Identification and functional relevance of de novo DNA methylation in cancerous B-cell populations

Abstract

Epigenetic remodeling is a hallmark of cancer, with the frequent acquisition of de novo DNA methylation in CpG islands. However, the functional relevance of de novo DNA methylation in cancer is less well defined. To begin to address this issue in B-cells, we used BeadArray assays to survey the methylation status of over 1,500 cancer-related CpG loci in two molecular subtypes of diffuse large B-cell lymphoma (ABC-DLBCL and GCB-DLBCL) and cognate normal B-cell populations. We identified 81 loci that showed frequent de novo DNA methylation in GCB-DLBCL and 67 loci that showed frequent de novo DNA methylation in ABC-DLBCL. These de novo methylated CpG loci included reported targets of polycomb repressive complexes (PRC) in stem cells. All candidate loci in GCB-DLBCL are proximal to genes that are poorly expressed or silent in purified normal germinal center (GC) B-cells. This is consistent with the hypothesis that de novo DNA methylation in cancer is more frequently involved in the maintenance rather than the initiation of gene silencing (de novo repression). This suggests that epigenetic switching occurs during tumorigenesis with de novo DNA methylation locking in gene silencing normally mediated by transcriptional repressors. Furthermore, we propose that similar to de novo genetic mutations, the majority of de novo DNA methylation events observed in tumors are passengers not causally involved in tumorigenesis.

FIG. 1

Hierarchical clustering of DNA methylation profiles in diffuse large B-cell lymphoma (DLBCL) and normal B-cells. Analyses were based on 690 autosomal loci proximal to or within 238 unique genes with no strong evidence of imprinting and having the fully methylated (SssI: M.SssI-treated whole genome amplification materials generated from human genomic DNA) and fully unmethylated (WGA: whole genome amplification from human genomic DNA) controls pass quality control metrics (see Methods). Hierarchical clustering was conducted using Pearson absolute distance and average-linkage.

FIG. 2

Bisulfite sequencing of CpG island proximal to CCNA1. Blackened and empty circles denote methylated and unmethylated CpG dinucleotides, respectively. The regions encompassing Illumina reaction CCNA1-1285 (CCNA1) was subject to bisulfite sequencing analysis in (A) GCB-DLBCL_13 and (B) peripheral B-cells activated by LPS treatment. Light gray and blackened circles denote unmethylated and methylated CpG dinucleotides, respectively. The CCNA1 amplicon spans nucleotide positions 35904243 – 35904473 of chromosome 13, based on the May 2004 human genome assembly provided at http://genome.ucsc.edu/.