Pirh2-dependent DNA damage in neurons induced by the G-quadruplex ligand pyridostatin

Noncanonical base pairing between four guanines (G) in single-stranded G-rich sequences forms a G-quartet, which self-stacks to create a four-stranded DNA structure known as the G-quadruplex (G4 or G4-DNA). In cancer cells, G4-DNA plays a role in regulating various DNA-dependent processes, including transcription, replication, and telomere function. However, the function of G4-DNA in neurons remains unclear. In this study, we conducted a genome-wide gene expression analysis (RNA-Seq) to identify genes influenced by a G4-DNA ligand, pyridostatin (PDS), in primary cultured neurons. PDS stabilizes G4 structures, enabling us to determine genes that are directly or indirectly regulated by G4-DNA.

We discovered that out of a total of 18,745 genes with measurable expression, 901 genes showed differential expression in neurons treated with PDS. Among these, 505 genes were downregulated, and 396 genes were upregulated. These genes were involved in networks related to p53 signaling, the immune response, learning and memory, and cellular senescence. Within the p53 network, the E3 ubiquitin ligase Pirh2 (Rchy1), which modulates DNA damage responses, was upregulated by PDS. Overexpressing Pirh2 ectopically led to the formation of DNA double-strand breaks, suggesting a novel DNA damage mechanism in neurons that is controlled by G4 stabilization. Pirh2 also downregulated DDX21, an RNA helicase responsible for unfolding G4-RNA and R-loops. Finally, we demonstrated that Pirh2 increased G4-DNA levels in the nucleolus of neuronal cells. Our findings reveal the genes responsive to PDS treatment and imply that similar transcriptional regulation could occur through endogenous G4-DNA ligands. These results also link G4-dependent regulation of transcription with DNA damage mechanisms in neuronal cells.