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.

项目摘要/摘要 哺乳动物的发展依赖于定义细胞命运的空间序列的转录程序。 虽然控制此类转录状态的机制尚不清楚，但研究的线索仍在研究中 非编码基因组中的增强子及其与基因基因座的长期相互作用，否则 线性尺度。这种接触由核蛋白的三维（3D）组织促进 动态调节的属性允许在胚胎发育过程中做出细胞命运决策。 Sox2是一个 转录因子（TF）对胚胎干细胞的自我更新和多能性至关重要（ESC）。在早期 胚胎发育，Sox2表达持续存在于神经祖细胞中，在这种神经祖细胞中也对他们的自我至关重要 更新和多稳定性，但没有在Mesendoderm中转录。在人类ESC（hESC）中，多能性 - 相关的TF，例如OCT4，与Sox2形成异栽培，被认为可以驱动Sox2转录 通过结合SOX2启动子。但是，Sox2转录选择性持续存在的机制 在没有多能TF的情况下，神经外胚层仍未知。我们的小组，对人类感兴趣 健康和疾病中的神经干细胞（NSC）生物学最近发现，在人类NSC中， Sox2受3D染色质循环距离基因座600 kb的新型增强剂调节。 重要的是，这种推定的增强剂在接触的hESC和中胚层祖细胞中受到压制 在远端增强子和启动子之间丢失。这些发现使我们假设，在 hESC与早期神经前体的分化，Sox2转录成为从头开始的 受发展调节的远处增强剂，该增强剂通过动态地对Sox2基因座发挥影响 染色质环的配置取决于染色质组织者CTCF。为了支持这一假设，我们 发现SOX2基因组邻域中关键CTCF DNA结合基序的CRISPR切除了这一点 3D染色质循环损害hESC的神经化和偏见hESC分化为内皮层命运的偏差 畸胎瘤分析。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人类神经发生中的激活？ 2）如何扰动 3D染色质折叠会影响神经发育，它如何调节增强剂活性？ 3）做sox2 根据蛋白质 - 蛋白质相互作用与 开发监管的TF？我们的研究将阐明以前未被认可但至关重要的远程 对神经发育必不可少的基因，SOX2和远处增强子之间的相互作用，这些基因是介导的 通过3D基因组架构的动态重组。阐明基本机制将使我们能够 将这些生物学概念应用于理解和治疗神经发育障碍。