Cellular Targets for Activation by the E2F1 Transcription Factor Include DNA Synthesis- and G₁/S-Regulatory Genes

Abstract

Although a number of transfection experiments have suggested potential targets for the action of the E2F1 transcription factor, as is the case for many transcriptional regulatory proteins, the actual targets in their normal chromosomal environment have not been demonstrated. We have made use of a recombinant adenovirus containing the E2F1 cDNA to infect quiescent cells and then measure the activation of endogenous cellular genes as a consequence of E2F1 production. We find that many of the genes encoding S-phase-acting proteins previously suspected to be E2F targets, including DNA polymerase alpha, thymidylate synthase, proliferating cell nuclear antigen, and ribonucleotide reductase, are indeed induced by E2F1. Several other candidates, including the dihydrofolate reductase and thymidine kinase genes, were only minimally induced by E2F1. In addition to the S-phase genes, we also find that several genes believed to play regulatory roles in cell cycle progression, such as the cdc2, cyclin A, and B-myb genes, are also induced by E2F1. Moreover, the cyclin E gene is strongly induced by E2F1, thus defining an autoregulatory circuit since cyclin E-dependent kinase activity can stimulate E2F1 transcription, likely through the phosphorylation and inactivation of Rb and Rb family members. Finally, we also demonstrate that a G1 arrest brought about by gamma irradiation is overcome by the overexpression of E2F1 and that this coincides with the enhanced activation of key target genes, including the cyclin A and cyclin E genes.

A role for Myc in facilitating transcription activation by E2F1

“…As shown in Figure 1b, ChIP analysis revealed that Myc bound to the E2F1, E2F2 and E2F3 promoters as early as 4 h following serum stimulation and persisted on these promoters through S phase (16 h). E2F1 protein was detected on the E2F1, E2F2 and E2F3 promoters at 12 and 16 h following serum stimulation, times corresponding to the previously documented initiation of E2F mRNA and protein accumulation (DeGregori et al, 1995;Leone et al, 1998). Examination of the levels of Myc and E2F1 in these nuclear fractions by western blot revealed that the patterns of Myc and E2F1 binding to E2F promoters coincided with the kinetics of protein accumulation for both proteins (Figure 1c).…”

Expression of thymidylate synthase in human cells is an early G1 event regulated by CDK4 and p16INK4A but not E2F

“…Previous reports using mouse and rat embryonic fibroblasts showed that ectopic expression of E2F1 and E2F2 leads to an increase in TS transcripts (DeGregori et al, 1995;Ishida et al, 2001). To further characterise the relationship between key effectors of G 1 /S transition and the regulation of human TS transcription, we infected HCT116 and MCF-7 cells with recombinant adenoviruses expressing E2F1, Dp1 and cyclin E. Western blot analysis revealed that, 24 h post-infection, both cyclin E and E2F1 were over-expressed in both cell lines ( Figure 4A and C).…”

“…As G 1 progresses, pRb is phosphorylated by these complexes (Weinberg, 1995;Blagosklonny and Pardee, 2002), ultimately leading to E2F release. E2F transcription factors play an essential role in cell-cycle progression and are directly involved in the regulation of genes required for the G 1 /S transition as well as genes related to DNA synthesis (DeGregori et al, 1995). E2F can bind DNA as a homodimers or heterodimer containing the co-activator Dp1 to activate the transcription of target genes such as cyclin E. Newly synthesised cyclin E then forms a complex with CDK2 to drive Sphase entry (Sauer and Lehner, 1995;Woo and Poon, 2003).…”

Some p53-binding proteins that can function as arbiters of life and death

“…Interestingly, the expression of E2F1-3 and cyclin A are precisely regulated during the cell cycle (reviewed in Loughan and La Thangue 6 ). The levels of E2F are high at the G 1 /S transition, 14,15 whereas cyclin A is high at mid-S to G 2 /M. 16 As cyclin A competes with p53 for binding to E2F1-3, 8 E2F has a window of opportunity to regulate p53 at the G 1 /S transition when E2F1-3 levels are high and the cyclin A and Ser315-phosphorylated p53 are relatively low.…”