Differential DNA methylation patterns of homeobox genes in proximal and distal colon epithelial cells

Abstract

Region and cell-type specific differences in the molecular make up of colon epithelial cells have been reported. Those differences may underlie the region-specific characteristics of common colon epithelial diseases such as colorectal cancer and inflammatory bowel disease. DNA methylation is a cell-type specific epigenetic mark, essential for transcriptional regulation, silencing of repetitive DNA and genomic imprinting. Little is known about any region-specific variations in methylation patterns in human colon epithelial cells. Using purified epithelial cells and whole biopsies (n= 19) from human subjects, we generated epigenome-wide DNA methylation data (using the HELP-tagging assay), comparing the methylation signatures of the proximal and distal colon. We identified a total of 125 differentially methylated sites (DMS) mapping to transcription start sites of protein-coding genes, most notably several members of the homeobox (HOX) family of genes. Patterns of differential methylation were validated with MassArray EpiTYPER. We also examined DNA methylation in whole biopsies, applying a computational technique to deconvolve variation in methylation within cell types and variation in cell-type composition across biopsies. Including inferred epithelial proportions as a covariate in differential methylation analysis applied to the whole biopsies resulted in greater overlap with the results obtained from purified epithelial cells compared with when the covariate was not included. Results obtained from both approaches highlight region-specific methylation patterns of HOX genes in colonic epithelium. Regional variation in methylation patterns has implications for the study of diseases that exhibit regional expression patterns in the human colon, such as inflammatory bowel disease and colorectal cancer.

Inflammation-associated DNA methylation patterns in epithelium of ulcerative colitis

“…S2A). Consistent with previous reports [31][32][33][34] a higher proportion of non-methylated CCGG sites (DNA methylation score 30) fell within the vicinity of the transcription start sites (TSS) of genes ( §2 kb). The distribution in this region was bimodal, whereas other genomic locations such as the gene body and intergenic regions were predominantly methylated ( Fig.…”

“…Results illustrated the relationship between CCGG rich and depleted regions and the relative DNA methylation state at these regions. As reported previously, 34 the proportion of CCGG sites was higher at the TSS, becoming relatively depleted up and downstream of the TSS. However, DNA methylation decreased in close proximity to TSS (mean score D 21.0 at the TSS) and then increased both up-and down-stream from each TSS peak (mean score D 74.1 §2 kb from TSS) ( Fig.…”

“…However, some variation may be attributable to the distinct epigenomic signatures observed in proximal and distal colonic regions; as identified previously. 34 This is reflected by 41 intersecting DMS mapping to 28 genes identified in normal epithelia and in the presence of disease (Table S3).…”

“…GO analysis was then performed, removing the overlapping DMS mapping to genes previously identified in proximal and distal epithelia, 34 thus identifying the enrichment of pathways, potentially more specific to inflammation. The enriched GO term that was identified was associated with bone morphogenesis (GO: 0060349, BH adjusted P D 0.09).…”

Shaping functional gut microbiota using dietary bioactives to reduce colon cancer risk

“…The Cancer Genome Anatomy Project (CGAP) functional grouping of the differentially expressed genes revealed major pathways implicated in colon cancer including epidermal growth factor (EGF), transforming growth factor beta (TGF-β), Wnt, Ras, insulin, and integrin signaling [77]. Differences in gene expression between the proximal and distal colon may result from the observed differential DNA methylation patterns in colonocytes [78]. Studies comparing cancers of the proximal and distal colon have revealed numerous differences in molecular and clinicopathological features, which reflect different susceptibilities to neoplastic transformation, such as the frequency of V-Ki-Ras2 Kirsten Rat Sarcoma 2 Viral Oncogene Homolog (KRAS) and B-Raf Proto-Oncogene, Serine/Threonine Kinase (BRAF) mutations, and the prevalence of MSI [79–81].…”