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

Hiroshi Hamada; Yuta Goto; Jun Arakawa; Erisa Murayama; Yui Ogawa; Midori Konno; Takahiro Oyama; Masashi Asai; Akira Sato; Sei-Ichi Tanuma; Fumiaki Uchiumi

doi:10.1093/jb/mvz047

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

J Biochem. 2019 Oct 1;166(4):363-373. doi: 10.1093/jb/mvz047.

Authors

Hiroshi Hamada¹, Yuta Goto¹, Jun Arakawa¹, Erisa Murayama¹, Yui Ogawa¹, Midori Konno¹, Takahiro Oyama^{2

3}, Masashi Asai¹, Akira Sato³, Sei-Ichi Tanuma^{3

4}, Fumiaki Uchiumi¹

Affiliations

¹ Department of Gene Regulation, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda-shi, Chiba-ken, Japan.
² Hinoki Shinyaku Co., Ltd, 9-6 Nibancho, Chiyoda-ku, Tokyo, Japan.
³ Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda-shi, Chiba-ken, Japan.
⁴ Genomic Medical Science, Research Institute of Science and Technology, Tokyo University of Science, Noda-shi, Chiba-ken, Japan.

PMID: 31199460
DOI: 10.1093/jb/mvz047

Abstract

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). We previously showed that 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 5'-upstream of the E2F family genes. The motifs are especially rich in the 5'-upstream of the E2F4 gene. In the present study, we constructed luciferase (Luc) expression vectors containing a 466 bp of the 5'-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.

Keywords: E2F; ETS; HL-60; TPA; differentiation.