Defining gene regulatory networks driving cortical evolution and brain development

项目摘要/摘要 神经精神障碍通常会影响我们最独特的认知和社交能力，这是 被认为是由于人类新皮质的扩张而发展起来的。独特的机制 在发育中的人类新皮质中协调皮质神经发生和分化，形成 这种扩张开始被描述出来。然而，尽管非编码基因调控的遗传变异 推动这些进化变化的是顺式基因调控元件，而不是蛋白质编码基因中的区域 (GRES)，包括启动子和增强子，以及控制皮质神经发生的转录因子(TF) 仍有待定性。为了开始研究这一未被充分研究的机制，我们和其他人利用 与基因并行分析染色质可及性和相互作用的下一代测序方法 表达来创建不同时空特异度的GRE图。我们之前确定了 数以千计的发育动态GRE及其可能的基因靶点 祖细胞与孕中期人类新皮质富含神经元的板层的比较，并从功能上验证了 选择性GREs在使用原代人类神经前体细胞的皮质神经发生中的作用。此外，我们发现， 人类获得的增强剂(HGE)，人类GRE的一个子集，比猕猴或老鼠更活跃 新皮质，调节富含外径向神经胶质(Org)的基因，它是一种神经前体，在大脑皮层中起着突出的作用 旋转术。这项工作支持这样一种假设，即人类发育动态的GREs和HGEs直接 基因表达程序控制对皮质至关重要的祖细胞库的增殖和分化 扩张。在这个提议中，我们试图检验这一假设，并从组织和基因水平上进行解析 细胞和基因异构体水平的分辨率图谱。我们将进行单核ATAC-SEQ以确定 并利用一种新的单细胞异构体测序(SCIso-seq)技术来研究 以前未被充分研究的基因调控机制--替代启动子的使用。这本新的地图集将向人们提供 我们使用CRISPR干扰从功能上定义GRE影响皮质神经发生的工作 (CRISPRi)包含捕获标签的文库，使转录组和sgRNA能够在 单细胞水平。最后，我们将定义和描述指导核扩散和核扩散平衡的因子 祖细胞的分化。这些结果将使我们能够确定基因组变异引起的细胞学基础 神经精神疾病的风险，并影响认知和大脑结构。这项工作将共同创造一个 人类发育过程中非编码GRE和TF的单细胞分辨功能注释 并阐明驱动皮质扩张的进化机制。总的来说，这项工作将提供一种 研究非编码遗传变异和细胞多样性的可扩展方法的蓝图。

PROJECT SUMMARY/ABSTRACT Neuropsychiatric disorders often affect our most distinguishing cognitive and social capabilities, which are thought to have developed as a result of the expansion of the human neocortex. The unique mechanisms orchestrating cortical neurogenesis and differentiation in the developing human neocortex forming the basis of this expansion are beginning to be described. However, although genetic variation in non-coding gene-regulatory regions, rather than in protein coding genes, drives these evolutionary changes, the cis gene regulatory elements (GREs), including promoters and enhancers, and the transcription factors (TFs) governing cortical neurogenesis remain to be characterized. To begin to investigate this understudied mechanism, we and others have leveraged next generation sequencing approaches to profile chromatin accessibility and interaction in parallel with gene expression to create GRE maps of varying levels of spatiotemporal specificity. We previously identified thousands of developmentally dynamic GREs and their putative gene targets by contrasting GRE activity in progenitor versus neuron-enriched laminae of mid-gestation human neocortex, and functionally validated the role of select GREs in cortical neurogenesis using primary human neural progenitor cells. Further, we found that human-gained enhancers (HGEs), a subset of GREs more active in the human than the macaque or mouse neocortex, regulate genes enriched in outer radial glia (oRG), a neural progenitor with prominent roles in cortical gyrification. This work supports the hypothesis that human developmentally dynamic GREs and HGEs direct gene expression programs controlling the proliferation and differentiation of progenitor pools key to cortical expansion. In this proposal, we seek to test this hypothesis and move from a tissue- and gene-level resolution atlas to a cellular- and gene isoform-level resolution atlas. We will perform single nucleus ATAC-seq to identify cell-specific GREs and leverage a novel single-cell isoform sequencing (scIso-seq) technology to investigate a previously understudied mechanism of gene regulation – alternative promoter usage. This new atlas will inform our work to functionally define the GREs impacting cortical neurogenesis at scale using CRISPR interference (CRISPRi) libraries containing capture tags enabling simultaneous reading of transcriptome and sgRNA at the single-cell level. Finally, we will define and characterize the TFs directing the balance of proliferation versus differentiation of progenitors. These results will enable us identify the cellular basis of genomic variation causing risk for neuropsychiatric disease, and influencing cognition and brain structure. Together this work will create a robust single cell-resolution functional annotation of non-coding GREs and TFs acting in developing human neocortex and elucidate evolutionary mechanisms driving cortical expansion. Broadly, this work will provide a blueprint for scalable approaches to study non-coding genetic variation and cellular diversity.