Molecular co-accessibility identifies coordinated regulation between distant cis-regulatory elements

In metazoans, gene expression is typically regulated by a cis-regulatory landscape (CRL) composed of a promoter and multiple enhancers. How these cis-regulatory elements (CREs) coordinate their function across large genomic distances remains unclear. For example, is the simultaneous activation of multiple enhancers required to promote transcription? Here, we combined single-molecule footprinting with long-read sequencing to quantify how often chromatin accessibility and transcription factor binding co-occur across entire CRLs in the Drosophila genome. Analysis of thousands of individual DNA molecules at each locus revealed that CREs form a specific network with shared single-molecule chromatin accessibility profiles. Co-accessibility is not limited to adjacent CREs and is frequently observed between CREs brought into proximity by chromatin looping. Co-accessible CREs exhibit strong coordination in their cell-type-specific accessibility, linking enhancer activity with transcriptional activation. Our data uncover dependencies between CREs genome-wide and suggest that coordinated enhancer activation is a widespread mechanism regulating gene expression.