PRC1-dependent H2A Monoubiquitination During Human Corticogenesis

人类皮质生成过程中 PRC1 依赖性 H2A 单泛素化

• 批准号: 10604561
• 负责人: Charles William Ryan
• 金额: $ 4.05万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10604561
• 关键词: 3-Dimensional; Accounting; Affect; Alleles; Apoptosis; Apoptotic; BMI1 gene; Binding; Binding Sites; Brain; Catalysis; Cell Cycle; Cerebrum; Child; Chromatin; Compensation; Complement; Complex; DNA Damage; DNA Repair; DNA Sequence Alteration; DNA replication fork; Data; Databases; Defect; Deposition; Developed Countries; Development; Disease; Dominant-Negative Mutation; Double Strand Break Repair; Environment; Future; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Transcription; Goals; Growth; Histone H2A; Histones; Human; Individual; Infant; Intellectual functioning disability; Ionizing radiation; Knock-out; Knowledge; Lead; Learning; Light; Literature; Lymphocyte; Mediating; Medical Genetics; Microcephaly; Mitotic; Modeling; Molecular; Monoubiquitination; Mutation; Neurodevelopmental Disorder; Neurons; Nucleosomes; Organoids; PRC1 Protein; Pathogenesis; Pathogenicity; Patients; Physicians; Population; Post-Translational Protein Processing; Process; Proliferating; Prosencephalon; Proteins; Reporting; Resolution; Response Elements; Role; Schizophrenia; Scientist; Site; Testing; Therapeutic; Training; Transcriptional Regulation; Ubiquitination; Variant; brain size; career; catalyst; cell type; clinically relevant; crosslink; developmental disease; early onset; experimental study; functional restoration; gene repression; genetic testing; genome integrity; genome sequencing; genome-wide; human embryonic stem cell; human embryonic stem cell line; insight; mouse model; mutant; nerve stem cell; neural; novel; postmitotic; premature; prevent; rapid growth; repaired; targeted treatment; trend; ubiquitin-protein ligase; whole genome

PROJECT SUMMARY Intellectual disability affects 2-3% of the population in industrialized nations, with genetic causes accounting for approximately half of these cases. Whole genome sequencing has led to the discovery of several novel pathogenic variants underlying neurodevelopmental disorders. Among these variants, proteins involved in chromatin post-translational modifications are highly represented. Variants in the polycomb repressive complex 1 (PRC1), the primary catalyst of histone 2A monoubiquitination (H2AUb1), illustrate this trend. RING1 and RNF2 are paralogues that serve as the constituent E3 ubiquitin ligase of PRC1, and missense variants in these genes that blunt H2AUb1 catalysis are associated with a neurodevelopmental disorder characterized by microcephaly, intellectual disability, and early onset schizophrenia. The goal of this proposal is to understand how pathogenic RING1 variants lead to disease genetically and molecularly. To explore the role of PRC1- dependent H2AUb1 in the context of human corticogenesis, we have generated isogenic human embryonic stem cell (hESC) lines harboring pathogenic variants in RING1 and subjected them to neural differentiation to generate 3D cerebral organoids. Previous studies suggest that PRC1-dependent H2AUb1 has roles in transcriptional repression and double strand break repair. Consistent with these previous reports, our preliminary data demonstrates that the RING1G284A/G284A variant diminishes global H2AUb1 levels, leads to increased expression of pro-apoptotic genes and decreased growth in cerebral organoids, and disrupts double strand break repair in neural progenitor cells (NPCs). Aim 1 will explore the genetic mechanism of RING1 pathogenic variants, as experiments will shed light onto whether pathogenesis is due to lost activity of wild type RING1 or due to a dominant negative effect of the mutant allele. Aim 2 will evaluate the molecular mechanism by which pathogenic RING1 variants cause microcephaly. Experiments will evaluate NPCs and organoids differentiated from hESCs harboring RING1 pathogenic variants for defects in DNA damage repair, increased rates of apoptosis, and the ability to silence transcription at sites of DNA damage. Mechanistic insights learned from these experiments will shed light onto the role of PRC1-dependent H2AUb1 in proliferating NPCs and in maintaining genome integrity more broadly. In addition, the results of these studies will inform future efforts to devise therapeutic strategies to restore these functions in individuals with pathogenic RING1 variants.

项目概要 智力障碍影响工业化国家 2-3% 的人口，其中遗传原因占主导地位 大约有一半的病例。全基因组测序发现了一些新的 神经发育障碍的致病变异。在这些变体中，涉及的蛋白质 染色质翻译后修饰的比例很高。多梳抑制复合体的变体 组蛋白 2A 单泛素化 (H2AUb1) 的主要催化剂 1 (PRC1) 说明了这一趋势。 RING1 和 RNF2 是旁系同源物，充当 PRC1 的组成 E3 泛素连接酶，以及这些中的错义变体 削弱 H2AUb1 催化作用的基因与神经发育障碍相关，其特征为 小头畸形、智力障碍和早发性精神分裂症。该提案的目标是了解 致病性 RING1 变异如何导致遗传和分子疾病。探索 PRC1- 的作用 在人类皮质发生的背景下依赖H2AUb1，我们已经产生了同基因人类胚胎 携带 RING1 致病性变异的干细胞 (hESC) 系，并对它们进行神经分化 生成 3D 大脑类器官。先前的研究表明 PRC1 依赖性 H2AUb1 在 转录抑制和双链断裂修复。与之前的报告一致，我们的 初步数据表明，RING1G284A/G284A 变体降低了整体 H2AUb1 水平，导致 促凋亡基因的表达增加，大脑类器官的生长减少，并破坏双 神经祖细胞 (NPC) 中的链断裂修复。目标1将探索RING1的遗传机制 致病变异，因为实验将揭示发病机制是否是由于野生型活性丧失所致 RING1 或由于突变等位基因的显性负效应。目标 2 将评估分子机制 致病性 RING1 变异导致小头畸形。实验将评估 NPC 和类器官 与携带 RING1 致病性变异的 hESC 不同，其 DNA 损伤修复缺陷增加 细胞凋亡率，以及在 DNA 损伤位点沉默转录的能力。学到的机制见解 这些实验将揭示 PRC1 依赖性 H2AUb1 在 NPC 增殖和 更广泛地维持基因组完整性。此外，这些研究的结果将为未来的努力提供信息 设计治疗策略来恢复具有致病性 RING1 变异的个体的这些功能。