Figure 2

Hes1 interacts with δ-catenin promoter. (A) EMSA to show direct binding of Hes1 to δ-catenin promoter. Lane 1, control (labeled δ-catenin promoter probe spanning the HLH motif, marked as ** in the Figure 1A); lane 2, labeled probe with WT-Hes1 overexpressed nuclear extracts, forming DNA-protein complexes (indicated by arrow); lane 3, unlabeled probe with E-box mutated, showing DNA-protein complexes; lane 4, unlabeled competitor + nuclear extracts + labeled probe, low dose unlabeled probe reduced the binding of protein-DNA complexes; lane 5, unlabeled competitor + nuclear extracts + labeled probe, high dose unlabeled probe inhibited the binding of protein-DNA complexes; lane 6-7, gel shift immune-assays were performed with the same labeled probe using an anti-Hes1 antibody. Incubating Hes1-overexpressed nuclear extracts with anti-Hes1 antibody before the addition of probe DNA inhibited protein-DNA complexes (lane 6). Incubating Hes1-overexpressed nuclear extracts with anti-Hes1 antibody after the addition of probe DNA disrupted the slowest moving protein-DNA complexes (lane 7, arrow). However, under this experimental condition, supershifts did not occur but the partially disrupted protein-DNA complex resulting fast moving band accumulation can be detected (lane 7, double arrows). (B) Anti-Hes1 chromosome immunoprecipitation (ChIP) of the δ-catenin promoter. Real-time qRT-PCR showed the signal relative to input chromatin. Compared with the negative control experiment using IgG, anti-Hes1 greatly recruited the δ-catenin promoter DNA. Anti-Histone H3 ChIP was performed as a positive control. *: p < 0.05. (C) Hes1 binding to E-boxes on δ-catenin promoter is important to negatively regulate E2F1 induced δ-catenin-luciferase reporter activity. Note: E2F1 expression elicited a strong δ-catenin-luciferase reporter activity in PC3 cells, whatever BK5 sequence was mutated (*p > 0.05). But when WT-Hes1 co-transfection with E2F1, it significantly suppressed E2F1 induced δ-catenin-luciferase reporter activity when wild type δ-catenin promoter sequence BK5 was employed compared to BK5 sequence mutated vector (*p < 0.05).