The cis-regulatory grammar and epigenetic control of human interneuron progenitor specification

人类中间神经元祖细胞规范的顺式调控语法和表观遗传控制

• 批准号: 10116764
• 负责人: Kristen L Kroll
• 金额: $ 52.38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-15 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10116764
• 关键词: Animals; Automobile Driving; Binding; Binding Sites; Biological Assay; Brain; Cerebral cortex; Chromatin; Chromatin Remodeling Factor; Code; Data; Development; Developmental Gene; Disease; Dissection; Dorsal; Embryo; Embryonic Development; Enzymes; Epigenetic Process; Epilepsy; Etiology; Fetal Development; Foundations; Ganglia; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genetic Variation; Genome; Genomics; Glutamates; Human; Human Genetics; Human Genome; Impairment; Intellectual functioning disability; Interneurons; Link; Location; Logic; Medial; Modeling; Molecular; Mutate; Mutation; Nervous system structure; Neurodevelopmental Disorder; Neurons; Pathogenicity; Process; Property; Prosencephalon; Regulation; Regulator Genes; Regulatory Element; Reporter; Resources; Role; Specific qualifier value; Testing; Untranslated RNA; Variant; Work; autism spectrum disorder; base; cell type; childhood epilepsy; chromatin remodeling; combinatorial; data resource; epigenetic regulation; epigenome; epigenome editing; excitatory neuron; gene repression; genetic variant; genome-wide; high throughput analysis; human embryonic stem cell; migration; nerve stem cell; neurodevelopment; neuron development; progenitor; programs; transcription factor; transcriptome

Control of gene expression involves interactions between genomic cis-regulatory elements (CREs) and the transcription factors that bind them, while chromatin modifiers also modulate genome access to control cell type specification during development. Defining these regulatory controls is important, as most human genetic variation linked to disease is in non-protein coding sequences, but the locations and functionality of CREs that specify many developing human cell types has not yet been defined. In the cerebral cortex, balanced development of inhibitory cortical interneurons (cINs) and excitatory neurons (cEXs) is required for proper function. cIN development is susceptible to perturbation to cause multiple neurodevelopmental disorders (NDDs), while NDD contributory mutations are found in many genes encoding chromatin modifiers, linking disrupted epigenetic regulation of cIN development to NDD etiology. However, most aspects of molecular regulation of human cIN development remain undefined, including which regulators are required, the CREs through which these regulators act and networks of gene expression under their control, effects of their disruption on cIN development, and contributions of human mutations in these genes and CREs to NDDs. To elucidate these, we use a directed differentiation model that mimics many aspects of human cIN specification and differentiation, is robust and experimentally manipulable, and has high utility for studying these processes. Here, we begin to define the central regulatory logic underlying the cIN developmental program and build a foundation for studying how its disruption contributes to NDD etiology. We first integrate several types of genome-wide data to define putative CREs controlling cIN specification. These data will be used to assess how pathogenic mutations in both CREs and in genes encoding chromatin modifiers disrupt cIN development to cause NDDs. We next explore roles for CHD2, a gene encoding a chromatin remodeler mutated to cause several NDDs: we define direct targets of CHD2 regulation, their dysregulation in the context of different pathogenic CHD2 gene variants, the epigenetic mechanisms involved, and effects of these CHD2 pathogenic mutations on development and function of cINs and cEXs. This work will elucidate both CHD2's required roles and mechanisms in cIN development and the basis of their disruption in NDDs. Finally, we conduct massively parallel reporter analysis: high throughput, quantitative, CRE activity testing is used to identify bona fide functional CREs, define cis-sequence requirements for CRE regulation during cIN specification, and compare CRE activity in cIN versus cEX progenitors and with single or combinatorial transcription factor binding site mutation. A subset of these CREs is then validated by epigenome editing. Together, this work will elucidate the cis-regulatory logic of a human cell type-specific gene regulatory program central to neurodevelopment and disease, while building a resource and experimental foundation for further dissection of how pathogenic variants both in human coding and non-coding genome sequences disrupt this program, contributing to NDDs.

基因表达的控制涉及基因组顺式调节元件（克雷斯）和转录因子之间的相互作用。 与之结合的转录因子，而染色质修饰剂也调节基因组进入控制细胞 开发过程中的类型规范。定义这些调控控制是重要的，因为大多数人类遗传 与疾病相关的变异是在非蛋白质编码序列中，但 还没有确定许多发育中的人类细胞类型。在大脑皮层中， 抑制性皮层中间神经元（cIN）和兴奋性神经元（cEX）的发育是正常的神经元发育所必需的。 功能cIN的发育容易受到干扰而导致多种神经发育障碍 （NDD），而NDD贡献突变被发现在许多基因编码染色质修饰剂，连接 破坏cIN发展对NDD病因的表观遗传调节。然而，分子的大多数方面 人类cIN发育的调控仍然不确定，包括需要哪些调控因子，克雷斯 通过这些调节剂的行为和网络的基因表达在其控制下，其影响， 破坏cIN发展，以及这些基因和克雷斯中的人类突变对NDD的贡献。到 为了阐明这些，我们使用了一个模拟人类cIN特化的许多方面的定向分化模型 和分化，是强大的和实验上可操作的，并具有很高的效用，研究这些过程。 在这里，我们开始定义cIN发展程序的核心调控逻辑，并建立一个 为研究其破坏如何促进NDD病因学奠定了基础。我们首先集成几种类型的 全基因组数据来定义控制cIN特化的推定克雷斯。这些数据将用于评估 克雷斯和编码染色质修饰剂的基因中的致病性突变破坏cIN的发育， 引起神经损伤。我们接下来探索CHD2的作用，CHD2是一种编码染色质重塑的基因， 几个NDD：我们定义了CHD2调节的直接靶点，它们在不同环境下的失调 致病性CHD2基因变异，所涉及的表观遗传机制，以及这些CHD2致病性 cIN和cEX的发育和功能突变。这项工作将阐明CHD2所需的角色 和cIN发展的机制及其在NDD中中断的基础。最后，我们进行大规模的 平行报告基因分析：高通量、定量、CRE活性检测用于识别真正的 功能性克雷斯，定义cIN规范期间CRE调节的顺式序列要求，并比较 cIN与cEX祖细胞中的CRE活性以及具有单个或组合转录因子结合位点 突变然后通过表观基因组编辑验证这些克雷斯的子集。总之，这项工作将阐明 神经发育中枢的人类细胞类型特异性基因调控程序的顺式调控逻辑， 疾病，同时建立一个资源和实验基础，进一步解剖如何致病 人类编码和非编码基因组序列中的变异破坏了这一程序，导致了NDD。