Abstract
BackgroundDistinctive structures called crypts harbor intestinal epithelial stem cells (IESCs) which generate progenitor and terminally differentiated cells in the intestinal epithelium. Mammalian IESCs and their daughter cells require the participation of DNA methylation and the transcription factor Sox9 for proliferation and differentiation. However, the association between Sox9 and DNA methylation in this process remains elusive.MethodsThe DNA methylation of small intestinal epithelial crypts in db/db mice was detected via combining methylated DNA immunoprecipitation with microarray hybridization. DNA methylation of Sox9 promoter in crypts and IESCs was validated using bisulfite sequence analysis. The target sequence of the transcription factor Sox9 in IESCs was investigated via chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq).ResultsIncreased Sox9 expression is accompanied by the loss of methylation in its promoter in IESCs. Sox9 targets the enhancers of the Wnt signaling pathway-related genes. Sox9 predominantly acts as a transcriptional activator at proximal enhancers of Wnt4, Tab2, Sox4, and Fzd8, but also functions as a potential transcriptional inhibitor at a distant enhancer of Cdk1. Lack of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway leads to the loss of intrinsic inhibitory action and ultimately produces overactivation of this pathway in db/db mice.ConclusionsOur study sheds light on the connections among DNA methylation, transcription factor modulation, and Wnt signaling in IESCs in the diabetic state. Hypomethylation in the Sox9 promoter is correlated to increased Sox9 expression in db/db IESCs. Although there is increased expression of Sox9 in db/db IESCs, the loss of Sox9 transcriptional activation in specific repressors of the Wnt signaling pathway might result in abnormalities in this pathway.
Highlights
Distinctive structures called crypts harbor intestinal epithelial stem cells (IESCs) which generate progenitor and terminally differentiated cells in the intestinal epithelium
We identified a total of 1216 group comparison-related differential methylation enrichment peak (DMEP), of which 760 were db/+_db/db and 456 were db/db_db/+ (Fig. 1c; Additional file 1: Table S5)
Hypomethylation in the Sry-box 9 (Sox9) promoter was correlated to its overexpression in IESCs Among the Wnt signaling pathway-related genes that were differentially expressed and differentially methylated, we identified Sox9, which is a transcription factor (TF) that plays a pivotal role in the regulation of Wnt signaling [7,8,9,10,11] and in the intestinal epithelium [22, 25]
Summary
Distinctive structures called crypts harbor intestinal epithelial stem cells (IESCs) which generate progenitor and terminally differentiated cells in the intestinal epithelium. The small intestinal epithelium has a distinctive organization, with intestinal epithelial stem cells (IESCs) harbored at the base of crypts. Recent studies using Sox chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIPseq) analysis have reported that Sox can activate canonical Wnt/β-catenin signaling in hepatocellular carcinoma [9] and prostate cancer [10]. In another Sox knockout mouse model, Wnt target genes appear to be unaltered in telogen hair follicles [11]. It remains unclear how Sox modulates IESCs via Wnt signaling pathways
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