Early Environment, Developmental Epigenetics, and Adult Colonic Diseases

项目总结 肠道微生物在出生后早期的定植在哺乳动物肠道发育中起着至关重要的作用。它 长期以来一直认为，DNA甲基化作为控制基因表达的表观遗传机制，是 参与肠道宿主-微生物的相互作用。生命中肠上皮细胞的高周转率 使肠道干细胞(ISCs)对肠道功能至关重要。然而，值得注意的是， 肠道微生物区系对DNA甲基化的影响机制 成年ISCs的行为仍然知之甚少。提高我们对这一过程的理解是根本 因为肠上皮是炎症和炎症的主要组织靶点之一 肿瘤的发生、DNA甲基化的异常以及肠道微生物区系的改变越来越多 被认为是疾病发病机制的关键。拟议的研究扩展了我们之前的工作，即 发现了3‘CpG岛(3’CGI)的一个子集，它们在小鼠的哺乳期间在ISCs中甲基化。 我们证明了3‘CGI甲基化传递与稳定基因相关的表观遗传记忆 成体ISCs的激活。此外，我们发现3‘CGI甲基化特别容易受到肠道的影响 微生物区系扰动。因此，拟议研究的目标是进一步阐明这种机制。 在ISCs发育过程中的表观遗传调控。我们的假设是后天建立的表观遗传记忆 通过3‘CGI甲基化为调节肠道宿主-微生物组相互作用提供了一条发育途径 会带来终生的功能后果。我们提出以下三个具体目标：(一)界定机制 通过3‘CGI甲基化调节肠道基因激活，(Ii)定义肠道 微生物区系调节3‘CGI甲基化，以及(Iii)定义微生物区系响应3’CGI的长期功能 甲基化。我们将利用最新的技术进步来隔离LGR5+ISCs；应用 最先进的技术，实现对微生物组的最终全基因组、公正的评估 对ISC表观基因组的影响；并使用尖端的器皿和CRISPR表观基因组编辑工具来解剖 微生物区系调控ISC功能的表观遗传机制。圆满完成 这些研究将对DNA甲基化在肠道中的功能作用产生重要的见解 发展，促进了我们对基因-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洞察力为发展提供了巨大的希望 关于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.