FIGURE 6.

HCF-1 is required for MLL5 recruitment to E2F1-responsive promoters. A, knockdown of HCF-1 in HeLa cells leads to down-regulation of E2F1 target genes. Two different lentivirus-based shRNAs target to an mRNA sequence starting 889 and 2018 bp downstream of the translation start site were used to knockdown HCF-1 expression in HeLa cells. Whole cell lysates were harvested at day 6 post-infection. The expression of endogenous HCF-1, E2F1, CYCLIN A, CDC2, and CDC6 were detected by polyclonal antibodies. Actin was used as a loading control. WB, Western blot. B, defective cell proliferation in HCF-1 knockdown cells. 1 × 10⁴ HeLa cells were seeded in 48-well plates at day 1, at each indicated time point, triplicates of cells from each group were harvested and counted in a hemocytometer. C, binding of the MLL5 protein to E2F1 target promoters were markedly reduced in HCF-1 knockdown cells. 3× FLAG-tagged MLL5 were transiently transfected in HEK293T cell lines that have been infected with lentivirus-based HCF-1 shRNA or scramble control, and the cells were harvested 72 h later. ChIP analyses were performed with an anti-FLAG antibody in HEK293T previously infected with lentivirus-based scramble control or HCF-1 shRNA. Two-tailed unpaired Student's t tests were performed, * p < 0.05.