Inflammation-associated DNA methylation patterns in epithelium of ulcerative colitis

Abstract

Aberrant DNA methylation patterns have been reported in inflamed tissues and may play a role in disease. We studied DNA methylation and gene expression profiles of purified intestinal epithelial cells from ulcerative colitis patients, comparing inflamed and non-inflamed areas of the colon. We identified 577 differentially methylated sites (false discovery rate <0.2) mapping to 210 genes. From gene expression data from the same epithelial cells, we identified 62 differentially expressed genes with increased expression in the presence of inflammation at prostate cancer susceptibility genes PRAC1 and PRAC2. Four genes showed inverse correlation between methylation and gene expression; ROR1, GXYLT2, FOXA2, and, notably, RARB, a gene previously identified as a tumor suppressor in colorectal adenocarcinoma as well as breast, lung and prostate cancer. We highlight targeted and specific patterns of DNA methylation and gene expression in epithelial cells from inflamed colon, while challenging the importance of epithelial cells in the pathogenesis of chronic inflammation.

Keywords: DNA methylation; inflammatory bowel disease; intestinal epithelial cell; transcriptome; ulcerative colitis.

Figure 1.

Classification of cellular proportions in non-inflamed and inflamed colonic regions. Intestinal epithelial cells (IECs) isolated from mucosal pinch biopsies are illustrated (A-D). IECs were labeled with fluorescent antibodies EpCAM and CD45 to distinguish cell populations. Representative histograms of EpCAM positive labeled cells (red) and its isotype control (blue) are illustrated in non-inflamed (A) and inflamed (C) regions. Quantification of the percentage of epithelial cells in the IEC isolate was then performed. Representative scatterplots of epithelial positive cells (upper left), CD45 positive cells (lower right) and double negative cells (lower left and upper right) are illustrated in non-inflamed (B) and inflamed (D) regions.

Figure 2.

Site-specific differential methylation between non-inflamed and inflamed colonic regions. DNA methylation values of DMS using a color scale from red (high DNA methylation) to yellow (low DNA methylation). Columns represent samples (n = 12 pairs) and rows represent all differentially methylated CCGG sites (n = 577) in inflamed (red) and non-inflamed samples (blue) (A). DMS mapping to active TSS regions (red) and enhancer regions (yellow) of HOXB3 (B), HOXB4 (C), and HOXB5 (D), as well as the ChIP-seq profiles of the set of histone marks assayed in colonic mucosa is shown. Below the histone marks, DMS (red) and all CCGG sites (green) mapping to each gene segment are also illustrated.

Figure 3.

Annotation of CCGG sites to genomic regions and ChromHMM states. The percentage of CCGG sites (gray bars) and differentially methylated sites (DMS) (red bars) annotating to TSS, intragenic and intergenic regions is shown (A). A greater number of DMS mapped to intragenic regions as opposed to TSS (promoter) regions. The total percentage of probes is greater than 100% as several probes were classified as belonging to more than one class of genomic region. The percentage of CCGG sites (gray bars) and DMS (red bars) mapping to 18 active and repressed genomic states in colonic mucosa using the ChromHMM model is shown (B). Using the genomic states defined by the ChromHMM model, significant enrichment of DMS, conditioned on the CCGG density at each genomic state was tested. P-values were determined using a Fisher exact test (*P<0.05, ** P<0.005, *** P<0.0005). Results indicate significant enrichment at enhancers, TSS regions, as well as repressed polycomb regions.

Figure 4.

Genome-wide and site-specific gene expression patterns in non-inflamed and inflamed colonic regions. Unsupervised hierarchal clustering of global gene expression profiles (A). Each column represents individual samples [I: Inflamed; NI: Non-inflamed, (n = 5 pairs)] and each row represents individual transcripts. The relative gene expression differences are expressed by a color gradient intensity scale ranging from yellow (low expression) to red (high expression). Volcano plot of differential expression analysis (B): Log fold change (logFC) between inflamed and non-inflamed samples (X-axis), –Log¹⁰ for identified non-significant (black) and significant (red) (Benjamini-Hochberg adjusted P of < 0.2) transcripts (Y-axis). Boxplot with overlaying stripchart representing the relative gene expression differences between inflamed (red) and non-inflamed (blue) samples for genes PRAC1 (C), HOXB13 (D), and PRAC2 (E).

Figure 5.

Integrative analysis of differential DNA methylation and gene expression patterns. DNA methylation patterns, gene expression patterns and spearman correlations for genes RARB (A) and FOXA2 (C) are illustrated. Left panel: bar plot and overlaying strip chart of DNA methylation levels between inflamed (red) and non-inflamed (blue) samples; middle panel: bar plot and overlaying strip chart of relative gene expression levels between inflamed (red) and non-inflamed (blue) samples; right panel: spearman correlation between DNA methylation levels (X-axis) and relative gene expression levels (Y-axis); P: P-value; rho: Spearman correlation value. DMS mapping to the active TSS (red) of RARB (B), bivalent enhancer (tan) and repressed polycomb regions (gray) of FOXA2 (D), as well as the ChIP-seq profiles of the set of 6 histone marks assayed in colonic mucosa is shown. Below the histone marks, DMS (red) and all CCGG sites (green) mapping to the given segment of RARB and FOXA2 are also illustrated.