Transcriptional regulation is essential for any gene expression including microRNA expression. MiR-1-1 and miR-133a-2 are essential microRNAs (miRs) involved in cardiac and skeletal muscle development and diseases. Early studies reveal two regulatory enhancers, an upstream and an intragenic, that direct the miR-1-1 and miR-133a-2 transcripts. In this study, we identify a unique serum response factor (SRF) binding motif within the enhancer through bioinformatic approaches. This motif is evolutionarily conserved and is present in a range of organisms from yeast, flies, to humans. We provide evidence to demonstrate that this regulatory motif is SRF-dependent in vitro by electrophoretic mobility shift assay, luciferase activity assay, and endogenous chromatin immunoprecipitation assay followed by DNA sequence confirmation, and in vivo by transgenic lacZ reporter mouse studies. Importantly, our transgenic mice indicate that this motif is indispensable for the expression of miR1-1/133a-2 in the heart, but not necessary in skeletal muscle, while the enhancer is sufficient for miR1-1/133a-2 gene expression in both tissues. The mutation of the motif alone completely abolishes miR-1-1/133a-2 gene expression in the animal heart, but not in the skeletal muscle. Our findings reveal an additional architecture of regulatory complex directing miR-1-1/133a-1 gene expression, and demonstrate how this intragenic enhancer differentially manages the expression of the two miRs in the heart and skeletal muscle, respectively.
References
[1]
Norman C, Runswick M, Pollock R, Treisman R (1988) Isolation and properties of cDNA clones encoding SRF, a transcription factor that binds to the c-fos serum response element. Cell 55: 989-1003. doi:10.1016/0092-8674(88)90244-9. PubMed: 3203386.
[2]
Treisman R (1994) Ternary complex factors: growth factor regulated transcriptional activators. Curr Opin Genet Dev 4: 96-101. doi:10.1016/0959-437X(94)90097-3. PubMed: 8193547.
[3]
Miano JM (2003) Serum response factor: toggling between disparate programs of gene expression. J Mol Cell Cardiol 35: 577-593. doi:10.1016/S0022-2828(03)00110-X. PubMed: 12788374.
[4]
Lee TC, Schwartz RJ (1992) Using proteases to avoid false identification of DNA-protein complexes in gel shift assays. BioTechniques 12: 486-490. PubMed: 1503744.
[5]
Li L, Liu Z, Mercer B, Overbeek P, Olson EN (1997) Evidence for serum response factor-mediated regulatory networks governing SM22alpha transcription in smooth, skeletal, and cardiac muscle cells. Dev Biol 187: 311-321. doi:10.1006/dbio.1997.8621. PubMed: 9242426.
Chen CY, Schwartz RJ (1996) Recruitment of the tinman homolog Nkx-2.5 by serum response factor activates cardiac alpha-actin gene transcription. Mol Cell Biol 16: 6372-6384. PubMed: 8887666.
[8]
Li S, Czubryt MP, McAnally J, Bassel-Duby R, Richardson JA et al. (2005) Requirement for serum response factor for skeletal muscle growth and maturation revealed by tissue-specific gene deletion in mice. Proc Natl Acad Sci U S A 102: 1082-1087. doi:10.1073/pnas.0409103102. PubMed: 15647354.
[9]
Niu Z, Yu W, Zhang SX, Barron M, Belaguli NS et al. (2005) Conditional mutagenesis of the murine serum response factor gene blocks cardiogenesis and the transcription of downstream gene targets. J Biol Chem 280: 32531-32538. doi:10.1074/jbc.M501372200. PubMed: 15929941.
[10]
Miano JM, Ramanan N, Georger MA, de Mesy Bentley KL, Emerson RL et al. (2004) Restricted inactivation of serum response factor to the cardiovascular system. Proc Natl Acad Sci U S A 101: 17132-17137. doi:10.1073/pnas.0406041101. PubMed: 15569937.
[11]
Parlakian A, Tuil D, Hamard G, Tavernier G, Hentzen D et al. (2004) Targeted inactivation of serum response factor in the developing heart results in myocardial defects and embryonic lethality. Mol Cell Biol 24: 5281-5289. doi:10.1128/MCB.24.12.5281-5289.2004. PubMed: 15169892.
[12]
Arsenian S, Weinhold B, Oelgeschl？ger M, Rüther U, Nordheim A (1998) Serum response factor is essential for mesoderm formation during mouse embryogenesis. EMBO J 17: 6289-6299. doi:10.1093/emboj/17.21.6289. PubMed: 9799237.
[13]
Wiebel FF, Rennekampff V, Vintersten K, Nordheim A (2002) Generation of mice carrying conditional knockout alleles for the transcription factor SRF. Genesis 32: 124-126. doi:10.1002/gene.10049. PubMed: 11857797.
[14]
Schratt G, Philippar U, Berger J, Schwarz H, Heidenreich O et al. (2002) Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells. J Cell Biol 156: 737-750. doi:10.1083/jcb.200106008. PubMed: 11839767.
[15]
Schratt G, Weinhold B, Lundberg AS, Schuck S, Berger J et al. (2001) Serum response factor is required for immediate-early gene activation yet is dispensable for proliferation of embryonic stem cells. Mol Cell Biol 21: 2933-2943. doi:10.1128/MCB.21.8.2933-2943.2001. PubMed: 11283270.
[16]
Weinhold B, Schratt G, Arsenian S, Berger J, Kamino K et al. (2000) Srf(-/-) ES cells display non-cell-autonomous impairment in mesodermal differentiation. EMBO J 19: 5835-5844. doi:10.1093/emboj/19.21.5835. PubMed: 11060034.
[17]
Chang J, Wei L, Otani T, Youker KA, Entman ML et al. (2003) Inhibitory cardiac transcription factor, SRF-N, is generated by caspase 3 cleavage in human heart failure and attenuated by ventricular unloading. Circulation 108: 407-413. doi:10.1161/01.CIR.0000084502.02147.83. PubMed: 12874181.
[18]
Yang X, Li Q, Lin X, Ma Y, Yue X et al. (2012) Mechanism of fibrotic cardiomyopathy in mice expressing truncated Rho-associated coiled-coil protein kinase 1. FASEB J 26: 2105-2116. doi:10.1096/fj.11-201319. PubMed: 22278938.
[19]
Lin X, Yang X, Li Q, Ma Y, Cui S et al. (2012) Protein Tyrosine Phosphatase-Like A Regulates Myoblast Proliferation and Differentiation through MyoG and the Cell Cycling Signaling Pathway. Mol Cell Biol 32: 297-308. doi:10.1128/MCB.05484-11. PubMed: 22106411.
[20]
Chen JF, Mandel EM, Thomson JM, Wu Q, Callis TE et al. (2006) The role of microRNA-1 and microRNA-133 in skeletal muscle proliferation and differentiation. Nat Genet 38: 228-233. doi:10.1038/ng1725. PubMed: 16380711.
[21]
Townley-Tilson WH, Callis TE, Wang D (2010) MicroRNAs. p. 1, 133 and 206: critical factors of skeletal and cardiac muscle development, function, and disease. Int J Biochem Cell Biol 42: 1252-1255.
[22]
Kusakabe R, Tani S, Nishitsuji K, Shindo M, Okamura K et al. (2013) Characterization of the compact bicistronic microRNA precursor, miR-1/miR-133, expressed specifically in Ciona muscle tissues. Gene Expr Patterns 13: 43-50. doi:10.1016/j.gep.2012.11.001. PubMed: 23159539.
[23]
Liu N, Bezprozvannaya S, Williams AH, Qi X, Richardson JA et al. (2008) microRNA-133a regulates cardiomyocyte proliferation and suppresses smooth muscle gene expression in the heart. Genes Dev 22: 3242-3254. doi:10.1101/gad.1738708. PubMed: 19015276.
[24]
Sayed D, Hong C, Chen IY, Lypowy J, Abdellatif M (2007) MicroRNAs play an essential role in the development of cardiac hypertrophy. Circ Res 100: 416-424. doi:10.1161/01.RES.0000257913.42552.23. PubMed: 17234972.
[25]
Carè A, Catalucci D, Felicetti F, Bonci D, Addario A et al. (2007) MicroRNA-133 controls cardiac hypertrophy. Nat Med 13: 613-618. doi:10.1038/nm1582. PubMed: 17468766.
[26]
Zhao Y, Samal E, Srivastava D (2005) Serum response factor regulates a muscle-specific microRNA that targets Hand2 during cardiogenesis. Nature 436: 214-220. doi:10.1038/nature03817. PubMed: 15951802.
[27]
Zhao Y, Ransom JF, Li A, Vedantham V, von Drehle M et al. (2007) Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2. Cell 129: 303-317. doi:10.1016/j.cell.2007.03.030. PubMed: 17397913.
[28]
Li Q, Lin X, Yang X, Chang J (2010) NFATc4 is negatively regulated in miR-133a-mediated cardiomyocyte hypertrophic repression. Am J Physiol Heart Circ Physiol 298: H1340-H1347. doi:10.1152/ajpheart.00592.2009. PubMed: 20173049.
[29]
Duisters RF, Tijsen AJ, Schroen B, Leenders JJ, Lentink V et al. (2009) miR-133 and miR-30 regulate connective tissue growth factor: implications for a role of microRNAs in myocardial matrix remodeling. Circ Res 104: 170-178. doi:10.1161/CIRCRESAHA.108.182535. PubMed: 19096030.
[30]
Matkovich SJ, Wang W, Tu Y, Eschenbacher WH, Dorn LE et al. (2009) MicroRNA-133a Protects Against Myocardial Fibrosis and Modulates Electrical Repolarization Without Affecting Hypertrophy in Pressure-Overloaded Adult Hearts. Circ Res, 106: 166–75. PubMed: 19893015.
[31]
Xiao J, Luo X, Lin H, Zhang Y, Lu Y et al. (2007) MicroRNA miR-133 represses HERG K+ channel expression contributing to QT prolongation in diabetic hearts. J Biol Chem 282: 12363-12367. doi:10.1074/jbc.C700015200. PubMed: 17344217.
[32]
Liu N, Williams AH, Kim Y, McAnally J, Bezprozvannaya S et al. (2007) An intragenic MEF2-dependent enhancer directs muscle-specific expression of microRNAs 1 and 133. Proc Natl Acad Sci U S A 104: 20844-20849. doi:10.1073/pnas.0710558105. PubMed: 18093911.
