Periodic cdc25C transcription is mediated by a novel cell cycle-regulated repressor element (CDE).

Abstract

We show that the cell cycle‐regulated transcription of the TATA‐less cdc25C gene in late S/G2 is largely mediated by a novel promoter element (CDE) located directly 5′ to one of the two major transcription initiation sites. Genomic dimethylsulfate footprinting experiments, using either synchronized or sorted normally cycling cells, show the formation in vivo of a CDE‐protein complex in both G0 and G1 cells and its dissociation in G2. Mutation of the CDE severely impairs cell cycle regulation of the cdc25C promoter and results in high expression in G0/G1, indicating that the CDE functions as a cell cycle‐regulated cis‐acting repressor element. Cell cycle regulation is also lost upon removal of the enhancer region located immediately upstream of the CDE, but is largely restored when this enhancerless minimal cdc25C promoter fragment is linked to the constitutive SV40 early enhancer. This indicates that the CDE is dependent on the presence of a transcriptional enhancer to effect cell cycle regulation. Our observations suggest that the periodic activation of the cdc25C gene in late S/G2 is brought about, at least in part, by a unique regulatory mechanism involving the cell cycle‐regulated dissociation of a repressor from the CDE.

The SV40 large T oncoprotein disrupts DNA-binding of the cell cycle-regulated transcriptional repressor CDF

“…This procedure yielded almost pure fractions of G 1 and G 2 cells (Figure 1). As previously reported (Lucibello et al, 1995), the CDE in cdc25C promoter was protected in WI38 cells in the G 1 phase, and this occupation is lost in G 2 (see insert in Figure 2). By contrast, in WI38VA13 cells the cdc25C CDE was unprotected in both G 1 and G 2 , although the interactions with constitutively bound NF-Y in the upstream region were una ected by expression of SV40 large T (Figure 2).…”

“…The increase in the normally cycling cells is presumably due to an e ect on the G 1 fraction (*60% of the cell population) where CDF-1 is active. Only a marginal e ect of SV-LT was seen with a reporter plasmid containing a mutated CDE (Lucibello et al, 1995;Zwicker et al, 1995b) (data not shown), indicating that CDF-1 is an essential target in the deregulation of the cdc25C promoter by the viral oncoprotein.…”

“…Another class of cell cycle genes is regulated by a mechanism of transcriptional repression that does not involve the transcription factor E2F. The proto-type of this group of genes is cdc25C (Lucibello et al, 1995), which is controlled by a novel repressor, termed CDF-1 (Liu et al, 1997). CDF-1 binds cooperatively to two contiguous elements, the CDE and CHR (Zwicker et al, 1995b) and seems to be unrelated to all known E2F and DP family members (Liu et al, 1997).…”

“…CDF-1 is also the major regulator of the cyclin A gene (Zwicker et al, 1995b). As shown by in vivo footprinting, occupation of the CDE and CHR elements is inversely correlated with promoter activity during the cell cycle, and mutations in either element lead to constitutive transcription (Lucibello et al, 1995;Zwicker et al, 1995b). It is however unclear what the e ect of viral oncoproteins on CDF-1 mediated repression is.…”

“…The cell cycle distributions were 79.6+2.7% G 0 /G 1 and 20.4+2.6% S/G 2 for cell transfected with the SV-LT expression vector, and 79.5+2.9% G 0 /G 1 and 20.5+2.9% S/G 2 Figure 1 FACS analysis of WI38 and WI38VA13 cells and sorted G 1 and G 2 fractions after staining with Hoechst 33258. The trypsinized cells were stained with Hoechst 33258 and sorted according to DNA content using a Becton-Dickinson FACStar Plus (Lucibello et al, 1995) Figure 2 Genomic DMS footprinting of the cdc25C promoter in sorted G 1 and G 2 fractions of WI38VA13 cells. The positions of the CDE and the NF-Y binding sites (Lucibello et al, 1995;Zwicker et al, 1995a) are indicated.…”