The role of EZH1 in neuronal differentiation and neurological disorders

项目摘要 智力残疾（ID）是由神经发育错误引起的终身疾病。我们最近发现 染色质修饰剂Zeste增强子同源物1中的功能获得和丧失（GOF和LOF）突变 （EZH 1）是导致10名儿童出现先前未诊断的智力残疾综合征的原因。EZH 1是一种 多梳抑制复合物2（PRC 2）的两种组蛋白H3赖氨酸27（H3 K27）甲基转移酶。的 另一个是EZH 2，长期以来一直被认为是H3 K27二和三甲基化（H3 K27 me 2/3）的主要负责人。 PRC 2介导的转录抑制，部分原因是EZH 1的催化活性较弱。ezh 2是 在分裂细胞中高度表达，其功能障碍导致神经祖细胞增殖和命运缺陷 规范和神经发育疾病。尽管有证据表明，EZH 1也表达在 发育和成人神经系统，其相关性和功能，在神经发育和稳态仍然 未知在初步工作中，我们发现EZH 1在人类大脑皮层中持续表达 在神经发生晚期，由于快速下降， EZH 2的表达。使用携带EZH 1 LOF和GOF突变的人胚胎干细胞（hESC）， 在单层和类器官培养中，我们发现了延迟的神经元分化的迹象。 在EZH 1 LOF中的早期分化和在EZH 1 GOF中的早期分化。然而，H3 K27 me 3的量 通过WB测量，在所有突变体和对照细胞系中显示出相似的水平。因此，我们假设 EZH 1通过非EZH 2冗余机制调节皮质神经发生时间， 随着神经发生的进展和EZH 2表达的下降。为了检验目标1中的假设，我们将确定 EZH 1在神经发生过程中的分化阶段特异性分子功能， EZH 1（SubAim1.1）的结合谱，其对H3 K27甲基化和转录调控的影响 2），以及PRC 2-EZH 1亚复合物（SubAim1.3）的组成随时间的变化。 分化在目标2中，我们将分析EZH 1突变在皮质神经发生时间和 EZH 1/2抑制剂对它们的能力，我们将剖析EZH 1突变驱动的起源和后果 通过将我们的类器官分析扩展到30、40、60和100天，神经发生时间失调（SubAim2.1）， 通过使用scRNAseq对组成和细胞类型特异性差异基因表达进行无偏定量， 的类器官（SubAim2.2），并评估EZH 1/2抑制剂在恢复细胞和 EZH 1 LOF和GOF突变引起的分子改变（SubAim2.3）。这些研究将揭示一个 目前，EZH 1在调节皮质神经发生和神经发育疾病中的作用被忽视 并可能为ID提供新的治疗靶点。

PROJECT SUMMARY Intellectual disabilities (ID) are lifelong conditions caused by neurodevelopmental errors. We recently identified gain and loss of function (GOF and LOF) mutations in the chromatin modifier Enhancer of Zeste Homologue 1 (EZH1) as the cause a previously undiagnosed intellectual disability syndrome in ten children. EZH1 is one of the two Histone H3 Lysine 27 (H3K27) methyltransferases of the Polycomb Repressive Complex 2 (PRC2). The other one, EZH2, has long been considered the main responsible for H3K27 di and trimethylation (H3K27me2/3) and PRC2 mediated transcriptional repression, in part owing to a weaker catalytic activity of EZH1. EZH2 is highly expressed in dividing cells, and its dysfunction leads to defects in neural progenitor proliferation and fate specification, and neurodevelopmental disease. Despite evidence indicating that EZH1 is also expressed in the developing and adult nervous system, its relevance and function in neural development and homeostasis remain unknown. In preliminary work, we found that EZH1 is expressed constantly across human cerebral cortex development and becomes the predominant paralogue by the late neurogenesis period owing to a rapid decline of EZH2 expression. Using human embryonic stem cells (hESC) carrying EZH1 LOF and GOF mutations, and their differentiation to cortical neurons in monolayer and organoid cultures, we found signs of delayed neuronal differentiation in EZH1 LOF and premature differentiation in EZH1 GOF. However, amounts of H3K27me3 measured by WB, showed similar levels across all the mutant and control cell lines. Thus, we hypothesize that EZH1 regulates cortical neurogenesis timing through a non EZH2 redundant mechanism that becomes dominant as neurogenesis progresses and EZH2 expression declines. To test this hypothesis in Aim 1 we will determine differentiation stage specific molecular functions of EZH1 during neurogenesis by defining the genomic binding profile of EZH1 (SubAim1.1), its effects on H3K27 methylation and transcriptional regulation (SubAim1.2), and the composition of PRC2-EZH1 subcomplexes (SubAim1.3) over time during neuronal differentiation. In Aim 2 we will dissect the effect of EZH1 mutations in cortical neurogenesis timing and the ability of EZH1/2 inhibitors to them we will dissect the origin and consequences of EZH1 mutation driven dysregulated neurogenesis timing by extending our organoid analysis to 30, 40, 60 and 100 days (SubAim2.1), by unbiased quantification of the composition and cell type specific differential gene expression using scRNAseq of the organoids (SubAim2.2), and assessing the potential of EZH1/2 inhibitors on restoring the cellular and molecular alterations caused by EZH1LOF and GOF mutations (SubAim2.3). These studies will uncover a currently disregarded role of EZH1 in the regulation of cortical neurogenesis and neurodevelopmental diseases and may provide new therapeutic targets for IDs.