HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways

by Conor W. Templeton, Laimonis A. Laimins

R-loops are trimeric nucleic acid structures that form when an RNA molecule hybridizes with its complementary DNA strand, displacing the opposite strand. These structures regulate transcription as well as replication, but aberrant R-loops can form, leading to DNA breaks and genomic instability if unresolved. R-loop levels are elevated in many cancers as well as cells that maintain high-risk human papillomaviruses. We investigated how the distribution as well as function of R-loops changed between normal keratinocytes and HPV positive cells derived from a precancerous lesion of the cervix (CIN I). The levels of R-loops associated with cellular genes were found to be up to 10-fold higher in HPV positive cells than in normal keratinocytes while increases at ALU1 elements increased by up to 500-fold. The presence of enhanced R-loops resulted in altered levels of gene transcription, with equal numbers increased as decreased. While no uniform global effects on transcription due to the enhanced levels of R-loops were detected, genes in several pathways were coordinately increased or decreased in expression only in the HPV positive cells. This included the downregulation of genes in the innate immune pathway, such as DDX58, IL-6, STAT1, IFN-β, and NLRP3. All differentially expressed innate immune genes dependent on R-loops were also associated with H3K36me3 modified histones. Genes that were upregulated by the presence of R-loops in HPV positive cells included those in the DNA damage repair such as ATM, ATRX, and members of the Fanconi Anemia pathway. These genes exhibited a linkage between R-loops and H3K36me3 as well as γH2AX histone marks only in HPV positive cells. These studies identify a potential link in HPV positive cells between DNA damage repair as well as innate immune regulatory pathways with R-loops and γH2AX/H3K36me3 histone marks that may contribute to regulating important functions for HPV pathogenesis.