DNA-binding E2F complexes have been identified throughout the mammalia
n cell cycle, including the transcriptionally inactive complexes with
pocket proteins, which occur early in the prereplicative G(1) phase of
the cycle, and the transactivating free E2F, which increases in late
G(1). Here, a regulatory B-myb promoter site was shown to bind with hi
gh affinity to free E2F and to E2F-pocket protein complexes in an indi
stinguishable way in vitro. In contrast, in vivo footprinting with NIH
3T3 cells demonstrated E2F site occupation specifically in early G(1)
, when the B-myb promoter is inactive. These observations indicate tha
t a novel mechanism governs E2F-DNA interactions during the cell cycle
and emphasize the relevance of E2F site-directed transcriptional repr
ession.