Accelerated evolution in the human lineage led to gain and loss of transcriptional enhancers in the RBFOX1 locus [midbrain]

A long-standing goal of evolutionary biology is to decode how changes in gene regulatory networks contribute to human-specific traits. Human Accelerated Regions (HARs) are prime candidates for driving gene regulatory modifications in human development. The RBFOX1 locus is densely populated with HARs, providing a set of potential regulatory elements that could have changed its expression in the human lineage. Here we examined the role of RBFOX1-HARs using transgenic zebrafish reporter assays and identified fifteen transcriptional enhancers that are active in the developing nervous system, ten of which displayed differential expression between the human and chimpanzee sequences. The engineered loss of two selected RBFOX1-HARs in knockout mouse models increased Rbfox1 expression at specific developmental stages and tissues in the brain, influencing the expression and splicing of a high number of Rbfox1 target genes. Our results provided insight into the spatial and temporal changes in gene expression driven by RBFOX1-HARs. Midbrains were extracted from Rbfox1 HAR.321/HAR.321 and Rbfox1 +/+ mice at 14.5 embryonic (E14.5) days, with five independent biological samples per genotype.演化生物学的长期核心目标之一，是解析基因调控网络的变化如何促成人类特异性性状的形成。人类加速区域（Human Accelerated Regions, HARs）是驱动人类发育过程中基因调控修饰的首要候选靶点。RBFOX1基因座上富集了大量HARs，为探究人类演化谱系中该基因的表达变化提供了一系列潜在调控元件。本研究通过转基因斑马鱼报告基因实验，对RBFOX1-HARs的功能进行了探究，最终鉴定出15个在发育神经系统中具有活性的转录增强子，其中10个在人类与黑猩猩的序列间呈现表达差异。在基因敲除小鼠模型中定向敲除两个筛选得到的RBFOX1-HARs后，Rbfox1在大脑特定发育阶段与组织中的表达水平显著上调，并影响了大量Rbfox1靶基因的表达与可变剪接。本研究结果为解析RBFOX1-HARs所介导的基因表达时空动态变化提供了重要见解。本研究于胚胎发育第14.5天（E14.5），从Rbfox1 HAR.321/HAR.321纯合敲除小鼠与Rbfox1野生型（+/+）小鼠中提取中脑组织，每个基因型设置5个独立生物学重复样本。