Targeted Nuclease Strategies for Mapping R-Loop Chromatin Structures.

R-loops are three stranded nucleic acid structures with essential roles in many nuclear processes. However, their unchecked accumulation as seen in some neurodevelopmental diseases and cancers and is associated with compromised genome stability. Genome-wide profiling of R-loops in normal cells and their comparison in disease states can help identify precise locations of pathogenic R-loops and advance efforts to attenuate deviant R-loops while preserving biologically important ones. Toward this, we have developed an antibody-independent R-loop detection strategy, BisMapR, that combines nuclease-based R-loop isolation with non-denaturing bisulfite chemistry to produce high-resolution, genome-wide R-loop profiles that retain strand information. Furthermore, BisMapR achieves greater resolution and is faster than existing strand-specific R-loop profiling strategies. We applied BisMapR to reveal discrete R-loop behavior at gene promoters and enhancers. We show that gene promoters exhibiting antisense transcription form R-loops in both directions. and uncover a subset of active enhancers that, despite being bidirectionally transcribed, form R-loops exclusively on one strand. Thus, BisMapR reveals a previously unnoticed feature of active enhancers and provides a tool to systematically examine their mechanisms in gene expression.