EMBO Reports

Lipid droplets (LDs) are unique organelles, surrounded by a phospholipid monolayer. They are present in most eukaryotic cells including the unicellular model organism S. cerevisiae. LDs store neutral lipids which serve as precursors for amphipathic membrane lipids and as an energy reserve. Loss of LDs in S. cerevisiae results in multiple cellular defects impairing lipid homeostasis and the biogenesis and function of other organelles. Here, we find that the expression levels of many proteins in isolated mitochondrial fractions are altered in cells that cannot synthesize neutral lipids and therefore lack LDs. In addition, among several downregulated proteins, we identified the previously uncharacterized Ylr001c (which we name Vlf1 for Vacuolar Lipophagy Factor 1). We show that Vlf1 is glycosylated and, in contrast to some previous reports, is actually localized to the vacuole. Furthermore, we demonstrate that changes in Vlf1 expression alter growth sensitivity to rapamycin, and detected a physical interaction of Vlf1 with Atg15, a lipase involved in autophagy. Additionally, we observe higher levels of autophagy/lipophagy in the absence of Vlf1 and a reduction upon overexpression of the protein. Taken together, the effects on lipohagy by Vlf1 makes it, according to our knowledge, the first vacuolar lipophagy regulator identified in S. cerevisiae.

Life on earth has always been exposed to the fluctuating Earth's magnetic field, but a magnetic sense affecting behavior has been debated for mammals. We here report that mice, kept under constant laboratory conditions, showed fluctuations in spontaneous behavioral activity with a periodicity of ~14 and ~28 days. This was confirmed in nine cohorts from four facilities on two continents, covering 3 to 41 months. Such oscillations were also maintained in brain Bmal1 knockout mice lacking circadian rhythms, suggesting independence of the circadian clock. The behavioral activity peaked around full and new Moon, and showed a strong alignment with the periodic geomagnetic fluctuations originating in the Earth's iono- and magnetosphere that are modulated by solar rotation and the orbital motion of the Moon. In the ultradian range, this alignment persisted in CRY1/2 knockout mice, suggesting that solar-lunar-driven geomagnetic fluctuations can modulate behavior rhythms independently of CRY1/2.

Macroautophagy is a conserved catabolic process that facilitates the degradation of cellular material by capturing it in double membrane vesicles termed autophagosomes. In Saccharomyces cerevisiae, selective macroautophagy is initiated by the scaffolding protein Atg11. Atg11 recruits the transmembrane protein Atg9, which resides in small vesicles, to autophagic cargo. Atg9 vesicles then fuse, forming the initial membrane sheet that expands into the autophagosomal membrane. While it is known that Atg9 interacts with Atg11 via a set of hydrophobic amino acids in the disordered N-terminus of Atg9, it is unclear how Atg11 mediates this interaction. To gain insight into this unknown aspect of autophagy initiation we utilized a combination of biochemical, structural, and cellular approaches. We demonstrate that the N-terminal domain (NTD) of Atg11 is the primary interaction site for Atg9, but the NTD requires clustering by the C-terminal region of Atg11 for its complete interaction with Atg9. We investigated the structure of the Atg11-NTD using cryo-EM which, in combination with AlphaFold modeling, revealed a positively charged binding pocket within the Atg11-NTD that is essential for Atg9 binding. Mutation of this conserved binding pocket leads to a loss of Atg9 binding in yeast and a reduction in the selective autophagy of mitochondria. Taken together, our results demonstrate the mechanism by which Atg11 recruits Atg9 to autophagy initiation sites.

Animal cells respond to hypoxic stress through cell-autonomous stabilization of HIF-1, a conserved transcription factor that mediates adaptation to low-oxygen environments. Although HIF-1 is essential for viability under hypoxia, its persistent activation during development causes miswiring defects in the nervous system, raising the question of how developing neurons balance the pro-survival benefits of HIF-1 stabilization against these associated risks. Here we show that C. elegans neurons address this challenge by mobilizing an internal promoter within the hif-1 locus to generate a dominant-negative isoform, HIF-1c, which acts in the nucleus to limit HIF-1/AHA-1 heterodimer formation. This buffering mechanism supports proper neuronal migration, axon guidance, and circuit formation and its loss phenocopies the defects seen when the ubiquitous HIF-1 degradation pathway is disrupted, confirming that HIF-1c serves as a critical layer of protection for the developing nervous system against hypoxic-induced errors. Lastly, we identify the retinoblastoma protein LIN-35 as a modulator of HIF-1 signaling in worms via control of hif-1c expression. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/745244v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@3ad347org.highwire.dtl.DTLVardef@4fd4forg.highwire.dtl.DTLVardef@1928804org.highwire.dtl.DTLVardef@11fd1e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Rubicon is a negative regulator of autophagy and the endolysosomal network (ELN) and an antagonist of the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Inhibition of Rubicon is considered a potential means to therapeutically upregulate autophagy and the ELN to treat Parkinsons disease and other conditions characterized by autophagic and ELN dysfunction. Rubicon is specific for the UVRAG-containing PI3KC3-C2 over the purely autophagic ATG14- containing PI3KC3-C1 complex. Here, we determined the high-resolution cryo-electron microscopy structure of PI3KC3-C2 in complex with the PI3KC3-binding domain (PIKBD) of Rubicon and compared it to cryo-EM structures of unbound PI3KC3-C2 and PI3KC3-C1. Rubicon binds directly to PI3KC3-C2 only via the BARA domain of the BECN1 subunit, which is common to both C1 and C2. The selectivity of Rubicon for the PI3KC3-C2 complex over the PI3KC3-C1 complex is attributed to a conformation of the BECN1BARA domain induced by UVRAG, rather than to direct contact with UVRAG or direct antagonism by the ATG14 subunit of PI3KC3-C1. Targeted disruption of the Rubicon:PI3K3-C2 structural interface by site-directed mutations enhances mitophagic activity in human epithelial cells to levels comparable to those observed in Rubicon knockout (KO) cells. Similarly, disruption of the interaction in Rubicon-overexpressing hippocampal neurons restored lysosomal flux to wild-type levels. These data show that suppressing the function of PI3K3- C2 can fully account for the negative regulatory effects of Rubicon in the autophagy and ELN pathways. Significance StatementEndolysosome maturation and autophagosome-lysosome fusion require the production of phosphatidylinositol 3-phosphate (PI(3)P) by the class III phosphatidylinositol 3-kinase complex II (PI3KC3-C2). Rubicon is a key negative regulator of endolysosomes and autophagy that suppresses PI3KC3-C2 activity. Here, we reveal in atomistic detail how Rubicon selectively recognizes PI3KC3-C2. Disrupting the Rubicon-PI3KC3-C2 interaction restores mitophagy and enhances lysosomal activity to the same extent as Rubicon gene deletion, establishing that PI3KC3-C2 inhibition fully accounts for the biological regulatory effects of Rubicon in the autophagy and lysosome pathways.

MTCH2 (mitochondrial carrier homolog 2) is a noncanonical member of the solute carrier family with five transmembrane (TM) helices, localized to the mitochondrial outer membrane. MTCH2's atypical topology creates a membrane-accessible hydrophilic groove, predicted to be necessary for its protein insertase and lipid scramblase activities. MTCH2 is linked to lipid metabolism and obesity and is required for starvation-induced mitochondrial hyperfusion. Here, we show that MTCH2 is a stable component of a complex containing the Armadillo (ARM) repeat-containing protein, ARMC1, and the DnaJ/Hsp40 chaperone protein, DNAJC11. Protein crosslinking, protein structural modeling, and molecular dynamics simulations demonstrate that the ARMC1 alpha-helical C-terminal domain (CTD) inserts into and stably interacts with the MTCH2 hydrophilic groove and blocks its lipid scramblase activity. We observe that starvation-induced mitochondrial fatty acid oxidation (FAO) is negatively regulated by the ARMC1-MTCH2 interaction. In ARMC1-deficient cells, FAO is stimulated compared to wild-type cells and lipid droplet abundance is significantly reduced. The altered lipid phenotype of ARMC1-/- cells is strictly dependent on MTCH2 and is reversed by ARMC1 expression in a manner dependent on its CTD. Beyond this metabolic axis, we also identify a function for ARMC1 in regulating lysosomal distribution and autophagic flux that is independent of its CTD and interaction with MTCH2. Thus, our data support a model in which the MTCH2-ARMC1 interaction functions as a metabolic switch during starvation to regulate the balance between fat storage and fat burning.

Polyphosphate (polyP), a linear polymer of orthophosphate residues, is enriched in secretory granules in specialised mammalian cell types including platelets and mast cells. Although polyP released during activation and degranulation of these cells has been shown to promote blood clotting and inflammation, little is known about the mechanisms governing polyP synthesis in these granules. In mice, the loss of IP6K1, an enzyme that catalyses the production of 5-InsP7, has been shown to result in depletion of platelet polyP and impaired hemostasis. Here, we use the rat mast cell line RBL-2H3 as a model to study the regulation of polyP synthesis in secretory granules. By monitoring real-time polyP synthesis in isolated mast cell granules, we demonstrate that ATP is the substrate fuelling granule polyP production. By the use of inhibitors, we show that accumulation of polyP in granules requires an intact transmembrane proton gradient maintained by vacuolar H+ATPase (V-ATPase). In RBL-2H3 cells, depletion of IP6K1 led to a substantial reduction in cellular polyP levels and defective accumulation of polyP, serotonin, and tryptase inside granules. Cells with reduced IP6K1 showed a profound loss of granule acidification, correlating with downregulated levels of V1 subunits of V-ATPase. Adding back active or catalytically inactive IP6K1 rescued the expression of V-ATPase V1 subunits, reversed granule deacidification, and restored polyP levels in IP6K1-depleted cells. Together, these data unveil a role for IP6K1 in maintaining granule pH and thereby supporting polyP synthesis in mammals.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with presequences which serve as targeting signals for the mitochondrial matrix, where they are cleaved by the mitochondrial processing peptidase (MPP). In this study, we comprehensively elucidated the role of the presequence and the mature part of mitochondrial precursors in the cytosol, by use of a cytosol-targeted MPP which prematurely processed mitochondrial precursors. Over time, cytoMPP resulted in mitochondrial depletion. However, the cellular response to cytoMPP was surprisingly different to that observed for other models of mitochondrial import inhibition. Cytosolic maturation rendered many proteins stable in the cytosol, indicating that their mature parts lack ubiquitination signals. Accordingly, cytoMPP did not induce the upregulation of the proteasome, which normally is a hallmark of mitochondrial dysfunction. Instead, cytoMPP elicited a heat shock response and impaired the sequestration of precursors in the cytosol. Our observations demonstrate that mitochondrial presequences are more than just address labels. Rather, they play an important role in quality control and orchestrate the cellular response to defects in mitochondrial protein import.

Endocytosis internalises nutrients, regulates extracellular signals, and recycles membrane components. Clathrin polymerises into a coat that shapes the endocytic vesicle from the plasma membrane. However, the role of clathrins dynamic assembly in the endocytic process remains unclear. We show, using two-colour fluorescence recovery after photobleaching assays in yeast, that the clathrin coat turns over rapidly in the early phase of endocytosis, dependent on the auxilin Swa2 and its ATPase. In the late phase the turnover is stopped by the coat protein Sla1. Regulated clathrin turnover is critical for the timing of endocytic progression and for controlling coat size. In the absence of this dynamic regulation the endocytic coats become abnormally large, resulting in the failure of the final actin-driven vesicle budding. These findings reveal that, in addition to its classic structural function, the dynamic properties of the clathrin lattice are critical for both the temporal and mechanical aspects of endocytosis.

The timing of DNA replication and centrosome duplication is tightly regulated with cell cycle progression to ensure the faithful duplication of the genome during cell division. Both DNA and centrosomes are licensed for replication in late telophase/early G1, replicated in S phase and segregated during mitosis; yet how defects in DNA replication licensing are coupled to centrosome homeostasis remains poorly understood. Here, we show that depletion of the replication licensing inhibitor Geminin in proliferating mouse embryonic fibroblasts induces robust centrosome amplification together with impaired primary cilium assembly. Rather than promoting whole-genome reduplication, knockdown of Geminin triggers a replication stress response, characterized by DNA damage accumulation throughout the cycle, and activation of an ATR-dependent DNA damage response. Mechanistically, Geminin depletion-induced replication stress activates the ATR-Chk1-Wee1 checkpoint axis prolonging G2 and leading to premature centriole disengagement and centrosome amplification. These findings identify replication stress as the signaling module that couples defective DNA replication licensing to centrosome amplification.

During eukaryotic cell division, kinetochores couple duplicated sister chromatids to mitotic spindle microtubules to mediate faithful chromosome segregation. Although the main kinetochore attachment sites to centromeric chromatin and microtubules are known, additional factors including microtubule-associated proteins are required for efficient chromosome biorientation and segregation in vivo. However, the roles and mechanisms of these factors in kinetochore function remain to be fully understood. Here, we characterise a previously unrecognised interaction between the microtubule plus-end tracking protein Bim1 and the outer kinetochore Ndc80 complex (Ndc80c) in S. cerevisiae. We show this interaction is mediated by a conserved SxIP motif within the intrinsically disordered Ndc80 N-terminus (Ndc80N), augmented by a secondary binding site containing an alpha-helical segment. This Ndc80 interaction with Bim1 increases the strength of Ndc80c-microtubule attachments. Phosphorylation of the Bim1-binding region of Ndc80N by the error correction Ipl1/Aurora B protein kinase alters its secondary structure and weakens the Bim1-Ndc80c interaction, providing a potential additional regulatory mechanism for how incorrect kinetochore-microtubule attachments are destabilised during error correction.

Zinc is an essential micronutrient at low concentrations, yet it becomes toxic at slightly higher ones. This is exploited by plants as an effective defensive strategy. However, the molecular components that are involved zinc-mediated immunity remain poorly defined. Here, we show that mixed-linked {beta}-1,3/1,4-glucans naturally occurring in microbial and grass cell walls and used as an agrobiological solution, trigger zinc accumulation in the Arabidopsis apoplast and upregulate the expression of the zinc transporters HMA2 and HMA4. This response occurs independently of salicylic acid, jasmonic acid and ethylene-mediated signalling pathways, but it requires the LysM receptor kinases CERK1, LYK4 and LYK5, indicating a specific pattern triggered immunity-associated mechanism. We further demonstrate that hma2hma4 mutants display constitutive activation of a broad set of defence-related genes, yet this transcriptional reprogramming is insufficient to confer resistance against the necrotrophic fungus Plectosphaerella cucumerina BMM. Moreover, metabolomic profiling highlights the contribution of specialized metabolites to this defective defence output. Altogether, our findings reveal that zinc-mediated toxicity constitutes a defence mechanism integrated into the immune response triggered by specific microbial or damage associated molecular patterns.

During eukaryotic cell division, kinetochores couple duplicated sister chromatids to mitotic spindle microtubules to mediate faithful chromosome segregation. Although the main kinetochore attachment sites to centromeric chromatin and microtubules are known, additional factors including microtubule-associated proteins are required for efficient chromosome biorientation and segregation in vivo. However, the roles and mechanisms of these factors in kinetochore function remain to be fully understood. Here, we characterise a previously unrecognised interaction between the microtubule plus-end tracking protein Bim1 and the outer kinetochore Ndc80 complex (Ndc80c) in S. cerevisiae. We show this interaction is mediated by a conserved SxIP motif within the intrinsically disordered Ndc80 N-terminus (Ndc80N), augmented by a secondary binding site containing an -helical segment. This Ndc80 interaction with Bim1 increases the strength of Ndc80c-microtubule attachments. Phosphorylation of the Bim1-binding region of Ndc80N by the error correction Ipl1/Aurora B protein kinase alters its secondary structure and weakens the Bim1-Ndc80c interaction, providing a potential additional regulatory mechanism for how incorrect kinetochore-microtubule attachments are destabilised during error correction.

Eukaryotic cells contain multiple biochemically distinct actin filament networks, which enable versatile functions of actin in a range of cellular processes. Yet, the mechanisms by which specific actin filament networks are assembled in a common cytoplasm remain elusive. Here, we investigated how functionally distinct actin nanoscale layers, specified by -actininand tropomyosin isoforms, Tpm1.6 and Tpm3.2, are assembled at focal adhesions. By combining genetic perturbations with mitochondrial-targeting of actin polymerases, we discovered that DAAM1 formin assembles Tpm3.2-actin filaments, whereas Ena/VASP family proteins polymerize -actinin cross-linked actin filament bundles at focal adhesions. Consequently, loss of DAAM1 dampened Tpm3.2 protein levels and impaired focal adhesion disassembly, thus phenocopying Tpm3.2-deficient cells. In contrast, Ena/VASP depletion led to defective focal adhesion maturation and loss of -actinin from focal adhesions. More broadly, our study highlights specific roles for formin and Ena/VASP family proteins in assembling biochemically and functionally distinct linear actin filament arrays in cells.

Successful oogenesis requires the precise coordination of nutrient uptake, storage, and utilization to meet the high metabolic demands of egg production. In mammals, fatty acid (FA) metabolism has emerged as a key driver of oocyte maturation; however, the mechanisms by which follicles regulate FA trafficking and utilization remain poorly understood across all systems. To address these issues, we leverage the genetic tractability of Drosophila melanogaster oogenesis. We found that nurse cell mitochondria are metabolically active and catabolize FA in a stage-dependent manner, with fatty acid oxidation (FAO) peaking during mid-oogenesis. By exposing explanted follicles to fluorescently labeled FAs, we monitored FA trafficking and found massive enrichment in lipid droplets. Mutants for the triglyceride lipase ATGL exhibited a reduction in both mitochondrial membrane potential and FAO, suggesting that mitochondria utilize FA from triglycerides stored in LDs. To determine the significance of this transient FA storage in LDs, we prevented the formation of nurse cell LDs with mutations in the triglyceride synthase DGAT1. The DGAT1 mutant follicles display excess accumulation of FAs in mitochondria, mitochondrial stress, and developmental arrest. We find that this mitochondrial dysfunction and follicle arrest are consequences of FA toxicity to mitochondria: limiting FA influx into follicles or FA import into mitochondria alleviates these defects. Our findings demonstrate that LD-derived FAs are actively mobilized to fuel the energy demands of oogenesis while LDs buffer against lipotoxicity, revealing a critical balance between FA storage and oxidation. These findings highlight LDs as central hubs regulating energy homeostasis and developmental progression in the follicle. Author SummaryOogenesis places extraordinary metabolic demands on the follicle, yet the energy source for follicle development is not well understood. In fruit flies, follicles take in large amounts of lipids from the hemolymph, the insect blood, and accumulate massive fat stores in the form of lipid droplets. Whether these stores are reserved for the embryo or already power oogenesis was unclear. Using mutants and fluorescent probes for metabolic activity, we found that some fatty acids are released from the lipid droplets and power energy production in mitochondria; this energy source is important for successful oogenesis. We then fed flies fluorescently labeled fatty acids and determined how these fatty acids travel when lipid droplet formation can occur versus when it is abolished. In the former case, fatty acids accumulate in lipid droplets; in the latter, they flood into mitochondria, causing mitochondrial dysfunction, reduced ATP levels, and follicle death. We can correct all these defects by limiting lipid influx specifically into mitochondria. Our findings reveal an important role for lipid droplets during oogenesis. They act as a metabolic buffer, supplying sufficient amounts of fatty acids to mitochondria for energy production while shielding the mitochondria from toxic lipid levels.

Mitochondria-targeted modulation of redox homeostasis has emerged as a promising strategy for controlling pathological cell proliferation. Here, we investigate the effects of Mitocurcumin in a Yorkie-driven intestinal stem cell tumor model in Drosophila. Using an integrative, genetically tractable approach combining in silico molecular modelling with in vivo functional analyses, we identify thioredoxin reductase (TrxR) as a conserved redox-associated target of Mitocurcumin. Docking and molecular dynamics simulations predict a stable interaction of Mitocurcumin with both Drosophila and mammalian TrxR homologs. Functionally, Mitocurcumin treatment reduces mitotic activity, elevates reactive oxygen species (ROS) selectively within escargot-positive intestinal stem cell population, enhances apoptosis in the tumor-bearing guts, and causes significant mitochondrial membrane depolarization. These cellular effects coincide with dose-dependent regression of Yorkie-induced intestinal hyperplasia. Despite mitochondrial functional impairment, mitochondrial morphology remains largely preserved, suggesting primary disruption of redox buffering rather than structural collapse. Metabolomic profiling of these guts further reveals remodelling of energy metabolism consistent with adaptive responses to oxidative stress. Importantly, Mitocurcumin alleviates tumor-associated organismal bloating and significantly extends lifespan indicating a previously uncharacterized systemic, organism-wide response to Mitocurcumin treatment in an in vivo scenario. Collectively, our findings establish TrxR-mediated redox regulation as a critical vulnerability in Yorkie-driven hyperproliferation and highlight the utility of Drosophila as an integrative in vivo platform for evaluating mitochondria-targeted bioactive molecules.

Life-long production of blood requires the preservation of hematopoietic stem cell (HSCs) regenerative capacity during inflammation. The cytokine, Thrombopoietin (THPO), is essential for HSC maintenance yet its role during inflammatory stress remains incompletely understood. Long-term repopulating potential was rapidly depleted in THPO-deficient HSCs upon poly(I:C) administration through inflammatory pyroptosis. Transcriptomic and chromatin accessibility analyses revealed constitutive interferon (IFN) pathway activation in THPO-deficient HSCs, characterized by enhanced STAT1 signaling, increased accessibility of STAT and IRF motifs, and elevated expression of IFN-stimulated genes. Lipidomic profiling further identified selective shifts in sphingomyelin (SM) species and enrichment of features associated with increased bilayer rigidity. THPO-deficient HSCs displayed elevated membrane SM incorporation, impaired membrane fluidity and altered membrane ultrastructure. Genetic ablation of Stat1 normalized membrane lipid abnormalities and reduced pyroptotic activation and restored HSC survival and regenerative function under inflammatory stress. Together, these findings identify a STAT1 and SM metabolism as critical THPO downstream to protect HSCs from inflammatory pyroptosis. Our results reveal membrane lipid homeostasis as a fundamental mechanism through which cytokine signaling safeguards HSC function during stress.

Nuclear pore complexes (NPCs) are nuclear envelope (NE)-embedded protein assemblies that mediate nucleocytoplasmic exchange and interact with the genome, including binding of an NPC component Nup93 to Polycomb chromatin domains. Here, we investigated the in vivo relevance of this relationship in Drosophila, which unusually contains two distinct paralogs of Nup93. Interestingly, we identified a Nup93-2-specific tumorigenic phenotype in larval wings, where depletion of Nup93-2, but not Nup93-1, led to tumor-like overgrowth, reminiscent of Polycomb mutations. Consistently, our transcriptomic analysis revealed a wide-spread loss of gene silencing in Nup93-2-depleted wings, particularly in a Nup93-bound Polycomb domain spanning genes for activators of JAK/STAT signaling. Nup93 paralogs were not found to differ in their effect on NPC biogenesis but strikingly, showed differences in subnuclear localization patterns. While Nup93-1 co-localized exclusively with fully assembled NPCs, Nup93-2 exhibited only partial co-localization and was found at additional NE locations in a tissue-specific manner. Together, our results identify an in vivo silencing role of a Nup93 paralog and suggest that Nup93-2 may form a unique NE-associated complex that targets a subset of Polycomb domains containing growth-promoting genes.

Stress granules (SGs) are cytoplasmic condensates that assemble under cellular stress, including viral infection. The best-characterized evasion strategy in SARS-CoV-2 infection involves the viral nucleocapsid (N) protein hijacking the SG core protein G3BP1, thereby preventing SG assembly. By performing network analysis of N protein interactomes, we identified CAPRIN1 as an underexplored candidate, despite its central role alongside G3BP1 in SG assembly. Here, we provide the first high-resolution spatial and functional data on CAPRIN1 in SARS-CoV-2 infection. Using ultrastructure expansion microscopy (U-ExM), we localized CAPRIN1 and viral egress markers to enlarged, CD63- and LAMP1-positive, multivesicular body (MVB)-like egress compartments, refining the current model of SARS-CoV-2 egress. CAPRIN1 was found on both the limiting membrane of these compartments and within the intraluminal vesicles, a pattern distinct from G3BP1. Knockout of CAPRIN1 increased infection-associated cell death, promoted an aberrant cell-death phenotype, and enhanced syncytia formation. Together, our data reveal distinct activities of CAPRIN1 during infection and identify the egress compartments as MVB-like, raising the possibility that SARS-CoV-2 repurposes more than one cellular pathway for egress.

Vascular Endothelial Zinc Finger 1 (VEZF1) is essential for embryonic development, but its role in pluripotency exit remains unclear. Previous work showed that Vezf1-deficient ESCs exhibit impaired differentiation, reduced Dnmt3b expression, and genome-wide hypomethylation. Here, we show that Vezf1-/- ESCs fail to efficiently repress the pluripotency transcriptional program during differentiation, a defect that persists after ectopic Dnmt3b expression. Genome-wide analysis revealed VEZF1 occupancy at regulatory regions of genes involved in several developmental signaling pathways, including MAPK, WNT, and Hippo, as well as at some pluripotency-associated genes. Many VEZF1-bound MAPK genes showed reduced expression in undifferentiated Vezf1-/-ESCs, suggesting that VEZF1 activity contributes to transcriptional competence required for efficient pluripotency exit. VEZF1 loss also led to widespread acquisition of new CTCF sites associated with developmental signaling, a subset of which overlapped VEZF1-bound regulatory regions. CTCF depletion had only limited effects on the expression of the VEZF1-bound MAPK genes examined, indicating that increased CTCF occupancy alone is insufficient to explain their reduced expression in Vezf1-/- ESCs. Together, our findings identify a DNMT3B-independent function of VEZF1 in facilitating the exit from pluripotency and establishing transcriptional competence for differentiation, while revealing a potential role for VEZF1 in regulating CTCF occupancy during developmental state transitions.