Cerebral organoid models of CHD2-associated intellectual and developmental disabilities

项目摘要 CHD 2中的致病性变体与智力和发育障碍（IDDs）相关，包括 自闭症谱系障碍、发育迟缓和早发性癫痫。染色体区解旋酶DNA 结合蛋白2（CHD 2）是一种染色质重塑蛋白，可改变染色质结构，导致 下游靶点的基因表达。关于CHD 2的变异如何导致IDDs，我们知之甚少。的 Chd 2缺陷的小鼠模型并不能重现人类患者中观察到的癫痫发作表型 CHD 2变异，表明脑中可能存在CHD 2的物种特异性功能。我假设 CHD 2对于调节参与灵长类特定大脑结构发育的基因非常重要， 例如脑室下外区。为了研究CHD 2在人类神经系统中的机制，我们 提出了一个三维脑类器官（CO）模型的CHD 2缺乏的发展。使用CRISPR/Cas9，我们 基因工程诱导多能干细胞（iPSC）系携带内源性形式的 具有Flag、HA和绿色荧光蛋白（GFP）标签的CHD 2（WTCHD 2-Flag-HA-GFP）。在我们的第一个目标中，我们建议 使用CRISPR/Cas9缺失CHD 2的一个拷贝，导致对于CHD 2单倍不足的iPSC（CHD 2 +/-Flag-L1）。 HA-GFP）。然后，WTCHD 2-Flag-HA-GFP iPSC和CHD 2 +/-Flag-HA-GFP iPSC将分化为前脑特异性细胞。 COs.我将确定CHD 2破坏后的神经发育缺陷，例如神经元发育的差异。 分化，增殖和形成不同的大脑皮层层，通过结合 免疫组织化学和细胞周期分析。在目标2中，我将鉴定在CHD 2中失调的基因 通过在WTCHD 2-Flag-HA-GFP和CHD 2 +/-Flag-HA-GFP CO中进行单细胞mRNA测序来破坏。在Aim中 3，我将通过进行染色质免疫沉淀，然后进行 对从WTCHD 2-Flag-HA-GFP CO解离的细胞进行下一代测序（ChIP-Seq）。然后我会将 CHD 2占据的结果与CHD 2破坏后哪些基因被错误调节的数据， 鉴定一组在人类神经元细胞中受CHD 2调控的候选基因。结合起来，这些 研究将导致在人类神经元细胞类型中识别CHD 2调节基因，揭示CHD 2调节的途径。 CHD 2参与了什么，并提高了我们对CHD 2背后的生物学机制的理解- 相关IDDs在未来，这种模式有可能通过尝试拯救 小分子的缺陷。

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.