Abstract
The cardiac homeobox protein Nkx2-5 is essential in cardiac development, and mutations in Csx (which encodes Nkx2-5) cause various congenital heart diseases1,2,3,4,5. Using the yeast two-hybrid system with Nkx2-5 as the 'bait', we isolated the T-box-containing transcription factor Tbx5; mutations in TBX5 cause heart and limb malformations in Holt-Oram syndrome6,7,8 (HOS). Co-transfection of Nkx2-5 and Tbx5 into COS-7 cells showed that they also associate with each other in mammalian cells. Glutathione S-transferase (GST) 'pull-down' assays indicated that the N-terminal domain and N-terminal part of the T-box of Tbx5 and the homeodomain of Nkx2-5 were necessary for their interaction. Tbx5 and Nkx2-5 directly bound to the promoter of the gene for cardiac-specific natriuretic peptide precursor type A (Nppa) in tandem, and both transcription factors showed synergistic activation. Deletion analysis showed that both the N-terminal domain and T-box of Tbx5 were important for this transactivation. A G80R mutation of Tbx5, which causes substantial cardiac defects with minor skeletal abnormalities in HOS, did not activate Nppa or show synergistic activation, whereas R237Q, which causes upper-limb malformations without cardiac abnormalities8, activated the Nppa promoter to a similar extent to that of wildtype Tbx5. P19CL6 cell lines9,10 overexpressing wildtype Tbx5 started to beat earlier and expressed cardiac-specific genes more abundantly than did parental P19CL6 cells, whereas cell lines expressing the G80R mutant did not differentiate into beating cardiomyocytes. These results indicate that two different types of cardiac transcription factors synergistically induce cardiac development.
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References
Komuro, I. & Izumo, S. Csx: a murine homeobox-containing gene specifically expressed in the developing heart. Proc. Natl. Acad. Sci. USA 90, 8145–8149 (1993).
Lints, T.J., Parsons, L.M., Hartley, L., Lyons, I. & Harvey, R.P. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119, 419–431 (1993).
Lyons, I. et al. Myogenic and morphogenetic defects in the heart tubes of murine embryos lacking the homeobox gene Nkx2-5. Genes Dev. 9, 1654–1666 (1995).
Schott, J.J. et al. Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281, 108–111 (1998).
Benson, D.W. et al. Mutations in the cardiac transcription factor NKX2.5 affect diverse cardiac developmental pathways. J. Clin. Invest. 104, 1567–1573 (1999).
Li, Q.Y. et al. Holt-Oram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family. Nature Genet. 15, 21–29 (1997).
Basson, C.T. et al. Mutations in human TBX5 cause limb and cardiac malformation in Holt-Oram syndrome. Nature Genet. 15, 30–35 (1997).
Basson, C.T. et al. Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome mutations. Proc. Natl. Acad. Sci. USA 96, 2919–2924 (1999).
Habara-Ohkubo, A. Differentiation of beating cardiac muscle cells from a derivative of P19 embryonal carcinoma cells. Cell Struct. Funct. 21, 101–110 (1996).
Monzen, K. et al. Bone morphogenetic proteins induce cardiomyocyte differentiation through the mitogen-activated protein kinase kinase kinase TAK1 and cardiac transcription factors Csx/Nkx-2.5 and GATA-4. Mol. Cell. Biol. 19, 7096–7105 (1999).
Bodmer, R. The gene tinman is required for specification of the heart and visceral muscles in Drosophila. Development 118, 719–729 (1993).
Azpiazu, N. & Frasch, M. Tinman and bagpipe: two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev. 7, 1325–1340 (1993).
Tanaka, M. et al. Vertebrate homologs of tinman and bagpipe: roles of the homeobox genes in cardiovascular development. Dev. Genet. 22, 239–249 (1998).
Tanaka, M., Chen, Z., Bartunkova, S., Yamasaki, N. & Izumo, S. The cardiac homeobox gene Csx/Nkx2.5 lies genetically upstream of multiple genes essential for heart development. Development 126, 1269–1280 (1999).
Kasahara, H. et al. Loss of function and inhibitory effects of human CSX/NKX2.5 homeoprotein mutations associated with congenital heart disease. J. Clin. Invest. 106, 299–308 (2000).
Hosoda, T. et al. Familial atrial septal defect and atrioventricular conduction disturbance associated with a point mutation in the cardiac homeobox gene CSX/NKX2-5 in a Japanese patient. Jpn. Circ. J. 63, 425–426 (1999).
Chen, C.Y. et al. Activation of the cardiac alpha-actin promoter depends upon serum response factor, Tinman homologue, Nkx-2.5, and intact serum response elements. Dev. Genet. 19, 119–130 (1996).
Skerjanc, I.S., Petropoulos, H., Ridgeway, A.G. & Wilton, S. Myocyte enhancer factor 2C and Nkx2-5 up-regulate each other's expression and initiate cardiomyogenesis in P19 cells. J. Biol. Chem. 273, 34904–34910 (1998).
Durocher, D., Charron, F., Warren, R., Schwartz, R.J. & Nemer, M. The cardiac transcription factors Nkx2-5 and GATA-4 are mutual cofactors. EMBO J. 16, 5687–5696 (1997).
Lee, Y. et al. The cardiac tissue-restricted homeobox protein Csx/Nkx2.5 physically associates with the zinc finger protein GATA4 and cooperatively activates atrial natriuretic factor gene expression. Mol. Cell. Biol. 18, 3120–3129 (1998).
Shiojima, I. et al. Context-dependent transcriptional cooperation mediated by cardiac transcription factors Csx/Nkx-2.5 and GATA-4. J. Biol. Chem. 274, 8231–8239 (1999).
Smith, J. T-box genes: what they do and how they do it. Trends Genet. 15, 154–158 (1999).
Gibson-Brown, J.J., Agulnik, S.I., Silver, L.M., Niswander, L. & Papaioannou, V.E. Involvement of T-box genes Tbx2-Tbx5 in vertebrate limb specification and development. Development 125, 2499–2509 (1998).
Takeuchi, J.K. et al. Tbx5 and Tbx4 genes determine the wing/leg identity of limb buds. Nature 398, 810–814 (1999).
Rodriguez-Esteban, C. et al. The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature 398, 814–818 (1999).
Horb, M.E. & Thomsen, G.H. Tbx5 is essential for heart development. Development 126, 1739–1751 (1999).
Koshiba-Takeuchi, K. et al. Tbx5 and the retinotectum projection. Science 287, 134–137 (2000).
Muller, C.W. & Herrmann, B.G. Crystallographic structure of the T domain-DNA complex of the Brachyury transcription factor. Nature 389, 884–888 (1997).
Liberatore, C.M., Searcy-Schrick, R.D. & Yutzey, K.E. Ventricular expression of Tbx5 inhibits normal heart chamber development. Dev. Biol. 223, 169–180 (2000).
Acknowledgements
We thank N. Mochizuki for technical advice, C. Masuo and K. Abe for technical assistance and J. Hiroi for encouragement. This work was supported in part by grants from the Japanese Ministry of Education, Science, and Culture; Mochida Memorial Foundation; and Kanae Foundation (Aventis).
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Hiroi, Y., Kudoh, S., Monzen, K. et al. Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation. Nat Genet 28, 276–280 (2001). https://doi.org/10.1038/90123
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DOI: https://doi.org/10.1038/90123
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