Figure 6

Hes-1 binds to the GADD45α promoter and blockade of Hes-1 SUMOylation reduces Hes-1 binding to GADD45α and decreases Hes-1 suppression of GADD45αexpression. (A) pGL3-GADD45α-P: cloned GADD45α promoter. N-boxes and primers used for Hes-1 ChIP assay were shown. (B) ChIP PCR was performed using Hes-1 antibody (or mouse IgG) and the primer for GADD45α promoter. The PCR product (left panel) and the result of ChIP real-time PCR (right panel) were shown. (C) Flag-Hes-1WT, Flag-Hes-1 3KR or Flag-SUMO-1-fusioned Hes-1WT was transfected to cells for ChIP PCR with anti-Flag antibody and primers for GADD45α promoter. Input: PCR product from input lysate. (D) Different doses of Flag-Hes-1WT or Flag-Hes-1 3KR was co-transfected with pGL3-GADD45α-P to cells for promoter activity assay. Different doses of Flag-Hes-1WT or Flag-Hes-1 3KR was transfected to cells and GADD45α mRNA (E) and protein (F) expression were analyzed by real-time PCR and immunoblot respectively, and the quantified results were shown. (G) Flag-Hes-1WT or Flag-Hes-1 3KR was transfected to cells with or without H₂O₂ treatment followed by immunoblot against GADD45α and Flag. The quantified result is shown below. (H) Two sets of Hes-1 siRNA and 0.4 μg of pGL3-GADD45α-P were co-transfected to cells for promoter activity assay. Two sets of Hes-1 siRNA were transfected to cells with or without the addition of H₂O₂ for GADD45α mRNA (I) and protein (J) expression analysis by real-time PCR and immunoblot against GADD45α and Flag respectively. Data for luciferase activity and western blot are expressed as that in Figure 4. Values for mRNA measure are averaged from two independent experiments with mean value for the control group normalized to 1.0. Other values are normalized to the control value. Each experiment was performed twice. * p < 0.05, ** p < 0.01 and # p < 0.001.