Developmentally Regulated Enhancers and Chromatin Architecture in Human Neurogenesis

项目摘要/摘要 哺乳动物的发育依赖于时空编排的转录程序，这些程序定义了细胞的命运。 虽然控制这种转录状态的机制仍不清楚，但从这项研究中出现了一些线索 非编码基因组内的增强子及其与基因座的远程相互作用 线性刻度。这种接触是由染色质的三维(3D)组织促进的，染色质是一种核物质 其动态调节允许细胞在胚胎发育期间决定命运的财产。SOX2是一种 转录因子(TF)对胚胎干细胞(ESCs)的自我更新和多能性至关重要。在早期 在胚胎发育过程中，SOX2的表达持续存在于神经前体细胞中，在那里它对其自身也是至关重要的 更新和多能性，但它不在中胚层转录。在人类胚胎干细胞(HESCs)中，多潜能 相关的转录因子，如OCT4，与SOX2形成异多聚体，并被认为推动SOX2转录 通过结合SOX2启动子。然而，SOX2转录选择性持续存在的机制 神经外胚层在缺乏多能性的转铁蛋白的情况下仍然未知。我们这个对人类感兴趣的团体 神经干细胞(NSC)在健康和疾病中的生物学，最近发现，在人类NSCs中，转录 SOX2受一种新型增强子的调控，该增强子位于距离基因位置600kb的位置，通过3D染色质环。 重要的是，这种假定的增强子在hESCs和中内胚祖细胞中受到抑制，其中接触 在远端增强子和启动子之间丢失。这些发现让我们假设，在 人类胚胎干细胞分化为早期神经前体，SOX2转录从头开始依赖于 发育调控的远端增强子，通过动态作用于SOX2基因座 根据染色质管理器CTCF配置的染色质循环。为了支持这一假设，我们 发现SOX2基因组邻域中关键的CTCFDNA结合基序的CRISPR切除破坏了这一点 3D染色质环损害人胚胎干细胞的神经化并使其向内皮细胞分化 畸胎瘤检测。拟议的研究计划将回答以下问题：1) 实验诱导人类神经发生中增强子的沉默和激活？2)扰动是如何 3D染色质折叠影响神经发育，以及它如何调节增强子活性？3)SOX2 作为增强子激活的有条件的启动子，取决于蛋白质与蛋白质的相互作用 发育调节因子？我们的研究将阐明以前未被认识到的但关键的长期 神经发育所必需的基因SOX2和远距离增强子之间的相互作用，它们是由 通过动态重组3D基因组结构。阐明潜在的机制将使我们能够 应用这些生物学概念来理解和治疗神经发育障碍。

PROJECT SUMMARY/ABSTRACT Mammalian development relies on spatiotemporally orchestrated transcriptional programs that define cell fates. While the mechanisms that govern such transcriptional states remain unclear, clues are emerging from the study of enhancers within the non-coding genome and their long-range interactions with gene loci otherwise distant on a linear scale. Such contacts are facilitated by the three-dimensional (3D) organization of chromatin, a nuclear property whose dynamic regulation allows for cell fate decisions during embryonic development. SOX2 is a transcription factor (TF) critical to the self-renewal and pluripotency of embryonic stem cells (ESCs). In early embryonic development, SOX2 expression persists in neural progenitors, where it is also crucial to their self- renewal and multipotency, but it is not transcribed in mesendoderm. In human ESCs (hESCs), pluripotency- associated TFs, such as OCT4, form heteromultimers with SOX2 and are thought to drive SOX2 transcription by binding the SOX2 promoter. However, the mechanism whereby SOX2 transcription selectively persists in neuroectoderm in the absence of pluripotency TFs remains unknown. Our group, which is interested in human neural stem cell (NSC) biology in health and disease, recently discovered that, in human NSCs, transcription of SOX2 is regulated by a novel enhancer located 600 kb away from the gene locus via 3D chromatin looping. Importantly, this putative enhancer is repressed in hESCs and mesendodermal progenitors where the contact between the distal enhancer and promoter is lost. These findings lead us to hypothesize that, during differentiation of hESCs to early neural precursors, SOX2 transcription becomes de novo dependent on a developmentally regulated distant enhancer, which exerts its effects on the SOX2 locus via dynamically configured chromatin looping dependent on the chromatin organizer CTCF. In support of this hypothesis, we found that CRISPR excision of critical CTCF DNA binding motifs in the SOX2 genomic neighborhood disrupt this 3D chromatin loop to impair neuralization of hESCs and bias hESC differentiation to endodermal fates in teratoma assays. The proposed research plan will answer the following questions: 1) What are the effects of experimentally induced enhancer silencing and activation in human neurogenesis? 2) How does perturbation of the 3D chromatin folding affect neural development and how does it modulate enhancer activity? 3) Does SOX2 act as a conditional initiator of enhancer activation depending on protein-protein interactions with developmentally regulated TFs? Our studies will shed light on previously unrecognized but critical long-range interactions between a gene essential to neural development, SOX2, and a distant enhancer, which are mediated by dynamic reorganization of 3D genome architecture. Elucidating the underlying mechanisms will allow us to apply these biological concepts toward understanding and treating neurodevelopmental disorders.