Making the Human Face: Elucidating the Role of Enhancers in Hominid Craniofacial Evolution

Abstract

There is increasing evidence that morphological differences among individuals of the same species and between species are driven by changes in gene expression mediated by non‐coding mutations that are enriched at enhancer elements. The identification and comparative evaluation of these cis‐regulatory variants specific to human facial morphology have been facilitated by several recent advances. Among these are access to high‐quality genome sequences, especially from archaic hominins such as the Neanderthals, by expanded catalogs of SNPs implicated in normal range human facial variation from Genome‐wide Association Studies (GWAS), and by the curation of active human and chimp Cranial Neural Crest Cell (CNCC) enhancers that are important for facial development. To identify functional variants that mediate change in enhancer activity in recent human evolution and normal range variation and to nominate their target genes we employed two methods: (1) STARR‐seq, an enhancer reporter assay that permits accurate, comparative analysis of regulatory activity across enhancer homologs and their constituent cis‐mutations, and (2) H3K27ac HiChIP, a method to characterize the three‐dimensional interactome between enhancers and their target genes.We performed STARR‐seq and HiChIP experiments in the in vitro derived CNCCs, and early proliferative cranial chondrocytes. Comparisons of enhancer activity among the orthologs present in homo‐ or hominin species uncovered several classes of enhancers with species‐ or clade‐ restricted activity. Furthermore, for enhancers associated with facial shape GWAS signals, we identified genetic variants associated with changes in regulatory activity. By incorporating HiChIP labeled target genes, we found that biased intra‐species and GWAS enhancer homologs target important developmental transcription factors and signaling molecules. Our experiments have begun to provide insights into how evolutionarily important and functionally relevant enhancer cis‐variants can modulate gene regulatory programs at the individual‐ or species‐centric level with an outcome on facial phenotypes.