Figure 7
Multiple downstream transcription factors contribute to transcriptional activation of UHRF1 and DNMT1 by the MEK/ERK pathway. (a,b) The diagrams illustrating the putative binding sites for transcription factors AML1a, E2F1, SP1, STAT3 and YY1 within the region of −5kb and + 3 kb around the transcriptional start site of UHRF1 gene (a) and DNMT1 gene (b). (c) qRT-PCR analysis showing efficient knockdown of corresponding transcription factors in HCT116 cells by specific siRNA against AML1a, E2F1, SP1, STAT3 and YY1, respectively. (d) qRT-PCR analysis showing the effect on UHRF1 and DNMT1 expression in HCT116 cells upon knockdown of each of the transcription factors. (e,f) ChIP analysis showing the effect of PD0325901 treatment on the binding of E2F1 and SP1 to two regions within the UHRF1 promoter (e) or DNMT1 promoter (f). IgG, control immunoglobulin. (g,h). Luciferase reporter assay showing that E2F1 and SP1 markedly enhanced transcriptional activation from the UHRF1 and DNMT1 promoters. The control PGL3-luc and UHRF1-luc (g) or DNMT1-luc (h) were cotransfected with or without an E2F1 or SP1 expression plasmid into HCT116 cells and treated with or without PD0325901 as indicated. Also shown were western blotting data for corresponding samples.
