Loss-of-Function Analyses of SETD1A in Human Neural Models

修改后的项目摘要/摘要部分 SETD1A罕见的功能丧失(LoF)突变与精神分裂症(SZ)密切相关，SZ是一种影响1%人口的衰弱精神疾病，以及其他严重的神经发育障碍。SETD1A编码组蛋白甲基转移酶复合体的一个组分，在赖氨酸4(H3K4)处产生单甲基化、双甲基化和三甲基化的组蛋白H3。H3K4三甲基化(H3K4me3)和H3K4me1分别是活性基因转录启动子和增强子的表观基因组标记。有趣的是，组蛋白甲基化也被认为是主要精神疾病常见的基于变异的全基因组关联研究中最丰富的基因途径之一。此外，最近一种带有SETD1A杂合敲除的小鼠模型显示出工作记忆缺陷，并显示出与神经发育障碍相关的转录变化，然而，似乎独立于H3K4me3机制。因此，SETD1A是否以及如何在人脑中引起与SZ相关的分子和细胞变化在很大程度上仍然不清楚。我们的中心假设是，人类诱导多能干细胞(HiPSC)来源的神经元和皮质器官概括了SETD1ALoF在人脑中与SZ相关的关键表观遗传学、分子和细胞特性。利用CRISPR/Cas9基因编辑，我们获得了携带SETD1A杂合LoF突变(位于不同遗传背景的外显子4和外显子16)的等基因HiPSC系。初步结果表明，突变系在皮质器官发育方面存在缺陷，早期发育阶段神经元分化较早。此外，对携带SETD1ALoF突变的HiPSC神经元的形态、电生理和转录分析表明，突触神经传递存在缺陷。有趣的是，来自突变系的3D皮质器官和2D培养物中显示差异表达的基因富含SZ和其他神经精神障碍/性状的常见GWAS风险变体，这表明主要精神疾病的SETD1ALoF和常见Gwas风险变体可能共享收敛路径。利用我们在HiPSC模型以及我们团队中的神经发生、突触生理学和功能基因组学方面的各自专业知识，我们建议表征人类神经系统中与SZ相关的LOF突变相关缺陷的分子和细胞机制。我们将在皮质类器官中鉴定与SETD1ALoF相关的特定细胞类型和发育阶段特定的细胞和分子表型，然后研究人类神经元中SETD1ALoF突变的突触表型(S)和相关的转录组变化。这项拟议的研究将使我们能够很好地控制评估SETD1ALoF突变对早期神经发育和突触特性缺陷潜在的分子和细胞机制的影响。

Modified Project Summary/Abstract Section Rare loss-of-function (LoF) mutations in SETD1A are strongly associated with schizophrenia (SZ), a debilitating mental disorder affecting 1% of the population, and other severe neurodevelopmental disorders. SETD1A encodes a component of the histone methyltransferase complex producing mono-, di, and trimethylated histone H3 at Lysine 4 (H3K4). H3K4 trimethylation (H3K4me3) and H3K4me1 are epigenomic marks of active gene transcriptional promoters and enhancers, respectively. Interestingly, histone methylation has also been suggested as one of the most enriched gene pathways in common variant-based genome-wide associations studies (GWAS) of major psychiatric disorders. Furthermore, a recent mouse model with heterozygous knockout of SETD1A exhibited working memory deficits and showed transcriptional changes that overlap with those implicated in neurodevelopmental disorders, however, seemingly independent from a H3K4me3 mechanism. Therefore, it remains largely unclear whether and how SETD1A causes SZ-relevant molecular and cellular changes in a human brain. Our central hypothesis is that human induced pluripotent stem cell (hiPSC)-derived neuronal cells and cortical organoids recapitulate key SZ-relevant epigenetic, molecular and cellular properties of SETD1A LoF in the human brain. Using CRISPR/Cas9 gene editing, we have generated isogenic hiPSC lines carrying heterozygous LoF mutations (in exon 4 and exon 16, on different genetic backgrounds) of SETD1A. Preliminary results showed that mutant lines were defective in cortical organoid development with premature neuronal differentiation at early developmental stages. Furthermore, morphological, electrophysiological and transcriptomic analyses of hiPSC neurons carrying SETD1A LoF mutation showed defective synaptic neurotransmission. Interestingly, genes showing differential expression in both 3D cortical organoids and 2D cultures from mutant lines are enriched for common GWAS risk variants of SZ and other neuropsychiatric disorders/traits, suggesting possible convergent pathways shared by SETD1A LoF and common GWAS risk variants of major psychiatric disorders. Leveraging our respective expertise in hiPSC models and neurogenesis, synaptic physiology and functional genomics within our team, we propose to characterize the molecular and cellular mechanisms underlying the deficits associated with SZ-associated LoF mutations in SETD1A in human neural systems. We will identify the cell-type-specific and developmental stage-specific cellular and molecular phenotypes associated with SETD1A LoF in cortical organoids, and then investigate the synaptic phenotype(s) of SETD1A LoF mutations in human neurons and associated transcriptome changes. The proposed study will enable us to perform a well-controlled assessment of the impact of SETD1A LoF mutations on the molecular and cellular mechanisms underlying deficits in early neurodevelopment and synaptic properties.