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

The National Institute of General Medical Sciences awarded The Wistar Institute of Anatomy and Biology $363,191 on April 1, 2026, under the Biomedical Research and Research Training program (CFDA 93.859) to conduct research on the role of non-canonical nucleic acid structures in genome regulation.

The funded work investigates how non-canonical DNA structures—specifically R-loops (three-stranded RNA-DNA hybrid structures formed during transcription) and G-quadruplexes (four-stranded guanine-rich structures)—regulate gene expression and genome organization in normal cells and contribute to disease when dysregulated. The research builds on prior discoveries identifying zinc finger domain-containing proteins that interact with R-loops and G-quadruplexes, including the protein ADNP, which resolves R-loops in vitro, and ATRX, which prevents R-loop formation. The upcoming award cycle will expand this work to identify broader roles for zinc finger proteins in genome organization through their R-loop and G-quadruplex interactions, determine genome-wide mechanisms of ATRX-mediated R-loop regulation and its impact on cellular localization, and elucidate how transcription-induced R-loops alter genome folding to drive lineage-specific gene expression during development.

Performance occurs in Philadelphia, Pennsylvania. The award runs through January 31, 2031.

Abstract

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.