Journal
NATURE BIOTECHNOLOGY
Volume 30, Issue 3, Pages 265-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.2136
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Funding
- National Human Genome Research Institute [HG003988]
- US National Institutes of Health (NIH) [DP5OD009145]
- National Institute of General Medical Sciences (NIGMS) [GM61390]
- National Institute of Child Health and Human Development (NICHD) [R01HD059862]
- University of California, San Francisco Liver Center [P30 DK026743]
- National Institute on Aging [AG039173]
- Achievement Rewards for College Scientists Foundation
- NIH [T32 GM007175]
- Amgen Research Excellence in Bioengineering and Therapeutic Sciences Fellowship
- CIHR
- Department of Energy, University of California [DE-AC02-05CH11231]
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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 similar to 22% affecting activity by >1.2-fold and similar to 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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