Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response [RNA-seq]
The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1? isoform, and while USP43 does not alter HIF-1? stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes.  Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia-dependent manner to increase the nuclear pool of HIF-1, independently of DUB activity. Together, our results unveil the DUB landscape in HIF signalling, and highlight the multifunctionality of DUBs, illustrating that they can provide important signalling roles outside of their catalytic activity. Overall design: To investigate the transcriptional responses of DUB genes in response to hypoxia, we generated RNA-Seq data sets of control and HIF1B KO cells from normoxic and hypoxic conditions. We also included USP43 KO samples for these conditions as this is a hypoxia-inducible DUB and a hit in our genetic screens.