Long range epigenetic silencing of chromosome 15q23 in human gastrointestinal cancer

Abstract

Cancer is a disease of genetic and epigenetic deregulation contributing to aberrant gene expression. Until recently, cancer associated epigenetic deregulation has been manifested as a local event affecting discrete genes. However, concurrent epigenetic silencing of a large chromosomal region encompassing multiple neighboring genes, termed as long range epigenetic silencing (LRES) is being newly identified. This kind of global epigenetic gene regulation may provide important implication in development of epigenetic based cancer treatment strategies. Therefor, we tried to identify novel LRES region in human gastrointestinal cancer and elucidate its importance in tumor progression. First, we compared microarray gene expression data of HCT116 and DKO to identify LRES region in human gastrointestinal cancer. As DKO is a derivative of HCT116 with DNMT1 and DNMT3b knocked out, difference between their gene expression implicates differential DNA methylation. Interestingly, we identified large chromosomal region with multiple genes at chromosome 15q23 showing low gene expression in HCT116 compared to DKO. Differential gene expression was confirmed with qRT-PCR. Contribution ofCpG island hypermethylation of gene promoters were verified with bisulfite sequencing. Histone modification, which is known as another important layer of epigenetic regulation, was also identified to regulate epigenetic silencing. Second, we examined the gene expression of 11 gastric cancer cells to verify whether the LRES was general event. We identified six gastric cancer cells showing comparable gene expression pattern with HCT116. To verify if LRES in gastric cancer cells were also regulated by DNA methylation and histone modification, we compared gene expression, methylation status, and histone modification of the genes within the LRES region. We confirmed that SNU601, which showed low level of gene expression along the LRES region, was associated with DNA methylation at gene promoters with MSP assay. SNU601 was also associated with low level of active histone marks, while repressive histone marks were enriched. Third, to validate how DNA methylation and histone modification interact to maintain silenced state of LRES region, we treated demethylating agent, 5-Aza-2-deoxycytidine (5-aza-CdR) to SNU601. We identified that gene restoration after the treatment was more likely a result of histone modification rather than demethylation of gene promoters. From these results we could suggest that maintaining DNA methylation contributes to inheriting stable silent state of LRES region, while plasticity of histone modification can result in transient gene expression change. Finally, to determine whether LRES of this region is clinically relevant event, we compared gene expression of five primary gastric tumor tissues with their normal pairs. We identified that genes within LRES region was silenced in primary gastric tissues. We concluded that this low gene expression was mediated by tumor specific DNA methylation at their promoters. Taken altogether, our results suggest a novel LRES region of chromosome 15q23 in human gastrointestinal cancer, mediated by tumor specific DNA hypermethylation and aberrant histone modification.