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Engineering 3-D Epigenome Topology with Light

利用光设计 3D 表观基因组拓扑

基本信息

批准号：
8955256
负责人：
Jennifer Elizabeth Phillips-Cremins
金额：
$ 240万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-30 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8955256
关键词：
3-Dimensional Astrocytes BHLH Protein Behavior Biological Assay Brain Cataloging Catalogs Cell Cycle Cell Nucleus Cells Chromosomes Clustered Regularly Interspaced Short Palindromic Repeats Computational algorithm Cues DNA Sequence Development Dimerization Disease Distal Employee Strikes Engineering Enhancers Epigenetic Process Eye Gene Expression Gene Targeting Genes Genetic Transcription Genome Genomics Goals Heterogeneity Individual Light Link Maps Modification Molecular Conformation Nature Nerve Degeneration Neuraxis Neurons Nuclear Optics Organism Phenotype Population Proliferating Proteins Resolution Series Structure System Testing Time Work biological systems cell type deep sequencing epigenome genome editing genomic tools innovation nerve stem cell optogenetics predictive modeling promoter public health relevance relating to nervous system response self-renewal spatiotemporal tool transcription factor

项目摘要

DESCRIPTION (provided by applicant): The Epigenome is not a static entity, but is dramatically altered in response to environmental and developmental cues throughout the lifetime of an organism. In the case of the developing central nervous system, multipotent neural progenitor cells (NPCs) undergo marked reconfiguration of epigenetic marks as they exit the cell cycle and differentiate into the diverse neural and glial phenotypes that make up the mammalian brain. The dynamic nature of the Epigenome is poorly understood, particularly at short time scales, due to severe limitations in heterogeneity and asynchrony of the large cell populations typically required for genomics assays. Here we propose to overcome these technical challenges by building powerful new optical tools for the precise control of transcription dynamics in the context of the three-dimensional nucleus. Specifically, we aim to build modular, light-inducible architectural proteins for precise spatiotemporal control over the dynamics of 3-D interactions between distal enhancers and promoters of genes essential for neural lineage commitment. A recent study described the striking observation that three basic helix-loop-helix transcription factors are dynamically expressed in an oscillatory manner in proliferating, self-renewing NPCs, whereas only one of these factors transitions to sustained expression upon terminal differentiation. Our own preliminary studies reveal that genes encoding these candidate oscillatory factors are (1) often localized at boundaries between topological sub-domains (sub-TADs) and (2) connected to distal enhancers throughout both adjacent sub-TADs through long-range 3-D interactions. We hypothesize that dynamic 3-D interactions between distal enhancers and promoters of neural-specific bHLH transcription factors might govern the oscillatory expression of these genes. We will test our hypothesis by generating high-resolution maps of higher-order genome folding in NPCs and NPC-derived terminally differentiated neurons and astrocytes around candidate oscillatory factors. We will employ a pipeline of customized computational algorithms to integrate genome folding maps with an annotated catalogue of cell type-specific enhancers to create predictive models for the propensity of an individual enhancer to form 3-D interactions with developmentally regulated neural genes. In parallel, we will build modular, light-activated looping systems with CRISPR/Cas9 gene targeting in combination with proteins that are able to undergo light-inducible dimerization. We will then undertake a series of studies exploring looping dynamics to understand the organizing principles governing neural cell fate commitment. This work is innovative because it studies Epigenome dynamics with a cross- disciplinary approach that combines CRISPR/Cas9 genome editing, principles from optogenetics and genomics tools for mapping 3-D genome topology (i.e. Chromosome-Conformation-Capture and deep sequencing). Discoveries made by this work should yield an unprecedented view into the currently unknown dynamic behavior of genome structure and how it is linked to cell fate transitions in the developing brain.

描述(申请人提供)：表观基因组不是一个静态的实体，而是在有机体的整个生命周期中，随着环境和发育线索的变化而急剧变化。在发育中的中枢神经系统中，多潜能神经前体细胞(NPC)在退出细胞周期并分化为组成哺乳动物大脑的不同神经和神经胶质表型时，经历了显著的表观遗传标记的重新配置。由于基因组分析通常所需的大细胞群体的异质性和异步性的严重限制，人们对表观基因组组的动态性质知之甚少，特别是在短时间尺度上。在这里，我们建议通过建立强大的新光学工具来克服这些技术挑战，以便在三维核的背景下精确控制转录动力学。具体地说，我们的目标是构建模块化的、光诱导的结构蛋白，用于精确的时空控制对神经谱系承诺至关重要的基因的远端增强子和启动子之间的三维相互作用的动力学。最近的一项研究描述了一个引人注目的观察结果，即三个基本的螺旋-环-螺旋转录因子在增殖、自我更新的NPC中以振荡的方式动态表达，而这些因子中只有一个在末端分化时过渡到持续表达。我们自己的初步研究表明，编码这些候选振荡因子的基因(1)通常定位在拓扑亚结构域(SubTADS)之间的边界上，(2)通过远程三维相互作用连接到相邻两个亚TADS的远端增强子。我们假设，神经特异性bHLH转录因子的远端增强子和启动子之间的动态三维相互作用可能控制这些基因的振荡表达。我们将通过生成鼻咽癌和鼻咽癌衍生的终末分化神经元和星形胶质细胞中围绕候选振荡因子的高阶基因组折叠的高分辨率地图来验证我们的假设。我们将使用一系列定制的计算算法来整合基因组折叠图谱和细胞类型特定增强子的注释目录，以创建单个增强子与发育调节的神经基因形成3-D交互作用的倾向性的预测模型。同时，我们将利用CRISPR/Cas9基因靶向与能够进行光诱导二聚的蛋白质相结合，建立模块化的光激活环路系统。然后，我们将进行一系列探索循环动力学的研究，以了解支配神经细胞命运承诺的组织原则。这项工作具有创新性，因为它结合了CRISPR/Cas9基因组编辑、光遗传学原理和绘制三维基因组拓扑的基因组学工具(即染色体构象捕获和深度测序)的跨学科方法来研究表观基因组动力学。这项工作的发现应该会为目前未知的基因组结构动态行为以及它如何与发育中的大脑中的细胞命运转换联系在一起提供一个前所未有的视角。

项目成果

期刊论文数量（7）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Detecting hierarchical genome folding with network modularity.

DOI：
10.1038/nmeth.4560
发表时间：
2018-03
期刊：
Nature methods
影响因子：
48
作者：
Norton HK;Emerson DJ;Huang H;Kim J;Titus KR;Gu S;Bassett DS;Phillips-Cremins JE
通讯作者：
Phillips-Cremins JE

CRISPR/Cas9 genome editing throws descriptive 3-D genome folding studies for a loop.