Genome-wide strand asymmetry in massively parallel reporter activity favors genic strands

Abstract

Massively parallel reporter assays (MPRAs) are useful tools to characterize regulatory elements in human genomes. An aspect of MPRAs that is not typically the focus of analysis is their intrinsic ability to differentiate activity levels for a given sequence element when placed in both of its possible orientations relative to the reporter construct. Here, we describe pervasive strand asymmetry of MPRA signals in data sets from multiple reporter configurations in both published and newly reported data. These effects are reproducible across different cell types and in different treatments within a cell type and are observed both within and outside of annotated regulatory elements. From elements in gene bodies, MPRA strand asymmetry favors the sense strand, suggesting that function related to endogenous transcription is driving the phenomenon. Similarly, we find that within Alu mobile element insertions, strand asymmetry favors the transcribed strand of the ancestral retrotransposon. The effect is consistent across the multiplicity of Alu elements in human genomes and is more pronounced in less diverged Alu elements. We find sequence features driving MPRA strand asymmetry and show its prediction from sequence alone. We see some evidence for RNA stabilization and transcriptional activation mechanisms and hypothesize that the effect is driven by natural selection favoring efficient transcription. Our results indicate that strand asymmetry is a pervasive and reproducible feature in MPRA data. More importantly, the fact that MPRA asymmetry favors naturally transcribed strands suggests that it stems from preserved biological functions that have a substantial, global impact on gene and genome evolution.

Transcription factor binding site orientation and order are major drivers of gene regulatory activity

“…Previous work has examined the effect of genomic element orientation on expression in both single locus experimental studies 7 , 32 and via high-throughput assays 9 , 13 , 18 , 33 . For example, using STARR-seq, it was shown that orientation of Alu repeat elements and DNA octamers are associated with expression differences 18 .…”

“…Previous work has examined the effect of genomic element orientation on expression in both single locus experimental studies 7 , 32 and via high-throughput assays 9 , 13 , 18 , 33 . For example, using STARR-seq, it was shown that orientation of Alu repeat elements and DNA octamers are associated with expression differences 18 . A previous MPRA identified that the orientation of the tested tiles influenced expression 17 , while in another MPRA experiment statistically significant differences in expression relative to the orientation of a set of core-promoter TFBS were shown 13 .…”

“…Massively parallel reporter assays (MPRAs) provide the ability to measure thousands of sequences for their regulatory activity by placing these sequences in front of a transcribed barcode 16 – 18 . Utilizing MPRA, we previously designed a library of 5,000 sequences in which we tested the effect of TFBS copy number, spacing and order 19 .…”

Advances in computational and experimental approaches for deciphering transcriptional regulatory networks

“…Standard reporter assays test whether a regulatory element activates a reporter gene in a one‐by‐one manner, potentially leading to bottlenecks, [ 2,104 ] whereas MRPAs enable the simultaneous assessment of thousands of cis ‐regulatory elements. [ 105–107 ] In MPRAs, candidate regulatory sequences are linked to unique barcodes and incorporated into classic promoter or enhancer reporter vectors, enabling the regulatory element to drive its own transcription and that of the associated barcode. Subsequently, next‐generation sequencing measures the barcode's expression and normalizes it to the genomic element's DNA abundance to represent cis ‐regulatory activity.…”