Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol alpha expression during early S phase

E2F-1, a transcription factor implicated in the activation of genes require d for S phase such as DNA pol alpha, is regulated by interactions with Rb a nd by cell-cycle dependent alterations in E2F-1 abundance. We have shown th at depletion of poly(ADP-ribose) polymerase (PARP) by antisense RNA express ion downregulates pol alpha and E2F-1 expression during early S phase. To e xamine the role of PARP in the regulation of pol alpha and E2F-1 gene expre ssion, we utilized immortalized mouse fibroblasts derived from wild-type an d PARP knockout (PARP-/-) mice as well as PARP-/- cells stably transfected with PARR cDNA [PARP-/-(+PARP)]. After release from serum deprivation, mild -type and PARP-/-(+PARP) cells, but not PARP-/- cells, exhibited a peak of cells in S phase by 16 h and had progressed through the cell cycle by 22 h, Whereas [H-3]thymidine incorporation remained negligible in PARP-/- cells, in vivo DNA replication maximized after 18 h in mild-type and PARP-/-(+PAR P) cells, To investigate the effect of PARP on E2F-1 promoter activity, a c onstruct containing the E2F-1 gene promoter fused to a luciferase reporter gene was transiently transfected into these cells, E2F-1 promoter activity in control and PARP-/-(+PARP) cells increased eightfold after 9 h, but not in PARP-/- cells. PARP-/- cells did not show the marked induction of E2F-1 expression during early S phase apparent in control and PARP-/-(+PARP) cell s. RT-PCR analysis and pol alpha activity assays revealed the presence of p ol alpha transcripts and a sixfold increase in activity in both wildtype an d PARP-/- (+PARP) cells after 20 h, but not in PARP-/- cells. These results suggest that PARP plays a role in the induction of E2F-1 promoter activity , which then positively regulates both E2F-1 and pol alpha expression, when quiescent cells reenter the cell cycle upon recovery from aphidicolin expo sure or removal of serum.