Massively parallel functional dissection of mammalian enhancers in vivo

The functional consequences of genetic variation in mammalian regulatory elements are poorly understood. We report the in vivo dissection of three mammalian liver enhancers at single nucleotide resolution via a massively parallelized reporter assay. For each enhancer, we synthesized a library of >100,000 mutant haplotypes with 2–3% divergence from wild-type. Each haplotype was linked to a unique sequence tag embedded within a transcriptional cassette. We introduced each enhancer library into mouse liver and measured the relative activities of individual haplotypes en masse by sequencing of the transcribed tags. Linear regression yielded highly reproducible estimates of the impact of every possible single nucleotide change on enhancer activity. The functional impact of most mutations was modest, with ~22% impacting activity by >1.2-fold, and only ~3% by >2-fold. These results suggest that mammalian enhancers are relatively robust to single nucleotide changes. Several, but not all positions with higher impact showed evidence for purifying selection, or co-localized with known liver-associated transcription factor binding sites, demonstrating the value of empirical high-resolution functional analysis.