A protein synthesis-dependent increase in E2F1 mRNA correlates with growth regulation of the dihydrofolate reductase promoter.

Abstract

Enhanced expression of genes involved in nucleotide biosynthesis, such as dihydrofolate reductase (DHFR), is a hallmark of entrance into the DNA synthesis (S) phase of the mammalian cell cycle. To investigate the regulated expression of the DHFR gene, we stimulated serum-starved NIH 3T3 cells to synchronously reenter the cell cycle. Our previous results show that a cis-acting element at the site of DHFR transcription initiation is necessary for serum regulation. Recently, this element has been demonstrated to bind the cloned transcription factor E2F. In this study, we focused on the role of E2F in the growth regulation of DHFR. We demonstrated that a single E2F site, in the absence or presence of other promoter elements, was sufficient for growth-regulated promoter activity. Next, we showed that the increase in DHFR mRNA at the G1/S-phase boundary required protein synthesis, raising the possibility that a protein(s) lacking in serum-starved cells is required for DHFR transcription. We found that, similar to DHFR mRNA expression, levels of murine E2F1 mRNA were low in serum-starved cells and increased at the G1/S-phase boundary in a protein synthesisdependent manner. Furthermore, in a cotransfection experiment, expression of human E2F1 stimulated the DHFR promoter 22-fold in serum-starved cells. We suggest that E2F1 may be the key protein required for DHFR transcription that is absent in serum-starved cells. Expression of E2F also abolished the serumstimulated regulation of the DHFR promoter and resulted in transcription patterns similar to those seen with expression of the adenoviral oncoprotein ElA. In summary, we provide evidence for the importance of E2F in the growth regulation of DHFR and suggest that alterations in the levels of E2F may have severe consequences in the control of cellular proliferation. A commonly employed model system for studying reentry into the proliferative state is serum starvation and subsequent stimulation of fibroblasts in culture. Addition of high concentrations of serum or growth factors to quiescent cells leads to a synchronously growing population in which transient activation and repression of growth-responsive genes can be monitored. Between 15 min and 4 h after serum stimulation, the immediate-early genes are expressed. These genes are transcribed in the presence of protein synthesis inhibitors, indicating that preexisting factors in quiescent cells are sufficient for activation of transcription (45). The early-response gene products include transcription factors such as Fos and EGR2. Several hours later (8 to 12 h after serum stimulation), a wave of gene expression requiring protein synthesis occurs at the transition from G, to DNA synthesis (S) phase. This protein synthesis requirement suggests that factors other than those present in quiescent cells are necessary for activation of transcription. Many late-serum-response gene products, such as DNA polymerase alpha, thymidine kinase, thymidylate synthase, carbamoyl phosphate synthase-aspartate carbamoyltransferasedihydrooro...

Life and death decisions by E2F-1

“…22 Subsequent studies have shown that E2F controls the transcription of cellular genes that are essential for cell division, such as enzymes involved in the biosynthesis of nucleotides. 23 In the 1990s, Nevins et al 24 deduced how E2F is regulated in normal cells by determining the mechanism of E2F activation by E1A. E1A caused a cellular protein to dissociate from E2F, which led them to show that E2F is inhibited by its association with Rb.…”

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

“…To investigate further the mechanism by which PARP contributes to regulation of pol a gene transcription during early S phase, we examined the e ect of PARP depletion by gene knockout on the abundance of E2F-1, a transcription factor that positively regulates the transcription of several genes whose products are required for DNA replication and cell growth; these genes include those encoding pol a, PCNA, dihydrofolate reductase, thymidine kinase, c-MYC, c-MYB, cyclin D, cyclin E, and E2F-1 itself (Blake and Azizkhan, 1989;DeGregori et al, 1995;Nevins, 1992;Pearson et al, 1991;Slansky et al, 1993). We previously demonstrated that PARP depletion by antisense RNA expression in 3T3-L1 cells prevents induction of E2F-1 expression during the early stages of di erentiation-linked DNA replication (SimbulanRosenthal et al, 1998a).…”

K-ras modulates the cell cycle via both positive and negative regulatory pathways

“…The transcriptional activity of members of the E2F family of transcription factors is regulated by multiple cell cycle-related events, such as increased expression (transcriptionally or post-transcriptionally) (Slansky et al, 1993), phosphorylation (for E2F-1, 2 and 3) by cyclin A/cdk2 complexes (Xu et al, 1994) and complex formation with pRb or pRb-related proteins p107 and p130. Therefore, increased expression of the E2F protein may not necessarily lead to increased transcriptional activity.…”