CHD8 regulates neurodevelopmental pathways associated with autism spectrum disorder in neural progenitors [ChIP-Seq]

Truncating mutations of CHD8, encoding a chromodomain helicase, and of many other genes with diverse functions, are strong-effect risk factors for autism spectrum disorder (ASD), suggesting multiple mechanisms of pathogenesis. We explored the transcriptional networks that CHD8 regulates in neural progenitor cells (NPCs) by reducing its expression and then integrating transcriptome sequencing (RNA-seq) with genome-wide CHD8 binding (ChIP-seq). Suppressing CHD8 to levels comparable with loss of a single allele caused altered expression of 1,756 genes, 64.9% of which were up-regulated. CHD8 showed widespread binding to chromatin, with 7,324 replicated sites that marked 5,658 genes. Integration of these data suggests that a limited array of direct regulatory effects of CHD8 produced a much larger network of secondary expression changes. Genes indirectly down-regulated (i.e., without CHD8 binding sites) reflect pathways involved in brain development, including synapse formation, neuron differentiation, cell adhesion, and axon guidance, whereas CHD8-bound genes are strongly associated with chromatin modification and transcriptional regulation. Genes associated with ASD were strongly enriched among indirectly down-regulated loci (p = 1.01x10-9) and CHD8-bound genes (p = 4.34x10-3), which align with previously identified co-expression modules during fetal development. We also find an intriguing enrichment of cancer related gene-sets among CHD8-bound genes (p < 1.9x10-11). In vivo suppression of chd8 in zebrafish produced macrocephaly comparable to that of humans with inactivating mutations. These data indicate that heterozygous disruption of CHD8 precipitates a network of gene expression changes involved in neurodevelopmental pathways in which many ASD-associated genes may converge on shared mechanisms of pathogenesis. ChIP-seq for CHD8 using three different antibodies, and the related protein CHD7, in human iPSC-derived NPCs treated with shRNA targeting GFP (which were used as control cells for an shRNA knockdown RNA-seq experiment that was part of the overall study)

CHD8（编码一类含染色质域的解旋酶）以及诸多功能各异的其他基因的截短突变，均为自闭症谱系障碍（ASD）的强效应风险因子，提示存在多种致病机制。我们通过敲低CHD8的表达水平，并将转录组测序（RNA-seq）与全基因组CHD8结合位点染色质免疫共沉淀测序（ChIP-seq）相结合，探究了CHD8在神经前体细胞（NPCs）中调控的转录网络。将CHD8表达抑制至与单等位基因缺失相当的水平后，共导致1756个基因的表达发生改变，其中64.9%的基因呈现上调。CHD8在染色质上存在广泛结合，共鉴定出7324个可重复结合位点，覆盖5658个基因。整合上述数据后发现，CHD8仅通过有限的直接调控作用，即可催生规模庞大的次级表达变化网络。间接下调的基因（即未结合CHD8的基因）富集于脑发育相关通路，包括突触形成、神经元分化、细胞黏附及轴突导向；而结合有CHD8的基因则与染色质修饰及转录调控显著相关。自闭症谱系障碍相关基因在间接下调位点（p = 1.01×10^-9）以及结合CHD8的基因集合（p = 4.34×10^-3）中均显著富集，这与此前鉴定的胎儿发育阶段共表达模块相符。我们还发现，结合CHD8的基因中存在与癌症相关基因集的显著富集（p < 1.9×10^-11），这一发现颇为引人关注。在斑马鱼体内抑制chd8的表达，可导致与携带失活突变的人类患者相似的巨头症。上述数据表明，CHD8的杂合缺失会引发一系列参与神经发育通路的基因表达变化，而诸多自闭症谱系障碍相关基因可能通过共同的致病机制汇聚于此。本研究针对采用靶向绿色荧光蛋白（GFP）的短发夹RNA（shRNA）处理的人类诱导多能干细胞（iPSC）分化而来的神经前体细胞（NPCs），使用三种不同抗体开展CHD8及相关蛋白CHD7的染色质免疫共沉淀测序（ChIP-seq）；该细胞系被用作本研究整体中一项shRNA敲低转录组测序实验的对照细胞。