PMC

Using MIRA-assisted microarray analysis, inflammation-dependent DNA methylation was determined by hybridization of ileum DNA from 28-day- and 8-month-old Gpx1/2-KO mice versus ileum DNA from control mice of the same age. Experiments were performed for mice on the B6 and B6;129 background. As a control for Gpx1/2-KO-specific DNA methylation in the intestinal epithelium, liver DNA was used. The number of methylated genes is indicated.

A, Comparison of genes affected by inflammation-dependent and tumor-associated DNA methylation revealed three gene groups: genes affected by inflammation-dependent DNA methylation (group A; example: CpG island at the 5′ end of Barhl2), genes affected by inflammation-dependent and tumor-associated DNA methylation (group B; example: CpG island inside the Itgb4 gene), and genes affected only by tumor-associated aberrant DNA methylation (group C; example: CpG island at the 3′ end of Pitx2 gene). The gel panels show COBRA analysis of genes falling into the three categories. B, Diagram indicating genes affected in common by aberrant DNA methylation in six different DKO ileum tumors. C, Comparison of genes affected by age-dependent, inflammation-dependent and tumor-associated DNA methylation. Genes affected by tumor-associated DNA methylation in different tumors are summarized together. D, Comparison of genes affected by age-dependent, inflammation-dependent and tumor-associated DNA methylation for each individual tumor.

A, Comparison of genes affected by inflammation-dependent DNA methylation in DKO ileum from mice with B6 and B6;129 genetic background at the age of 8 months. B, DNA methylation analysis of the Robo1, Parc and Lepr CpG islands in control and DKO B6 mice by COBRA. Using gene-specific primers, bisulfite converted DNA was amplified. After cutting with enzyme (BstUI, TaqIα or HpyCH4IV) recognizing CpG sites, mock (−) and enzyme-digested (+) PCR products were fractionated by size on a 2% agarose gel. In vitro methylated mouse DNA (M) served as a positive control. C, DNA methylation patterns of the Robo1, Parc and Lepr CpG islands in control and DKO mice with B6;129 genetic background were analyzed by COBRA. D, Comparison of genes affected by age-dependent and inflammation-dependent DNA methylation in DKO ileum from mice with B6 and B6;129 genetic background at the age of 8 months.

Chromatin immunoprecipitation was conducted with ileal epithelial cells of 8-month-old wildtype and Gpx-1/2-KO mice. A, The Venn diagram summarizes the data obtained from CpG island arrays. Of the 249 genes that underwent DNA methylation in the ileum of DKO mice, 146 were marked by H3K27me3 in the ileum of control mice. 46 of these genes lost the Polycomb mark in the ileum of Gpx-1/2-KO mice. B, The RT-PCR data show verification of H3K27me3 loss in the ileum of DKO mice versus wildtype mice for the genes indicated. Asterisks indicate statistically significant differences (*, p<0.01; **, p<0.001). Hoxa10 promoter primers were used as positive control for binding of H3K27me3, H19 primers were used as a positive control for H3K27me3 and H3K9me3. The Gapdh promoter was used as a negative control. Binding to IgG antibodies was used as background control. Data shown are relative to “Input” (100%). C, Analysis of H3K9me3 in the ileum of control and DKO mice. D, Nonspecific IgG was used as a control.

A, Robo1; B, Gpc6; C, Gabrg3. Marked isoforms and genes are according to the UCSC Genome Browser. The “Methylation” bar represents DNA regions where inflammation-associated DNA methylation occurs according to the MIRA microarray experiments. CpG islands and localization of COBRA primers are indicated. Using gene-specific primers, bisulfite-converted DNA was amplified. The numbers refer to mouse identification numbers. These were the same mice used in the microarray approach (). After cutting with enzymes (BstUI, TaqIα or HpyCH4IV) recognizing CpG dincucleotides, mock (−) and enzyme-digested (+) PCR products for individual mouse tissue samples were fractionated by size on a 2% agarose gel. In vitro CpG-methylated mouse DNA (M) served as a positive control. Cleavage indicates DNA methylation. Additionally, PCR products were cloned into the pGEMTeasy vector and sequenced. White and black circles represent unmethylated and methylated CpG sequences, respectively.