Non-monotonic regulation of gene expression, neural progenitor fate and brain growth by the chromatin remodeller CHD8

Heterozygous CHD8 mutations are associated with autism and macrocephaly with high penetrance in the human population. The reported mutations may have loss-of-function (haploinsufficient), hypomorphic or dominant negative effects on protein function. To determine the effects of reducing CHD8 protein function below haploinsufficient levels on brain development, we established a Chd8 allelic series in the mouse. Chd8 heterozygous mice exhibited relatively subtle brain overgrowth and little gene expression changes in the embryonic neocortex. In comparison, mild Chd8 hypomorphs displayed significant postnatal lethality, with surviving animals exhibiting more pronounced brain hyperplasia, and significantly altered expression of over 2000 genes. Autism-associated genes were downregulated and neural progenitor proliferation genes upregulated. Severe Chd8 hypomorphs displayed even greater transcriptional dysregulation, affecting genes and pathways that largely overlapped with those dysregulated in the mild hypomorphs. By contrast, homozygous, conditional deletion of Chd8 in early neuronal progenitors resulted in the induction of p53 target genes, cell cycle exit, apoptosis and pronounced brain hypoplasia. Intriguingly, increased progenitor proliferation in hypomorphs was primarily restricted to TBR2+ intermediate progenitors, suggesting critical roles for CHD8 in regulating the expansion of this population. Given the importance of these progenitors in human cortical growth, this observation suggests that human brain development might be more sensitive to CHD8 deficiency than the mouse. We conclude that brain development is acutely sensitive to CHD8 dosage and that the varying sensitivities of different progenitor populations and cellular processes to CHD8 dosage can result in non-linear effects on gene transcription and brain growth. Cortical mRNA profiles of control and Chd8 heterozygous mice at E12.5, and control and Chd8 conditional knockout mice at E10.5, were generated by sequencing on the Hiseq 4000杂合型CHD8突变与自闭症及人群中高外显率的巨头症密切相关。已报道的此类突变可对蛋白质功能产生功能丧失（haploinsufficient，单倍体剂量不足）、功能减退（hypomorphic）或显性负效作用。为探究将CHD8蛋白功能降低至单倍体剂量不足水平以下对大脑发育的影响，我们在小鼠中构建了Chd8等位基因系列（allelic series）。Chd8杂合小鼠在胚胎新皮层中仅表现出相对轻微的脑过度生长，且基因表达变化幅度极小。与之相比，轻度Chd8功能减退突变体（hypomorphs）出现显著的产后致死现象，存活个体则表现出更为明显的脑增生，且超过2000个基因的表达发生显著改变。自闭症相关基因表达下调，而神经祖细胞增殖相关基因表达上调。重度Chd8功能减退突变体的转录失调程度更为严重，其受影响的基因与通路在很大程度上与轻度功能减退突变体中失调的分子重叠。与之形成鲜明对比的是，在早期神经元祖细胞中条件性敲除（conditional knockout）Chd8的纯合小鼠，会诱导p53靶基因表达、细胞周期退出、细胞凋亡，并出现显著的脑发育不全。有趣的是，功能减退突变体中祖细胞增殖增加主要局限于TBR2+中间祖细胞（TBR2+ intermediate progenitors），这表明CHD8在调控该类细胞群的扩增中发挥关键作用。鉴于此类祖细胞在人类皮层发育中的重要性，该观察结果提示人类大脑发育对CHD8缺陷的敏感性可能高于小鼠。我们得出结论：大脑发育对CHD8的剂量极为敏感，且不同祖细胞群与细胞过程对CHD8剂量的不同敏感性，可对基因转录与脑生长产生非线性影响。本研究通过Hiseq 4000测序平台，获取了胚胎第12.5天的对照组与Chd8杂合小鼠，以及胚胎第10.5天的对照组与Chd8条件性敲除小鼠的皮层mRNA表达谱。