Hypoxia-mediated regulation of DDX5 through decreased chromatin accessibility and post-translational targeting restricts R-loop accumulation

Local hypoxia (low oxygen) occurs in most solid tumors and is associated with aggressive disease and therapy resistance. Widespread changes in gene expression play a critical role in the biological response to hypoxia. However, most of the prior research has focused on hypoxia-inducible genes in hypoxia as opposed to those which are decreased. Using ATACseq, we demonstrate that chromatin accessibility is decreased in an oxygen-dependent manner, predominantly at gene promoters and specific pathways are impacted including DNA repair, splicing and the R-loop interactome. As R-loops accumulate in hypoxic conditions we hypothesized that an underlying mechanism could be the repression of the R-loop interactome. One of the genes with decreased chromatin accessibility in hypoxia was DDX5, encoding the RNA helicase, DDX5, which showed reduced expression in various cancer cell lines in hypoxic conditions, tumor xenografts and in patient samples with hypoxic tumors. In addition, we identified TRIM5 as a novel hypoxia inducible E3 ligase which targets DDX5 for proteasomal degradation independently of changes at mRNA levels. We demonstrate multiple mechanisms contributing to reduced DDX5 expression in hypoxic conditions. Most interestingly, we found that when DDX5 is rescued in hypoxia, R-loop levels accumulate further, therefore demonstrating that hypoxia-mediated repression of DDX5 restricts R-loop accumulation. Together these data support the hypothesis that a critical part of the biological response to hypoxia is the repression of multiple R-loop processing factors, however, as shown for DDX5, their role is specific and distinct.