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Submitted on July 2, 2004
Accepted on October 22, 2004
Department of Molecular Cell Biology, Department of Medicine, Baylor Breast Center, Baylor College of Medicine, Houston TX
* To whom correspondence should be addressed. E-mail: pbrown{at}breastcenter.tmc.edu.
There is a growing body of literature supporting estrogen's ability to affect gene expression through a "non-classical" pathway, in which ER modulates the activity of other transcription factors such as AP-1, Sp-1 or NF-
B. We hypothesized that many estrogen-induced genes are dependent on AP-1 for their expression and that these genes can be identified using genomic strategies. Using cells expressing an inducible cJun dominant negative, we studied the estrogen induction of genes under conditions in which AP-1 was normal or blocked. We show that the expression of AP-1-dependent genes was inhibited by the cJun dominant negative and that AP-1 blockade does not affect mRNA ER
expression or estrogen induction of ERE activity. Using a microarray approach we then identified 20 new estrogen-induced/AP-1-dependent genes. These estrogen-induced/AP-1-dependent genes contain a higher frequency of consensus AP-1 sites in their promoters and have increased sensitivity to the AP-1 stimulant tetradecanoyl phorbol acetate (TPA) when compared with estrogen-induced genes whose expression was not affected by AP-1 blockade. We also show estrogen and AP-1 dependent recruitment of ER, SRC-1 and p300 to the promoter of these genes by chromatin immunoprecipitation. These studies demonstrate that microarrays can be used in a "reverse genetics" approach to predict the functional promoter structure of large numbers of genes that are regulated by multiple transcription factors.
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