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(Received for publication, July 24, 1996, and in revised form, November 2, 1996)
From the Previously genomic DNase I footprinting showed
changes in protein binding to two overlapping E2F sites correlates with
activation of dhfr gene expression at the G1/S
boundary of the Chinese hamster cell cycle (Wells, J., Held, P.,
Illenye, S., and Heintz, N. H. (1996) Mol. Cell. Biol. 16, 634-647). Here gel mobility and antibody supershift assays were used
to relate changes in the components of E2F DNA binding complexes in
cell extracts to repression and induction of dhfr gene
expression. In extracts from log phase cells, E2F complexes contained
predominantly E2F-4 and E2F-2 in association with DP-1, and DNA binding
assays showed complexes containing E2F-2 preferentially interact with
only one of the two overlapping E2F sites. In serum
starvation-stimulation experiments, arrest in G1 by low
serum was accompanied by decreased levels of dhfr mRNA
and the appearance of an E2F-4·DP-1·p130 complex. After serum
stimulation, induction of dhfr gene expression was preceded
by loss of the p130 complex in mid G1 and coincided with marked increases in two free E2F·DP-1 complexes in late
G1, one of which contained E2F-4 and a second which
contained an unidentified E2F. We suggest activation of
dhfr gene expression after serum stimulation requires at
least two temporally distinct processes, relief of p130-mediated
repression and subsequent activation of transcription by free E2F.
Volume 272, Number 7,
Issue of February 14, 1997
pp. 4483-4492
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
§
,
,
,
Department of Pathology,
§ Program in Cell and Molecular Biology, University of
Vermont College of Medicine, Burlington, Vermont 05405 and the
** Laboratory of Molecular Oncology, Massachusetts General Hospital
Cancer Center, Charlestown, Massachusetts 02129
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