Regulation of <i>DSCC1</i> promoter activity by E2F transcription factor.

<p>(A) Nucleotide sequence of the −10 to +90 bp region human <i>DSCC1</i>. Three putative E2F binding motifs are underlined. (B) pDSCC1-1023/+109 was transiently transfected with pRL-TK and pcDNA3 HA-E2Fs into SW480, or with pRL-TK and pcDNA3 HA-E2F1 (0.01-1 µg) into SW480 and HCT116 cells. (C) pDSCC1-10/+109 or the shorter promoter constructs was transfected with E2F1 or the empty vector into SW480 cells. (D) Site-directed mutation analysis of putative E2F binding sites in the proximal promoter region. pDSCC1-10/+109 or its mutant clones was transfected with E2F1 or the empty vector into SW480 cells. The data represents mean ± SD from three independent experiments. Promoter activity indicates the relative luciferase unit or the fold induction over empty vector transfectant. (E) Chromatin immunoprecipitation was performed using anti-E2F1 antibody. The precipitated DNAs were subjected to the amplification of <i>DSCC1</i> promoter by quantitative PCR. To ascertain the specific binding to the EBS, the amplification of a distal upstream region in the <i>DSCC1</i> promoter was used for normalization. A significant difference was determined by t-test. (F) HCT116 cells were transfected with control or E2F1 siRNA (25 nM) for 48 h. <i>DSCC1</i> expression was detected by quantitative PCR. A significant difference was determined by t-test. (G) SW480 cells were transfected with control or E2F1 siRNA (25 nM), followed 8 h later by transfection with reporter plasmid (pDSCC1-10/+109) and E2F1 expression vector or the empty vector. After 48 h, luciferase activity was measured. The data represents mean ± SD from three independent experiments.