Article

Massively parallel functional dissection of mammalian enhancers

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
Nature Biotechnology (Impact Factor: 39.08). 02/2012; 30(3):265-70. DOI: 10.1038/nbt.2136
Source: PubMed

ABSTRACT The functional consequences of genetic variation in mammalian regulatory elements are poorly understood. We report the in vivo dissection of three mammalian enhancers at single-nucleotide resolution through a massively parallel reporter assay. For each enhancer, we synthesized a library of >100,000 mutant haplotypes with 2-3% divergence from the wild-type sequence. 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 the transcribed tags. Linear regression analysis yielded highly reproducible estimates of the effect of every possible single-nucleotide change on enhancer activity. The functional consequence of most mutations was modest, with ∼22% affecting activity by >1.2-fold and ∼3% by >2-fold. Several, but not all, positions with higher effects 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.

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    • "Individual nucleotides were perturbed for only a handful of putative enhancers in a directed way (Ernst et al. 2011), limiting our understanding of the role of individual regulatory motifs and motif positions in establishing enhancer activity. This situation is remedied by recently developed massively parallel reporter assays (Melnikov et al. 2012; Patwardhan et al. 2012; Sharon et al. 2012; Arnold et al. 2013) that take advantage of large-scale sequencing to simultaneously measure the reporter activity of thousands of enhancer variants. However, these assays have only been used to dissect four human and one mouse enhancers, leaving open the question of what fraction of genome-wide regulatory predictions can be experimentally validated at the single-nucleotide level. "
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