HPV induced R-loop formation represses innate immune gene expression while activating DNA damage repair pathways [gH2AX ChIP seq]

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-b, 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 gH2AX 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 gH2AX/H3K36me3 histone marks that may contribute to regulating important functions for HPV pathogenesis Overall design: Formaldehyde was added to 1 × 107 cells to a final concentration of 1% for 10 min at room temperature. Excess formaldehyde was quenched upon adding 0.125M glycine before then washing samples with PBS. Cells were then incubated in collection buffer (0.1M TrisHCl pH 9.4 and 10mM DTT containing Roche Protease Inhibitor Cocktail) for 10min on ice. Cells were then collected and spun down before being sequentially washed and incubated with NCP1 (10mM EDTA, 0.5mM EGTA, 10mM HEPES pH 6.5, 0.25% Triton X100) and NCP2 (1mM EDTA, 0.5mM EGTA, 10mM HEPES, and 200mM NaCl) before being lysed in 0.5% Empigen BB, 1% SDS, 10mM EDTA, 50mM Tris HCl pH 8.0 containing Roche Protease Inhibitor Cocktail for 30 min on ice. Samples were then sonicated using a Bioruptor (Diagenode) on high power, 30 s on/90 s off cycles for 20 min. After sonication, Input DNA was removed before loading the samples into preblocked magnetic beads in IP buffer containing 2 ug of the gH2AX antibody. Immunoprecipitations were allowed to incubate overnight at 4 °C while rotating. The next day, samples were washed 8 times with RIPA buffer for 5 min while rotating. One wash in TE buffer was performed before samples were eluted for 10 min at 65 °C in 10% sodium dodecyl sulfate (SDS), 10 mM Tris pH 7.4, 50 mM ethylenediaminetetraacetic acid (EDTA). DNA was purified from these elutions using a PCR purification kit (Qiagen) and stored at -20 °C.