Mechanisms of De Novo Methylation in Cancer

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DESCRIPTION (provided by applicant): The objectives of this grant, which has been funded for almost 30 years, have been to understand the mechanisms for the establishment and inheritance of DNA methylation patterns and to develop drugs which can interfere with cytosine methylation and reactivate silenced genes. This research has led to the recent approval by the FDA of two DNA demethylating agents (5-aza-CR and 5-aza-CdR) for the treatment of myeloid dysplastic syndrome. In the next five year period of the project, we hope to take advantage of epigenomic analysis to understand how DNA methylation patterns are established and maintained by an interaction between DNA methyltransferases and specific chromatin components. To do this, we have developed a custom NimbleGen array allowing for the analyses of nucleosomes, histone modifications and DNA methylation in an integrated way at 1,800 transcription start sites (TSS) in normal and transformed cells. In Specific Aim 1, we will utilize the tiling array to map nucleosomes in both normal (PrECs) and transformed prostate cancer cells (PC3). We shall then determine how interfering with DNA methylation pharmacologically in PC3 cells or genetically in HCT116 colon cancer cells alters the distribution of histone marks focusing on the histone H3-K27me3 mark applied by the polycomb repressive complex 2 (PRC2). In Specific Aim 2, we shall determine how the epigenome is reorganized during the restoration of DNA methylation to HCT116 derivatives (DKO) in which two of the three DNA methyltransferases (DNMT1 and DNMT3B) have been genetically knocked down. In Specific Aim 3, we will follow-up on our new results which show the strong anchoring of the de novo methyltransferases DNMT3A and 3B to nucleosomes. We wish to determine how the enzymes interact with nucleosomes so that we can understand how specific patterns are established. In Specific Aim 4, we will continue our quest to develop DNA demethylating drugs which are more stable than those currently approved by the FDA for cancer treatment and which are able to reverse aberrant DNA methylation, histone modifications and nucleosome positioning. Achievement of these aims should have major impact in our understanding of the epigenetics of cancer and have direct relevance to new strategies to treat and prevent cancer. PUBLIC HEALTH RELEVANCE: It has become clear over the last few years that the abnormal silencing of genes by somatically heritable epigenetic processes, can contribute directly to the formation of human cancers. Although we know that altered patterns of DNA methylation play a fundamental role in the silencing of tumor suppressor genes, we do not know how these altered patterns are set up or how normal patterns are established and inherited during human development. Recent excitement in the field has focused on the potential role of gene silencing mechanisms involving the polycomb repressive complexes (PRCs), which are essential for normal development and have recently been found also to play a role in inactivating tumor suppressor genes. These PRCs can directly silence genes by themselves and also somehow set up genes for more permanent silencing induced by DNA methylation. Recently, the FDA has approved two DNA demethylating agents and one histone deacetylase inhibitor for the treatment of particular kinds of cancer.