The goal of this project is to determine the mechanism by which cAMP(cyclic AMP) signaling pathways or histone deacetylase (HDAC) inhibitors lead to transcriptional repression of the mouse mammary tumor virus (MMTV) promoter in the physiological context of 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 complex chromatin. One question of interest is whether the two templates are regulated through different cAMP-dependent mechanisms. One possibilibty is that the differences in response to cAMP signaling are due to the same cAMP-initiated cascade of events having two different outcomes due to the distinct structures of the templates. Alternatively, the structure of the template may determines which cascade targets it. These models might be used to explain cell type-specific differences in regulation of the same promoter. If the chromatin structure of the promoter is in distinct configurations in two different cell types, the promoter may respond differently to the same extracellular signal. Distinguishing between the two possibilities requires understanding the mechanism by which these distinct transcriptional outcomes occur. In the past year we have focused on signaling-induced changes in histone modifications at the MMTV promoter in ordered chromatin. By using chromatin immunoprecipitation assays, we have determined that cAMP signaling leads to the deacetylation of histones in the MMTV promoter region. Our current efforts are directed toward defining other changes in histone modification (methylation or phosphorylation) which may occur in response to cAMP signaling. In addition we are working out techniques to examine cAMP-induced changes in the binding of various transcription factors, coactivators, corepressors, and histone deacetylases. Another approach to determining the factors involved in cAMP regulation of MMTV is to use dominant negative expression to inhibit the activities of various factors known to be involved in this signaling pathway. We have determined that the cAMP-regulated transcription factor, CREB, is not involved in the MMTV response and our current work is focused on p300 and CBP, important cofactors thought to be integral players in a number of signaling pathways. Our interest in transcriptional repression led us to investigate the mechanism by which the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) represses the MMTV promoter in chromatin. HDAC inhibitors are clinically relevant drugs but little is known about the mechanisms by which they modulate gene expression. One hypothesis is that they activate genes through increased histone acetylation whereas nothing is known about their repressive action. Our studies in the last year have determined that TSA represses MMTV transcription directly and rapidly, but has no effect on chromatin remodeling at the promoter. Chromatin immunoprecipitation assays have shown that the acetylation of histones at the MMTV promoter does not increase, but rather decreases after TSA treatment. Thus histones are not the only mediators of the response to these drugs. In collaboration with Dr. Jane Trepel, we are examining the effect of other HDAC inhibitors on MMTV transcription. These studies will provide important insights into how these drugs work in affecting physiological promoter templates.
| Rodriguez-Collazo, Pedro; Snyder, Sara K; Chiffer, Rebecca C et al. (2008) cAMP signaling induces rapid loss of histone H3 phosphorylation in mammary adenocarcinoma-derived cell lines. Exp Cell Res 314:1-10 |
| Mulholland, Niveen M; Snyder, Sara K; Kolla, Sarah S et al. (2003) Chromatin-dependent regulation of the MMTV promoter by cAMP signaling is mediated through distinct pathways. Exp Cell Res 287:361-73 |
