Abstract
DNA:RNA hybrids play key roles in both physiological and disease states by regulating chromatin and genome organization. Their homeostasis during cell differentiation and cell plasticity remains elusive. Using an isogenic human stem cell platform, we systematically characterize R-loops, DNA methylation, histone modifications, and chromatin accessibility in pluripotent cells and their lineage-differentiated derivatives. We confirm that a portion of R-loops formed co-transcriptionally at pluripotency genes in pluripotent stem cells and at lineage-controlling genes in differentiated lineages. Notably, a subset of R-loops maintained after differentiation are associated with repressive chromatin marks on silent pluripotency genes and undesired lineage genes. Moreover, in reprogrammed pluripotent cells, cell-of-origin-specific R-loops are initially present but are resolved with serial passaging. Our analysis suggests a multifaceted role of R-loops in cell fate determination that may serve as an additional layer of modulation on cell fate memory and cell plasticity.