The role of non-canonical nucleic acid structures in genome regulation

Nuclear processes such as transcription, replication, and repair disrupt the double stranded nature of DNA to result in the formation of non-canonical structures, the most prevalent being R-loops and G-quadruplexes (G4s). R-loops are three-stranded nucleic acid structures, which occur co-transcriptionally and comprise an RNA-DNA hybrid and a displaced single strand of DNA. The displaced ssDNA can form G4s if it contains spaced stretches of guanine nucleotides. G4 formation can facilitate the formation and stabilization of R-loops. Both R-loops and G4s have important roles in normal cellular function but are also deregulated in several developmental disorders and cancers. Our long-term goal is to elucidate how R-loops and G4 levels are managed in normal cells, how they regulate gene expression and genome organization, and how their misregulation contributes to various diseases. We discovered an enrichment for zinc finger domain containing proteins at R-loops and characterized one such factor, ADNP, to show that ADNP can resolve R-loops in vitro and that its loss results in accumulation of R-loops in vivo. We also identified a role for R-loops and G4s in genome organization by showing that these structures promote CTCF binding genome-wide, providing a new model for how transcription-induced R-loops can alter genome folding and contribute to lineage-specific gene expression programs during development. Through our work on another R-loop regulator, ATRX, we showed that while ATRX cannot resolve R-loops in vitro, its RNA binding activity can prevent R-loops from forming in vitro. In the next award cycle, we propose to build on these studies to: 1) identify a broader role for zinc finger proteins in genome organization through their interactions with R-loops and G4s; 2) determine the extent and mechanisms of ATRX-mediated R-loop regulation genome-wide and the impact on the localization of epigenetic regulators; and 3) elucidate the contribution of R- loops and G4s on the spatial organization of the genome. Project Number: 1R35GM161559-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Kavitha Sarma | Institution: WISTAR INSTITUTE, PHILADELPHIA, PA | Award Amount: $363,191 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MGG-F (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11260496