Cerebral organoid models of CHD2-associated intellectual and developmental disabilities

项目总结 CHD2的致病变异与智力和发育障碍(IDDS)有关，包括 自闭症谱系障碍、发育迟缓和早发性癫痫。染色体结构域解旋酶DNA 结合蛋白2(CHD2)是一种染色质重塑蛋白，可以改变染色质结构，导致改变 下游靶标的基因表达。关于CHD2中的变异是如何导致IDDS的，人们知之甚少。这个 CHD2缺乏的小鼠模型不能概括人类癫痫患者的癫痫表型 CHD2变异，表明CHD2在大脑中可能具有物种特有的功能。我假设 CHD2对于调节涉及灵长类特定大脑结构发育的基因将是重要的， 比如外脑室下区。为了研究CHD2在人类神经系统中的作用机制，我们 建议建立CHD2缺乏症的3D脑器官(CO)模型。使用CRISPR/CAS9，我们 已经通过基因工程获得了一种诱导多能干细胞(IPSC)系，该系携带内源性形式的 带有Flag、HA和绿色荧光蛋白(GFP)标签的CHD2(WTCHD2-Flag-HA-GFP)。在我们的第一个目标中，我们建议 使用CRISPR/CAS9删除CHD2的一个拷贝，导致IPSC对CHD2的单倍体不足(CHD2/-标志- HA-GFP)。WTCHD2-Flag-HA-GFP iPSCs和CHD2/-Flag-HA-GFP iPSCs将被区分为前脑特异的 因为。我将确定CHD2中断时的神经发育缺陷，例如神经元的差异 分化、增殖和形成不同的大脑皮层，通过结合 免疫组织化学和细胞周期分析。在目标2中，我将识别CHD2的错误调控基因 中断，通过在WTCHD2-Flag-HA-GFP和CHD2/-Flag-HA-GFP Cos中执行单细胞mRNA测序。在AIM 3，我将通过执行染色质免疫沉淀来确定全基因组CHD2的占有率 对WTCHD2-Flag-HA-GFP Cos分离的细胞进行下一代测序(ChIP-Seq)。然后我就会关联起来 CHD2占用的结果与CHD2中断时基因被错误调控的数据一起 在人类神经细胞中识别一组受CHD2调控的候选基因。加在一起，这些 研究将导致在人类神经细胞类型中识别CHD2调节的基因，揭示 哪些CHD2参与其中，并提高我们对CHD2背后的生物学机制的理解- 关联的IDD。在未来，这种模式有可能通过试图拯救 小分子缺陷。

PROJECT SUMMARY Pathogenic variants in CHD2 are associated with intellectual and developmental disabilities (IDDs), including autism spectrum disorder, developmental delay, and early onset epilepsy. Chromodomain helicase DNA binding protein 2 (CHD2) is a chromatin remodeling protein that alters chromatin structure, leading to changes in gene expression of downstream targets. Little is known about how variants in CHD2 lead to IDDs. The mouse model of Chd2 deficiency does not recapitulate the seizure phenotype seen in human patients with CHD2 variants, indicating that there may be species-specific functions of CHD2 in the brain. I hypothesize that CHD2 will be important for regulating genes involved in the development of primate-specific brain structures, such as the outer subventricular zone. To study the mechanism of CHD2 in a human neuronal system, we propose the development of a 3D cerebral organoid (CO) model of CHD2 deficiency. Using CRISPR/Cas9, we have genetically engineered an induced pluripotent stem cell (iPSC) line carrying an endogenous form of CHD2 with Flag, HA, and green fluorescent protein (GFP) tags (WTCHD2-Flag-HA-GFP). In our first aim, we propose deletion of one copy of CHD2 using CRISPR/Cas9, resulting in iPSCs haploinsufficient for CHD2 (CHD2+/-Flag- HA-GFP). WTCHD2-Flag-HA-GFP iPSCs and CHD2+/-Flag-HA-GFP iPSCs will then be differentiated into forebrain specific COs. I will determine neurodevelopmental defects upon CHD2 disruption, such as differences in neuronal differentiation, proliferation, and formation of distinct cerebral cortex layers, through a combination of immunohistochemistry and cell cycle analyses. In Aim 2 I will identify genes misregulated upon CHD2 disruption, by performing single-cell mRNA sequencing in WTCHD2-Flag-HA-GFP and CHD2+/-Flag-HA-GFP COs. In Aim 3, I will determine genome-wide CHD2 occupancy, by performing chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) on cells dissociated from WTCHD2-Flag-HA-GFP COs. I will then correlate the results of CHD2 occupancy with the data on which genes are misregulated upon CHD2 disruption to identify a set of candidate genes that are regulated by CHD2 in human neuronal cells. Combined, these studies will lead to identification of CHD2-regulated genes in human neuronal cell types, revealing pathways in which CHD2 is involved and improving our understanding of the biological mechanisms behind CHD2- associated IDDs. In the future this model has the potential for drug development by attempting to rescue defects with small molecules.