Organizational principles and functional role of 3D enhancer hubs in cell fate decisions

项目摘要 胚胎发育和组织动态平衡依赖于细胞类型特异性转录的严格调控 特定转录因子(TF)及其目标增强子的程序。出现了三个- 三维(3D)基因组组织作为转录控制层的重要一层，强调了这样一个事实 了解增强子如何与靶基因沟通以协调转录活动需要 3D核拓扑学知识。在最近的一项研究中，我们捕捉到了三维空间的剧烈重新布线 H3K27ac HiChIP对体细胞和胚胎干细胞(ESCs)的调控作用及鉴定 复杂的3D“增强子中心”，其中增强子与多个高表达基因在空间上聚集在一起 在茎的调节中具有已知或预测的功能。遗传或表观遗传调节 ESCs中的增强子导致所有中枢连接基因的下调和部分分化，这 支持这些结构节点在基因协同调节和细胞识别中的重要作用。此外，我们 提供了蛋白质组学和遗传学证据，证明KLF家族的转录因子在组织和 ESC中3D增强子中心的调控和确定的候选辅助因子。基于这些结果，我们 假设3D增强器集线器充当细胞身份的建筑“总部”，其中细胞 特定类型的基因被特定的转录调节因子隔离，以便于协调 基因表达。在这里，我们将在小鼠胚胎干细胞和早期胚胎干细胞的情况下检验这一假设。 使用体外和体内方法决定发育细胞的命运。具体地说，我们的目标是(1) 系统增强子和枢纽内的基因，以确定对多能性的功能后果 转录网络和ESC身份的稳定性，(2)决定关键的蛋白质因子和活性 其控制增强子中枢形成和功能，以及(3)识别和表征3D增强子中枢，该3D增强中枢 对每个早期发育命运的获取和维持都至关重要。成功完成 我们的目标将提供对3D增强中心的组织和监管的机械性见解，确定 它们在控制细胞命运中的作用，并通过针对关键结构来揭示工程细胞身份的新方法 节点和因素。

Project Summary Embryonic development and tissue homeostasis rely on the tight regulation of cell-type specific transcriptional programs by particular transcription factors (TFs) and their target enhancers. The emergence of three- dimensional (3D) genome organization as an important layer of transcriptional control, stresses the fact that understanding how enhancers communicate with target genes to coordinate transcriptional activity requires knowledge of the 3D nuclear topology. In a recent study, we captured a drastic rewiring of three-dimensional regulatory contacts between somatic cells and embryonic stem cells (ESCs) by H3K27ac HiChIP and identified complex 3D “enhancer hubs”, where enhancers are spatially clustered with multiple highly-expressed genes with known or predicted functions in regulation of stemness. Genetic or epigenetic modulation of such enhancers in ESCs resulted in downregulation of all hub-connected genes and partial differentiation, which supports a vital role for these architectural nodes in gene coregulation and cell identity. In addition, we provided proteomics and genetic evidence that KLF family TFs play an important role in the organization and regulation of 3D enhancer hubs in ESCs and identified candidate cofactors. Based on these results, we hypothesize that 3D enhancer hubs function as architectural “headquarters” of cell identity, where cell type-specific genes are sequestered by specific transcriptional regulators to facilitate coordinated gene expression. Here, we will test this hypothesis both in the contexts of mouse ESCs, and of early developmental cell fate decisions using in vitro and in vivo approaches. Specifically, we aim to (1) target systematically enhancers and genes within hubs to determine the functional consequences on the pluripotent transcriptional network and the stability of ESC identity, (2) determine the critical protein factors and activities that control enhancer hub formation and functionality and (3) identify and characterize 3D enhancer hubs that are critical for acquisition and maintenance of each of the early developmental fates. Successful completion of our aims will offer mechanistic insights into the organization and regulation of 3D enhancer hubs, determine their role in cell fate control and reveal novel ways for engineering cell identity by targeting critical architectural nodes and factors.