Massively parallel functional dissection of mammalian enhancers in vivo

Abstract

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.

Variant Interpretation: Functional Assays to the Rescue

“…13,[55][56][57] For example, MPRAs have been used to quantify the effects of more than 100,000 variants of three liver enhancers. 58 MPRAs have also been used to simultaneously test thousands of variants associated with eQTLs 22 or variants in linkage disequilibrium with lead SNPs from GWASs for red blood cell traits. 59 Another noteworthy adaptation of MPRAs is population-scale self-transcribing active regulatory region sequencing (POPSTARR), in which candidate regulatory elements from numerous individuals are cloned via DNA sequence capture and tested in parallel.…”

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

“…Whereas classically enhancer-reporter assays consist of cloning each enhancer one by one, first in vitro, later in vivo (Banerji et al 1981;O'Kane and Gehring 1987;Chiocchetti et al 1997;Dailey 2015), now hundreds to thousands of enhancers can be tested in parallel (Patwardhan et al 2009(Patwardhan et al , 2012Kwasnieski et al 2012;Melnikov et al 2012;Arnold et al 2013;Kheradpour et al 2013;Smith et al 2013;White et al 2013;Vanhille et al 2015). These methods can be broadly categorized in two groups, namely, massively parallel reporter assays (MPRA) utilizing barcodes as a measure of activity of synthesized enhancer fragments (Patwardhan et al 2009(Patwardhan et al , 2012Kwasnieski et al 2012;Melnikov et al 2012;Kheradpour et al 2013;Smith et al 2013;White et al 2013) and self-transcribing active regulatory region sequencing (STARR-seq) (Arnold et al 2013;Vanhille et al 2015).…”

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

“…This deletion removed the SCR, a recently described Sox2 specific enhancer in ES cells 18,22 . Although the introduction of the large deletion was greatly reduced on the Cast allele, three clones (1,11,75) were identified with a large deletion on the Cast allele ( Figure 5). Of these three clones two (1, 11) contained 3' break points within 50 bp of the 3' gRNA target region.…”

“…Enhancer predictions are commonly based on histone modification marks, mediator-cohesin complexes and binding of cell type-specific transcription factors [7][8][9][10] . Validation of predicted enhancers is most often done through a vector based assay in which the enhancer activates expression of a reporter gene [11][12] . These data provide valuable information about the regulatory potential of putative enhancer sequences but do not reveal their function in their endogenous genomic context or identify the genes they regulate.…”