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

Abstract

Genome-wide epigenomic maps have revealed millions of putative enhancers and promoters, but experimental validation of their function and high-resolution dissection of their driver nucleotides remain limited. Here, we present HiDRA (High-resolution Dissection of Regulatory Activity), a combined experimental and computational method for high-resolution genome-wide testing and dissection of putative regulatory regions. We test ~7 million accessible DNA fragments in a single experiment, by coupling accessible chromatin extraction with self-transcribing episomal reporters (ATAC-STARR-seq). By design, fragments are highly overlapping in densely-sampled accessible regions, enabling us to pinpoint driver regulatory nucleotides by exploiting differences in activity between partially-overlapping fragments using a machine learning model (SHARPR-RE). In GM12878 lymphoblastoid cells, we find ~65,000 regions showing enhancer function, and pinpoint ~13,000 high-resolution driver elements. These are enriched for regulatory motifs, evolutionarily-conserved nucleotides, and disease-associated genetic variants from genome-wide association studies. Overall, HiDRA provides a high-throughput, high-resolution approach for dissecting regulatory regions and driver nucleotides.

Transcription imparts architecture, function, and logic to enhancer units

“…If transcription is spurious and unimportant to enhancer function, core promoter sequences should be dispensable. To answer this question, we re-analyzed the "Highresolution Dissection of Regulatory Activity" (HiDRA) dataset 37 , which uses the STARR-seq assay on Analysis of Transposase-Accessible Chromatin (ATAC-seq) fragments. This impressively comprehensive dataset quantifies enhancer activity from 100-600 bp fragments enriched within DHSs, thus dissecting potential enhancer elements genome-wide.…”

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

“…Additionally, MPRAs test sequences cloned in one of two possible orientations, effectively assuming that enhancer activity is independent of orientation. Finally, while larger sheared genomic DNA fragments 12 , PCR amplicons 15 or captured sequences 21,22 have been used in MPRAs, most studies using MPRAs synthesize libraries of putative enhancers on microarrays, and are therefore limited to testing shorter sequences (typically less than 200 bp).…”

Resolving mechanisms of immune-mediated disease in primary CD4 T cells

“…To assess for enhancer activity within each disease-associated locus, we used the sharpr2 package 68 in R (version 3.5.1). This was used because: (1) the tiled regions were of different sizes, (2) the offset between constructs (50bp) was not a factor of the length of genomic sequence (114bp), (3) this method facilitated inclusion of the reference allele constructs from SNPs to improve coverage within the locus (since these constructs also contained the reference genomic sequence at the sites of SNPs), and (4) none of the co-ordinates of the regions on their respective chromosomes overlapped.…”