DNA methylation, through DNMT1, has an essential role in the development of gastrointestinal smooth muscle cells and disease [miRNA-Seq]

DNA methylation is a key epigenetic modification that can regulate gene expression. Genomic DNA hypomethylation is commonly found in many gastrointestinal (GI) diseases. Dysregulated gene expression in GI smooth muscle cells (GI-SMC) can lead to motility disorders. However, the consequences of genomic DNA hypomethylation within GI-SMC are still elusive. Utilizing a Cre-lox murine model, we have generated SMC-restricted DNA methyltransferase 1 (Dnmt1) knockout (KO) mice and analyzed the effects of Dnmt1 deficiency. Dnmt1-KO pups are born smaller than their wild type littermates, have shortened GI tracts, lose peristaltic movement due to loss of the tunica muscularis in their intestine, causing massive intestinal dilation, and death around post-natal day 21. Within smooth muscle tissue, significant CpG hypomethylation occurs across the genome at promoters, introns and exons. Additionally, there is a marked loss of differentiated SMC markers (Srf, Myh11, miR-133, miR-143/145), an increase in pro-apoptotic markers (Nr4a1, Gadd45g), loss of cellular connectivity, and an accumulation of coated vesicles within SMC. Interestingly, we observed consistent abnormal expression patterns of enzymes involved in DNA methylation between both ¬Dnmt1-KO mice and diseased human GI tissue. These data demonstrate that DNA hypomethylation in embryonic SMC, via congenital Dnmt1 deficiency, contributes to massive dysregulation of gene expression and is lethal to GI-SMC. These results suggest that Dnmt1 has a necessary role in the embryonic, primary development process of SMC with consistent patterns being found in human GI diseased tissue. mRNA and DNA methyation profiles of Dnmt1 WT and KO jejunum smooth muscle were generated by deep sequencing using Illumina Hiseq2000.DNA甲基化（DNA methylation）是一类关键的表观遗传修饰，可调控基因表达。基因组DNA低甲基化常见于多种胃肠道（GI）疾病。胃肠道平滑肌细胞（GI-SMC）的基因表达失调可引发胃肠道运动功能障碍。然而，GI-SMC内基因组DNA低甲基化所产生的生物学效应仍有待阐明。本研究利用Cre-lox小鼠模型，构建了平滑肌细胞特异性DNA甲基转移酶1（Dnmt1）敲除（KO）小鼠，并分析了Dnmt1缺失的生物学影响。结果显示，Dnmt1-KO幼鼠出生时体型较野生型同窝小鼠更小，胃肠道长度缩短，因肠道肌层丢失而丧失蠕动功能，进而引发严重的肠道扩张，并在出生后第21天左右死亡。在平滑肌组织中，全基因组范围内的启动子、内含子与外显子区域均出现显著的CpG低甲基化。此外，分化型平滑肌细胞标志物（Srf、Myh11、miR-133、miR-143/145）的表达显著下调，促凋亡标志物（Nr4a1、Gadd45g）的表达上调，细胞连接丧失，且平滑肌细胞内出现有被囊泡聚集现象。值得注意的是，我们观察到Dnmt1-KO小鼠与患病人类胃肠道组织中，参与DNA甲基化的酶的表达模式均存在一致的异常。上述数据表明，通过先天性Dnmt1缺失导致的胚胎期平滑肌细胞DNA低甲基化，会引发严重的基因表达失调，并对GI-SMC产生致死效应。本研究结果提示，Dnmt1在平滑肌细胞的胚胎原发性发育过程中发挥不可或缺的作用，且该作用模式在人类胃肠道病变组织中同样存在。本研究通过Illumina Hiseq2000平台开展深度测序，获取了Dnmt1野生型（WT）与敲除型（KO）小鼠空肠平滑肌的mRNA及DNA甲基化谱。