p53-mediated regulation of LINE1 retrotransposon-derived R-loops

Long interspersed nuclear element 1 (LINE1/L1) retrotransposons, which comprise 17% of the human genome, typically remain inactive in healthy somatic cells but are reactivated in several cancers. We previously demonstrated that p53 silences L1 transposons in human somatic cells, potentially acting as a tumor-suppressive mechanism. However, the precise molecular mechanisms underlying p53-mediated repression of L1 and its life cycle intermediates remain unclear. In this study, we used DNA-RNA immunoprecipitation-sequencing experiments to investigate RNA-DNA hybrids, which are key intermediates formed during L1 retrotransposition. Our findings reveal that L1 mRNA-genomic DNA (cis L1 R-loops) and L1 mRNA-complementary DNA (trans L1 R-loops) hybrids are upregulated in p53^-/- cells. This increase is synergistic with L1 activation by histone deacetylase (HDAC) inhibitors (HDACi). However, treatment with a reverse transcriptase inhibitor reduces this accumulation, indicating that retrotransposition activity plays a significant role in R-loop accumulation. Interestingly, in WT cells, hyperactivated L1 transposons are suppressed upon HDACi withdrawal. L1 suppression in WT cells coincided with the recruitment of repressive marks, specifically H3K9me3 and H3K27me3, simultaneously preventing the addition of activating marks like H3K4me3, and H3K9ac at the L1 5'UTR. Mechanistically, we demonstrate that p53 cooperates with histone methyltransferases SETDB1 and G9A to deposit H3K9me3 marks at the L1 promoter, thereby silencing transposons. This study is the first to reveal novel roles of p53 in preventing the formation of L1-derived RNA-DNA hybrids (R-loops) and suppression of hyperactivated L1 elements by cooperating with histone methyltransferases, underscoring its critical role in maintaining genomic stability.