Deubiquitinating enzyme mutagenesis screens identify a USP43 driven HIF-1 transcriptional response [CRISPR]

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: explore the contribution of DUBs to HIF signalling using CRISPR/Cas9 mutagenesis screens

缺氧诱导因子（Hypoxia Inducible Factors, HIFs）的泛素化及蛋白酶体介导降解是后生动物氧感知通路的核心事件，但去泛素化酶（deubiquitinating enzymes, DUBs）在HIF信号通路中的调控作用仍不甚明确。本研究采用定制化DUBs单引导RNA（single guide RNA, sgRNA）文库开展CRISPR/Cas9诱变筛选，以解析DUBs在HIF信号通路中的参与方式。在明确参与HIF激活或抑制的DUBs的同时，本研究鉴定出USP43是高效激活HIF应答所必需的一种DUB。缺氧调控的USP43可选择性结合HIF-1α亚型；尽管USP43不会改变HIF-1α的稳定性，但它能促进HIF-1α的核聚集及其与靶基因的结合。从机制层面来看，USP43以缺氧依赖的方式与14-3-3蛋白结合，从而在不依赖DUB催化活性的情况下增加HIF-1的核内储备。综上，本研究揭示了HIF信号通路中的DUB调控网络，并凸显了DUBs的多功能性，表明其可在催化活性之外发挥重要的信号调控功能。研究整体设计：通过CRISPR/Cas9诱变筛选探究DUBs对HIF信号通路的调控贡献。