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P53-dependent R-loop formation and HPV pathogenesis

ABSTRACT: R-loops are trimeric RNA: DNA hybrids that are important physiological regulators of transcription; however, their aberrant formation or turnover leads to genomic instability and DNA breaks. High-risk human papillomaviruses are the causative agents of genital as well as oropharyngeal cancers and exhibit enhanced amounts of DNA breaks. The levels of R-loops were found to be increased up to 50-fold in cells that maintain high-risk HPV genomes and were readily detected in squamous cell cervical carcinomas in vivo but not in normal cells. The high levels of R-loops in HPV positive cells were present on both viral and cellular sites together with RNase H1, an enzyme that controls their resolution. Depletion of RNase H1 in HPV positive cells further increased R-loop levels, resulting in impaired viral transcription and replication and reduced expression of DNA repair genes such as FANCD2 and ATR, which are necessary for viral functions. Overexpression of RNase H1 decreased total R-loop levels, reducing DNA breaks by over 50%. Furthermore, increased RNase H1 expression leads to reduced R-loop levels, blocking viral transcription and replication while enhancing the expression of factors in the innate immune regulatory pathway. This suggests that maintaining elevated R-loop levels is important for the HPV life cycle. The E6 viral oncoprotein was found to be responsible for inducing high levels of R-loops by inhibiting p53’s transcriptional activity. Our studies indicate that high R-loop levels are critical for HPV pathogenesis and that this depends on suppressing the p53 pathway.

ORGANISM(S): Homo sapiens

PROVIDER: GSE240391 | GEO | 2023/08/08

REPOSITORIES: GEO

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Project description:Strong cellular stress causes wide-spread perturbations in the transcriptome and in several types of DNA/RNA interactions, and through incompletely understood pathways to formation of cytoplasmic stress granules (SGs). We have investigated the relationships between strong transcriptional induction, RNA:DNA hybrid stretches (R-loops), and SG formation under severe hyperosmotic and glucose stress. Several mutations affecting DNA processing proteins, including the DNA polymerase subunit Pol32, confer SG formation defects. Severe stress increased R loop levels globally. We found that facilitating removal of R-loops by RNase H overexpression, with activity towards RNA:DNA hybrids, accelerated and enhanced transcriptional induction of stress-activated genes. Thus, overexpression of RNase H1, but not RNase H2, reduced R-loops globally around the 1 h mark. Remarkably, it also reduced SG formation. We performed a genome-wide analysis of the induction or repression kinetics of gene expression under severe stress conditions. RNase H1 overexpression reduced R-loops locally in highly transcribed stress-affected genes, as expected. Notably, it also increased expression of several stress-induced genes. Conversely, in cells where R-loops are not efficiently resolved, transcriptional induction of the same genes under stress was muted and occurred with a delay. Thus, in pol32∆ mutants, where SG accumulation is delayed, R-loop levels are elevated, and induction of stress genes suppressed. The pol32∆ mutants are also refractory to the effects of RNase H1 overexpression on stress gene induction, R-loop resolution, and SG formation, indicating that Pol32 may act downstream of Rnh1 in the regulation of these three processes. These findings demonstrate an unexpected link between R-loops and formation of SGs. Together, these observations indicate that under stress, strong transcriptional induction of specific genes or genomic regions causes R-loop accumulation, which then requires RNase H1 activity for resolution. If unresolved, the accumulated R-loops impede continued stress-induced transcription, and delay or prevent SG formation.

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P53-dependent R-loop formation and HPV pathogenesis

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