lentiMPRA and MPRAflow for high-throughput functional characterization of gene regulatory elements

Abstract

Massively parallel reporter assays (MPRAs) can simultaneously measure the function of thousands of candidate regulatory sequences (CRSs) in a quantitative manner. In this method, CRSs are cloned upstream of a minimal promoter and reporter gene, alongside a unique barcode, and introduced into cells. If the CRS is a functional regulatory element, it will lead to the transcription of the barcode sequence, which is measured via RNA sequencing and normalized for cellular integration via DNA sequencing of the barcode. This technology has been used to test thousands of sequences and their variants for regulatory activity, to decipher the regulatory code and its evolution, and to develop genetic switches. Lentivirus-based MPRA (lentiMPRA) produces 'in-genome' readouts and enables the use of this technique in hard-totransfect cells. Here, we provide a detailed protocol for lentiMPRA, along with a user-friendly Nextflow-based computational pipeline-MPRAflow-for quantifying CRS activity from different MPRA designs. The lentiMPRA protocol takes~2 months, which includes sequencing turnaround time and data processing with MPRAflow.

Thecis-regulatory effects of modern human-specific variants

“…We amplified this library of sequences, each along with a minimal promoter and barcode. We then inserted these constructs into the lentiMPRA vector, so that the barcode, which is the readout of activity, is located within the 5’UTR of the reporter gene and is transcribed if the assayed sequence is an active regulatory element 32 . We associated each sequence with multiple barcodes to achieve a high number of independent replicates of expression per sequence, thereby reducing potential site-of-integration effects.…”

“…We analyzed variants that likely emerged and reached fixation or near fixation along the modern human lineage (yellow) and that were not polymorphic in any other ape or archaic genome (green) (top). The modern and archaic human variants and their surrounding 200 bp were synthesized, cloned into barcoded expression constructs and infected in triplicates into three human cell lines using a chromosomally integrating vector, following the lentiMPRA protocol 32 (see methods). We compared the activity (RNA/DNA) of the modern and archaic human constructs to identify variants promoting differential expression using MPRAnalyze 40 (bottom).…”

“…2b), with the lower correlation scores being in ESC, likely due to our use of lower multiplicity of infection (MOI) in these cells due to their increased sensitivity to lentivirus infection. High barcode and read coverage in MPRA generally provides increased power to detect differences in allelic expression 32, 45 . Thus, to determine how variability depended on our barcode counts, we downsampled the number of barcodes per sequence and calculated the RNA/DNA ratio at each step for each of the three replicates.…”

“…Thus, MPRA is an effective highthroughput tool to identify variants underlying divergent regulation, especially in organisms where experimental options are limited [28][29][30][31] . Here, we conducted a lentivirus-based MPRA (lentiMPRA 32 ) on the 14,042 fixed or nearly fixed single-nucleotide variants that emerged along the modern human lineage. We generated a library of both the derived (modern human) and ancestral (archaic human and ape) sequences of each locus and expressed them in three human cell types: embryonic stem cells (ESCs), neural progenitor cells (NPCs), and primary fetal osteoblasts.…”

Multinomial Convolutions for Joint Modeling of Sequence Motifs and Enhancer Activities

“…Massively parallel reporter assays (MPRAs) hold great promise in eliciting transcriptional regulatory mechanisms in a biological system of interest [16,54,55] . Modeling MPRA datasets can identify the putative transcription factors (TFs) acting on the assayed sequences, the regulatory roles of these TFs and their relative influence on transcriptional regulation.…”