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Fig. 3. Tbx2 is upregulated in Tbx20 mutants and is a direct target for repression by Tbx20. (A-F) Whole-mount RNA in situ and section analysis for Tbx2 expression in Tbx20-null embryos and wild-type littermates. Arrows indicate the corresponding heart regions in wild type and mutant embryos. OFT, outflow tract; A/V, atrioventricular canal. (G-L) Whole-mount RNA in situ demonstrates downregulation of Nppa, chisel and cited 1 in Tbx20 mutants. (M-P) Expression of Tbx20 and Tbx3 is unaffected. Arrows indicate the corresponding heart regions in wild type and mutant embryos. (Q) ChIP analysis with embryonic heart extracts revealed binding of Tbx20 to region containing conserved T-box sites within Tbx2 promoter (lane 1, primer P-813, P-573). ChIP analysis with primers against an unrelated promoter region revealed no Tbx20 recruitment (lane 2) (see Materials and methods for primers). No recruitment was found with beads, IgG or H₂O. (R) Co-transfection of Tbx20 expression vector (500 ng) with Tbx2 promoter-luciferase constructs (100 ng) into HEK293 cells demonstrates repression by Tbx20, which was abrograted by mutation (Mu) of conserved T-box elements within the Tbx2 promoter. ^*P<0.05, paired t-test.

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