Fig. 6. Human ANF promoter-luciferase construct was transfected to COS7 cells along with various expression plasmids. Tbx5 and Nkx2.5 activated this promoter weakly (4.7- and 3.7-fold, respectively). As reported, co-expression of these factors synergistically activated this promoter (11.6-fold). Addition of zebrafish tbx20 expression vector repressed Tbx5-mediated activation (1.6-fold), although this did not affect Nkx2.5-mediated activation (5.2-fold). Interestingly, Tbx20 abrogated efficiently the synergistic activation by Tbx5 and Nkx2.5 (7.0-fold). When another heart-specific factor GATA4 was expressed, the ANF promoter was activated weakly (3.5-fold). When GATA4 and Tbx5 were coexpressed, robust activation was observed (25.3-fold). Tbx20 abrogated this strong synergistic activation efficiently (3.2-fold), whereas Tbx20 did not affect the ANF activation by GATA4 alone (2.5 fold). Tbx20 alone did not affect the ANF promoter activity (1.3 fold).