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Submitted on September 30, 2003
Accepted on November 17, 2003
1 Department of Obstetrics & Gynecology, Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston, Texas 77555-1062
* To whom correspondence should be addressed. E-mail: msoloff{at}utmb.edu.
The specific binding of transcription factors to DNA has been shown to be inhibited by chromatin structure and increased by cooperative interactions with other proteins. Consequently, in situ analysis using chromatin immunoprecipitation offers the most accurate view of transcriptional control. Transient transfection studies and in vitro analyses of interleukin-1 induced cox-2 transcription in a number of cell types have indicated regulation by either NF-
B or C/EBP
, or both acting cooperatively. To determine the mechanisms of COX-2 induction in cultured human myometrial cells in situ, we examined the crosslinking of the RelA subunit of NF-B and C/EBP to the cox-2 promoter and flanking sequences. As a control, we inspected the interaction of these transcription factors with the IL-8 gene, which has been shown in other cell types to be activated by the cooperative interaction of NF-B and C/EBP. Indeed, both transcription factors were crosslinked to the il-8 promoter after IL-1 treatment, but only RelA was crosslinked to cox-2 DNA. The il-8 promoter was also found to physically interact with proteins crosslinked to sites further upstream. IL-1 treatment also increased polymerase II crosslinking to both promoters, and increased histone H4 acetylation at specific sites. These results indicate that modification of chromatin structure is part of the response to IL-1 stimulation. Chromatin immunoprecipitation thus provides critical insight into the mechanisms of COX-2 and IL-8 expression in human myometrial cells.
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