Characterization of the human E2F4 promoter region and its response to 12- O -tetradecanoylphorbol-13-acetate

The E2F transcription factors (TFs), which control the progression of the cell cycle in response to DNA damage and various stresses, are known to interact with a tumour suppressor, Retinoblastoma 1 (RB1). The response of the human RB1 promoter to a 12-O-tetradecanoylphorbol-13-acetate (TPA) in HL-60 cells is mediated by a duplicated GGAA motif, which is also present in the 50-upstream of the E2F family genes. The motifs are especially rich in the 50-upstream of the E2F4 gene. In the present study, we constructed luciferase expression vectors containing a 466 bp of the 50-upstream of the human E2F4 gene. The transfection of this plasmid and deletion/mutation-introduced derivatives into HL-60 cells and a Luc reporter assay showed that duplicated and triplicated GGAA (TTCC) motifs in the E2F4 promoter respond to TPA. As expected, electrophoretic mobility shift assay indicated that SPI1 (PU.1) binds to the GGAA motif-containing element. A quantitative RT-PCR and western blotting showed that the E2F4 transcripts and its encoding proteins accumulate during the differentiation of HL-60 into macrophage-like cells. In contrast, the expression of the E2F1 gene and the protein, which possibly acts as a cell cycle accelerator, was greatly diminished.