RT Journal Article SR Electronic T1 Functional Characterization of Enhancer Evolution in the Primate Lineage JF bioRxiv FD Cold Spring Harbor Laboratory SP 283168 DO 10.1101/283168 A1 Jason C. Klein A1 Aidan Keith A1 Vikram Agarwal A1 Timothy Durham A1 Jay Shendure YR 2018 UL http://biorxiv.org/content/early/2018/03/15/283168.abstract AB Background Enhancers play an important role in morphological evolution and speciation by controlling the spatiotemporal expression of genes. Due to technological limitations, previous efforts to understand the evolution of enhancers in primates have typically studied many enhancers at low resolution, or single enhancers at high resolution. Although comparative genomic studies reveal large-scale turnover of enhancers, a specific understanding of the molecular steps by which mammalian or primate enhancers evolve remains elusive.Results We identified candidate hominoid-specific liver enhancers from H3K27ac ChIP-seq data. After locating orthologs in 11 primates spanning ∼40 million years, we synthesized all orthologs as well as computational reconstructions of 9 ancestral sequences for 348 “active tiles” of 233 putative enhancers. We concurrently tested all sequences (20 per tile) for regulatory activity with STARR-seq in HepG2 cells, with the goal of characterizing the evolutionary-functional trajectories of each enhancer. We observe groups of enhancer tiles with coherent trajectories, most of which can be explained by one or two mutational events per tile. We quantify the correlation between the number of mutations along a branch and the magnitude of change in functional activity. Finally, we identify 57 mutations that correlate with functional changes; these are enriched for cytosine deamination events within CpGs, compared to background events.Conclusions We characterized the evolutionary-functional trajectories of hundreds of liver enhancers throughout the primate phylogeny. We observe subsets of regulatory sequences that appear to have gained or lost activity at various positions in the primate phylogeny. We use these data to quantify the relationship between sequence and functional divergence, and to identify CpG deamination as a potentially important force in driving changes in enhancer activity during primate evolution.