Identifying transcription factor-bound activators and silencers in the chromatin accessible human genome using ATAC-STARR-seq

Abstract

Massively parallel reporter assays 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 a multi-omic approach that quantifies 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 design 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. We demonstrate that activators and silencers represent distinct functional groups that are enriched for unique sets of TF motifs and are marked by specific combinations of histone modifications. Using Tn5 cut-sites retained by the ATAC-STARR library, we performed TF footprinting and stratified these groups by the presence of specific TF footprints that are supported by chromatin immunoprecipitation data. We found that activators and silencers clustered by distinct TF footprint combinations are enriched for distinct gene regulatory pathways, and thus, represent distinct gene regulatory networks of human lymphoblastoid cell function. Altogether, these data highlight the multi-facted capabilities of ATAC-STARR-seq to comprehensively investigate the regulatory landscape of the human genome all from a single DNA fragment source.