The goal of this project is to determine the mechanism by which activation of cAMP signaling pathways leads to transcriptional repression of the mouse mammary tumor virus (MMTV)promoter in ordered chromatin. Our previous work has shown that cAMP signaling causes repression of the MMTV promoter in a glucocorticoid-independent fashion only when it is incorporated into ordered, replicating chromatin in cultured cells. In contrast, a transiently-transfected MMTV promoter construct which does not have an organized nucleoprotein structure is activated by cAMP signaling. These results emphasize the importance of studying transcriptional regulation of genes in their native setting of ordered and complex chromatin. We have determined that the repression is associated with the inhibition of binding by Oct1 and the basal transcription machinery to the MMTV promoter. In addition, the use of dominant negative repressors of the cAMP-responsive CREB protein has allowed us to conclude that CREB is not likely to be involved in the repressive signal. Our current work focuses on two situations in which cAMP-induced repression at the MMTV promoter is abrogated. First, we have found that increasing cellular levels of histone acetylation through the use of histone deacetylase inhibitors leads to relief of cAMP-induced repression. This observation indicates that cAMP signaling may result in an inhibition of histone acetylation at the MMTV promoter. We are currently testing this hypothesis by doing chromatin immunoprecipitation assays with antibodies against acetylated forms of histones. Expression of the adenovirus E1A 13S protein also relieves repression by cAMP signaling. This protein interacts with several histone acetyltransferases and is thought to stimulate transcription at target genes through interaction with transcription factors bound to promoters. We are carrying out a mutational analysis of the 13S E1A protein to determine which interactions are necessary for relief of repression. This knowledge will provide insight into the nature of the signaling pathway leading to the repression and the possible targets at the MMTV promoter.
- National Institutes of Health: $118,914.00
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