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PATWARDHAN, Rupali P; HIATT, Joseph B; AHITUV, Nadav; PENNACCHIO, Len A; SHENDURE, Jay; WITTEN, Daniela M; KIM, Mee J; SMITH, Robin P; MAY, Dalit; LEE, Choli; ANDRIE, Jennifer M; LEE, Su-In; COOPER, Gregory M
Nature biotechnology, 03/2012, Letnik: 30, Številka: 3Journal Article
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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