Nucleosomes Correlate with In Vivo Progression Pattern of De Novo Methylation of p16 CpG Islands in Human Gastric Carcinogenesis

BACKGROUND: The exact relationship between nucleosome positioning and methylation of CpG islands in human pathogenesis is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In 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/SIGNIFICANCE: It 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

Polycomb CBX7 Directly Controls Trimethylation of Histone H3 at Lysine 9 at the p16 Locus

BACKGROUND: H3K9 trimethylation (H3K9me3) and binding of PcG repressor complex-1 (PRC1) may play crucial roles in the epigenetic silencing of the p16 gene. However, the mechanism of the initiation of this trimethylation is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we found that upregulating the expression of PRC1 component Cbx7 in gastric cancer cell lines MGC803 and BGC823 led to significantly suppress the expression of genes within the p16-Arf-p15 locus. H3K9me3 formation was observed at the p16 promoter and Regulatory Domain (RD). CBX7 and SUV39H2 binding to these regions were also detectable in the CBX7-stably upregulated cells. CBX7-SUV39H2 complexes were observed within nucleus in bimolecular fluorescence complementation assay (BiFC). Mutations of the chromodomain or deletion of Pc-box abolished the CBX7-binding and H3K9me3 formation, and thus partially repressed the function of CBX7. SiRNA-knockdown of Suv39h2 blocked the repressive effect of CBX7 on p16 transcription. Moreover, we found that expression of CBX7 in gastric carcinoma tissues with p16 methylation was significantly lower than that in their corresponding normal tissues, which showed a negative correlation with transcription of p16 in gastric mucosa. CONCLUSION/SIGNIFICANCE: These results demonstrated for the first time, to our knowledge, that CBX7 could initiate H3K9me3 formation at the p16 promoter

Correlation between <i>H. pylori</i> infection and the average proportion of methylated-CpG site at each CpG site within <i>p16</i> CpG island.

<p>(<b>A</b>) Result of 289 bp PCR products of <i>H. pylori</i>-specific <i>23S rDNA</i> from 14 representative gastric samples detected as previous report <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035928#pone.0035928-Liu1" target="_blank">[22]</a>; (<b>B</b>) The methylation proportion of each <i>p16</i> CpG site in the <i>H. pylori</i>-positive normal/gastritis biopsies (<i>N</i> = 13) and the <i>H. pylori</i>-negative samples (<i>N</i> = 7); The gray ovals represent the nucleosome position; (<b>C</b>) The methylation proportion of each <i>p16</i> CpG site in the <i>H. pylori</i>-positive GC samples (<i>N</i> = 17) and the <i>H. pylori</i>-negative GC samples (<i>N</i> = 9).</p

Chromatin accessibility determined by quantitative PCR across the <i>p16</i> promoter.

<p>(<b>A</b>) Genomic organization of the <i>p16</i> promoter and exon-1 region; Predicted nucleosome occupancy in A375 cells are depicted as black peaks and the fragment without nucleosome signal are depicted as inverse gray peaks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035928#pone.0035928-Luo1" target="_blank">[29]</a>. The previously reported nucleosome position (three ovals) and location of the 150 bp-MSP amplicon (black line) are also illustrated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035928#pone.0035928-Fatemi1" target="_blank">[15]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035928#pone.0035928-Herman2" target="_blank">[17]</a>. (<b>B</b>) Amplicons of the chromatin accessibility measured through a set of quantitative PCR assays were used to quantify the mono-nucleosomal DNA partially digested using <i>MNase</i> (Horizontal lines). The detected nucleosomes are marked using gray ovals. Positions of these CpG sites in the <i>p16</i> genomic sequence corresponding to the fragment displayed in panel A are indicated by thin vertical lines, and the transcription start site (TSS) is represented as a bent arrow. (<b>C</b>) The relative enrichment of various fragments within <i>p16</i> CpG island in <i>p16</i>-methylated AGS and <i>p16</i>-active MGC803 cells with and without digestion using 0.1 U <i>MNase</i> for 5 min. Relative enrichment was calculated and normalized according to the relative copy number (2^−ΔCt; ΔCt = Ct^Uncut−Ct^Cut) for each primer set <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035928#pone.0035928-Eads2" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035928#pone.0035928-Bai2" target="_blank">[26]</a>. The average value and standard deviation of four quantitative PCR reactions are displayed. We reported the <i>p16</i> methylation and expression data of these two cells previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035928#pone.0035928-Li1" target="_blank">[13]</a>.</p

Comparison of methylation status of <i>p16</i> CpG island in gastric carcinomas (GC), chronic gastritis, and normal gastric mucosa using sMSP-sequencing.

<p>(<b>A</b>) Proportion of clone groups containing different number of methylated-CpG sites within the bisulfite-sequenced clones; (<b>B</b>) The proportion of clones containing ≥6 methylated-CpGs in sMSP clones from GCs (16.7%) was significantly higher than that from gastritis (8.3%) or normal biopsies (7.5%) (77/460 vs. 18/217 or 5/67, <i>p</i> = 0.003 or 0.050). The fully methylated-CpG cluster in the proximal promoter (PP)-nucleosomal region was observed only in GC samples. Most <i>p16</i> molecules with promoter methylation are fully methylated. (<b>C</b>) The average methylation frequency of each CpG site within the 45 tested CpGs in the 83 bp-MSP-positive samples (<i>n</i> = 32) and the negative samples (<i>n</i> = 19). The G in the CpG site-10 is a G/A polymorphism.</p

Comparison of the proportion of methylation at individual CpG site in the <i>p16</i> CpG islands in GCs and gastritis/normal biopsies containing or not containing <i>H. pylori</i> (<i>Hp</i>)-specific DNA.

a, b, c, d, e, f<p>Wann Whitney test, <i>P</i> = 0.012, 0.040, 0.023, 0.010, 0.018, 0.003, 0.034.</p

Results of bisulfite-sequencing of the 588 bp fragment of the <i>p16</i> CpG islands.

<p>(<b>A</b>) Sequences of the methylation-positive and negative clones from the 588 bp unbiased PCR products amplified using a CpG-free primer set and bisulfite-treated genomic DNA templates of two representative gastric carcinoma tissues containing methylated <i>p16</i> by the 115 bp MethyLight assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035928#pone.0035928-Zhou1" target="_blank">[11]</a>; (<b>B</b>) Sequences of the methylation-positive and negative clones from the 588 bp sMSP products. Purple bars, methylated CpG sites; Blue bars, seeding methylated CpG sites detected in the unbiased PCR products; Green bars, seeding methylated CpG sites used to design the sMSP primers. Each row presents one clone; Clone markers of methylation-positive <i>p16</i> molecules (containing more than three methylated-CpG sites) are highlighted with blue color. The standard sequence of the fully methylated <i>p16</i> amplicon and the primer-matched regions in each assay are demonstrated on the top part.</p

Relationship between nucleosome positioning and methylation of each CpG site within <i>p16</i> CpG island in human gastric mucosa samples (<i>N</i> = 51).

<p>(<b>A</b>) The detected nucleosome occupancy within a <i>p16</i> CpG island as displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035928#pone-0035928-g001" target="_blank">Figure 1B</a>; three seeding methylation CpG sites at 46, 47, and 48 locations are marked with purple color; (<b>B</b>) A gray-graded representation of the average methylation density at individual CpG sites within the <i>p16</i> promoter and exon-1 region based on the results of all informative clones obtained from each sample using the sMSP-sequencing assay; The methylation density of each CpG site in each tested sample was labeled, 0.1∼0.7 mean 10%∼70%; (<b>C</b>) The positive rate of methylated-CpG at each CpG site in the sequenced gastric tissue samples with various pathological changes. *, The positive rate at the site-5 in GCs is statistically significantly higher than in gastritis and normal samples (Fish exact test, <i>P</i><0.02). #, The positive rate at the site-12 and 13 GCs is significantly higher than in gastritis and normal when the proportion value of each sample was used in the Mann Whitney test (<i>P</i> = 0.043, two-sides).</p