Fig. 7

smarcal1 is a direct target of E2F6. (A) Demonstration of E2Fs binding to SMARCAL1 promoter by EMSA. EMSA using nuclear protein extracts from E2Fs-dp1 co-transfected 293T cells shows that E2F-dp1 complexes bound to E2F sites in SMARCAL1 promoter. 200 times unlabeled probe was used to compete with the biotin labeled probe. The black arrowhead indicates the band of specific DNA–protein complexes. The asterisk indicates the band of the unbound probe. (B) Reporter gene assay shows SMARCAL1 transactivity was specifically inhibited by E2F6. E2F6-dp1 co-transfected with SMARCAL1 reporter plasmids and renilla luciferase activity was measured as transfection control. P1: pGL3-SMARCAL1 promoter1 containing E2F binding sites. P2: pGL3-SMARCAL1 promoter2 without E2F binding sites.∗P < 0.05. (C) RT-PCR analysis of SMARCAL1 expression in E2F6 transfected 293T cells. The empty vector of pcDNA3.1 was transfected as a control. PCR was performed for 26 cycles. (D) Real time PCR analysis of mRNA levels in 293T cells in (C). The results represent three experiments. β-actin mRNA was measured as an internal control. Data are presented as means ± SE. ∗P < 0.05. (E) Embryos injected with E2F6 mRNA (200 pg per embryo) showed gross morphology defects similar to smarcal1 morphants at 2 dpf. Human SMARCAL1 mRNA (200 pg per embryo) was used as control. (F) Example of E2F6 mRNA injected embryos. White arrowhead in the bottom image indicates the heart edema. (G) E2F6 mRNA injection inhibits smarcal1 mRNA expression in zebrafish embryos at 24 hpf but not at 48 hpf. Human SMARCAL1 mRNA (200 pg per embryo) was used as control. (H) Real time PCR analysis of mRNA levels in embryos in (G). The results represent three experiments. β-actin mRNA was measured as an internal control. Data are presented as means ± SE. ∗P < 0.05.