Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA.
Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA; Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.
Am J Hum Genet. 2022 Nov 3;109(11):2049-2067. doi: 10.1016/j.ajhg.2022.09.015. Epub 2022 Oct 24.
Point mutations and structural variants that directly disrupt the coding sequence of MEF2C have been associated with a spectrum of neurodevelopmental disorders (NDDs). However, the impact of MEF2C haploinsufficiency on neurodevelopmental pathways and synaptic processes is not well understood, nor are the complex mechanisms that govern its regulation. To explore the functional changes associated with structural variants that alter MEF2C expression and/or regulation, we generated an allelic series of 204 isogenic human induced pluripotent stem cell (hiPSC)-derived neural stem cells and glutamatergic induced neurons. These neuronal models harbored CRISPR-engineered mutations that involved direct deletion of MEF2C or deletion of the boundary points for topologically associating domains (TADs) and chromatin loops encompassing MEF2C. Systematic profiling of mutation-specific alterations, contrasted to unedited controls that were exposed to the same guide RNAs for each edit, revealed that deletion of MEF2C caused differential expression of genes associated with neurodevelopmental pathways and synaptic function. We also discovered significant reduction in synaptic activity measured by multielectrode arrays (MEAs) in neuronal cells. By contrast, we observed robust buffering against MEF2C regulatory disruption following deletion of a distal 5q14.3 TAD and loop boundary, whereas homozygous loss of a proximal loop boundary resulted in down-regulation of MEF2C expression and reduced electrophysiological activity on MEA that was comparable to direct gene disruption. Collectively, these studies highlight the considerable functional impact of MEF2C deletion in neuronal cells and systematically characterize the complex interactions that challenge a priori predictions of regulatory consequences from structural variants that disrupt three-dimensional genome organization.
点突变和结构变异直接破坏 MEF2C 的编码序列与一系列神经发育障碍（NDD）有关。然而，MEF2C 杂合不足对神经发育途径和突触过程的影响尚不清楚，也不清楚调控其的复杂机制。为了探索与改变 MEF2C 表达和/或调控的结构变异相关的功能变化，我们生成了一系列 204 个人类诱导多能干细胞（hiPSC）衍生的神经干细胞和谷氨酸能诱导神经元的等位基因系列。这些神经元模型携带 CRISPR 工程突变，包括 MEF2C 的直接缺失或拓扑关联域（TAD）和包含 MEF2C 的染色质环边界的缺失。对突变特异性改变的系统分析，与暴露于每个编辑相同 guide RNA 的未编辑对照进行对比，表明 MEF2C 的缺失导致与神经发育途径和突触功能相关的基因的差异表达。我们还发现通过多电极阵列（MEA）测量的突触活性显著降低。相比之下，我们观察到在删除远端 5q14.3 TAD 和环边界后，对 MEF2C 调控破坏有强大的缓冲作用，而近端环边界的纯合缺失导致 MEF2C 表达下调和 MEA 上的电生理活性降低，与直接基因破坏相当。总的来说，这些研究强调了 MEF2C 缺失在神经元细胞中的巨大功能影响，并系统地表征了挑战结构变异对三维基因组组织破坏的调控后果的先验预测的复杂相互作用。
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