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Molecular and Cellular Biology, December 1999, p. 8393-8399, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Coexamination of Site-Specific Transcription Factor
Binding and Promoter Activity in Living Cells
Kathryn E.
Boyd
and
Peggy J.
Farnham*
McArdle Laboratory for Cancer Research,
University of Wisconsin Medical School, Madison, Wisconsin 53706
Received 15 July 1999/Returned for modification 27 August
1999/Accepted 20 September 1999
Previously, we have used a chromatin cross-linking and
immunoprecipitation protocol for the analysis of Myc and USF binding to
the cad promoter. The adaptation of this technique for the study of mammalian transcription factors was a big step
forward in the analysis of transcription factor family member
specificity, allowing for the first time a definitive knowledge of
which factor binds to a promoter region under normal
physiological conditions. However, due to limitations of the assay,
our previous studies could not definitively prove that both Myc and USF
bound to the exact same site on the cad promoter, nor could
we directly correlate loss of in vivo binding of a particular factor
with loss of transcriptional activity. Therefore, we have further
modified the chromatin immunoprecipitation protocol to alleviate these
problems. We have now shown that it is possible to coexamine
growth-regulated transcriptional activity and promoter occupancy by
using stably integrated promoter constructs. We show that both Myc and
USF bind to the exact same E box on the cad promoter,
suggesting that competition between these two factors for a single site
occurs in living cells. We also find that cad promoter
constructs that retain USF binding but lose Myc binding in vivo no
longer display an increase in transcriptional activity in mid- to late
G1 phase of the cell cycle. Finally, we propose that cell
cycle-regulated transcriptional activation of the cad
promoter may be a stochastic, rather than a predetermined, process.
*
Corresponding author. Mailing address: 1400 University
Ave., McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, WI 53706. Phone: (608) 262-2071. Fax: (608) 262-2824. E-mail:
farnham{at}oncology.wisc.edu.

Present address: Department of Pathology, Yale University, New
Haven,
Conn.
Molecular and Cellular Biology, December 1999, p. 8393-8399, Vol. 19, No. 12
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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