Early Environment, Developmental Epigenetics, and Adult Colonic Diseases

项目摘要 出生后早期肠道微生物定植在哺乳动物肠道发育中起着至关重要的作用。它 长期以来，人们一直认为DNA甲基化作为一种控制基因表达的表观遗传机制， 参与肠道宿主-微生物相互作用。肠上皮细胞在整个生命周期中的高周转率 使肠道干细胞（ISCs）对肠道功能至关重要。然而，值得注意的是， 肠道微生物群对DNA甲基化的影响的潜在机制，以调节出现和 成年ISCs的行为仍然知之甚少。提高我们对这一过程的理解至关重要 因为肠上皮是炎症的主要组织靶之一， 肿瘤发生，异常DNA甲基化以及肠道微生物群的改变越来越多地被发现。 被认为是疾病发病机制的关键。拟议的研究扩展了我们以前的工作， 鉴定了在小鼠哺乳期期间在ISC中甲基化的3' CpG岛（3' CGI）的子集。 我们证明了3' CGI甲基化传递了与稳定基因相关的表观遗传记忆， 在成人ISCs中激活。此外，我们发现3' CGI甲基化是唯一易受肠 微生物群扰动。因此，本研究的目的是进一步阐明其机制 表观遗传调控在发展ISCs。我们的假设是出生后建立的表观遗传记忆 通过3' CGI甲基化提供了调节肠道宿主-微生物组相互作用的发育途径 会导致终生的功能性后果我们提出以下三个具体目标： 3' CGI甲基化调节肠道基因激活的机制，（ii）定义肠道基因激活的机制。 微生物群调节3' CGI甲基化，和（iii）定义微生物群响应性3' CGI的长期功能 甲基化我们将利用最新的技术进步，实现Lgr 5 + ISC的隔离;应用 最先进的技术，以实现最终的全基因组，微生物组的公正评估 对ISC表观基因组的影响;并使用尖端的类器官和CRISPR表观基因组编辑工具来解剖 微生物群介导的调节ISC功能的表观遗传机制。圆满完成 这些研究将有助于深入了解DNA甲基化在肠道疾病中的功能作用， 发展，推进我们对基因与环境相互作用的分子基础的理解， 肠子此外，从这些研究中获得的机械见解为发展提供了巨大的希望 肠道疾病的干预和治疗。

PROJECT SUMMARY Gut microbial colonization in early postnatal life plays a crucial role in mammalian intestinal development. It has long been postulated that DNA methylation, as an epigenetic mechanism to control gene expression, is involved in intestinal host-microbial interactions. The high turnover of intestinal epithelial cells throughout life makes intestinal stem cells (ISCs) critically important for gut function. Remarkably, however, how the mechanisms underlying the effects of the gut microbiota on DNA methylation to regulate the emergence and behavior of adult ISCs remain poorly understood. Improving our understanding of this process is fundamental for human health because intestinal epithelium is one of the major tissue targets of inflammation and tumorigenesis, and aberrant DNA methylation as well as alterations of the gut microbiota are increasingly recognized to be critical for disease pathogenesis. The proposed research extends our previous work that identified a subset of 3’ CpG islands (3’ CGIs) that are methylated in ISCs during the suckling period in mice. We demonstrated that 3’ CGI methylation transmits an epigenetic memory associated with stable gene activation in adult ISCs. In addition, we discovered that 3’ CGI methylation is uniquely vulnerable to gut microbiota perturbations. Therefore, the goal of the proposed research is to further elucidate the mechanisms of epigenetic regulation in developing ISCs. Our hypothesis is that postnatally established epigenetic memory by 3’ CGI methylation provides a developmental pathway for regulating intestinal host-microbiome interactions with lifelong functional consequences. We propose the following three specific aims: (i) Define the mechanism by which 3’ CGI methylation regulates intestinal gene activation, (ii) Define the mechanism by which the gut microbiota regulates 3’ CGI methylation, and (iii) Define the long-term function of microbiota-responsive 3’ CGI methylation. We will capitalize on recent technological advancements enabling isolation of Lgr5+ ISCs; apply state-of-the-art techniques to achieve the ultimate genome-wide, unbiased assessment of the microbiome effects on the ISC epigenome; and use cutting-edge organoid and CRISPR epigenome editing tools to dissect the microbiota-mediated epigenetic mechanisms that regulate ISC function. The successful completion of these studies will yield important insights into the functional role of DNA methylation during intestinal development, advancing our understanding of the molecular basis of gene-environment interactions in the intestine. Furthermore, the mechanistic insight gained from these studies offers great promise for development of interventions and treatments of intestinal diseases.