Engineering 3-D Epigenome Topology with Light

 描述（由申请人提供）：表观基因组不是一个静态实体，而是在生物体的整个生命周期中响应环境和发育线索而发生显著改变。在发育中的中枢神经系统的情况下，多能神经祖细胞（NPC）经历表观遗传标记的显著重构，因为它们退出细胞周期并分化成构成哺乳动物大脑的不同神经和神经胶质表型。表观基因组的动态性质知之甚少，特别是在短时间尺度上，这是由于基因组学测定通常所需的大细胞群体的异质性和重复性的严重限制。在这里，我们建议克服这些技术挑战，建立强大的新的光学工具的精确控制的转录动力学的背景下的三维细胞核。具体来说，我们的目标是建立模块化的，光诱导的建筑蛋白质精确的时空控制的动态3-D之间的相互作用的远端增强子和启动子的基因对神经谱系的承诺。最近的一项研究描述了一个惊人的观察结果，即三个基本的螺旋-环-螺旋转录因子在增殖的自我更新的NPC中以振荡的方式动态表达，而这些因子中只有一个在终末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）通常位于拓扑子结构域（sub-TADs）之间的边界，（2）通过长距离3-D相互作用连接到两个相邻的sub-TADs的远端增强子。我们推测，动态的3-D之间的相互作用的远端增强子和启动子的神经特异性bHLH转录因子可能会支配这些基因的振荡表达。我们将通过生成候选振荡因子周围NPC和NPC衍生的终末分化神经元和星形胶质细胞中高阶基因组折叠的高分辨率地图来测试我们的假设。我们将采用定制的计算算法的管道整合基因组折叠图谱与细胞类型特异性增强子的注释目录，以创建预测模型，用于预测单个增强子与发育调节的神经基因形成3-D相互作用的倾向。同时，我们将构建模块化的光激活循环系统，其中CRISPR/Cas9基因靶向与能够进行光诱导二聚化的蛋白质相结合。然后，我们将进行一系列的研究，探索循环动力学，以了解管理神经细胞命运承诺的组织原则。这项工作是创新的，因为它研究了表观基因组动力学与跨学科的方法，结合CRISPR/Cas9基因组编辑，光遗传学的原则和基因组学工具，用于映射3-D基因组拓扑结构（即染色体构象捕获和深度测序）。这项工作所取得的发现应该产生一个前所未有的观点，以了解目前未知的基因组结构的动态行为，以及它是如何与发育中的大脑中的细胞命运转变相联系的。

 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.