Chemical Genetic Dissection of SWI/SNF Chromatin Remodeling Complex Functions in Cerebral Cortex Development

大脑皮层发育中 SWI/SNF 染色质重塑复杂功能的化学遗传学解析

• 批准号: 10660367
• 负责人: Thomas S Vierbuchen
• 金额: $ 54.22万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660367
• 关键词: ARID1A gene; Adopted; Affect; Alleles; Biological Models; Brain; Cell Fate Control; Cell Proliferation; Cells; Cerebral cortex; Child; Chromatin Remodeling Factor; Complement; Complex; Data; Defect; Development; Developmental Process; Disease; Dissection; Embryo; Environment; Exhibits; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genetic study; Genomic approach; Goals; Human Genetics; In Vitro; Individual; Intellectual functioning disability; Knowledge; Link; Loss of Heterozygosity; Measures; Meta-Analysis; Methods; Modeling; Molecular; Mus; Mutate; Mutation; Neurodevelopmental Disorder; Neuronal Differentiation; Neurons; Nucleosomes; Organoids; Phenocopy; Pluripotent Stem Cells; Predisposition; Prosencephalon; Protocols documentation; Recurrence; Regulatory Element; Reproducibility; Research Personnel; Resolution; Role; Therapeutic; Time; Transcription Regulatory Protein; autism spectrum disorder; cell fate specification; cell type; chemical genetics; developmental disease; epigenomics; experimental study; gene regulatory network; genetic approach; in vivo; insight; interest; loss of function; loss of function mutation; mouse model; neurogenesis; novel; novel therapeutic intervention; novel therapeutics; postmitotic; protein degradation; rapid technique; stem cell model; temporal measurement; tool; transcriptomics

PROJECT SUMMARY/ABSTRACT Intellectual Disability (ID) and Autism Spectrum Disorders (ASD) are the most common developmental disorders, affecting 3-4% of children in the U.S, with few therapeutic options. Although insights into the mechanisms that cause these heterogeneous disorders remains very limited, genetic studies of ID/ASD have revealed a central role for mutations in genes encoding transcriptional regulatory proteins, including multiple subunits of the SWI/SNF ATP-dependent nucleosome remodeling complex (BAF complexes). For example, heterozygous loss-of-function mutations in Arid1b, the largest subunit of the canonical BAF complex (cBAF), are among the most frequent mutations observed in de novo ID/ASD cases. However, the function of ARID1B/cBAF complexes in gene regulation during normal brain development and the specific developmental processes that are disrupted by Arid1b loss-of-function mutations remain significant gaps in knowledge. Characterizing the specific functions of transcriptional regulatory complexes in cell type-specific gene regulation in the dynamic and heterogeneous cellular environment of the embryonic brain remains difficult using current model systems and experimental tools. Our long-term goal is to develop pluripotent stem cell- based model systems and experimental tools to characterize gene regulatory networks that control cell fate specification during brain development. Towards this end, my lab recently developed a robust, reproducible protocol to make forebrain organoids from mouse pluripotent stem cells. This reduced complexity model maintains key features of the developing brain and can enable experimental approaches that are not possible in vivo. Here, we propose to 1) perform the first in depth transcriptomic and epigenomic characterization of cerebral cortex development in our novel mouse organoid model using single cell genomics approaches, 2) define the impact of Arid1b loss-of-function mutations on cortical development in vivo and in organoids, 3) implement chemical genetic approaches (dTAG) to parse stage-specific effects of ARID1B loss, 4) define direct effects of ARID1B loss on gene regulation during cortical development, 5) determine which gene expression changes are reversible upon reintroduction of ARID1B into post-mitotic cortical neurons. These data will help to establish mouse cortical organoids as a model system that can complement and extend upon in vivo approaches for studying molecular and cellular mechanisms of brain development. Our findings will provide new insight into the mechanisms by which loss-of-function mutations in Arid1b give rise to changes in gene regulation during early stages of cortical neurogenesis and reveal specific genes, cell types, and developmental stages that are susceptible to reduction of cBAF complexes. Given the relevance of this complex to common developmental disorders, our findings may also reveal novel therapeutic opportunities for ID and ASD.

项目摘要/摘要 智力残疾（ID）和自闭症谱系障碍（ASD）是最常见的发展 疾病影响美国3-4％的儿童，几乎没有治疗选择。虽然对 引起这些异质性疾病的机制仍然非常有限，ID/ASD的遗传研究已有 揭示了在编码转录调节蛋白的基因中突变的核心作用，包括多个 SWI/SNF ATP依赖性核小体重塑复合物（BAF复合物）的亚基。例如， ARID1B的杂合丧失功能突变，ARID1B是规范BAF复合物（CBAF）的最大亚基， 是在从头/ASD病例中观察到的最常见的突变之一。但是， 正常脑发育过程中基因调节中的ARID1B/CBAF复合物和特定的发育 受ARID1B功能丧失突变破坏的过程在知识中仍然存在很大的差距。 表征细胞类型特异性基因中转录调节复合物的特定功能 在胚胎大脑的动态和异质细胞环境中调节仍然困难 使用当前的模型系统和实验工具。我们的长期目标是发展多能干细胞 基于模型系统和实验工具来表征控制细胞命运的基因调节网络 大脑发育过程中的规格。为此，我的实验室最近开发了可重现的强大，可重复的 从小鼠多能干细胞中制造前脑器官的方案。这种降低的复杂性模型 保持发育中的大脑的关键特征，并可以实现无法实现的实验方法 体内。在这里，我们提议1）执行第一个深度转录组和表观基因组表征 使用单细胞基因组学方法的新型小鼠器官模型中的脑皮质发育，2） 定义ARID1B功能丧失突变对体内和类器官中皮质发育的影响，3） 实施化学遗传方法（DTAG）以解析ARID1B损失的特异性效应，4）定义 ARID1B损失对皮质发育过程中基因调节的直接影响，5）确定哪个基因 将ARID1B重新引入有丝分裂后皮质神经元后，表达变化是可逆的。这些 数据将有助于建立鼠标皮质器官作为模型系统，可以补充并扩展 体内方法研究脑发育的分子和细胞机制。我们的发现会 提供有关ARID1B功能丧失突变的机制的新见解 皮质神经发生早期的基因调节，并揭示特定的基因，细胞类型和 容易减少CBAF复合物的发育阶段。考虑到这一点的相关性 复杂到常见的发育障碍，我们的发现也可能揭示出新的治疗机会 ID和ASD。