Massively parallel functional dissection of mammalian enhancers

Department of Genome Sciences, University of Washington, Seattle, Washington, USA.
Nature Biotechnology (Impact Factor: 41.51). 02/2012; 30(3):265-70. DOI: 10.1038/nbt.2136
Source: PubMed


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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    • "The method in this paper allows the regulatory element to be placed at the discretion of the experimenter. Additionally, the previously published library construction methods (Kwasnieski et al., 2012; Patwardhan et al., 2012; Melnikov et al., 2012; Kheradpour et al., 2013; Arnold et al., 2013) require microbial propagation of DNA libraries whereas we present a simpler entirely in vitro strategy. The work presented here is the first implementation of a massively parallel reporter assay to study cis-regulatory activity during an environmental stress response. "
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    ABSTRACT: Here we present a genome-wide method for de novo identification of enhancer regions. This approach enables massively parallel empirical investigation of DNA sequences that mediate transcriptional activation and provides a platform for discovery of regulatory modules capable of driving context-specific gene expression. The method links fragmented genomic DNA to the transcription of randomer molecule identifiers and measures the functional enhancer activity of the library by massively parallel sequencing. We transfected a Drosophila melanogaster library into S2 cells in normoxia and hypoxia, and assayed 4,599,881 genomic DNA fragments in parallel. The locations of the enhancer regions strongly correlate with genes up-regulated after hypoxia and previously described enhancers. Novel enhancer regions were identified and integrated with RNAseq data and transcription factor motifs to describe the hypoxic response on a genome-wide basis as a complex regulatory network involving multiple stress-response pathways. This work provides a novel method for high-throughput assay of enhancer activity and the genome-scale identification of 31 hypoxia-activated enhancers in Drosophila .
    Full-text · Article · Dec 2015 · PeerJ
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    • "Previous studies have described distributions of mutational effects for several prokaryotic CREs both in vitro (Maerkl and Quake 2007, 2009; Geertz et al. 2012) and in vivo (Patwardhan et al. 2009; Kinney et al. 2010; Brewster et al. 2012; Sharon et al. 2012; Kosuri et al. 2013). As these studies predominantly focused on characterizing general relationships between trans factors (TFs and RNAP) and their cis binding sites, the analysis of interactions between individual cis mutations has been limited (Kwasnieski et al. 2012), partly due to restrictions in the techniques used (Melnikov et al. 2012; Patwardhan et al. 2012). Understanding the dependence of the effect of a mutation on the genetic background in which it appears, a phenomenon termed epistasis (Fisher 1918; Phillips 2008) is critical to understanding adaptation and the engineering of synthetic promoters with specific properties (Kinkhabwala and Guet 2008). "
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    ABSTRACT: Changes in gene expression are an important mode of evolution, however the proximate mechanism of these changes is poorly understood. In particular, little is known about effects of mutations within cis binding sites for transcription factors, or the nature of epistatic interactions between these mutations. Here, we tested the effects of single and double mutants in two cis binding sites involved in the transcriptional regulation of the Escherichia coli araBAD operon, a component of arabinose metabolism, using a synthetic system. This system decouples transcriptional control from any post-translational effects on fitness, allowing a precise estimate of the effect of single and double mutations, and hence epistasis, on gene expression. We found that epistatic interactions between mutations in the araBAD cis regulatory element are common, and that the predominant form of epistasis is negative. The magnitude of the interactions depended on whether the mutations are located in the same or in different operator sites. Importantly, these epistatic interactions were dependent on the presence of arabinose, a native inducer of the araBAD operon in vivo, with some interactions changing in sign (e.g., from negative to positive) in its presence. This study thus reveals that mutations in even relatively simple cis regulatory elements interact in complex ways such that selection on the level of gene expression in one environment might perturb regulation in the other environment in an unpredictable and uncorrelated manner.
    Preview · Article · Nov 2015 · Molecular Biology and Evolution
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    • "Alternatively, use barcoding and subassembly strategies to exceed the sequence length of sequencing technologies. A barcode is a short (10–20 bp) degenerate sequence cloned near the mutated gene, which acts as a molecular tag to enable the assembly of the full-length variable region (Hiatt et al. 2010;Patwardhan et al. 2012). The advantages of barcoding and assembly include reduced sequencing costs, as each variable region is sequenced and assigned to a barcode only once. "
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    ABSTRACT: Deep mutational scanning is a highly parallel method that uses high-throughput sequencing to track changes in >10(5) protein variants before and after selection to measure the effects of mutations on protein function. Here we outline the stages of a deep mutational scanning experiment, focusing on the construction of libraries of protein sequence variants and the preparation of Illumina sequencing libraries. © 2015 Cold Spring Harbor Laboratory Press.
    Full-text · Article · Aug 2015 · Cold Spring Harbor Protocols
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