The role of EZH1 in neuronal differentiation and neurological disorders

项目总结 智力障碍(ID)是由神经发育错误造成的终生疾病。我们最近确认了 ZEST同源物1染色质修饰增强子的功能获得和功能丧失(GOF和LOF)突变 (EZH1)是10名儿童以前未被诊断出的智力残疾综合征的原因。EZH1是 多梳抑制复合体2(PRC2)的两个组蛋白H3赖氨酸27(H3K27)甲基转移酶。这个 另一种是EZH2，长期以来一直被认为是导致H3K27二甲基化和三甲基化(H3K27me2/3)的主要原因。 PRC2介导转录抑制，部分原因是EZH1的催化活性较弱。EZH2是 在分裂细胞中高表达，其功能障碍导致神经前体细胞增殖和命运的缺陷 规范，和神经发育疾病。尽管有证据表明EZH1也在 发育中的和成人神经系统，其在神经发育和动态平衡中的相关性和功能 未知。在初步工作中，我们发现EZH1在人的大脑皮层中持续表达 发育，并在神经发生后期由于迅速下降而成为主要的并列 EZH2的表达。使用携带EZH1 LOF和GOF突变的人胚胎干细胞(HESC)，以及 在单层和类器官培养中，我们发现了神经元延迟分化的迹象。 EZH1 LOF的分化和EZH1 GOF的早分化。然而，H3K27me3的数量 通过WB测量，在所有突变和对照细胞系中显示出相似的水平。因此，我们假设 EZH1通过主导的非EZH2冗余机制调节皮质神经发生的时间 随着神经发生的进展，EZH2的表达下降。为了在目标1中检验这一假设，我们将确定 从基因组角度研究EZH1在神经发生过程中的分化阶段特异性分子功能 EZH1(SubAim1.1)结合谱及其对H3K27甲基化和转录调控的影响 (SubAim1.2)，以及PrC2-EZH1亚复合体(SubAim1.3)在神经元发育过程中随时间的变化 差异化。在目标2中，我们将剖析EZH1突变在皮质神经发生时间和 EZH1/2抑制剂对它们的作用我们将剖析EZH1突变驱动的起源和后果 通过将我们的有机物分析延长至30、40、60和100天(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量化 并评估EZH1/2抑制剂在修复细胞和 EZH1LOF和GOF突变引起的分子改变(SubAim2.3)。这些研究将揭示一种 目前忽视EZH1在皮质神经发生和神经发育疾病调控中的作用 可能为酒精性肠炎提供新的治疗靶点。

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.