A systematic comparison reveals substantial differences in chromosomal versus episomal encoding of enhancer activity

Abstract

Candidate enhancers can be identified on the basis of chromatin modifications, the binding of chromatin modifiers and transcription factors and cofactors, or chromatin accessibility. However, validating such candidates as bona fide enhancers requires functional characterization, typically achieved through reporter assays that test whether a sequence can increase expression of a transcriptional reporter via a minimal promoter. A longstanding concern is that reporter assays are mainly implemented on episomes, which are thought to lack physiological chromatin. However, the magnitude and determinants of differences in cis-regulation for regulatory sequences residing in episomes versus chromosomes remain almost completely unknown. To address this systematically, we developed and applied a novel lentivirus-based massively parallel reporter assay (lentiMPRA) to directly compare the functional activities of 2236 candidate liver enhancers in an episomal versus a chromosomally integrated context. We find that the activities of chromosomally integrated sequences are substantially different from the activities of the identical sequences assayed on episomes, and furthermore are correlated with different subsets of ENCODE annotations. The results of chromosomally based reporter assays are also more reproducible and more strongly predictable by both ENCODE annotations and sequence-based models. With a linear model that combines chromatin annotations and sequence information, we achieve a Pearson's R 2 of 0.362 for predicting the results of chromosomally integrated reporter assays. This level of prediction is better than with either chromatin annotations or sequence information alone and also outperforms predictive models of episomal assays. Our results have broad implications for how cis-regulatory elements are identified, prioritized and functionally validated.

Systematic dissection of genomic features determining transcription factor binding and enhancer function

“…We demonstrate that (i) the PPARγ motif affinity (and not cooperative elements in the immediately flanking sequence) largely determines PPARγ binding to genomic sequences when removed from their chromatin context; (ii) enhancer activity depends not only on PPARγ binding but also on a network of 20-30 TF motifs in the flanking sequence that have distinct quantitative contributions to expression; and (iii) various pairs of motifs interact in additive, inhibitory, and synergistic ways with varying constraints on motif positioning. Although in this study we measured enhancer activity in an episomal context, a recent study found that enhancer activity was highly concordant between episomal and genomic contexts (r = 0.86 across 2,236 candidate enhancers vs. 0.90-0.98 for replicates within each context) (93). Importantly, our results show that PPARγ binding and enhancer activity are independently regulated.…”

The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription

“…To disentangle both activities, we developed a new dual transgenic assay that can measure enhancer and promoter activity at the same genomic location in the same embryos such that the timing as well as tissue specificity of both activities can be directly compared. Transgenic assays have the advantage of being able to measure regulatory activity at the endogenous levels of TFs and within a consistent chromatinized context-two properties that have a major impact on both enhancer and promoter activity (Archer et al 1992;Jeong and Stein 1994;Hebbar and Archer 2008;Inoue et al 2017). The readout (in situ hybridization) provides both spatial and temporal information at single-cell resolution, although it is difficult to derive quantitative information on activity-a clear disadvantage compared with in vitro reporter assays.…”

“…However, these assays function outside of a normal chromatinized context, an important property known to affect both promoter (transcriptional initiation) (Archer et al 1992) and enhancer (Inoue et al 2017) activity through various mechanisms (Jeong and Stein 1994;Hebbar and Archer 2008). We therefore set out to assess the ability of enhancers to act as promoters in vivo in transgenic embryos using a stably integrated reporter that does not contain a core promoter.…”

Using GWAS to identify novel therapeutic targets for osteoporosis

“…Currently, most experimental strategies rely on reporter assays that assess regulatory function of variants using very artificial systems that investigate sequences outside of their native chromosomal context 34 . There are, however, new methods, such as using lentiviral based reporter assays that integrate into the genome that are very promising 107 . Additionally, CRISPR/Cas9 based genome-editing approaches that allow one to modify individual variants in human cells are becoming more efficient and will likely to play a large role in causal variant and gene discovery moving forward 108,109 .…”