Overlapping DNA methylation dynamics in mouse intestinal cell differentiation and early stages of malignant progression. Mus musculus

Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of carcinogenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2positive), differentiated cells (EphB2negative), ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of carcinogenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes regulated by PRC2 and associated with the intestinal stem cell signature. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+mice, but at lower levels than advanced cancers. Taking into account the parallelisms between human and mouse intestinal carcinogenesis, this study provides a reference framework to interpret the alterations found in human cancer. Overall design: We have analyzed ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Samples were in triplicates, except for tissue adjacent to adenoma (in duplicates).

肠道隐窝细胞分化与肿瘤发生的小鼠模型，已被用于解析这两类过程的潜在分子机制。DNA甲基化（DNA methylation）是关键的表观遗传标记，在细胞身份维持、分化程序及癌症发生中发挥重要作用。为探究细胞分化与恶性转化的动态变化，本研究比对了小鼠小肠隐窝及肿瘤发生早期阶段的DNA甲基化谱。本研究采用DNA甲基化全基因组分析结合基因表达芯片技术，对以下样本进行比对分析：肠道隐窝干细胞（EphB2阳性，EphB2positive）、分化细胞（EphB2阴性，EphB2negative）、ApcMin/+腺瘤及对应癌旁正常组织，同时纳入小肠、大肠样本与结肠癌细胞系CT26。与肿瘤晚期阶段相比，小肠隐窝分化与肿瘤发生早期阶段的DNA甲基化变化较少且幅度相对轻微。两类过程共有的高甲基化位点大多集中于启动子与增强子邻近区域，且富集于受PRC2调控、并与肠道干细胞特征相关的基因中。高甲基化进程呈渐进式：与肠道干细胞相比，分化细胞中的高甲基化水平极低，而在肿瘤晚期阶段则达到完全甲基化。低甲基化则在细胞分化与肿瘤中呈现不同的特征谱：ApcMin/+小鼠腺瘤旁的非肿瘤组织中已存在低甲基化，但其水平低于晚期癌症样本。鉴于人类与小鼠肠道肿瘤发生过程存在相似性，本研究为解读人类癌症中发现的表观遗传改变提供了参考框架。实验整体设计：本研究分析了ApcMin/+腺瘤及对应癌旁正常组织、小肠与大肠样本，以及结肠癌细胞系CT26。所有样本均设置生物学重复，其中腺瘤旁组织重复数为2次，其余样本重复数为3次。