Developmentally Regulated Enhancers and Chromatin Architecture in Human Neurogenesis

项目概要/摘要 哺乳动物的发育依赖于时空协调的转录程序来定义细胞的命运。 虽然控制这种转录状态的机制仍不清楚，但从这项研究中已经出现了一些线索 非编码基因组内的增强子及其与基因位点的远程相互作用， 一个线性刻度。这种接触是由染色质的三维（3D）组织促进的， 在胚胎发育过程中，其动态调节允许细胞命运决定的性质。SOX 2是一个 转录因子（TF）对胚胎干细胞（ESC）的自我更新和多能性至关重要。月初 在胚胎发育中，SOX 2的表达在神经祖细胞中持续存在，在那里它对它们的自我发育也至关重要。 更新和多能性，但在中内胚层中不转录。在人ESC（hESC）中，多能性- 相关的TF，如OCT 4，与SOX 2形成异源多聚体，并被认为驱动SOX 2转录 通过结合SOX 2启动子。然而，SOX 2转录选择性持续存在的机制， 在不存在多能性TF的情况下，神经外胚层的功能仍然未知。我们的研究小组对人类 神经干细胞（NSC）生物学在健康和疾病，最近发现，在人类神经干细胞，转录 SOX 2是由一个新的增强子位于600 kb以外的基因座通过3D染色质环调控。 重要的是，这种假定的增强子在hESC和中内胚层祖细胞中被抑制， 远端增强子和启动子之间的连接丢失。这些发现使我们假设，在 在hESC分化为早期神经前体细胞的过程中，SOX 2转录变得从头依赖于一个神经元的转录。 发育调节的远端增强子，其通过动态地对SOX 2基因座发挥作用。 依赖于染色质组织者CTCF的配置的染色质成环。为了支持这一假设，我们 发现CRISPR切除SOX 2基因组附近的关键CTCF DNA结合基序破坏了这一点。 3D染色质环损害hESC的神经化并使hESC分化偏向内胚层命运， 畸胎瘤测定。拟议的研究计划将回答以下问题：1） 实验诱导的增强子沉默和激活在人类神经发生？2)微扰是如何 3D染色质折叠影响神经发育及其如何调节增强子活性？3)SOX2 作为增强子激活的条件引发剂，取决于蛋白-蛋白相互作用， 发育调节型TF我们的研究将揭示以前未被认识但关键的长距离 神经发育所必需的基因SOX 2和远端增强子之间的相互作用， 通过3D基因组结构的动态重组。阐明潜在的机制将使我们能够 将这些生物学概念应用于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.