Biology subjects

Dellaire, G.

Publications and source records attributed to Dellaire, G..

11 recordsLinked to original sources

bioRxiv2025

Epigenetic Control of TERRA by FTSJ3 is Critical for Telomerase-Driven Cancers

Telomerase reverse transcriptase (hTERT) overexpression, a hallmark of most cancers, drives tumorigenesis by enabling limitless replicative potential. Direct targeting of hTERT is challenging, necessitating alternative strategies. Through genome-wide synthetic dosage lethality (SDL) screening in cancer models, including patient-derived organoids, we identify FTSJ3, an RNA 2-O-methyltransferase, as a critical vulnerability in hTERT-overexpressing cells. FTSJ3 methylates telomeric repeat-containing RNA (TERRA), a modification essential for recruiting SUV39H1 to telomeric ends to mediate H3K9 trimethylation and establish stable heterochromatin. Loss of FTSJ3 disrupts this cascade, impairing H3K9 trimethylation, HP1-alpha recruitment, and telomeric heterochromatin maintenance. Notably, this reveals an unexpected dependency on TERRA methylation for telomeric heterochromatin stability in hTERT-driven cancers. Non-malignant cells, lacking telomerase activity and de novo telomere repeat synthesis, are unaffected by FTSJ3 suppression. Our findings establish the FTSJ3/TERRA/SUV39H1 axis as a critical mechanism supporting telomeric heterochromatin stability in hTERT-driven cancers. This telomere-directed epigenetic strategy provides a robust framework for translational therapeutic innovation.

cancer biology↗

bioRxiv2025

Telomeric SUMO level influences the choices of APB formation pathways and ALT efficiency

Many cancers use an alternative lengthening of telomeres (ALT) pathway for telomere maintenance. ALT telomeric DNA synthesis occurs in ALT telomere-associated PML bodies (APBs). However, the mechanisms by which APBs form are not well understood. Here, we monitored the formation of APBs with time-lapse imaging employing CRISPR knock-in to track the promyelocytic leukemia (PML) protein at endogenous levels. We found APBs form via two pathways: telomeres recruit PML proteins to nucleate PML bodies de novo, or telomeres fuse with preformed PML bodies. Both nucleation and fusion of APBs require interactions between SUMO and SUMO interaction motifs (SIMs). Moreover, APB nucleation is associated with higher levels of SUMOs and SUMO-mediated recruitment of DNA helicase BLM, resulting in more robust telomeric DNA synthesis. Finally, further boosting SUMO levels at telomeres enhances APB nucleation, BLM enrichment, and telomeric DNA synthesis. Thus, high SUMO levels at telomeres promote APB formation via nucleation, resulting in stronger ALT activity.

cell biology↗

bioRxiv2024

The evolutionarily conserved PRP4K-CHMP4B/vps32 splicing circuit regulates autophagy

The pre-mRNA processing factor 4 kinase (PRP4K) is an essential gene in animal cells, making interrogation of its function challenging. Here, we report the first knockout model for PRP4K in the social amoeba Dictyostelium discoideum, revealing a new function in splicing events controlling autophagy. When prp4k knockout amoebae underwent multicellular development, we observed defects in differentiation linked to abnormal autophagy and aberrant secretion of stalk cell inducer c-di-GMP. Autophagosome-lysosome fusion was found to be impaired after PRP4K loss in both human cell lines and amoebae. Mechanistically, PRP4K loss results in mis-splicing and reduced expression of the ESCRT-III gene CHMP4B in human cells and its ortholog vps32 in Dictyostelium, and re-expression of CHMP4B or Vps32 cDNA (respectively) restored normal autophagosome-lysosome fusion in PRP4K-deficient cells. Thus, our work reveals a novel PRP4K-CHMP4B/vps32 splicing circuit regulating autophagy that is conserved over at least 600 million years of evolution.

cell biology↗

bioRxiv2024

SUMO Promotes DNA Repair Protein Collaboration to Support Alterative Telomere Lengthening in the Absence of PML

Alternative lengthening of telomeres (ALT) pathway maintains telomeres in a significant fraction of cancers associated with poor clinical outcomes. A better understanding of ALT mechanisms can provide a basis for developing new treatment strategies for ALT cancers. SUMO modification of telomere proteins plays a critical role in the formation of ALT telomere-associated PML bodies (APBs), where telomeres are clustered and DNA repair proteins are enriched to promote homology-directed telomere DNA synthesis in ALT. However, whether and how SUMO contributes to ALT beyond APB formation remains elusive. Here, we report that SUMO promotes collaboration among DNA repair proteins to achieve APB-independent telomere maintenance. By using ALT cancer cells with PML protein knocked out and thus devoid of APBs, we show that sumoylation is required for manifesting ALT features, including telomere clustering and telomeric DNA synthesis, independent of PML and APBs. Further, small molecule-induced telomere targeting of SUMO produces signatures of phase separation and ALT features in PML null cells in a manner depending on both sumoylation and SUMO interaction with SUMO interaction motifs (SIMs). Mechanistically, SUMO-induced effects are linked to the enrichment of DNA repair proteins, including Rad52, Rad51AP1, and BLM, to the SUMO-containing telomere foci. Finally, we find that Rad52 can undergo phase separation, enrich SUMO on telomeres, and promote telomere DNA synthesis in collaboration with the BLM helicase in a SUMO-dependent manner. Collectively, our findings suggest that, in addition to forming APBs, SUMO also promotes collaboration among DNA repair proteins to support telomere maintenance in ALT cells. Given the promising effects of sumoylation inhibitors in cancer treatment, our findings suggest their potential use in perturbing telomere maintenance in ALT cancer cells.

cell biology↗

bioRxiv2023

Exploring the interactome of PML nuclear subdomains during fatty acid stress using APEX2-mediated proximity labeling

When exposed to excess fatty acids, specific cell types produce nuclear lipid droplets (nLDs) that associate with promyelocytic leukemia (PML) protein to form Lipid Associated PML Structures (LAPS) that are enriched in lipid biosynthetic enzymes but deficient in canonical proteins associated with PML nuclear bodies (PML NBs). To identify the PML interactome during lipid stress, we employed proximity-dependent biotin identification (BioID) in U2OS cells expressing PMLI and PMLII fused to the ascorbate peroxidase APEX2 and cultured in the absence or presence of oleate to enhance lipid droplet formation. The resulting interactome included proteins enriched under oleate-treated conditions, such mitogen activated protein kinase-activated protein kinase 2 (MK2), ESCRT proteins and the COPII vesicle transport proteins Sec23B, Sec24A and USO1. COPII proteins co-localized with both PML-NBs and LAPS but were selectively enriched in PML-NBs following oleate treatment. The nuclear localization of USO1 was uniquely dependent on PML expression. Thus, the APEX2-PML proximity interactome implicates PML domains in the nuclear function of a non-canonical network of COPII vesicle trafficking proteins. SUMMARY STATEMENTThis is the first study to utilize APEX2 proximity labelling to identify the protein interactome of PML nuclear substructures and how interactions are modified under conditions of fatty acid-induced stress.

cell biology↗

bioRxiv2022

PML and PML-like exonucleases restrict retrotransposons in jawed vertebrates

We have uncovered a novel role for the promyelocytic leukemia (PML) gene and novel PML-like DEDDh exonucleases in the maintenance of genome stability through the restriction of LINE-1 (L1) retrotransposition in jawed vertebrates. Although the PML tumour suppressor protein in mammals is SUMOylated and forms nuclear bodies, we found that the spotted gar PML ortholog and related proteins in fish are not SUMOylated and function as cytoplasmic DEDDh exonucleases. In contrast, more closely related avian and turtle PML proteins are predicted to be SUMOylated and localized both to the cytoplasm and to nuclear bodies. We also identified PML-like exon 9 (Plex9) genes in teleost fishes that encode exonucleases sharing homology to gar PML. In an example of convergent evolution and akin to TREX1, gar PML and zebrafish Plex9 proteins suppressed L1 retrotransposition and could complement TREX1 knockout in mammalian cells. We also characterized the first non-mammalian TREX1 homologs in axolotl. Following export to the cytoplasm, the human PML-I isoform also restricted L1 through its conserved C-terminus and suppressed CGAS activation. Thus, PML first emerged as a cytoplasmic suppressor of retroelements, and this function is retained in amniotes despite its role in the assembly of nuclear bodies and the acquisition of SUMO-modification.

molecular biology↗

bioRxiv2020

Pandemic danger to the deep: the risk of marine mammals contracting SARS-CoV-2 from wastewater

We are in unprecedented times with the ongoing COVID-19 pandemic. The pandemic has impacted public health, the economy and our society on a global scale. In addition, the impacts of COVID-19 permeate into our environment and wildlife as well. Here, we discuss the essential role of wastewater treatment and management during these times. A consequence of poor wastewater management is the discharge of untreated wastewater carrying infectious SARS-CoV-2 into natural water systems that are home to marine mammals. Here, we predict the susceptibility of marine mammal species using a modelling approach. We identified that many species of whale, dolphin and seal, as well as otters, are predicted to be highly susceptible to infection by the SARS-CoV-2 virus. In addition, geo-mapping highlights how current wastewater management in Alaska may lead to susceptible marine mammal populations being exposed to the virus. Localities such as Cold Bay, Naknek, Dillingham and Palmer may require additional treatment of their wastewater to prevent virus spillover through sewage. Since over half of these susceptibility species are already at risk worldwide, the release of the virus via untreated wastewater could have devastating consequences for their already declining populations. For these reasons, we discuss approaches that can be taken by the public, policymakers and wastewater treatment facilities to reduce the risk of virus spillover in our natural water systems. Thus, we indicate the potential for reverse zoonotic transmission of COVID-19 and its impact on marine wildlife; impacts that can be mitigated with appropriate action to prevent further damage to these vulnerable populations.

ecology↗

bioRxiv2020

PRP4K is a haploinsufficient tumour suppressor negatively regulated during epithelial-to-mesenchymal transition

The pre-mRNA processing factor 4 kinase (PRP4K, also known as PRPF4B) is an essential gene. However, reduced PRP4K expression is associated with aggressive breast and ovarian cancer phenotypes including taxane therapy resistance, increased cell migration and invasion in vitro and cancer metastasis in mice; results consistent with PRP4K being a haploinsufficient tumour suppressor. Increased cell migration and invasion is associated with epithelial-to-mesenchymal transition (EMT), but how reduced PRP4K levels affect normal epithelial cell migration or EMT has not been studied. Depletion of PRP4K by small hairpin RNA (shRNA) in non-transformed mammary epithelial cell lines (MCF10A, HMLE) reduced or had no effect on 2D migration in the scratch assay but resulted in greater invasive potential in 3D transwell assays. Depletion of PRP4K in mesenchymal triple negative breast cancer cells (MDA-MB-231) resulted in both enhanced 2D migration and 3D invasion, with 3D invasion correlated with higher fibronectin levels in both MDA-MB-231 and MCF10A cells and without changes in E-cadherin. Induction of EMT in MCF10A cells, by treatment with WNT-5a and TGF-{beta}1, or depletion of eukaryotic translation initiation factor 3e (eIF3e) by shRNA, resulted in significantly reduced PRP4K expression. Mechanistically, induction of EMT by WNT-5a/TGF-{beta}1 reduced PRP4K transcript levels, whereas eIF3e depletion led to reduced PRP4K translation. Finally, reduced PRP4K levels after eIF3e depletion correlated with increased YAP activity and nuclear localization, both of which are reversed by overexpression of exogenous PRP4K. Thus, PRP4K is a haploinsufficient tumour suppressor negatively regulated by EMT, that when depleted in normal mammary cells can increase cell invasion without inducing full EMT.

cancer biology↗

bioRxiv2020

Lipid-associated PML domains regulate CCTα, Lipin1 and lipid homeostasis

Nuclear LDs (nLDs) originate at the inner nuclear membrane by a mechanism that involves the promyelocytic leukemia (PML) protein. Here we demonstrate that nLDs in oleate-treated U2OS cells are associated with Lipid-Associated PML (LAP) domains that differ from canonical PML nuclear bodies by the relative absence of SUMO1, SP100 and DAXX. nLDs were also enriched in CTP:phosphocholine cytidylyltransferase (CCT), the phosphatidic acid phosphatase Lipin1 and diacylglycerol (DAG). High resolution imaging revealed that LAP domains and CCT occupy distinct polarized regions on nLDs, and that loss of LAP domains in PML knockout U2OS cells reduced the recruitment of CCT onto nLDs by its amphipathic -helical M-domain. The association of Lipin1 and DAG with nLDs was also LAP domain-dependent. The disruption of CCT and Lipin1 localization on nLDs in PML knockout cells resulted in the inhibition of phosphatidylcholine and triacylglycerol synthesis indicating that LAP domains are a unique PML subdomain involved in nLD assembly and regulation of lipid metabolism.

cell biology↗

bioRxiv2018

Novel strand exchange activity of the human PALB2 DNA Binding Domain and its critical role for DNA repair in cells

Breast cancer associated proteins 1 and 2 (BRCA1, -2) and partner and localizer of BRCA2 (PALB2) protein are tumor suppressors linked to a spectrum of malignancies, including breast cancer and Fanconi anemia. They stimulate RAD51 recombinase during homology-directed repair (HDR). Along with being a hub for a protein interaction network, PALB2 interacts with DNA. The mechanism of PALB2 DNA binding and its function are poorly understood. We identified a major DNA-binding site in PALB2, mutation of which reduces the RAD51 foci formation and the overall HDR efficiency in cells by 50%. PALB2 N-terminal DNA-binding domain (N-DBD) stimulates the RAD51 strand exchange reaction. Surprisingly, it promotes the strand exchange without RAD51. Moreover, N-DBD stimulates the inverse strand exchange and can use both DNA and RNA substrates. Our data reveal a versatile DNA interaction property of PALB2 and demonstrate a critical role of PALB2 DNA binding for chromosome repair in cells.

biochemistry↗

bioRxiv2017

A marker-free co-selection strategy for high efficiency human genome engineering

Targeted genome editing using engineered nucleases facilitates the creation of bona fide cellular models for biological research and may be applied to human cell-based therapies. Broadly applicable and versatile methods for increasing the levels of gene editing in cell populations remain highly desirable due to the variable efficiency between distinct genomic loci and cell types. Harnessing the multiplexing capabilities of CRISPR-Cas9 and Cpf1 systems, we designed a simple and robust co-selection strategy for enriching cells harboring either nuclease-driven non-homologous end joining (NHEJ) or homology-directed repair (HDR) events. Selection for dominant alleles of the endogenous sodium-potassium pump (Na+,K+-ATPase) that render cells resistant to ouabain is used to enrich for custom modifications at another unlinked locus of interest, effectively increasing the recovery of engineered cells. The process was readily adaptable to transformed and primary cells, including hematopoietic stem and progenitor cells (HSPCs). The use of universal CRISPR reagents and a commercially available small molecule inhibitor streamlines the incorporation of marker-free genetic changes in human cells.

molecular biology↗