Abstract
BackgroundThe exact relationship between nucleosome positioning and methylation of CpG islands in human pathogenesis is unknown.Methodology/Principal FindingsIn the present study, we characterized the nucleosome position within the p16 CpG island and established a seeding methylation-specific PCR (sMSP) assay based on bisulfite modification to enrich the p16 alleles containing methylated-CpG at the methylation “seeding" sites within its intron-1 in gastric carcinogenesis. The sMSP-positive rate in primary gastric carcinoma (GC) samples (36/40) was significantly higher than that observed in gastritis (19/45) or normal samples (7/13) (P<0.01). Extensive clone sequencing of these sMSP products showed that the density of methylated-CpGs in p16 CpG islands increased gradually along with the severity of pathological changes in gastric tissues. In gastritis lesions the methylation was frequently observed in the region corresponding to the exon-1 coding-nucleosome and the 5′UTR-nucleosome; the methylation was further extended to the region corresponding to the promoter-nucleosome in GC samples. Only few methylated-CpG sites were randomly detected within p16 CpG islands in normal tissues. The significantly inversed relationship between the p16 exon-1 methylation and its transcription was observed in GC samples. An exact p16 promoter-specific 83 bp-MSP assay confirms the result of sMSP (33/55 vs. 1/6, P<0.01). In addition, p16 methylation in chronic gastritis lesions significantly correlated with H. pylori infection; however, such correlation was not observed in GC specimens.Conclusions/SignificanceIt was determined that de novo methylation was initiated in the coding region of p16 exon-1 in gastritis, then progressed to its 5′UTR, and ultimately to the proximal promoter in GCs. Nucleosomes may function as the basic extension/progression unit of de novo methylation of p16 CpG islands in vivo.
Highlights
Methylation of CpG islands around transcription start sites (TSS) represses gene expression epigenetically and plays crucial roles in cell differentiation, development, and pathogenesis
We found that amplicons of primer-2/4/7/8/9 sets were highly resistant to micrococcal nuclease (MNase) digestion in the AGS cell line suggesting the presence of a nucleosome core in these regions
The hypersensitive region around the TSS was not detectable in the p16-unmethylated MGC803 cells, which suggests the existence of a transcription factor complex
Summary
Methylation of CpG islands around transcription start sites (TSS) represses gene expression epigenetically and plays crucial roles in cell differentiation, development, and pathogenesis. It has previously been reported that nucleosome positioning may influence DNA methylation patterns throughout the genome, and DNA methyltransferases preferentially target nucleosomebound DNA in the Arabidopsis thaliana and human embryonic stem cells [1]. The mechanistic details describing the role of nucleosome positioning in relation to DNA methylation are still unknown. Attempts to characterize exact aberrant methylation and its extension patterns within CpG islands in human tissue samples, especially in the early stage of carcinogenesis, have not been successful for variety of reasons. The exact relationship between nucleosome positioning and methylation of CpG islands in human pathogenesis is unknown
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
