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Molecular and Cellular Biology, August 2000, p. 5797-5807, Vol. 20, No. 16
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Target Gene Specificity of E2F and Pocket Protein Family Members in Living Cells

Julie Wells, Kathryn E. Boyd,dagger Christopher J. Fry,Dagger Stephanie M. Bartley, and Peggy J. Farnham*

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706

Received 30 November 1999/Returned for modification 19 January 2000/Accepted 6 May 2000

E2F-mediated transcription is thought to involve binding of an E2F-pocket protein complex to promoters in the G0 phase of the cell cycle and release of the pocket protein in late G1, followed by release of E2F in S phase. We have tested this model by monitoring protein-DNA interactions in living cells using a formaldehyde cross-linking and immunoprecipitation assay. We find that E2F target genes are bound by distinct E2F-pocket protein complexes which change as cells progress through the cell cycle. We also find that certain E2F target gene promoters are bound by pocket proteins when such promoters are transcriptionally active. Our data indicate that the current model applies only to certain E2F target genes and suggest that Rb family members may regulate transcription in both G0 and S phases. Finally, we find that a given promoter can be bound by one of several different E2F-pocket protein complexes at a given time in the cell cycle, suggesting that cell cycle-regulated transcription is a stochastic, not a predetermined, process.

* Corresponding author. Mailing address: McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, WI 53706. Phone: (608) 262-2071. Fax: (608) 262-2824. E-mail: farnham{at}oncology.wisc.edu.

dagger Present address: Department of Pathology, Yale University School of Medicine, New Haven, CT 06520.

Dagger Present address: Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, MA 01605.

Molecular and Cellular Biology, August 2000, p. 5797-5807, Vol. 20, No. 16
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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