Cell Cycle Regulation of the Murine Cyclin E Gene Depends on an E2F Binding Site in the Promoter

Abstract

Cyclin E controls progression through the G 1 phase of the cell cycle in mammalian fibroblasts and potentially in many other cell types. Cyclin E is a rate-limiting activator of cdk2 kinase in late G 1 . The abundance of cyclin E is controlled by phase-specific fluctuations in the mRNA level; in mammalian fibroblasts, mRNA is not detected under conditions of serum starvation and is accumulated upon serum stimulation, with expression starting in mid-G 1 . Here, we report the cloning of the murine cyclin E promoter. We isolated a 3.8-kb genomic fragment that contains several transcriptional start sites and confers cell cycle regulation on a luciferase reporter gene. This fragment also supports transcriptional activation by adenovirus E1A, a known upstream regulator of cyclin E gene expression. An E2F binding site which is required for G 1 -specific activation of the cyclin E promoter in synchronized NIH 3T3 cells was identified in this fragment.Regulation of cell cycle progression in mammalian cells depends on the sequential activation of a series of cyclin-dependent kinases. The catalytic activity of a given cyclin-dependent kinase subunit depends on its correct posttranslational modification and on its association with the appropriate cyclin, which acts as a regulatory subunit (reviewed in reference 28). Thus, phase-specific fluctuations in the abundance of various cyclins constitute one mechanism by which the timing of cyclindependent kinase activation is controlled. It was shown that phase-specific transcriptional activation of cellular genes contributes to the control of cell cycle progression; in particular, heterodimeric transcription factor E2F/DP (referred to as E2F [17]) mediates the activation of several cellular genes at the G 1 -to-S transition (27). More recently, it was shown that E2F binding sites determine activation at the G 1 -to-S transition of the genes encoding E2F-1 (9, 11), a subunit of the E2F/DP heterodimer, and cyclin A (26), a key regulator of the G 1 -to-S transition (24, 33), indicating a regulatory link between the ability of a cell to enter S phase and the appropriate activation of E2F during G 1 .Cyclin E (13, 18) is expressed in mid-G 1 ; it associates with and activates cdk2 kinase (6,14). Conditional overexpression of cyclin E results in a decrease in the length of the G 1 interval, consistent with an acceleration of the G 1 -to-S transition (25, 31). Microinjection of anti-cyclin E antibodies during G 1 inhibits entry into S phase in normal human fibroblasts (22), and genetic evidence suggests that cyclin E controls S phase entry during early embryogenesis in Drosophila melanogaster (12). These findings strongly suggest that cyclin E and its associated kinase are centrally involved in the decision to enter S phase in most if not all eukaryotic cells. Cyclin E mRNA is absent from serum-starved normal fibroblasts and appears in mid-G 1 (13). Regulation of cyclin E gene expression appears to be involved in the transformation of mammalian cells by viral oncogenes, since a rapid ind...

[Lys61]N-Ras is able to induce full activation and nuclear accumulation of Cdk4 in NIH3T3 cells

“…The presence of oncogenic Ras does not alter the amount of cyclin E either in absence or in presence of FCS. Since the cyclin E promoter contains E2F binding sites (Botz et al, 1996), the lack of induction of cyclin E expression by oncogenic Ras is consistent with the ®nding that Ras cannot inactivate pRb. Our results show that in serum-starved cells cdk2 is associated with cyclin E and that this association increases 9 h after serum stimulation.…”

“…The hypophosphorylated form of these proteins binds the transcription factor E2F, and upon phosphorylation, E2F is released and can activate transcription of S phase-speci®c genes (Draetta, 1994;Lam and La Thangue, 1994;Mittnacht, 1998). Among the genes containing E2F-binding sites in their promoter regions there are those coding for enzymes and proteins that are essential for DNA replication such as DNA polymerase a, thymidin kinase, ribonucleotide reductase, cyclin A and cyclin E (Botz et al, 1996;Dalton, 1992;Dou et al, 1994;Geng et al, 1996;Zwicker et al, 1995). The activation of cyclin D/cdk4 and phosphorylation of pRb is thus a limiting step for G1 progression.…”

The AP-1 transcription factor regulates breast cancer cell growth via cyclins and E2F factors

“…Cyclin E is a direct target gene of E2F1 (DeGregori et al, 1995;Botz et al, 1996). We found that Tam67 suppressed cyclin E mRNA at all time points examined, showing a 30-60% suppression (P ¼ 0.0005) (Figures 5a and b).…”

Malignant transformation by cyclin E and Ha-Ras correlates with lower sensitivity towards induction of cell death but requires functional Myc and CDK4

“…The promotor of the cyclin E gene has been isolated and analysed by several groups and was shown to contain multiple potential E2F binding sites. Transcriptional regulation indeed depends on the presence of these E2F binding sites and on the presence and function of Rb (Botz et al, 1996;Geng et al, 1996;Ohtani et al, 1995). In addition to the regulation of cyclin E or cyclin D expression through the modulation of the promotor, the enzymatic activity of D-and E-type cyclin/CDK complexes is regulated independently on a posttranscriptional level.…”