Abstract 4230: A novel method for sequence- and strand-specific, genome-wide 5-hmC profiling.

Abstract

5-Hydroxymethylcytosine (5-hmC) is a new epigenetic hallmark rapidly getting much interest as the subject of mapping and sequencing work. While the exact function of 5-hmC is not fully understood, it is likely to regulate gene expression via active DNA de-methylation. Previous studies have shown it may play an important role in cell differentiation and carcinogenesis. Cells that are more stem- and progenitor-like have greatly reduced levels of 5-hmC compared with more differentiated cells. Similarly, tumor cells display less 5-hmC than their normal counterparts. This was not associated with either grade or stage, suggesting that global loss of 5-hmC may be an early event in carcinogenesis. To date, several methods have been developed to profile 5-hmC at the genomic level: most are enrichment-based and utilize antibodies to 5-hmC or its modified forms or make use of bioorthogonal labeling and pull-down of glycosylated 5-hmC. The caveat is that these approaches are low resolution and that they require large amounts of input genomic DNA. Initial efforts for detection of 5-hmC at single-base resolution require several micrograms of DNA, require parallel or subtractive sequencing, and employ successive chemical treatments that can degrade DNA and hinder sequencing. Here we report on a new method that by combining modification-sensitive restriction enzymes with massively parallel (“next-generation”) sequencing approaches, genome-wide 5-hmC can be mapped at single site resolution from low (100 ng) DNA inputs. Importantly, this method can be used for strand-specific localization of 5-hmC as well as direct identification of single nucleotide polymorphisms (SNPs) within the sequencing reads. Data can also be compared directly with single-base resolution DNA methylation data from reduced-representation bisulfite sequencing (RRBS) for simultaneous 5-mC and 5-hmC profiling from the same sample. 5-mC and 5-hmC of human brain DNA using this combined method indicates unique distributions of the 5-hmC modification. This new method should provide a unique tool in enhancing our understanding of the interplay of genetic and epigenetic regulations in carcinogenesis. Citation Format: XueGuang Sun, Adam Petterson, Hunter Chung, Xi Yu Jia, Marc E. Van Eden. A novel method for sequence- and strand-specific, genome-wide 5-hmC profiling. [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 4230. doi:10.1158/1538-7445.AM2013-4230