Sox9 transcriptionally regulates Wnt signaling in intestinal epithelium stem cells under hypomethylated crypts in diabetic state

Mammalian intestinal epithelium stem cells (IESCs) and their daughter cells require the participation of DNA methylation and the transcription factor Sox9 for proliferation and differentiation. Combining methylated DNA immunoprecipitation with microarray hybridization, we demonstrate that hypomethylation in promoter participates in the aberrant formation of crypts in diabetic db/db mice through ectopic Wnt signaling. More importantly, increased expression of Sox9 is accompanied by the loss of methylation in its promoter in IESCs. Using ChIP-seq analysis for Sox9 in IESCs, we demonstrate that Sox9 primarily targets the enhancers of Wnt signaling pathway-related genes. Sox9 is not only predominately acting as a transcriptional activator at proximal enhancer but also as a potential transcriptional inhibitor at distant enhancer. Lack of Sox9 transcriptional activation in specific repressors of Wnt signaling pathway results in loss of intrinsic inhibitory action and ultimately produces over-activation of this pathway in diabetes mellitus. A total of six samples are included in the experiment for promoter DNA methylation analysis, in which three are experimental group definded as db/db mice and three are control group definded as db/+ mice. The mice were maintained under a 12 h light/12 h dark cycle in a specific pathogen-free animal facility. The experiments were conducted using 16-week-old male homozygous mice, which were maintained for 8 weeks with hyperglycemia (random blood glucose level ? 16.7 mmol/l) prior to euthanasia. Identical genetic background BKS heterozygote db/+ mice (random blood glucose level < 11.1 mmol/l) were used as the control. All experiments with mice were approved by the Animal Care Committee of Sun Yat-Sen University (Permit Number: 201412000091).