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

修改项目摘要/摘要部分 SETD 1A中罕见的功能丧失（LoF）突变与精神分裂症（SZ）（一种影响1%人口的衰弱性精神障碍）和其他严重神经发育障碍密切相关。SETD 1A编码组蛋白甲基转移酶复合物的组分，在赖氨酸4（H3 K4）处产生单甲基化、二甲基化和三甲基化组蛋白H3。H3 K4三甲基化（H3 K4 me 3）和H3 K4 me 1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的常见变异全基因组关联研究（GWAS）中最丰富的基因途径之一。此外，最近的小鼠模型与杂合敲除SETD 1A表现出工作记忆缺陷，并显示转录变化，与那些涉及神经发育障碍重叠，然而，似乎独立于H3 K4 me 3机制。因此，目前尚不清楚SETD 1A是否以及如何在人脑中引起SZ相关的分子和细胞变化。我们的中心假设是，人类诱导多能干细胞（hiPSC）衍生的神经元细胞和皮质类器官重现了人脑中SETD 1A LoF的关键SZ相关表观遗传、分子和细胞特性。使用CRISPR/Cas9基因编辑，我们已经产生了携带SETD 1A杂合LoF突变（在外显子4和外显子16中，在不同的遗传背景上）的等基因hiPSC系。初步结果表明，突变株系在皮层类器官发育中有缺陷，在早期发育阶段具有过早的神经元分化。此外，携带SETD 1A LoF突变的hiPSC神经元的形态学、电生理学和转录组学分析显示突触神经传递缺陷。有趣的是，在3D皮质类器官和来自突变系的2D培养物中显示差异表达的基因富集了SZ和其他神经精神疾病/性状的常见GWAS风险变体，这表明SETD 1A LoF和主要精神疾病的常见GWAS风险变体共享的可能的会聚途径。利用我们团队在hiPSC模型和神经发生，突触生理学和功能基因组学方面的各自专业知识，我们建议表征与人类神经系统中SETD 1A中SZ相关LoF突变相关的缺陷的分子和细胞机制。我们将确定与皮质类器官中的SETD 1A LoF相关的细胞类型特异性和发育阶段特异性细胞和分子表型，然后研究人类神经元中SETD 1A LoF突变的突触表型和相关的转录组变化。这项拟议的研究将使我们能够对SETD 1A LoF突变对早期神经发育和突触特性缺陷的分子和细胞机制的影响进行良好的对照评估。

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.