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 ΔHAR.321/ΔHAR.321 and Rbfox1 +/+ mice at 14.5 embryonic (E14.5) days, with five independent biological samples per genotype.