Functional analyses of three Csx/Nkx-2.5 mutations that cause human congenital heart disease

A homeodomain-containing transcription factor Csx/Nkx-2.5 is an important r egulator of cardiogenesis in mammals. Three different mutants, Gln170ter (d esignated A) and Thr178Met (designated B) in the helix 2 of the homeodomain and Gln198ter mutation (designated C) just after homeodomain, have been re ported to cause atrial septal defect with atrial ventricular block. We here examined the functions of these three mutants of Csx/Nkx-2.5. The atrial n atriuretic peptide (ANP) promoter was activated by wild type Csx/Nkx-2.5 (W T, similar to8-fold), B (similar to2-fold), and C (similar to6-fold) but no t by A. When A, B, or C was cotransfected into COS-7 cells with the same am ount of WT, WT-induced activation of the ANP promoter was attenuated by A a nd B (A > B), whereas C further enhanced the activation. Immunocytochemical analysis using anti-Myc tag antibody indicated that transfected Myc-tagged WT, B, and C were localized in the nucleus of both COS-7 cells and cardiom yocytes of neonatal rats, whereas A was distributed diffusely in the cytopl asm and nucleus in COS-7 cells. Electrophoretic mobility shift assay showed that Csx/Nkx-2.5-binding sequences were bound strongly by WT and C, weakly by B, but not by A Immunoprecipitation and GST pull-down assay revealed th at WT and all mutants interacted with GATA-4, The synergistic activation of the ANP promoter by WT and GATA-4 was further enhanced by C but was inhibi ted by A and B, In the cultured cardiomyocytes, overexpression of C but not WT, A, or B, induced apoptosis, These results suggest that although the th ree mutants induce the same cardiac phenotype, transactivation ability and DNA binding ability are different among the three mutants and that apoptosi s may be a cause for C-induced cardiac defect.