Tyler J. Hansen1 and Emily Hodges1,2 1Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA; 2Vanderbilt Genetics Institute, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA Corresponding author: emily.hodgesvanderbilt.edu Abstract

Massively parallel reporter assays (MPRAs) test the capacity of putative gene regulatory elements to drive transcription on a genome-wide scale. Most gene regulatory activity occurs within accessible chromatin, and recently described methods have combined assays that capture these regions—such as assay for transposase-accessible chromatin using sequencing (ATAC-seq)—with self-transcribing active regulatory region sequencing (STARR-seq) to selectively assay the regulatory potential of accessible DNA (ATAC-STARR-seq). Here, we report an integrated approach that quantifies activating and silencing regulatory activity, chromatin accessibility, and transcription factor (TF) occupancy with one assay using ATAC-STARR-seq. Our strategy, including important updates to the ATAC-STARR-seq assay and workflow, enabled high-resolution testing of ∼50 million unique DNA fragments tiling ∼101,000 accessible chromatin regions in human lymphoblastoid cells. We discovered that 30% of all accessible regions contain an activator, a silencer, or both. Although few MPRA studies have explored silencing activity, we demonstrate that silencers occur at similar frequencies to activators, and they represent a distinct functional group enriched for unique TF motifs and repressive histone modifications. We further show that Tn5 cut-site frequencies are retained in the ATAC-STARR plasmid library compared to standard ATAC-seq, enabling TF occupancy to be ascertained from ATAC-STARR data. With this approach, we found that activators and silencers cluster by distinct TF footprint combinations, and these groups of activity represent different gene regulatory networks of immune cell function. Altogether, these data highlight the multilayered capabilities of ATAC-STARR-seq to comprehensively investigate the regulatory landscape of the human genome all from a single DNA fragment source.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.276766.122.

Freely available online through the Genome Research Open Access option.

Received March 25, 2022. Accepted July 11, 2022.