NAT10-Dependent N4-acetylcytidine (ac4C) on R-loops Drive Glioblastoma Tumorigenesis and Stemness [RRCHIP]

In this study, we found that glioblastoma stem cells (GSCs) maintain a highly active R-loop state compared to their differentiated progeny and normal human stem cells. Genome-wide mapping via RR-ChIP-seq revealed cell-specific enrichment and spatial accumulation of R-loops at promoter-proximal regions in GSCs, correlating with active transcription and open chromatin states.

To identify key regulators, we profiled the R-loop interactome and identified N-Acetyltransferase 10 (NAT10), an established RNA ac4C-modifying enzyme, as a high-affinity R-loop-binding protein in GSCs. Driven by transcriptional activation via OLIG1, NAT10 is overexpressed in both GSCs and GBM. ac4C RIP-seq on R-loop RNA revealed abundant ac4C-modified R-loops that regulated the GSC genome. Mechanistically, NAT10 catalyzes widespread ac4C deposition on the RNA stand of R-loops, stabilizing promoter-associated R-loops, and facilitating an open chromatin state to sustain GSC self-renewal through controlling key stemness regulators such as the transcription factor EGR1.