Biology subjects

Wit, N.

Publications and source records attributed to Wit, N..

7 recordsLinked to original sources

bioRxiv2026

Regulation of proteasome activation by a ubiquitin-independent feedback mechanism

The ubiquitin-proteasome system is the major pathway for selective protein degradation in eukaryotic cells. Some proteins are degraded by the proteasome without ubiquitination, but the prevalence and underlying mechanisms remain poorly understood. Here, we use pulsed-SILAC proteomics to systematically identify proteins undergoing ubiquitin-independent proteasomal degradation (UbInPD). We identify the paralogous proteasome activation factors ZFAND5 and ZFAND6 (ZFAND5/6) and show that ZFAND5 contains a ubiquitin-independent degron that can both promote its rapid turnover and allosterically activate the proteasome. These findings support a model in which proteasome activation and activator degradation are coupled through a ubiquitin-independent feedback mechanism. We further show that ZFAND5/6, together with the scaffold protein p62, restrain NF-{kappa}B activation in the TLR4 pathway. We link this regulation to the degradation of UBCH5c, an abundant E2 ubiquitin-conjugating enzyme that can initiate or prime ubiquitin chain synthesis. Our findings expand the known landscape of UbInPD and reveal unexpected links between proteasome activation, E2 enzyme regulation, and inflammatory signalling.

biochemistry↗

bioRxiv2025

Mapping the genetic landscape of iron metabolism uncovers the SETD2 methyltransferase as a modulator of iron flux.

Cellular iron levels must be tightly regulated to ensure sufficient iron for essential enzymatic functions whilst avoiding the harmful generation of toxic species. Here, to better understand how iron levels are controlled, we carry out genome-wide mutagenesis screens in human cells. Alongside mapping known components of iron-sensing, we determine the relative contributions of iron uptake, iron recycling, ferritin breakdown, and mitochondrial flux in controlling the labile iron pool. We also identify SETD2, a histone methyltransferase, as a chromatin modifying enzyme that controls intracellular iron availability through ferritin breakdown. Functionally, we show that SETD2 inhibition or cancer-associated SETD2 mutations render cells iron deficient, thereby driving resistance to ferroptosis, and potentially explaining how some tumours evade anti-tumoural immunity.

cell biology↗

bioRxiv2024

VHL synthetic lethality screens uncover CBF-beta as a negative regulator of STING

Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor {beta} (CBF-{beta}) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-{beta}. Mechanistically, CBF-{beta} loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-{beta} at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-{beta} in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling.

cancer biology↗

bioRxiv2024

Hypoxia promotes airway differentiation in the human lung epithelium

Human early embryos develop under physiological hypoxia, but how hypoxia regulates human organogenesis remains little known. We have investigated oxygen availability effects on the human lung epithelium using organoids. We find first-trimester lung epithelial progenitors remain undifferentiated under normoxia, but spontaneously differentiate towards multiple airway cell types and inhibit alveolar differentiation under hypoxia. Using chemical and genetic tools, we demonstrate that hypoxia-induced airway differentiation is dependent on HIF (Hypoxia-Inducible Factor) pathways, with HIF1 and HIF2 differentially regulating fate decisions. Transcription factors KLF4 and KLF5 are direct targets of the HIF pathway and promote progenitor differentiation to basal and secretory cells. Chronic hypoxia also induces transdifferentiation of human alveolar type 2 cells to airway cells via the HIF pathway, mimicking alveolar bronchiolization processes in lung disease. Our results reveal roles for hypoxia and HIF signalling during human lung development and have implications for aberrant cell fate decisions in chronic lung diseases.

developmental biology↗

bioRxiv2024

An essential role for RNA binding by Periphilin in silencing by the HUSH complex

The human silencing hub (HUSH) complex is a transcription-dependent, epigenetic repressor complex that provides a genome-wide immunosurveillance system for the recognition and silencing of newly-integrated retroelements. The core HUSH complex of TASOR, MPP8 and Periphilin, represses these retroelements through SETDB1-mediated H3K9me3 deposition and MORC2-dependent chromatin compaction. HUSH-dependent silencing is RNA-mediated, yet no HUSH components contain any RNA-binding domain. Here we used an unbiased approach to identify which HUSH component was able to bind RNA and determine whether RNA-binding was essential for HUSH function. We identify Periphilin as the major RNA-binding component of the HUSH complex and show that Periphilins N-terminal domain is essential for both RNA binding and HUSH function. Periphilin binding to RNA was independent of its interaction with TASOR or MPP8, as its N-terminal domain was sufficient for RNA targeting. The artificial tethering of Periphilin to a HUSH-insensitive, nascent transcript, enabled the HUSH-dependent silencing of the transcript. This tethering of Periphilin allowed the RNA-binding region of Periphilin to be removed such that only its C-terminal domain was required for oligomerisation and interaction with TASOR. We therefore show that Periphilin is the predominant RNA-binding protein of the HUSH complex and this RNA-binding is essential for HUSH activity.

genetics↗

bioRxiv2024

Deubiquitinating enzyme mutagenesis screens identify a USP43 driven 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, 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 and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results unveil the DUB landscape in HIF signalling, and highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.

cell biology↗

bioRxiv2021

Multiple 9-1-1 complexes promote homolog synapsis, DSB repair, and ATR signaling during mammalian meiosis

DNA damage response mechanisms have meiotic roles that ensure successful gamete formation. While completion of meiotic double-strand break (DSB) repair requires the canonical RAD9A-RAD1-HUS1 (9A-1-1) complex, mammalian meiocytes also express RAD9A and HUS1 paralogs, RAD9B and HUS1B, predicted to form alternative 9-1-1 complexes. The RAD1 subunit is shared by all predicted 9-1-1 complexes and localizes to meiotic chromosomes even in the absence of HUS1 and RAD9A. Here we report that testis-specific RAD1 disruption resulted in impaired DSB repair, germ cell depletion and infertility. Unlike Hus1 or Rad9a disruption, Rad1 loss also caused defects in homolog synapsis, ATR signaling and meiotic sex chromosome inactivation. Comprehensive testis phosphoproteomics revealed that RAD1 and ATR coordinately regulate numerous proteins involved in DSB repair, meiotic silencing, synaptonemal complex formation, and cohesion. Together, these results establish critical roles for both canonical and alternative 9-1-1 complexes in meiotic ATR activation and successful prophase I completion.

genetics↗