Abstract
Abstract DNA methyltransferases (DNMTs) are responsible for establishing (DNMT3A, DNMT3B, DNMT3L) and maintaining (DNMT1) DNA methylation genome-wide. Hypomethylation of repetitive sequences and transposable elements coupled with gene-specific promoter hypermethylation events contribute to the genomic instability and loss of tumor suppressor gene transcription observed in cancer. Regulation of aberrant methylation in cancer remains poorly understood. The aim of our study was to identify unique and overlapping target sites for each of the DNMTs to better understand regulation of normal and aberrant DNA methylation. We hypothesized that acute depletion of the DNMTs (individual and combination) mediated by siRNA technology in NCCIT human embryonic carcinoma cells would result in both distinct and broad changes in DNA methylation patterning. Genome-wide methylation was assayed using the HumanMethylation450 Bead Chip (450K array) allowing for specific CpG site methylation status determination. Select target sites were verified by bisulfite genomic sequencing. DNMT1 knockdown samples (individual/combination) revealed genome-wide hypomethylation, with the strongest demethylation occurring in gene bodies, 3′UTR, and intergenic sequences. Interestingly, only the DNMT1 individual knockdown showed significant hypermethylation in gene promoters. DNMT3 knockdowns showed more specific changes in DNA methylation, but surprisingly, more hypermethylation events occurred than hypomethylation at CpG dinucleotides. Hypermethylation observed in DNMT3 knockdown occurred primarily in gene bodies and 3′UTR, and overlapped significantly with those genes that become hypomethylated in DNMT1 knockdown, indicating a potential cross-regulatory role for the DNMTs to maintain proper regulation of DNA methylation at specific gene termini. Conversely, gene promoters were targeted for hypomethylation in DNMT3 knockdown, and did not significantly overlap with genes that become hypermethylated in DNMT1 knockdown. Of particular interest was that DNMT3B knockdown resulted in widespread non-CpG hypomethylation. In contrast, DNMT3L knockdown showed non-CpG hypermethylation, indicating a potential mechanism for regulation of non-CpG methylation where DNMT3B is responsible for non-CpG methylation, and DNMT3L acts to restrict DNMT3B's activity at non-CpG dinucleotides. Our results reveal a complex view of DNA methylation regulation, in which DNMTs not only target specific sites for methylation, but also cooperate to establish and maintain proper levels of DNA methylation at CpG and non-CpG dinucleotides. Moving forward, we believe our results will provide the framework needed to define the regulatory mechanisms by which DNA methylation is conferred and ultimately develop therapeutic strategies to correct aberrant methylation events that occur in cancer. Citation Format: Rochelle L. Tiedemann, Jeong-Hyeon Choi, Keith D. Robertson. Acute depletion reveals novel divisions of labor among human DNA methyltransferases in cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B34.
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