Figure 3.

USF2 binds to unmethylated E-box in the PDYN promoter CGI and activates gene transcription. (A) A putative CpG-containing TF binding sites (TF-BSs) in the PDYN promoter CGI identified by TRANSFAC. The CpG 12 is located in E-box sequence (CACGTG). (B and C) Methylation-sensitive DNA-binding factor interacting with the CGI E-box was identified by EMSA in nuclear extract of RFB (B) and human dlPFC (C). Labeled probe: unmethylated (UM) CGI E-box oligonucleotide. Competitors: UM- and methylated (M) CGI E-box oligonucleotides; and wild-type (E-box wt) and mutant (E-box mut) E-box oligonucleotides. Filled arrowhead shows the specific complex. (D and E) The CGI E-box-binding factor is identified as USF2 in RFB (D) and human dlPFC (E) in supershift and depletion experiment with antibodies against c-Myc, Max, Snai 1, USF1, and USF2. IgG, control IgG. Filled and open arrowheads show the specific and supershifted complex. (F) Binding of USF2 ectopically produced in SK-N-MC cells to the CGI E-box is inhibited by E-box methylation or hydroxymethylation. Labeled probes: E-box UM, M, or hydrohymethylated (HM) CGI oligonucleotides. ΔB-USF2, a dominant negative mutant lacking DNA-binding domain. (G and H) Luciferase reporter assays in SK-N-MC cells. Schematics of human PDYN promoter and luciferase reporter constructs are shown. Luc, luciferase coding sequence. Cells were transfected with USF2 (+) or ΔB-USF2 (ΔB) expressing vectors, or mock (−). RLU, relative light units normalized to “renilla” luciferase activity in (G) and to mock in (H). In (H), the unmethylated (UM) or methylated (M) CGI-wt inserted in pCpGL-basic, a CpG-free reporter vector, was transfected. (I) Expression of the endogenous PDYN gene is activated in SK-N-MC cells transfected with USF2- but not with ΔB-USF2-expressing vector, or mock transfected cells. The bar graphs show averages of 3 independent experiments and the errors of the means. *P < 0.05, **P < 0.01, ***P < 0.001.