Tbx5 associates with Nkx2-5 and synergistically promotes cardiomyocyte differentiation

The cardiac homeobox protein Nkx2-5 is essential in cardiac development, an d mutations in Csx (which encodes Nkx2-5) cause various congenital heart di seases(1-5). Using the yeast two-hybrid system with Nkx2-5 as the 'bait', w e isolated the T-box-containing transcription factor Tbx5; mutations in TBX 5 cause heart and limb malformations in Holt-Gram syndrome(6-8) (HOS). Co-t ransfection of Nkx2-5 and Tbx5 into COS-7 cells showed that they also assoc iate with each other in mammalian cells. Glutathione S-transferase (GST) 'p ull-down' assays indicated that the N-terminal domain and N-terminal part o f 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, an d both transcription factors showed synergistic activation. Deletion analys is 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-lim b malformations without cardiac abnormalities(8). activated the Nppa promot er to a similar extent to that of wildtype Tbx5. P19CL6 cell lines(9,10) ov erexpressing wildtype Tbx5 started to beat earlier and expressed cardiac-sp ecific genes more abundantly than did parental P19CL6 cells, whereas cell l ines expressing the G80R mutant did not differentiate into beating cardiomy ocytes. These results indicate that two different types of cardiac transcri ption factors synergistically induce cardiac development.