TBR1 Regulates Autism Risk Genes in the Developing Neocortex. Mus musculus

Exome sequencing studies have identified multiple genes harboring de novo loss-of-function (LoF) variants in individuals with autism spectrum disorders (ASD), including TBR1, a master regulator of cortical development. We performed ChIP-seq for TBR1 during mouse cortical neurogenesis and show that TBR1-bound regions are enriched adjacent to ASD genes. ASD genes were also enriched among genes that are differentially expressed in Tbr1 knockouts, which together with the ChIP-seq data, suggests direct transcriptional regulation. Of the 9 ASD genes examined, 7 were misexpressed in the cortices of Tbr1 knockout mice, including 6 with increased expression in the deep cortical layers. ASD genes with adjacent cortical TBR1 ChIP-seq peaks also showed unusually low levels of LoF mutations in a reference human population and among Icelanders. We then leveraged TBR1 binding to identify an appealing subset of candidate ASD genes. Our findings highlight a TBR1-regulated network of ASD genes in the developing neocortex that are relatively intolerant to LoF mutations, indicating that these genes may play critical roles in normal cortical development. Overall design: ChIP-seq for Tbr1 on mouse whole cortex dissected from embryonic day 15.5 (E15.5) embryos

外显子组测序研究已在自闭症谱系障碍（autism spectrum disorders, ASD）患者中鉴定出多个携带新生功能丧失（de novo loss-of-function, LoF）变异的基因，其中包括TBR1——大脑皮层发育的主控调控因子。本研究在小鼠皮层神经发生过程中针对TBR1开展了染色质免疫沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）实验，结果显示TBR1结合区域在自闭症谱系障碍基因的邻近区域显著富集。在Tbr1基因敲除小鼠中差异表达的基因集合内，自闭症谱系障碍基因同样呈现显著富集态势，结合上述染色质免疫沉淀测序结果，可提示TBR1对这些基因存在直接转录调控作用。在所检测的9个自闭症谱系障碍基因中，有7个在Tbr1敲除小鼠的皮层组织中表达异常，其中6个在皮层深层的表达水平出现上调。在皮层区域存在邻近TBR1染色质免疫沉淀测序峰的自闭症谱系障碍基因，在参考人类人群以及冰岛人群中均表现出异常低的功能丧失变异携带率。随后我们依托TBR1结合特征，筛选出了一组极具研究价值的自闭症谱系障碍候选基因。本研究结果揭示了发育中新皮层中受TBR1调控的自闭症谱系障碍基因网络，这类基因相对难以耐受功能丧失变异，提示它们在正常皮层发育过程中发挥关键作用。
实验整体设计：对取自胚胎第15.5天（embryonic day 15.5, E15.5）小鼠的全皮层组织开展TBR1染色质免疫沉淀测序实验。