Hypermethylation of promoter CpG islands plays a prominent role in cancer. In partnership with alterations in histone acetylation/methylation, this epigenetic event establishes a repressive chromatin structure that leads to silencing of key cancer-related genes. The occurrence of DNA methylation within the genome is not random, but rather patterns of methylation are generated that are gene and tumor type specific. How DNA methylation patterns are established is still poorly understood. Since various transcriptional factors or regulators are found in association with DNA methyltransferases (DNMTs) in vivo, we hypothesize that: 1) Oncogenic transcription factors can recruit DNMTs to target gene promoters and define a unique epigenetic signature in tumor cells; 2) Dissecting such complex epigenetic hierarchy will identify novel molecular targets for diagnosis, prognosis and therapeutic intervention. To test our hypothesis, we developed a high throughput technique for genome wide analysis of DNA methylation associated with specific proteins such as histones, transcription factors or any DNA binding proteins. The new approach named ChlP-Chop-DMH will combine both genome wide location analysis (also known as ChlP-on-Chip) and Differential Methylation Hybridization (DMH) analysis, two emerging technologies used in epigenetic research. The proposed method has distinct advantages over current protocols: first, this method directly examines the in vivo interaction of specific proteins with methylated DNA throughout the genome; second, this method may uncover novel biological properties of transcription factors; third, this method can be applied to discover novel epigenetic biomarkers relevant to tumorigenesis. In preliminary studies, we have verified the utility of this method with methylated histone H3 at lysine 9 and lysine 4 in human cancer cells. In the R21 phase, we will continue minor refinement of the method and pursue three aims: 1) Improve and optimize the ChlP-Chop-DMH method for analyzing genome wide association of DNA methylation with histone modification; 2) Utilize the proposed method to investigate the association of DNA methylation with chromatin remodeling factors: 3) Show proof-of-concept using the array to examine primary non-Hodgkin's lymphomas (NHLs). In this development phase we will focus on the sensitivity, reproducibility and accuracy of the proposed method. In the R33 phase, our goal is to utilize the technology to test biological hypotheses. We will fully implement the method and pursue these aims: 1) Discover epigenetic target genes associated with known oncogenic transcription factors c-Myc and BCL6: 2) Validate the identified epigenetic targets and investigate the regulatory role of the associated oncogenic transcription factors. This systematic approach will provide a powerful tool for future mechanistic studies as well as cancer diagnosis. ? ? ?
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