Characterization of sequence determinants of enhancer function using natural genetic variation

Sequence variation in enhancers that control cell-type-specific gene transcription contributes significantly to phenotypic variation within human populations. However, it remains difficult to predict precisely the effect of any given sequence variant on enhancer function due to the complexity of DNA sequence motifs that determine transcription factor (TF) binding to enhancers in their native genomic context. Using F₁-hybrid cells derived from crosses between distantly related inbred strains of mice, we identified thousands of enhancers with allele-specific TF binding and/or activity. We find that genetic variants located within the central region of enhancers are most likely to alter TF binding and enhancer activity. We observe that the AP-1 family of TFs (Fos/Jun) are frequently required for binding of TEAD TFs and for enhancer function. However, many sequence variants outside of core motifs for AP-1 and TEAD also impact enhancer function, including sequences flanking core TF motifs and AP-1 half sites. Taken together, these data represent one of the most comprehensive assessments of allele-specific TF binding and enhancer function to date and reveal how sequence changes at enhancers alter their function across evolutionary timescales.