INO80 regulates promoter-associated R-loops to coordinate transcription and maintain genome stability in embryonic stem cells

Embryonic stem cells (ESCs) possess a distinctive cell cycle structure and chromatin landscape that necessitate precise coordination between transcription and DNA replication. The chromatin remodeler INO80 regulates both transcription and genome stability; however, its role in R-loop homeostasis in ESCs remains incompletely understood. R-loops, which are formed by RNA:DNA hybrids, play regulatory roles but can also induce replication stress and genomic instability if unregulated. We investigated the effects of INO80 loss on R-loop dynamics, replication stress responses, and transcriptional regulation in mouse ESCs. INO80-deficient cells exhibited a global reduction in steady-state R-loops under normal conditions, with the most pronounced loss at promoters enriched for pluripotency-associated transcription factors. R-loop levels were restored under hydroxyurea-induced replication stress, indicating that INO80 is dispensable for stress-induced R-loop accumulation but is required for maintaining physiological R-loops. Cleavage Under Targets and Tagmentation and chromatin immunoprecipitation sequencing revealed that several R-loop regions overlap with INO80-binding sites and are linked to transcriptional activation and genome maintenance. Transcriptome analysis showed that INO80-stabilized R-loops are associated with genes involved in cell cycle progression, DNA repair, and chromatin organization, whereas INO80 loss results in altered transcriptional programs, G1 arrest, and increased apoptosis. These findings indicate that INO80 stabilizes functional R-loops that support transcriptional regulation and genomic integrity. Our study identifies a previously unrecognized role for INO80 in preserving functional R-loops in ESCs. By stabilizing promoter-associated R-loops and coordinating transcription during cell cycle progression, INO80 maintains both transcriptional regulation and genome stability. These findings underscore the importance of chromatin remodeling in maintaining R-loop homeostasis and suggest that INO80 dysfunction may have implications for stem cell identity and genomic integrity.