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 chromatin structure in understanding transcriptional regulation of genes targeted by signaling pathways. In the case of histone deacetylase (HDAC) inhibitors, these clinically-relevant drugs repress MMTV promoter activity regardless of chromatin context. However, the inhibition of histone deacetylases leads to a general increase in histone acetylation, an event which has been tightly correlated with transcriptional activity. The existence of genes which are transcriptionally repressed by these drugs indicates that we do not fully understand their mode of action. One question of interest is whether the two structurally-distinct MMTV 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 determined that regulation of the two MMTV templates by cAMP is mechanistically-distinct. The template in disorganized, accessible chromatin is activated by cAMP in a protein kinase A (PKA)-dependent but CREB-independent fashion. Interestingly, p300 plays a role in this process and must be targeted by cAMP signaling independently of CREB. We are currently focused on determining how p300 functions in this process. The cAMP-induced repression of the MMTV template in organized chromatin is dependent on PKA but does not require CREB or p300. Instead, chromatin immunoprecipitation assays indicate that cAMP induces the deacetylation and dephosphorylation of histones in the MMTV promoter region, with the changes being particularly pronounced in a region of the promoter known to contain negative regulatory elements. These results indicate the cAMP-dependent recruitment of corepressor complexes containing chromatin modifying activities. Our current efforts are directed towards identifying these complexes. We have determined that HDAC inhibitors repress MMTV transcription by a mechanism which does not involve either chromatin remodeling or histone modification. In addition, we have shown that the MMTV promoter is a primary target for these drugs and therefore, a good model for studying their mode of action. Because histone acetylation at the MMTV promoter is unaffected by these drugs, we conclude that the ultimate target is a nonhistone protein. Our current efforts are directed at determining the sequences in the MMTV promoter which are crucial for regulation by HDAC inhibitors. We are also developing a pull-down assay with promoter fragments to assay for HDAC inhibitor-induced changes in factor binding or factor acetylation. These studies will provide important insights into how these drugs work in affecting physiological promoter templates. In the process of studying histone modification at the MMTV promoter in response to signaling pathways, we determined that cAMP signaling causes a drastic reduction in bulk levels of histone H3 phosphorylation in our breast adenocarcinoma cell lines. Since this particular modification has been associated with both transcriptional activity and proper progression through mitosis, we are very interested in understanding the mechanism of this dephosphorylation and its effects on cell biology. Current efforts are aimed towards determining which potential sites of phosphorylation on H3 are affected and whether phosphorylation of any other histones is also changed in response to cAMP signaling. We are also exploring any changes in cell cycle regulation.
| 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 |
