Resolving systematic errors in widely used enhancer activity assays in human cells

Abstract

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that two previous observations relating to plasmid-transfection into human cells render such assays unreliable: (1) the function of the bacterial plasmid origin-of-replication (ORI) as conflicting core-promoter and (2) the activation of a type-I-interferon (IFN-I) response. These problems cause strongly confounding false-positives and -negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core-promoter and by inhibiting two IFN-I-inducing kinases. This corrects luciferase assays and enables genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells, and are key to the characterization of human enhancers.

A systematic evaluation of the design, orientation, and sequence context dependencies of massively parallel reporter assays

“…They are as follows, with the numbers corresponding to the vertical order on the left side of Figure 1: (1) the "classic" MPRA assay design, using the pGL4.23c vector, wherein the enhancer library resides upstream of a minimal promoter, and the associated barcodes reside in the 3′ UTR of the reporter gene ("pGL4") 10,24 . (2-3) the Self-Transcribing Active Regulatory Region Sequencing (STARR-seq) assay design, wherein the enhancer library resides in the 3′ UTR of the reporter gene, either as originally described ("HSS") 12 or a newer version that uses the bacterial origin of replication for transcription initiation ("ORI") 25 . In both cases, we introduce barcodes immediately adjacent to the location of the enhancers in the 3' UTR in order to facilitate consistent procedures with other assays.…”

“…Readymix (Kapa Biosystems) with a 65 °C annealing temperature and 30 s extension. These primers amplify both candidate enhancers and previously assigned degenerate barcodes and add homology arms to the ORI vector (Addgene 99296) 25 . We followed the reaction in real time with Sybr Green (Thermo fisher scientific) and stopped the reaction before plateauing, at 13 cycles.…”

Transcription imparts architecture, function, and logic to enhancer units

“…Numerous high-throughput sequencing methods identify enhancers using either plasmid or integrated reporter constructs and are collectively known as massively parallel reporter assays (MPRAs). While these assays offer unprecedented throughput for surveying genome function, their technical biases and limitations are a focus of ongoing research and optimization [23][24][25] . For example, most published MPRAs have been limited to short synthetic sequences (50-150 bp), despite the precise size of genomic enhancers remaining unknown 11 .…”

STARR‐seq and UMI‐STARR‐seq: Assessing Enhancer Activities for Genome‐Wide‐, High‐, and Low‐Complexity Candidate Libraries

“…The transfection of plasmid DNA triggers a type I interferon (INF‐I) response in many mammalian and human cell lines via the cGAS/STING pathway (Muerdter et al., ; Paludan & Bowie, ), making it necessary to evaluate the cell line of interest prior to performing STARR‐seq [see Muerdter et al. ()]. An active interferon response would lead to false‐positive and false‐negative enhancer activities (see Background Information for details).…”