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.
Highlights
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
The experimental component of High-resolution Dissection of Regulatory Activity (HiDRA) is the combination of ATAC-seq and STARR-seq (i.e., ATAC-STARR-seq): fragments are enriched from open chromatin and regulatory regions using ATAC-seq (Assay for TransposaseAccessible Chromatin with highthroughput sequencing) and subsequently cloned into the 3′ untranslated region (3′ UTR) of a reporter gene on the self-transcribing enhancer reporter vector used in Self-Transcribing Active Regulatory Region sequencing (STARR-seq)[13,16,21]
Our HiDRA library covers 4486 predicted enhancers and 9631 predicted promoters (“Active Transcription Start Site (TSS)” state5,6,14) with more than 10 unique fragments (Fig. 1c, colored lines), a ~130-fold and ~210-fold enrichment compared with 35 enhancer and 46 promoter regions expected to be covered by chance at the same coverage
Summary
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. 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, evolutionarilyconserved nucleotides, and disease-associated genetic variants from genome-wide association studies. We apply HiDRA to infer genome-wide regulatory activity across ~7 million DNA fragments preferentially selected from accessible chromatin in the GM12878 lymphoblastoid cell line, resulting in ~65,000 discrete genomic regions showing significant regulatory function. These are enriched for endogenous active histone marks (including H3K9ac, H3K27ac), regulatory sequence motifs, and regions bound by immune regulators. HiDRA provides a general, scalable, and high-throughput approach for the high-resolution experimental dissection of regulatory regions and driver nucleotides in the context of human biology and disease
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