Fig. 5. Tbx2 directly binds Nmyc1 and represses its expression in regions of relatively low proliferation within the heart. (A-H) Regions of relatively low proliferation; outflow tract (OFT) and atrioventricular canal (A/V) are indicated by arrows. Corresponding sections (C,D; G,H; K) are shown progressively from anterior to posterior, respectively. Expression of Nmyc1 (A-D) and cyclin A2 (E-H) is downregulated in Tbx20-null mutants. Section analysis of wild-type littermates reveals regional differences in expression of Nmyc1 (C) and cyclin A2 (Ccna2) (G), with relatively low levels in OFT and A/V. (I-K) Wild-type Tbx2 expression is complementary to that of Nmyc1 and cyclinA2. (L) Expression of phosphorylated histone H3 in wild-type embryos revealed regions of low proliferation within developing heart. The left, middle and right panels show right, frontal and left side views, respectively. (M) ChIP analysis with embryonic heart extracts demonstrated recruitment of Tbx2 and Tbx20 to regions containing T-box consensus sites within intron 1 of the Nmyc1 gene (lane 1, primer P-4030, P-4330; lane 2, primer P-4630, P-4930). ChIP analysis with primers against an unrelated promoter region revealed no Tbx2 recruitment (lane 3) (see Materials and methods for primers). No recruitment was found with IgG. (N) Co-transfections of Tbx2 or Tbx20 expression vectors with Nmyc1 intron 1-luciferase reporter into HEK293 cells demonstrated repression or activation, respectively. ^**P<0.005, paired t-test; ^*P<0.05, paired t-test. (O) Co-transfections of Tbx2 expression vector with Nmyc1 intron 1-luciferase reporter into HEK293 cells demonstrated dose-dependent repression by Tbx2. ^**P<0.005, paired t-test.