Identification and Characterization of Novel Isoforms of Human DP-1 DP-1α REGULATES THE TRANSCRIPTIONAL ACTIVITY OF E2F1 AS WELL AS CELL CYCLE PROGRESSION IN A DOMINANT-NEGATIVE MANNER

Abstract The cell cycle-regulating transcription factors DP-1 and E2F form a heterodimeric complex and play a central role in cell cycle progression. Two different DP subunits (DP-1 and DP-2) exist in humans. In this study, we identified two novel DP-1 isoforms (DP-1α and DP-1β) and characterized their structure and function. DP-1α is composed of 278 amino acids and lacks a portion of the C-terminal heterodimerization domain, whereas DP-1β is composed of 357 amino acids with a frameshift that causes truncation of the C-terminal domain. Yeast two-hybrid and immunoprecipitation assays demonstrated that DP-1α binding to E2F1 was significantly reduced as compared with that of wild-type DP-1 or DP-1β. Immunofluorescence analysis revealed that the subcellular localization of both DP-1 isoforms changed from the cytoplasm to the nucleus in HEK 293 cells cotransfected with E2F1 and wild-type DP-1 or DP-1β. However, such a translocation for DP-1α was barely observed. Reverse transcription-PCR results showed that the three DP-1 isoforms are expressed ubiquitously at equal levels in several normal human tissues. We also demonstrated the expression of these isoforms at the protein level by Western blotting. Interestingly, we observed a significant decrease in transcriptional activity, a marked delay of cell cycle progression, and an inhibition of cell proliferation in DP-1α-transfected HEK 293 cells. Together, the results of the present study suggest that DP-1α is a novel isoform of DP-1 that acts as a dominant-negative regulator of cell cycle progression.