Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response

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, we conduct CRISPR/Cas9 mutagenesis screens using a bespoke DUBs sgRNA library 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-1alpha isoform. While USP43 does not alter HIF-1alpha stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia- and phosphorylation-dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.