Developmentally Regulated Enhancers and Chromatin Architecture in Human Neurogenesis

人类神经发生中的发育调控增强子和染色质结构

• 批准号: 10682470
• 负责人: Dimitris G. Placantonakis
• 金额: $ 53.7万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10682470
• 关键词: 3-Dimensional; ATAC-seq; Affect; Architecture; Attenuated; Binding; Biological; Biological Assay; CRISPR-mediated transcriptional activation; Cells; Chromatin; Chromatin Loop; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA Binding; Data; Development; Disease; Distal; Distant; Embryonic Development; Enhancers; Etiology; Excision; Genes; Genetic Enhancer Element; Genetic Transcription; Genome; Genomics; Health; Hi-C; Human; Impairment; In Vitro; Mediating; Methylation; Microscopic; Molecular; Neighborhoods; Nervous System; Neurodevelopmental Disorder; Neuroectoderm; Nuclear; Process; Property; Proteins; Regenerative Medicine; Regulation; Repression; Research; Specific qualifier value; Teratoma; Testing; Transcript; Untranslated RNA; bisulfite sequencing; cell fate specification; cell type; chromatin immunoprecipitation; chromatin modification; chromosome conformation capture; embryonic stem cell; epigenetic silencing; genomic locus; human embryonic stem cell; in vivo; interest; multidisciplinary; nerve stem cell; neural; neurodevelopment; neurogenesis; novel; pluripotency; progenitor; programs; promoter; protein protein interaction; self-renewal; single-cell RNA sequencing; spatiotemporal; stem cell biology; stem cell differentiation; transcription factor; translational potential

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.

项目概要/摘要 哺乳动物的发育依赖于时空精心编排的转录程序，这些转录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，但研究中正在出现线索 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，否则基因位点就很遥远 线性标度。染色质的三维 (3D) 组织促进了这种接触，染色质是核 其动态调节允许胚胎发育过程中细胞命运决定的特性。 SOX2 是 转录因子（TF）对于胚胎干细胞（ESC）的自我更新和多能性至关重要。早在 在胚胎发育过程中，SOX2 的表达持续存在于神经祖细胞中，这对于神经祖细胞的自我调节也至关重要。 更新和多能性，但它不在中内胚层中转录。在人类 ESC (hESC) 中，多能性 相关的 TF，例如 OCT4，与 SOX2 形成异多聚体，并被认为驱动 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 缺乏多能性转录因子的神经外胚层仍然未知。我们小组对人类感兴趣 神经干细胞（NSC）生物学在健康和疾病中的作用，最近发现，在人类 NSC 中， SOX2 通过 3D 染色质环由距离基因位点 600 kb 的新型增强子调节。 重要的是，这种假定的增强子在 hESC 和中内胚层祖细胞中受到抑制，其中接触 远端增强子和启动子之间丢失。这些发现使我们推测，在 hESCs 分化为早期神经前体细胞时，SOX2 转录从头开始依赖于 发育调节的远程增强子，通过动态地对 SOX2 基因座发挥作用 配置的染色质环取决于染色质组织者 CTCF。为了支持这个假设，我们 发现 CRISPR 切除 SOX2 基因组附近的关键 CTCF DNA 结合基序破坏了这一点 3D 染色质环会损害 hESC 的神经化并使 hESC 分化为内胚层命运 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的增强子沉默和激活？ 2）如何扰动 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 是否有效 作为增强子激活的条件启动子，取决于蛋白质与蛋白质的相互作用 发育调节的 TF？我们的研究将揭示以前未被认识但至关重要的远程 神经发育必需基因 SOX2 和远程增强子之间的相互作用，由介导 通过 3D 基因组架构的动态重组。阐明根本机制将使我们能够 应用这些生物学概念来理解和治疗神经发育障碍。