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Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation

Nature Genetics volume 28, pages 276–280 (2001)Cite this article

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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Figure 1: Association of Tbx5 and Nkx2-5.
Figure 2: Transactivation of Nppa by Tbx5 and Nkx2-5.
Figure 3: Binding of Tbx5 and Nkx2-5 to the Nppa promoter.
Figure 4: Expression of cardiac genes in P19CL6 cell lines.

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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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Authors and Affiliations

  1. Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo, Japan

    Yukio Hiroi, Koshiro Monzen, Yuichi Ikeda & Ryozo Nagai

  2. Department of Cardiovascular Medicine, Kanazawa Medical University, Ishikawa, Japan

    Sumiyo Kudoh

  3. International Medical Center of Japan, Tokyo, Japan

    Yoshio Yazaki

  4. Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba, Japan

    Issei Komuro

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  1. Yukio Hiroi
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Correspondence to Issei Komuro.

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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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