Abstract 2970: Acute depletion of DNA methyltransferases reveals unique and overlapping target sites in cancer.

Abstract

Abstract DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) DNA methylation to regulate gene transcription and promote overall genome stability. Global changes in DNA methylation, such as hypomethylation of repetitive sequences and hypermethylation of tumor supressor gene promoters, are commonly observed in various types of cancer; however, the DNMTs’ contribution to this aberrant methylation remains largely unknown. In this study, we aim to identify unique and overlapping target sites for each of the DNMTs in order to better understand aberrant DNA methylation in cancer that will ultimately permit development of new therapeutic strategies. We utilize siRNA-mediated knockdown technology to acutely deplete the mRNA for each of the DNMTs in both individual and combinatorial fashion in NCCIT embryonal carcinoma cells. Subsequently, DNA methylation is observed genome-wide in each DNA sample using two different methodologies: (1) Methyl-CpG Binding Domain (MBD)-seq, which identifies regions of the genome that are enriched for DNA methylation, and (2) Illumina Infinium HumanMethylation450 BeadChip (450K array) allowing for specific CpG site methylation status determination. Aligned MBD-seq sequences for individual DNMT knockdowns are analyzed by read coverage nomalization within 100Kbp windows and using various peak-calling algorithms (e.g. MACS, BALM). For the 450K array, DNA samples for both individual and combination knockdowns undergo bisulfite conversion and array processing; β-values for each CpG site are derived from the array signal intensities using GenomeStudio and R/Bioconductor (minfi). Genome-wide DNA methylation analysis reveals that DNMT1 depletion, both individual and in combination with knockdown of other DNMTs, results in global demethylation among all genomic features. Interestingly, a small population of genes exhibit hypermethylation in gene-promoter CpG islands for DNMT1 (individual only) depletion. In contrast, de novo methyltransferase depletion (individual and combination) shows more specific demethylation effects. In particular, hypomethylation events resulting from DNMT3B depletion (individual and combination (not 3B+3L)) occur within gene bodies and largely outside of CpG islands and flanking regions (shores, shelves). Additionally, a number of hypermethylation events occurring within the 3’UTR region of genes are observed in de novo methyltransferase depleted samples. We anticipate further analysis will reveal unique and overlapping target sites for each of the DNMTs that will lay the ground-work necessary to characterize and understand DNMT recruitment both in normal and cancerous tissues. Citation Format: Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Acute depletion of DNA methyltransferases reveals unique and overlapping target sites in cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2970. doi:10.1158/1538-7445.AM2013-2970