Histone deacetylase (HDAC) inhibitors represent an exciting new class of anti-cancer drugs which induce cell cycle arrest and either apoptosis or differentiation in cultured cells and display anti-tumor activity in animal studies. They are currently being used in clinical trials to treat various human cancers. These drugs inhibit the enzymes which remove acetyl groups from histones, resulting in large increases in cellular histone acetylation. This modification has long been associated with transcriptional activity, leading to the hypothesis that these drugs generally increase the expression of genes. However, microarray analyses have repeatedly shown that HDAC inhibitors repress as many genes as they activate, even upon short term treatment. These results indicate that histone deacetylases play both positive and negative roles in transcriptional regulation. However, the mechanisms by which they promote transcription are largely unknown. Some of the genes known to be down-regulated by histone deacetylase inhibitors are important for maintenance of cell proliferation, such as c-myc and cyclin D1. The well-characterized MMTV promoter is known to be repressed by HDAC inhibitors. Our recent studies have provided important insights into the mechanism by which this occurs. Surprisingly, the repression does not involve changes in histone acetylation, suggesting that histone deacetylases target non-histone proteins at the MMTV promoter. Mutational analysis of the promoter shows that the repression is mediated through the core promoter region, including the TATA and initiator elements. Our hypothesis is that histone deacetylases regulate the function of some component of the basal transcription machinery at the MMTV promoter thereby directly regulating transcriptional initiation. Consistent with this, chromatin immunoprecipitation (ChIP) analysis shows that the unphosphorylated, initiating form of RNA polymerase II is rapidly lost from the promoter following treatment with HDAC inhibitors. To better understand the mechanism of repression, we are taking two approaches. First, we will continue to use ChIP analysis to determine patterns of factor association with the promoter in the presence and absence of HDAC inhibitors. We are particularly interested in the complexes and factors which associate with the core promoter such as the mediator complex and the general transcription factors such as TFIID, TFIIB, and TFIIE. In addition, we are developing an in vitro transcription system which will be used to study the mechanism of repression. We have already succeeded in recapitulating the repression in vitro and will use a depletion and add back approach to determine the target of the HDAC at the basal MMTV promoter. A review of the literature on acetylation of basal transcription machinery components shows that very little is known. As a complementary approach to studying the effects of HDACs on transcription we are screening these components for acetylation. Initially we are focusing on TFIID, a complex of 15 proteins which binds directly to the core promoter and serves as a platform for the assembly of the transcriptional initiation complex. We are purifying an epitope-tagged version of the complex and screening it for acetylated components in the presence and absence of HDAC inhibitors using both anti-acetyllysine antibodies and mass spectroscopy. Our preliminary results indicate that at least two components of TFIID are acetylated. We hope to collaborate with others and take the same approach in identifying acetylated components of the mediator complex. Eventually these studies would be directed at understanding the function of these acetylation events.
- National Institutes of Health: $49,548.00
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