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: 10640960
• 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/10640960
• 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; 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; Neurodevelopmental Disorder; Neurons; Pathogenicity; Predisposition; Process; Property; Prosencephalon; Regulation; Regulator Genes; Regulatory Element; Reporter; Repression; Resources; Role; Specific qualifier value; Testing; Untranslated RNA; Variant; Work; autism spectrum disorder; cell type; childhood epilepsy; chromatin remodeling; combinatorial; data integration; data resource; directed differentiation; epigenetic regulation; epigenome; epigenome editing; excitatory neuron; gene network; gene repression; genetic variant; genome-wide; 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.

基因表达的调控涉及基因组顺式调控元件(CRE)和 结合它们的转录因子，而染色质修饰物也调节基因组访问以控制细胞 开发过程中的类型规范。确定这些监管控制是重要的，因为大多数人类基因 与疾病相关的变异是在非蛋白质编码序列中，但Cres的位置和功能 具体说明许多发育中的人类细胞类型还没有定义。在大脑皮层，平衡 抑制性皮质中间神经元(CIN)和兴奋性神经元(CEXs)的发育是正常的 功能。CIN的发育容易受到干扰而导致多种神经发育障碍 虽然在许多编码染色质修饰物的基因中发现了与NDD有关的突变，但 破坏了CIN发育对NDD病因学的表观遗传调控。然而，分子的大部分方面 对人类CIN发育的调节仍然没有定义，包括需要哪些调节器 通过这些调控因子的作用和它们控制的基因表达网络，它们的影响 对CIN发育的干扰，以及这些基因和CRE的人类突变对NDDS的贡献。至 为了阐明这些，我们使用了一个定向分化模型，该模型模仿了人类CIN规范的许多方面 和差异性，是稳健的和可实验操作的，对研究这些过程具有很高的实用价值。 在这里，我们开始定义CIN发展计划背后的中央监管逻辑，并建立一个 为研究其中断如何导致新城疫病因学奠定了基础。我们首先集成了几种类型的 全基因组数据以定义可能控制CIN规范的Cres。这些数据将被用来评估 Cres和染色质修饰物编码基因的致病突变扰乱了CIN的发展 因为NDDS。接下来，我们探索CHD2的作用，这是一种编码染色质重构体的基因，突变后导致 几种非脱氧核糖核酸：我们定义了CHD2调控的直接靶点，它们在不同背景下的调控失调 致病CHD2基因变异、涉及的表观遗传学机制以及这些致病基因的作用 Cins和cexs基因的发育和功能突变。这项工作将阐明CHD2的S所需的角色 以及在CIN发展中的机制及其在NDDS中被破坏的基础。最后，我们进行了大规模的 平行报告分析：使用高通量、定量、Cre活性测试来识别真实性 FunctionalCres，定义CIN规范期间Cre调节的顺式序列要求，并比较 具有单一或组合转录因子结合位点的CIN祖细胞和CEX祖细胞的Cre活性 突变。然后通过表观基因组编辑来验证这些CRE的子集。总之，这项工作将阐明 人类细胞类型特异性基因调控程序的顺式调控逻辑对神经发育和 疾病，同时为进一步剖析致病机理建立资源和实验基础 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NDDS。