Aberrant gene silencing resulting from alterations in DNA methylation and chromatin structure plays an important role in the inactivation of tumor suppressor and other genes in human cancers. Our long term goals are to investigate the molecular mechanisms underlying these gene silencing events, and to study how the epigenetic silencing of certain genes contributes to human carcinogenesis. Emerging work over the last several years points to local chromatin environment as a critical factor in determining the epigenetic fate of CpG islands during development and transformation. During the last funding period we have studied the interdependent relationship between DNA methyltransferases and histone modifying complexes, and their role in promoting or preventing epigenetic silencing in cancer. We have uncovered a novel role for histone H4 modifications (H4K16 Ac; H4K20me3) and the complexes that catalyze these marks, in regulation of RNA polymerase II pausing dynamics at CpG island promoters. Specifically, we find that the deposition of H4K20 methylation by the SUV420H2 histone methyltransferase imposes a block to promoter escape by Pol II through inhibition of the hMOF-mediated acetylation of H4K16, acting as a switch to enforce Pol II promoter-proximal pausing. Further, we have identified a subset of genes marked by H4K20me3 in normal cells that are highly prone to aberrant DNA methylation in primary non-small cell lung cancers. These findings support the model that a critical event in tumorigenesis is the replacement of chromatin-based repression mechanisms by the more stable and heritable repression imposed by DNA methylation. The goal of this proposal is to test the hypothesis that local targeting of SUV420H2 and H4K20me3-enforced Pol II pausing places certain genes at increased risk of epigenetic silencing in cancer. Using a combination of directed gene studies and global analyses, we will assess whether, and by what mechanism, SUV420H2 and H4K20me3-mediated repression promotes aberrant DNA methylation at methylation-prone CpG island promoters. We will directly test the impact of altered Pol II pausing dynamics and destabilization of paused Pol II on aberrant DNA methylation at CpG island promoters. A transgene approach will be employed to determine whether the aberrant targeting of SUV420H2 and H4K20me3-mediated repression is sufficient to drive ectopic Pol II pausing and aberrant DNA methylation at a unique chromosomal locus. Finally, in a combination of knock down experiments, chromatin immunoprecipitation and novel GRO-seq technology, we will explore the relationship between divergent transcription, SUV420H2-mediated H4K20me3 and the spread of DNA methylation at CpG island promoters prone to aberrant methylation in cancer cells. There is currently a great deal of interest in the application of epigenetic therapy in cancr treatment. The results of our studies will provide important insight into the basic mechanisms driving epigenetic silencing in cancer, and will also provide a framework for the development of improved strategies for reprogramming the cancer epigenome.