PRC1-dependent H2A Monoubiquitination During Human Corticogenesis

项目摘要 在工业化国家，智力残疾影响着2-3%的人口，遗传原因占 这些案件的一半左右。全基因组测序已经导致了几个新的发现， 神经发育障碍的致病性变异。在这些变异体中， 染色质翻译后修饰是高度代表性的。多梳抑制复合体的变体 1（PRC 1）作为组蛋白2A单泛素化（H2 AUb 1）的主要催化剂，说明了这一趋势。RING 1和 RNF 2是作为PRC 1的组成性E3泛素连接酶的旁系同源物，并且这些中的错义变体 钝化H2 AUb 1催化的基因与神经发育障碍相关，其特征在于 小头畸形、智力残疾和早发性精神分裂症。本提案的目的是了解 致病性RING 1变异如何在遗传和分子上导致疾病。探讨PRC 1的作用- 在人类皮质生成的背景下，我们已经产生了同基因人类胚胎干细胞， 在RING 1中携带致病性变体的人胚胎干细胞（hESC）系，并使它们进行神经分化， 生成3D脑类器官。先前的研究表明PRC 1依赖的H2 AUb 1在以下方面发挥作用： 转录抑制和双链断裂修复。与以前的报告一致，我们 初步数据表明，RING 1G 284 A/G284 A变体降低了全球H2 AUb 1水平，导致 促凋亡基因表达增加，脑类器官生长减少， 神经祖细胞（NPC）中链断裂修复。目的1探讨RING 1的遗传机制 致病性变体，因为实验将揭示致病性是否是由于野生型的活性丧失所致 RING 1或由于突变等位基因的显性负效应。目的2将评估分子机制 致病性RING 1变异体通过其导致小头畸形。实验将评估NPC和类器官 与携带RING 1致病性变体的hESC在DNA损伤修复中的缺陷相比， 细胞凋亡率，以及在DNA损伤位点沉默转录的能力。机械学的见解 这些实验将揭示PRC 1依赖性H2 AUb 1在NPC增殖和增殖中的作用。 更广泛地维持基因组的完整性。此外，这些研究的结果将为今后的努力提供参考， 设计治疗策略，以恢复具有致病性RING 1变异体的个体的这些功能。

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.