小鼠多潜能干细胞染色质三维空间构象调控干性基因表达的分子机制

Different cell types exhibit a characteristic higher-order nuclear architecture. However, very little is known about how that interchromosomal interactions are organized, the epigenetic features that govern higher-order chromosome interaction or the role those interactions play in gene regulation. Somatic cell reprogramming, in which somatic cells are converted to pluripotent cells, provides a good model to address these questions. We performed circular chromosome conformation capture (4C) combined with next-generation sequencing using the Oct4 enhancer as bait to identify the Oct4 enhancer interactome in both human and mouse ES cells.We hypothesize that PSCs possess a unique higher-order chromosome structure that facilitates expression of pluripotency genes, and that somatic cell reprogramming requires establishment of that structure. We will characterize the signature of higher-order chromosome structure of mouse pluripotent stem cells and determine epigenetic features of Oct4, Sox2, and Nanog enhancer interactomes in mouse fibroblasts, neural stem cells, iPS cells and ES cells, and determine how interchromosomal modulates pluripotency gene expression. In addition, we will determine how higher-order chromosome structure is established when mouse somatic cells are converted to pluripotent stem cells and if the establishment of interchromosomal interaction is important for somatic cell reprogramming. These studies should further our understanding of higher-order chromosome structure in mouse cells.Mechanistic insights gained from these studies will be useful for application of PSCs to regenerative medicine.

不同细胞类型具有独特的细胞核三维空间构象。然而，我们对染色体间相互作用的建立机制、高级染色体相互作用的表观遗传学特征以及染色体间相互作用在基因调节中的作用了解甚少。体细胞重编程为解决以上问题提供了良好的模型。本课题组以Oct4增强子作为模板，通过环形染色体构象捕获及下一代测序技术，测定了人类和小鼠ES细胞中Oct4增强子染色质互作谱。我们推测多能干细胞具有促进干性基因表达的独特高级染色体结构，且此结构为体细胞重编程所必需。本课题将在小鼠成纤维细胞、神经干细胞、iPS细胞和ES细胞中检测Oct4、Sox2和Nanog增强子相互作用的表观遗传特征，并探究染色体相互作用调控干性基因表达的机制。此外，我们将进一步研究体细胞重编程过程中高级染色体结构的建立机制及其重要性。本研究为小鼠高级染色体结构提供了新颖的的见解，为体细胞重编程在转化医学中的应用提供了理论依据。

神经发生是神经祖细胞（neural progenitor cell, NPC）产生神经元和神经胶质细胞的复杂过程。 根据发育阶段，NPC可以自我更新或分化以产生不同类型的神经元和神经胶质后代。 精确控制这种平衡以确保神经系统的正常发育并维持成年大脑的体内平衡。Kruppel样因子4（Kruppel-like factor 4, Klf4）是一种含锌指蛋白，主要作为转录因子调控了多种细胞生理学过程，可以调节胚胎干细胞的自我更新，并与Oct4，Sox2和c-Myc一起将体细胞重编程为诱导的多能干细胞。我们认为klf4还起到了转录因子之外的重要作用。本项目中，我们证明了神经前体细胞（neural progenitor cells, NPCs）中的Klf4基因缺失会增加小鼠神经发生，同时降低其自我更新能力。