LINE-1 transposon derived DNA lesions reshape the chromatin landscape to promote genome instability

Abstract Aberrant expression of Long Interspersed Element-1 (LINE-1/L1) retrotransposons is increasingly linked to genomic instability in cancer, particularly in the context of compromised p53 function. The endonuclease (EN) domain of the LINE-1 ORF2 protein (ORF2p) generates DNA strand breaks, yet its role in shaping downstream chromatin and transcriptional responses remains poorly defined. Here, we investigate the impact of ORF2p-EN activity on DNA damage response signalling and epigenomic remodelling in A375 melanoma cells using a L1 expression system. ORF2p-EN led to activation of ATM dependent DNA damage signalling, evidenced by increased γH2AX accumulation. The EN induced DNA damage showed enrichment of yH2AX at fragile hotspot regions, consistent with localized DNA damage. We also observed increase in levels of the DNA repair factor XRCC5, suggesting a possible engagement of non-homologous end joining pathway. EN-induced DNA damage correlated with increased P300 activity and enhanced H3K27ac enrichment at regulatory regions, linking DNA damage signalling with chromatin acetylation. Pharmacological inhibition of ATM attenuated both γH2AX accumulation and H3K27ac levels, supporting a model in which chromatin remodelling occurring downstream of DNA damage signalling. Collectively, our findings position ORF2p endonuclease activity as an initiating source of DNA damage that is followed by chromatin remodelling and transcriptional reprogramming, with p53 acting as a critical modulator of this axis. These results provide mechanistic insight into how LINE-1 activation may contribute to oncogenic gene expression programs in cancer.