Twist1, a basic helix-loop-helix transcription factor, is expressed in mesenchymal precursor populations during embryogenesis and in metastatic cancer cells. In the developing heart, Twist1 is highly expressed in endocardial cushion (ECC) valve mesenchymal cells and is down regulated during valve differentiation and remodeling. Previous studies demonstrated that Twist1 promotes cell proliferation, migration, and expression of primitive extracellular matrix (ECM) molecules in ECC mesenchymal cells. Furthermore, Twist1 expression is induced in human pediatric and adult diseased heart valves. However, the Twist1 downstream target genes that mediate increased cell proliferation and migration during early heart valve development remain largely unknown. Candidate gene and global gene profiling approaches were used to identify transcriptional targets of Twist1 during heart valve development. Candidate target genes were analyzed for evolutionarily conserved regions (ECRs) containing E-box consensus sequences that are potential Twist1 binding sites. ECRs containing conserved E-box sequences were identified for Twist1 responsive genes Tbx20, Cdh11, Sema3C, Rab39b, and Gadd45a. Twist1 binding to these sequences in vivo was determined by chromatin immunoprecipitation (ChIP) assays, and binding was detected in ECCs but not late stage remodeling valves. In addition identified Twist1 target genes are highly expressed in ECCs and have reduced expression during heart valve remodeling in vivo, which is consistent with the expression pattern of Twist1. Together these analyses identify multiple new genes involved in cell proliferation and migration that are differentially expressed in the developing heart valves, are responsive to Twist1 transcriptional function, and contain Twist1-responsive regulatory sequences.
References
[1]
Thisse B, el Messal M, Perrin-Schmitt F (1987) The twist gene: isolation of a Drosophila zygotic gene necessary for the establishment of dorsoventral pattern. Nucleic Acids Res 15: 3439–3453.
[2]
Chen ZF, Behringer RR (1995) twist is required in head mesenchyme for cranial neural tube morphogenesis. Genes Dev 9: 686–699.
[3]
Firulli BA, Redick BA, Conway SJ, Firulli AB (2007) Mutations within helix I of Twist1 result in distinct limb defects and variation of DNA binding affinities. J Biol Chem 282: 27536–27546.
[4]
Chakraborty S, Wirrig EE, Hinton RB, Merrill WH, Spicer DB, et al. (2010) Twist1 promotes heart valve cell proliferation and extracellular matrix gene expression during development in vivo and is expressed in human diseased aortic valves. Dev Biol 347: 167–179.
[5]
Ansieau S, Morel AP, Hinkal G, Bastid J, Puisieux A (2010) TWISTing an embryonic transcription factor into an oncoprotein. Oncogene 29: 3173–3184.
[6]
Hinton RB Jr, Lincoln J, Deutsch GH, Osinska H, Manning PB, et al. (2006) Extracellular matrix remodeling and organization in developing and diseased aortic valves. Circ Res 98: 1431–1438.
[7]
Shelton EL, Yutzey KE (2007) Tbx20 regulation of endocardial cushion cell proliferation and extracellular matrix gene expression. Dev Biol 302: 376–388.
[8]
Shelton EL, Yutzey KE (2008) Twist1 function in endocardial cushion cell proliferation, migration, and differentiation during heart valve development. Dev Biol 317: 282–295.
[9]
Combs MD, Yutzey KE (2009) VEGF and RANKL regulation of NFATc1 in heart valve development. Circ Res 105: 565–574.
[10]
Chakraborty S, Cheek J, Sakthivel B, Aronow BJ, Yutzey KE (2008) Shared gene expression profiles in developing heart valves and osteoblast progenitor cells. Physiol Genomics 35: 75–85.
[11]
Barnes RM, Firulli AB (2009) A twist of insight - the role of Twist-family bHLH factors in development. Int J Dev Biol 53: 909–924.
[12]
Connerney J, Andreeva V, Leshem Y, Mercado MA, Dowell K, et al. (2008) Twist1 homodimers enhance FGF responsiveness of the cranial sutures and promote suture closure. Dev Biol 318: 323–334.
[13]
Mori M, Nakagami H, Koibuchi N, Miura K, Takami Y, et al. (2009) Zyxin mediates actin fiber reorganization in epithelial-mesenchymal transition and contributes to endocardial morphogenesis. Mol Biol Cell 20: 3115–3124.
[14]
Oshima A, Tanabe H, Yan T, Lowe GN, Glackin CA, et al. (2002) A novel mechanism for the regulation of osteoblast differentiation: transcription of periostin, a member of the fasciclin I family, is regulated by the bHLH transcription factor, twist. J Cell Biochem 86: 792–804.
[15]
Alexander NR, Tran NL, Rekapally H, Summers CE, Glackin C, et al. (2006) N-cadherin gene expression in prostate carcinoma is modulated by integrin-dependent nuclear translocation of Twist1. Cancer Res 66: 3365–3369.
[16]
Loots GG, Ovcharenko I (2004) rVISTA 2.0: evolutionary analysis of transcription factor binding sites. Nucleic Acids Res 32: W217–221.
[17]
Ho Sui SJ, Mortimer JR, Arenillas DJ, Brumm J, Walsh CJ, et al. (2005) oPOSSUM: identification of over-represented transcription factor binding sites in co-expressed genes. Nucleic Acids Res 33: 3154–3164.
[18]
Jegga AG, Sherwood SP, Carman JW, Pinski AT, Phillips JL, et al. (2002) Detection and visualization of compositionally similar cis-regulatory element clusters in orthologous and coordinately controlled genes. Genome Res 12: 1408–1417.
[19]
Gotea V, Ovcharenko I (2008) DiRE: identifying distant regulatory elements of co-expressed genes. Nucleic Acids Res 36: W133–139.
[20]
Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S (1983) In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96: 191–198.
[21]
Plageman TF Jr, Yutzey KE (2004) Differential expression and function of Tbx5 and Tbx20 in cardiac development. J Biol Chem 279: 19026–19034.
[22]
Meyers SG, Corsi AK (2010) C. elegans twist gene expression in differentiated cell types is controlled by autoregulation through intron elements. Dev Biol 346: 224–236.
[23]
Evans-Anderson HJ, Alfieri CM, Yutzey KE (2008) Regulation of cardiomyocyte proliferation and myocardial growth during development by FOXO transcription factors. Circ Res 102: 686–694.
[24]
Sengupta A, Molkentin JD, Yutzey KE (2009) FOXO transcription factors promote autophagy in cardiomyocytes. J Biol Chem 284: 28319–28331.
[25]
Mandel EM, Kaltenbrun E, Callis TE, Zeng XX, Marques SR, et al. (2010) The BMP pathway acts to directly regulate Tbx20 in the developing heart. Development 137: 1919–1929.
[26]
Combs MD, Braitsch CM, Lange AW, James JF, Yutzey KE (2011) NFATC1 promotes epicardium-derived cell invasion into myocardium. Development 138: 1747–1757.
[27]
Zhou J, Rappaport EF, Tobias JW, Young TL (2006) Differential gene expression in mouse sclera during ocular development. Invest Ophthalmol Vis Sci 47: 1794–1802.
[28]
Wang X, Wang RH, Li W, Xu X, Hollander MC, et al. (2004) Genetic interactions between Brca1 and Gadd45a in centrosome duplication, genetic stability, and neural tube closure. J Biol Chem 279: 29606–29614.
[29]
Ma L, Lu MF, Schwartz RJ, Martin JF (2005) Bmp2 is essential for cardiac cushion epithelial-mesenchymal transition and myocardial patterning. Development 132: 5601–5611.
[30]
Ehrman LA, Yutzey KE (1999) Lack of regulation in the heart forming region of avian embryos. Dev Biol 207: 163–175.
[31]
Lincoln J, Alfieri CM, Yutzey KE (2004) Development of heart valve leaflets and supporting apparatus in chicken and mouse embryos. Dev Dyn 230: 239–250.
[32]
Martin KA, Walsh K, Mader SL (1994) The mouse creatine kinase paired E-box element confers muscle-specific expression to a heterologous promoter. Gene 142: 275–278.
[33]
Bulger M, Groudine M (2010) Enhancers: the abundance and function of regulatory sequences beyond promoters. Dev Biol 339: 250–257.
[34]
Connerney J, Andreeva V, Leshem Y, Muentener C, Mercado MA, et al. (2006) Twist1 dimer selection regulates cranial suture patterning and fusion. Dev Dyn 235: 1345–1357.
[35]
Castanon I, Von Stetina S, Kass J, Baylies MK (2001) Dimerization partners determine the activity of the Twist bHLH protein during Drosophila mesoderm development. Development 128: 3145–3159.
[36]
Capparuccia L, Tamagnone L (2009) Semaphorin signaling in cancer cells and in cells of the tumor microenvironment–two sides of a coin. J Cell Sci 122: 1723–1736.
[37]
Zhou Y, Gunput RA, Pasterkamp RJ (2008) Semaphorin signaling: progress made and promises ahead. Trends Biochem Sci 33: 161–170.
[38]
Feiner L, Webber AL, Brown CB, Lu MM, Jia L, et al. (2001) Targeted disruption of semaphorin 3C leads to persistent truncus arteriosus and aortic arch interruption. Development 128: 3061–3070.
[39]
Vincentz JW, Barnes RM, Rodgers R, Firulli BA, Conway SJ, et al. (2008) An absence of Twist1 results in aberrant cardiac neural crest morphogenesis. Dev Biol 320: 131–139.
[40]
Kawaguchi J, Kii I, Sugiyama Y, Takeshita S, Kudo A (2001) The transition of cadherin expression in osteoblast differentiation from mesenchymal cells: consistent expression of cadherin-11 in osteoblast lineage. J Bone Miner Res 16: 260–269.
[41]
Vallin J, Girault JM, Thiery JP, Broders F (1998) Xenopus cadherin-11 is expressed in different populations of migrating neural crest cells. Mech Dev 75: 171–174.
[42]
Shiota M, Izumi H, Onitsuka T, Miyamoto N, Kashiwagi E, et al. (2008) Twist promotes tumor cell growth through YB-1 expression. Cancer Res 68: 98–105.
[43]
Rosemary Siafakas A, Richardson DR (2009) Growth arrest and DNA damage-45 alpha (GADD45alpha). Int J Biochem Cell Biol 41: 986–989.
[44]
Kardassis D, Murphy C, Fotsis T, Moustakas A, Stournaras C (2009) Control of transforming growth factor beta signal transduction by small GTPases. Febs J 276: 2947–2965.
[45]
Hegedus Z, Czibula A, Kiss-Toth E (2007) Tribbles: a family of kinase-like proteins with potent signalling regulatory function. Cell Signal 19: 238–250.
[46]
Medema JP, Schuurhuis DH, Rea D, van Tongeren J, de Jong J, et al. (2001) Expression of the serpin serine protease inhibitor 6 protects dendritic cells from cytotoxic T lymphocyte-induced apoptosis: differential modulation by T helper type 1 and type 2 cells. J Exp Med 194: 657–667.
[47]
Wirrig EE, Hinton RB, Yutzey KE (2011) Differential expression of cartilage and bone-related proteins in pediatric and adult diseased aortic valves. J Mol Cell Cardiol 50: 561–569.
[48]
Wirrig EE, Yutzey KE (2011) Transcriptional regulation of heart valve development and disease. Cardiovasc Pathol 20: 162–167.
