De novo enhancer function in divergent hominin haplotypes

Identifying functional changes in gene regulatory elements in the human lineage is essential to understanding how humans emerged as a species. We performed genome-wide comparative genomic analysis of humans and great apes to identify 1581 Human Ancestor Quickly-Evolved Regions (HAQERs), which we identify as the fastest-evolved regions of the human genome (hg38). To detect how rapid sequence divergence contributed to divergent gene regulatory functionality in HAQERs, we designed in vivo STARR-seq as a multiplex single-cell assay of enhancer activity in the developing mouse brain. We tested multiple haplotypes from hominins (Human/Neanderthal/Denisova) and non-hominins (Chimpanzee and the inferred Human-Chimpanzee ancestor) for 13 HAQERs to assess their enhancer activity in the developing brain. We found significant differences in enhancer activity between hominins and non-hominins in 8 of these sequences. In this assay, STARR-seq plasmids along with an EGFP transfection reporter plasmid are delivered to the developing mouse cerebral cortex via in utero electroporation. Following 18 hours of incubation, mouse brains are harvested and GFP+ cells are isolated via Fluorescent Activated Cell Sorting. Single cell sequencing is then performed to recover gene expression, EGFP transfection reporter plasmid expression, and STARR-seq reporter expression. We performed two independent STARR-seq experiments (V4.2 = E14.5 injection, V4.1 = E15.5 injection)  with the same input library, which we sequenced for input normalization.