Figure 4.

AP2α transactivates the Dnmt3a promoter by directly binding to a small proximal promoter region. (a) Schematic diagram of promoter segments of the murine Dnmt3a gene (−1.7 to +0.1 kb) inserted in the pGL3 basic luciferase vector. (b) A series promoter segments of the Dnmt3a gene were co-transfected with pRL-TK (Renilla) in 3T3-L1 preadipocytes at ci0 h. Results were expressed as firefly luciferase activity normalized to renilla luciferase activity. Error bars indicate S.D. (n=3). Statistical significance is indicated: ***P<0.001. (c) ChIP analysis of AP2α binding to the target region on the Dnmt3a promoter (−0.6 to −0.4 kb). Chromatin samples of 3T3-L1 preadipocytes at ci48 h were subjected to ChIP assays by using an AP2α antibody and normal rabbit IgG as a control. An upstream region (−1.1 to −0.9 kb) and a downstream region (−0.1 to +0.1 kb) in the Dnmt3a promoter were used as negative controls. (d) EMSA assay on the left panel (lane 1–12) showed the binding of endogenously expressed AP2α to the target region on the Dnmt3a promoter under the condition of biotin-labeled DNA probes and cold competitors. The right panel (lane 13–16) showed the supershift (SS) results of endogenously expressed AP2α in 3T3-L1 preadipocytes at ci48 h by anti-AP2α antibody. Addition of anti-AP2α antibody to the reaction resulted in the formation of the SS complex in lane 15. (e) Inactivation of the Dnmt3a promoter by mutating AP2α binding site in the Dnmt3a promoter. 3T3-L1 preadipocytes at ci0 h were co-transfected with a luciferase reporter plasmid of the Dnmt3a promoter (wt or mut) and pRL-TK. Results were expressed as firefly luciferase activity normalized to renilla luciferase activity. Error bars indicate S.D. (n=3). Statistical significance is indicated: ***P<0.001