Figure 1.
Characterization of methylation and expression of p16 in the fusion cell lines.
(A) The methylation states of p16 CpG islands in the fusion monoclones were analyzed using DHPLC (left). The proportion of methylated-p16 in the monoclone-E10 was stably maintained at passages 45, 50, 55, and 60 (right). (B) RT-PCR shows varying mRNA expression of p16 in the fusion monoclones and their parental MGC803 and AGS cell lines. (C) Confocal immunocytochemistry staining shows varying P16 expression in the various cell lines.
Figure 2.
Bisulfite sequencing of p16 alleles in the fusion cells and their parental cells.
(A) Bisulfite clone sequencing of the 392 bp fragment of the p16 CpG island in AGS and MGC803 cells; Each green bar represents a CpG site. Nucleosomal location in exon-1 is also marked (26). (B and C) Bisulfite sequencing in the fusion monoclone-E3 and subclones. Focal methylation changes (yellow-shadowed); methylated CpG sites (red dot); unmethylated CpG sites (open dot); focally methylated- or demethylated-p16 molecules (black arrows). (D) Homeostatic methylation model for a single CpG island in the tetraploid cells.
Figure 3.
Bisulfite sequencing of p16 alleles in HCT116 cells.
(A) Bisulfite clone sequencing of the 392 bp fragment of the p16 CpG island in HCT116 cells. Wild-type (deletion)/mutant (G-insertion) p16 alleles; focally demethylated-p16 molecules (black arrows); focally de novo methylated molecules (red arrows). (B) Homeostatic methylation model for a single CpG island in the diploid cells.
Figure 4.
p16 chromatin hydroxymethylation characterization.
(A) Immuno-precipitation and PCR analysis of synthetic and cellular 5hmC-containing p16 sequences. The AGS and fusion monoclone-D3 and E3 cells show significant levels of 5hmC. (B) TAB-seq analysis reveals completely hydroxymethylated-p16 alleles in the fusion subclones, but not the parental cells. (C) TAB-seq analysis of HCT116 cells shows the majority of the wildtype p16 alleles are completely hydroxymethylated.
Figure 5.
Analysis of hydroxymethylation in the sense-strand of p16 CpG island in HCT116 cells.
(A) Location of the 369 bp amplicon of the sense-strand p16 exon-1. (B) Detection of the 369 bp PCR products using DHPLC (sizing mode) at 48°C. (C) Detection of hydroxymethylated molecules amplified from the p16 sense-strand after TAB modification using DHPLC (FL detector) at the partial denaturing temperature 55°C. Under this condition, the hydroxymethylated- and unhydroxymethylated-p16 molecules are partially and completely denatured, respectively. The regular bisulfite-treated templates are used as reference controls. (D) Results of TAB-sequencing for the 369 bp PCR products amplified from HCT116 cells.
Figure 6.
Allele specific RT-PCR analysis of p16 mRNA of cells.
(A) RT-PCR clone sequencing of the control 293T cell line (del) shows wildtype p16 mRNA. The HCT116 (G-ins) cell line revealed only mutant p16 mRNA clones. (B) Clone sequencing results show both wildtype and mutant p16 mRNA.
Figure 7.
Characterization of histone modifications in the p16 chromatin by ChIP-PCR assays.
(A) The active H3K4me3 level within the p16 CpG islands in the fusion subclones and MGC803 cells was significantly higher than AGS cells, especially in the exon-1 region (monoclones vs. AGS, P<0.01). (B) The repressive H3K9me3 level in the fusion subclones and AGS cells was significantly higher than MGC803 cells, especially in the promoter region (monoclones vs. MGC803, P<0.01).