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

In quiescent cells, cdc2 mRNA is almost undetectable. Stimulation of c ells to reenter the cell cycle results in induction of cdc2 expression , beginning at the G(1)-to-S transition and reaching maximum levels du ring late S and G(2) phases. To investigate cdc2 transcriptional regul ation throughout cell cycle progression, we monitored protein-DNA inte ractions by in vivo footprinting along 800 bp of the human cdc2 promot er in quiescent fibroblasts and at different time points following ser um stimulation. We found 11 in vivo protein-binding sites, but no prot ein binding was observed at a high-affinity E2F site that had previous ly been implicated in cdc2 regulation, Nine of the identified in vivo binding sites (among them were two inverted CCAAT boxes, two Spl sites , and one ets-2 site) bind transcription factors constitutively throug hout the cell cycle, However, gt 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 G(0)- and G(1)-specific protein complex disappears at the -20 element at the beg inning of S phase, This sequence deviates at one base position from kn own E2F consensus binding sites, We found that the major E2F activity in human fibroblasts contains E2F-4 and p130, The -20 element of the c dc2 gene specifically interacts with a subset of E2F-4-p130 complexes present in G(0) cells but does not interact with S-phase-specific E2F complexes. Transient-transfection experiments with wild-type and mutan t cdc2 promoter constructs indicate that the -20 element is involved i n suppressing cdc2 activity in quiescent cells. We suggest that the pr esence of the p130-E2F-4 complex in G(0)/G(1) blocks access of compone nts of the basal transcription machinery or prevents transactivation b y the constitutively bound upstream activator proteins.