In Vivo Structure of the Human cdc2 Promoter: Release of a p130–E2F-4 Complex from Sequences Immediately Upstream of the Transcription Initiation Site Coincides with Induction of cdc2 Expression

Abstract

In quiescent cells, cdc2 mRNA is almost undetectable. Stimulation of cells to reenter the cell cycle results in induction of cdc2 expression, beginning at the G1-to-S transition and reaching maximum levels during late S and G2 phases. To investigate cdc2 transcriptional regulation throughout cell cycle progression, we monitored protein-DNA interactions by in vivo footprinting along 800 bp of the human cdc2 promoter in quiescent fibroblasts and at different time points following serum stimulation. We found 11 in vivo protein-binding sites, but no protein binding was observed at a high-affinity E2F site that had previously been implicated in cdc2 regulation. Nine of the identified in vivo binding sites (among them were two inverted CCAAT boxes, two Sp1 sites, and one ets-2 site) bind transcription factors constitutively throughout the cell cycle. However, at two elements located at positions -60 and -20 relative to the transcription start site, the binding pattern changes significantly as the cells are entering S phase. A G0- and G1-specific protein complex disappears at the -20 element at the beginning of S phase. This sequence deviates at one base position from known E2F consensus binding sites. We found that the major E2F activity in human fibroblasts contains E2F-4 and p130. The -20 element of the cdc2 gene specifically interacts with a subset of E2F-4-p130 complexes present in G0 cells but does not interact with S-phase-specific E2F complexes. Transient-transfection experiments with wild-type and mutant cdc2 promoter constructs indicate that the -20 element is involved in suppressing cdc2 activity in quiescent cells. We suggest that the presence of the p130-E2F-4 complex in G0/G1 blocks access of components of the basal transcription machinery or prevents transaction by the constitutively bound upstream activator proteins.

p130/E2F4 Binds to and Represses the cdc2 Promoter in Response to p53

“…The CDE in the cdc2 promoter overlaps a putative E2F-binding site. Binding of p130/E2F4 to this element was correlated with repression of the promoter in G 0 -arrested cells (54). The factor CDF-1 can also bind to the CDE/CHR elements of the cdc2 promoter (56).…”

“…To detect proteins binding to the cdc2 R box, gel mobility shift assays were carried out as described previously, with an oligonucleotide spanning this region (54). Briefly, cells were lysed for 20 min on ice in buffer I, containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl 2 , 10 mM KCl, 0.3 M sucrose, 0.1 mM EGTA, 0.5% Nonidet P-40, 0.5 mM DTT, 0.5 mM PMSF, FIG.…”

Cell Cycle Regulation of the Human Polo-like Kinase (PLK) Promoter

“…In late G 1 , transcription of E2F-regulated genes such as DHFR, E2F-1, and DNA polymerase is activated following dissociation of E2F/DP complexes from RB (15)(16)(17). Similarly, transcription of cdc2 is repressed during G 1 by binding of E2F-4-p130 complexes at the CHR/CDE region (18).…”

The E2F–Cdc2 Cell-Cycle Pathway Specifically Mediates Activity Deprivation-Induced Apoptosis of Postmitotic Neurons

“…Members of the E2F family of transcription factors bind the EBE in proliferating cells (Tommasi and Pfeifer, 1995;Takahashi et al, 2000;Rayman et al, 2002). We therefore tested whether the EBE within the cdc2 promoter binds to an E2F family member in granule neurons.…”