., Dunwoodie, S. U., and Hardeman, E. C. (1991) Coordination of skeletal muscle gene expression occurs late in mammalian development. Dev. Biol. 146, 167–178.
Jin, J.-P., Huang, Q.-Q., Yen, H.-L, and Lin, J. J.-C. (1992) Complete nucleotide sequence and structural organization of rat cardiac troponin T gene. A single gene generates embryonic and adult isoforms via developmentally regulated alternative splicing. J. Mol. Biol. 227, 1269–1276.
Long, C. S. and Ordahl, C. P. (1988) Transcriptional repression of an embryo-specific muscle gene. Dev. Biol. 127, 228–234.
Tymms, M. J. (2000) Transcription Factor Protocols. Humana Press, Totowa, NJ.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc.
About this protocol
Cite this protocol
Lin, J.JC., Grosskurth, S.E., Harlan, S.M., Gustafson-Wagner, E.A., Wang, Q. (2007). Characterization of cis-Regulatory Elements and Transcription Factor Binding. In: Zhang, J., Rokosh, G. (eds) Cardiac Gene Expression. Methods in Molecular Biology, vol 366. Humana Press. https://doi.org/10.1007/978-1-59745-030-0_10
Download citation
DOI: https://doi.org/10.1007/978-1-59745-030-0_10
Publisher Name: Humana Press
Print ISBN: 978-1-58829-352-7
Online ISBN: 978-1-59745-030-0
eBook Packages: Springer Protocols