High-resolution genome-wide functional dissection of transcriptional regulatory regions in human

Abstract

Abstractselected from accessible chromatin in the GM12878 lymphoblastoid cell line. By design, accessibilityselected fragments were highly overlapping (up to 370 per region), enabling us to pinpoint driver regulatory nucleotides by exploiting subtle differences in reporter activity between partially-overlapping fragments, using a new machine learning model SHARPR2. Our resulting maps include ~65,000 regions showing significant enhancer function and enriched for endogenous active histone marks (including H3K9ac, H3K27ac), regulatory sequence motifs, and regions bound by immune regulators. Within them, we discover ~13,000 high-resolution driver elements enriched for regulatory motifs and evolutionarily-conserved nucleotides, and help predict causal genetic variants underlying disease from genome-wide association studies. Overall, HiDRA provides a general, scalable, high-throughput, and high-resolution approach for experimental dissection of regulatory regions and driver nucleotides in the context of human biology and disease.peer-reviewed)

Dissecting the regulatory activity and sequence content of loci with exceptional numbers of transcription factor associations

“…This association was significantly stronger in loci proximal (<5kb) to transcription start sites than in loci located more distally. Both ChIP-seq and DHS motif-defined ( Figure 2D) associations correlated strongly with activity in previous high-throughput reporter assays of ~2000 selected loci in HepG2 and in ATAC-seq fragments in GM12878 ( Figure 2E) (Inoue et al 2017;Wang et al 2018). For both reporter assay datasets, the number of DAP associations represents a specific, quantitative marker of regulatory activity that compares favorably to commonly-used markers of promoter or enhancer activity ( Figure 2D-E).…”

“…GM12878 High Resolution Dissection of Regulatory Assay (HiDRA) data was obtained from previously published work hosted at the GEO accession GSE104001 in the file GSE104001_HiDRA_counts_per_fragmentgroup.txt (Wang et al 2018 (Mifsud et al 2015). We used the TS5_GM12878_promoter-other_significant_interactions.txt file for analysis.…”

High throughput characterization of genetic effects on DNA:protein binding and gene transcription

“…Recently, high-throughput assays have been used to assess the enhancer function of genomic regions [18,19], the allelic effects on gene expression for naturally occurring variation in 104 regulatory regions [20], fine-map variants associated with gene expression in lymphoblastoid cell lines (LCLs) and HepG2 [21], and fine-map variants associated with red blood cell traits in GWAS [22]. In addition to using reporter assays to measure enhancer function on gene expression, there are several methods to directly measure binding affinity of DNA sequences for specific transcription factors.…”

“…Our protocol also has the advantage of being highly streamlined. Unlike STARR-seq, our method does not require preparation of DNA regions for use in the assay, such as whole genome fragmentation [18], or targeting regions [19,27], while, similar to STARR-seq, it requires only a single cloning and transformation step. Because the UMIs are inserted after transfection, there are no additional bottleneck issues (due to library complexity) in the cloning and transformation steps.…”

Identification of essential regulatory elements in the human genome

“…Genome-wide epigenomic maps have revealed hundreds of thousands regions showing signatures of enhancers, promoters, and other gene-regulatory elements 9 . However, the highresolution dissection of driver nucleotides as well as the characteristics of essentiality of those regions remain limited at present 10 . There are multiple sources of data (biochemical, genetic, evolutionary) that convey functional information on the non-coding genome 11 .…”