Defining gene regulatory networks driving cortical evolution and brain development

定义驱动皮质进化和大脑发育的基因调控网络

• 批准号: 10616776
• 负责人: Luis de la Torre-Ubieta
• 金额: $ 67.94万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10616776
• 关键词: ATAC-seq; Adult; Affect; Atlases; Automobile Driving; Biochemical; Biological; Biological Assay; Brain; CISH gene; CRISPR interference; CRISPR/Cas technology; Cell Nucleus; Cells; ChIP-seq; Chromatin; Chromosome Mapping; Code; Cognition; Cognitive; Data; Development; Disease; Engineering; Enhancers; Equilibrium; Evolution; Excision; Experimental Models; FGFR2 gene; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Determinism; Genetic Variation; Genome; Genomics; Human; Human Development; Impairment; LYN gene; Length; Libraries; Macaca; Maps; Mass Spectrum Analysis; Methodology; Molecular; Morphogenesis; Mus; Neocortex; Neuroglia; Neurons; Nuclear; Nucleic Acid Regulatory Sequences; Outcome; Phenotype; Pregnancy; Proliferating; Protein Isoforms; Proteins; Proteomics; Radial; Reading; Regulator Genes; Regulatory Element; Research Personnel; Resolution; Risk; Role; Specificity; Structure; Technology; Testing; Therapeutic; Tissues; Untranslated RNA; Validation; Work; brain size; cell type; falls; gene regulatory network; gene repression; genetic variant; genomic variation; insight; neocortical; nerve stem cell; neurogenesis; neuropsychiatric disorder; next generation sequencing; novel; progenitor; programs; promoter; social; spatiotemporal; transcription factor; transcriptome

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.

项目总结/摘要 神经精神疾病通常会影响我们最显着的认知和社交能力，这些能力是 被认为是人类大脑皮层扩张的结果。独特的机制 在发育中的人类新皮层中协调皮层神经发生和分化，形成了 这种扩张开始被描述。然而，尽管非编码基因调控的遗传变异 区域，而不是在蛋白质编码基因，驱动这些进化的变化，顺式基因调控元件 包括启动子和增强子在内的GRES以及控制皮质神经发生的转录因子（TF） 仍有待鉴定。为了开始研究这种未充分研究的机制，我们和其他人利用了 分析染色质可接近性和与基因平行的相互作用的下一代测序方法 表达来创建不同时空特异性水平的GRE图。我们之前发现 成千上万的发育动态GRES及其推定的基因靶点， 祖细胞与孕中期人类新皮质的神经元富集板层，并在功能上验证了 使用原代人神经祖细胞选择GRES在皮质神经发生中的作用。此外，我们发现， 人类获得的增强子（HGE），一种在人类中比猕猴或小鼠中更活跃的GRE子集 新皮层，调节基因丰富的外放射状胶质细胞（oRG），神经祖细胞与突出的作用，在皮层 螺旋化这项工作支持这一假设，即人类发展动态GRES和HGE直接 控制祖细胞池增殖和分化的基因表达程序是皮质神经元 扩张.在这项提案中，我们试图验证这一假设，并从组织和基因水平的分辨率 图谱到细胞和基因亚型水平分辨率图谱。我们将进行单核ATAC-seq来鉴定 细胞特异性GRES，并利用一种新的单细胞亚型测序（scIso-seq）技术来研究 先前未充分研究的基因调控机制-替代启动子的使用。这本新地图集将告诉 我们的工作是使用CRISPR干扰在功能上定义影响皮层神经发生的GRES CRISPRi文库包含捕获标签，使得能够同时阅读转录组和sgRNA。 单细胞水平。最后，我们将定义和描述指导增殖与 祖细胞的分化。这些结果将使我们能够确定基因组变异的细胞基础， 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 非编码GRES和TF在发育人类中作用的稳健的单细胞分辨率功能注释 新皮层和阐明进化机制驱动皮质扩张。总的来说，这项工作将提供一个 研究非编码遗传变异和细胞多样性的可扩展方法的蓝图。

项目成果

Developmental isoform diversity in the human neocortex informs neuropsychiatric risk mechanisms.

人类新皮质的发育亚型多样性揭示了神经精神风险机制。

• DOI: 10.1101/2023.03.25.534016
• 发表时间: 2023
• 期刊: bioRxiv : the preprint server for biology
• 作者: Patowary,Ashok;Zhang,Pan;Jops,Connor;Vuong,CelineK;Ge,Xinzhu;Hou,Kangcheng;Kim,Minsoo;Gong,Naihua;Margolis,Michael;Vo,Daniel;Wang,Xusheng;Liu,Chunyu;Pasaniuc,Bogdan;Li,JingyiJessica;Gandal,MichaelJ;delaTorre-Ubieta,Luis
• 通讯作者: delaTorre-Ubieta,Luis

Defining the evolutionary and gene regulatory logic of vertebrate neuronal diversity.

定义脊椎动物神经元多样性的进化和基因调控逻辑。

• DOI: 10.1016/j.neuron.2021.12.013
• 发表时间: 2022
• 期刊: Neuron
• 影响因子: 16.2
• 作者: delaTorre-Ubieta,Luis