DNA methyltransferase 3A controls intestinal epithelial barrier function and regeneration in the colon [RNA-seq]

Genetic variants in the DNMT3A locus have been associated with inflammatory bowel disease (IBD). DNMT3A is part of the epigenetic machinery physiologically involved in DNA methylation. We show that DNMT3A plays a critical role in maintaining intestinal homeostasis and gut barrier function. DNMT3A expression is downregulated in intestinal epithelial cells (IECs) from IBD patients and upon TNF treatment in murine intestinal organoids. Ablation of DNMT3A in Caco-2 cells results in global DNA hypomethylation, which is linked to impaired regenerative capacity, transepithelial resistance and intercellular junction formation. Genetic deletion of Dnmt3a in IECs (Dnmt3aΔIEC) in mice confirms the phenotype of an altered epithelial ultrastructure with shortened apical-junctional complexes, reduced Goblet cell numbers and increased intestinal permeability in the colon in vivo. Dnmt3aΔIEC mice suffer from increased susceptibility to experimental colitis, characterized by reduced epithelial regeneration. These data demonstrate a critical role for DNMT3A in orchestrating intestinal epithelial homeostasis and response to tissue damage and suggest an involvement of impaired epithelial DNMT3A function in the etiology of IBD. For the in vitro experiment with Caco-2 cells, 4 groups have been analyzed with 4 replicates each. For in vivo experiment using purified intestinal epithelial cells from Dnmt3a △IEC in steady state, 4 wild type mice and 4 Dnmt3a △IEC mice were used.

DNMT3A基因座的遗传变异与炎症性肠病（inflammatory bowel disease，IBD）相关。DNMT3A是表观遗传调控系统（epigenetic machinery）的组成部分，生理条件下参与DNA甲基化过程。本研究证实，DNMT3A在维持肠道稳态与肠屏障功能中发挥关键作用。炎症性肠病患者的肠上皮细胞（intestinal epithelial cells，IECs）以及经肿瘤坏死因子（tumor necrosis factor，TNF）处理的小鼠肠道类器官中，DNMT3A的表达均被下调。在Caco-2细胞中敲除DNMT3A会导致全基因组DNA低甲基化，这与细胞再生能力受损、跨上皮电阻降低以及细胞间连接形成异常相关。体内实验证实，小鼠肠上皮细胞特异性缺失Dnmt3a（Dnmt3aΔIEC）会引发上皮超微结构改变，具体表现为顶膜连接复合物缩短、杯状细胞（Goblet cell）数量减少以及结肠肠道通透性升高。Dnmt3aΔIEC小鼠对实验性结肠炎的易感性升高，其特征为上皮再生能力减弱。上述实验数据证实，DNMT3A在协调肠道上皮稳态与组织损伤应答过程中发挥关键作用，同时提示上皮细胞DNMT3A功能受损可能参与炎症性肠病的发病机制。针对Caco-2细胞的体外实验共设置4组，每组设置4个生物学重复。针对稳态条件下取自Dnmt3aΔIEC小鼠与野生型小鼠的纯化肠上皮细胞的体内实验，每组分别使用4只野生型小鼠与4只Dnmt3aΔIEC小鼠。