Jason Ernst, Alexandre Melnikov, Xiaolan Zhang, Li Wang, Peter Rogov et al.
Massively parallel reporter assays (MPRA) enable nucleotide-resolution dissection of transcriptional regulatory regions, such as enhancers, but only few regions at a time. Here, we present a combined experimental and computational approach, Sharpr-MPRA, that allows high-resolution analysis of thousands of regions simultaneously. Sharpr-MPRA combines dense tiling of overlapping MPRA constructs with a probabilistic graphical model to recognize functional regulatory nucleotides, and to distinguish activating and repressive nucleotides, using their inferred contribution to reporter gene expression. We use Sharpr-MPRA to test 4.6 million nucleotides spanning 15,000 putative regulatory regions tiled at 5-nucleotide resolution in two human cell types. Our results recover known cell type-specific regulatory motifs and evolutionarily-conserved nucleotides, and distinguish known activating and repressive motifs. Our results also show that endogenous chromatin state and DNA accessibility are both predictive of regulatory function in reporter assays, identify retroviral elements with activating roles, and uncover ‘attenuator’ motifs with repressive roles in active chromatin.
Jason Ernst, Alexandre Melnikov, Xiaolan Zhang, Li Wang, Peter Rogov, Tarjei S. Mikkelsen, Manolis Kellis. Genome-scale high-resolution mapping of activating and repressive nucleotides in regulatory regions. 2016-10-03. https://doi.org/10.1038/nbt.3678
The minimal TATA promoter scale-up MPRA was assayed in K562 across two 244K array designs and two biological replicates. Each of 15,720 selected 295-bp regions was tiled with overlapping 145-bp reporter constructs at 5-bp offsets; this table contains the authors' inferred base-resolution minP scores.
The SV40 promoter scale-up MPRA was assayed in HepG2 across two 244K array designs and two biological replicates. Each of 15,720 selected 295-bp regions was tiled with overlapping 145-bp reporter constructs at 5-bp offsets; this table contains the authors' inferred base-resolution SV40P scores.
The SV40 promoter scale-up MPRA was assayed in K562 across two 244K array designs and two biological replicates. Each of 15,720 selected 295-bp regions was tiled with overlapping 145-bp reporter constructs at 5-bp offsets; this table contains the authors' inferred base-resolution SV40P scores.
The minimal TATA promoter scale-up MPRA was assayed in HepG2 across two 244K array designs and two biological replicates. Each of 15,720 selected 295-bp regions was tiled with overlapping 145-bp reporter constructs at 5-bp offsets; this table contains the authors' inferred base-resolution minP scores.
The published combinedP SHARPR-MPRA output for HepG2 combines the minP and SV40P scale-up reporter measurements from two 244K array designs and two biological replicates per promoter. The table contains inferred activity at every base of the 295-bp regions that passed package-level data-availability QC.
A low-resolution episomal MPRA tested 250 H3K27ac-marked candidate regulatory regions in K562. Each 385-bp region was represented by nine overlapping 145-bp constructs spaced at 30-bp offsets, with 24 independent 10-nt barcodes per tile and two biological transfection replicates.
A low-resolution episomal MPRA tested 250 H3K27ac-marked candidate regulatory regions in HepG2. Each 385-bp region was represented by nine overlapping 145-bp constructs spaced at 30-bp offsets, with 24 independent 10-nt barcodes per tile and two biological transfection replicates.
The published combinedP SHARPR-MPRA output for K562 combines the minP and SV40P scale-up reporter measurements from two 244K array designs and two biological replicates per promoter. The table contains inferred activity at every base of the 295-bp regions that passed package-level data-availability QC.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 7 files (ZIP)GSE71279_family.soft.gzHEPG2_SHARPR-MPRA_scores.zipK562_SHARPR-MPRA_scores.zipPilot_counts_sequences.zipPilot_normalized.zipScaleup_counts_sequences.zipsource_manifest.txt