Aberrant DNA Methylation of Ribosomal RNA Genes in Human Cancer

Abstract

Aberrant regulation of rRNA genes has been reported in human cancers. The aim of this study was to analyze the epigenetic regulation of rRNA genes in lung cancer and to correlate methylation levels of the promoter, 18S and 28S regions with different chromatin states. Here we report, that methylation of rDNA ranged from 10% to 30% at individual CpG sites in the promoter region. In primary lung cancers a 1.2-fold increased rDNA methylation was observed at 19 analyzed CpGs (p < 0.001). Moreover, we report an increased methylation level of rDNA towards the 28S region of rRNA genes. This hypermethylation was more pronounced in cancer cell lines compared to primary tissue. To analyze the methylation status of protein-enriched rDNA, we utilized a technique that combines ChIP and bisulfite sequencing. Pol I- and CTCF-associated rDNA exhibited reduced methylation levels compared to global rDNA. Histone H1- associated promoter regions showed a 1.5-fold increase in methylation levels compared to H3-associated rDNA. In DNMT1 knock out cells a strong reduction of methylation of 18S and 28S regions was found in comparison to wild type HCT116 cells (2.7- and 1.4-fold reduction, respectively). In double DNMT1 and DNMT3B knock out cells no substantial rDNA methylation was detected. Independent of this hypomethylation, 28S level and number of rDNA repeats were constant in wild type, single knock out and double knockout HCT116 cells. Our data suggest that aberrant methylation of rDNA occurs in human cancer and that rRNA gene activity can be modulated in a constant manner independent of the level of methylated rDNA.