Histone Modification and Changes in Chromatin: Silencing of Tumor Suppressor Gene

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The genomic DNA of eukaryotes must be highly compacted for packaging within the nuclei of cells. This compaction is achieved through the formation of a complex nucleoprotein structure known as chromatin. Chromatin is a highly dynamic structure that exerts a powerful influence over cellular processes that involve accessing chromosomal DNA. The primary protein components of chromatin are the core histones H2A, H2B, H3 and H4. The post-translational modification of the core histones is an important mechanism by which chromatin structure is regulated. The core histones have been shown to undergo several types of modification such as acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation. In addition, these modifications are often found at multiple sites in the core histones. The main focus of our current proposal is to use the sensitive technique of mass spectrometry as a foundation for the comprehensive identification, mapping and characterization of histone post-translational modifications. Our initial aim in these studies is to use mass spectrometry to analyze core histones isolated from bovine thymus. This will allow for the identification and mapping of the complete spectrum of histone posttranslational modifications present in higher eukaryotic chromatin. As novel sites of modification are identified, the characterization of their in vivo function will be done in yeast, taking advantage of the unique ability to genetically manipulate the core histones in this organism. Many observations point to the critical role that the proper regulation of chromatin structure plays in preventing the uncontrolled cell growth characteristic of cancer. Therefore, we propose experiments that seek to identify characteristic alterations in the extent or pattern of histone modifications that correlate with Chronic Lymphocytic Leukemia. As described in other sections of this program project, the modification of chromatin structure is being explored as a potential chemotherapeutic strategy. Small molecule inhibitors of histone deacetylases have shown potential in controlling the growth and differentiation of cancer cells. To most effectively utilize these compounds, it is vital to more precisely characterize the alterations in histone modifications that are induced by these drugs. Therefore, we propose to use mass spectrometry to comprehensively characterize the changes in histone modification that result from the treatment of leukemia cells with chemotherapeutic agents that target chromatin structure.