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

项目摘要/摘要 智力障碍(ID)和自闭症谱系障碍(ASD)是最常见的发育 疾病，在美国影响着3%-4%的儿童，几乎没有治疗选择。尽管对 导致这些异质性疾病的机制仍然非常有限，ID/ASD的遗传学研究 揭示了编码转录调节蛋白的基因突变的核心作用，包括 SWI/SNFATP依赖的核小体重塑复合体(BAF复合体)的亚单位。例如, 典型BAF复合体(CBAF)最大亚单位Arid1b的杂合性功能丧失突变， 是在新生ID/ASD病例中观察到的最常见的突变之一。然而，它的功能 ARID1B/cBAF复合体在正常脑发育和特定发育过程中的基因调控 被Arid1b功能缺失突变破坏的过程仍然是知识的重大缺口。 细胞类型特异性基因转录调控复合体的特殊功能 对胚胎大脑动态和异质细胞环境的调节仍然很困难 使用现有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控网络 在大脑发育过程中的规范。为此，我的实验室最近开发了一种健壮的、可重现的 从小鼠多能干细胞中制造前脑有机物质的方案。这种降低了复杂性的模型 保持大脑发育中的关键特征，并能够实现不可能的实验方法 在活体内。在这里，我们建议1)进行第一次深入的转录和表观基因组学表征 用单细胞基因组学方法在我们的新的小鼠器官模型中发育大脑皮质，2) 确定Arid1b功能缺失突变对体内和有机体内皮质发育的影响，3) 实施化学遗传方法(DTag)来分析ARID1B丢失的特定阶段影响，4)定义 ARID1B缺失对皮质发育过程中基因调控的直接影响，5)决定哪个基因 当ARID1B重新导入有丝分裂后皮质神经元时，表达变化是可逆的。这些 数据将有助于建立小鼠皮质器官作为一个模型系统，可以补充和扩展 研究大脑发育的分子和细胞机制的活体方法。我们的发现将 提供了对Arid1b功能缺失突变引起 皮质神经发生早期阶段的基因调控，并揭示特定的基因、细胞类型和 易受cBAF复合体还原影响的发育阶段。考虑到这件事的相关性 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。

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.