Phosphotyrosine signaling plays a critical role in many biological processes, from cell proliferation to immune response. Despite its importance, proteomic studies of tyrosine phosphorylation have been limited in scale a...Phosphotyrosine signaling plays a critical role in many biological processes, from cell proliferation to immune response. Despite its importance, proteomic studies of tyrosine phosphorylation have been limited in scale and throughput due to the need for specialized enrichment with costly reagents and labor-intensive protocols. To address these challenges, we developed R2HaPpY, a phosphotyrosine enrichment method that combines highly simplified phosphotyrosine superbinder reagent preparation and automated high-throughput enrichment. Our new reagent binds phosphotyrosine peptides at higher efficiency than other enrichment reagents and reduces both cost and preparation time by 20-fold. We generalized the R2HaPpY method to samples of low and high levels of phosphotyrosine. We benchmarked biological application to study EGF signaling dynamics in HeLa cells. Using only ~1 mg of input peptides, we detect and quantify 1651 unique phosphotyrosine sites. These include 878 regulated pY sites, many of which are novel or not previously annotated as EGF-responsive. Our results reveal differential temporal regulation and represent the largest phosphotyrosine dataset of cellular response to EGF stimulation to date. This streamlined, cost-effective, and sensitive method enables quantitative mapping of tyrosine phosphorylation dynamics at a scale of hundreds of samples, facilitating integration of phosphotyrosine signaling into multiomic studies across diverse biological systems and disease states.
Plasmodium parasites, the causative agents of malaria, undergo complex replication within vertebrate and insect hosts, presenting unique opportunities for therapeutic intervention. A key challenge during these replicatio...Plasmodium parasites, the causative agents of malaria, undergo complex replication within vertebrate and insect hosts, presenting unique opportunities for therapeutic intervention. A key challenge during these replication events, i.e., schizogony in vertebrate red blood cells and sporogony in oocysts within mosquitos, is ensuring the faithful partitioning of nuclei and organelles into the numerous daughter cells that form at once from a single parental cell. While nuclear microtubule-organizing centers, or centriolar plaques (CPs), have been hypothesized to play a central role in this process, the molecular mediators linking CPs and organelles remain incompletely defined. Here, we characterize the roles of two striated fiber assemblin (SFA) homologs, SFA1 and SFA2, in replication of Plasmodium falciparum and Plasmodium berghei across two hosts. We show that these SFAs form a physical bridge between the CP and the nascent apical poles of daughter cells, facilitating high-fidelity progeny formation during schizogony and sporogony. Loss of SFA function disrupts merozoite and sporozoite formation, with profound consequences for transmission. These findings identify striated fiber assemblins as essential organizers of parasite morphogenesis and potential targets for anti-malarial therapies.
Acin-Perez R, Assali EA, Veliova M
… +16 more, Ngo J, Brownstein AJ, Villalobos F, Petcherski A, Hernansanz-Agustin P, Kim-Vasquez D, Xu S, Tamboline M, Silva RM, Upcher A, Shu C, Ferriss DE, Liesa M, Enriquez JA, Sekler I, Shirihai OS
Adrenergic stimulation of brown adipocytes induces a robust detachment of mitochondria from lipid droplets (LD), which is followed by lipolysis and lipid catabolism. However, the signals inducing mitochondria attachment...Adrenergic stimulation of brown adipocytes induces a robust detachment of mitochondria from lipid droplets (LD), which is followed by lipolysis and lipid catabolism. However, the signals inducing mitochondria attachment or detachment, and their role in lipid metabolism, remain unknown. Here, we reconstituted mitochondria-LD interaction in brown adipocyte tissue (BAT) ex vivo. We find that removal of mitochondria from lipid droplets permits higher lipolytic activity of recombinant lipases. Testing the effect of thermogenic secondary messengers and metabolites on attachment and detachment identified elevated mitochondrial matrix calcium as a potent inducer of detachment. Further, deletion of the mitochondrial sodium/calcium exchanger, NCLX, resulted in reduced attachment and increased detachment, while activation of NCLX increased attachment. We find that elevated matrix calcium causes detachment by inducing architectural transformation of peridroplet mitochondria (PDM) from their typical LD-surface-bound crescent shape into a round shape. PDE2A inhibition activates NCLX and increases PDM content in BAT in vitro and in vivo. We conclude that a surge in mitochondrial matrix calcium ions serves as a potent signal to induce mitochondrial detachment from lipid droplets, thereby facilitating lipolysis.
Accurate transition into mitosis driven by cyclin B1-CDK1 activity is essential to avoid chromosome segregation errors and preserve genome integrity. How this activity is spatially controlled to trigger mitotic onset rem...Accurate transition into mitosis driven by cyclin B1-CDK1 activity is essential to avoid chromosome segregation errors and preserve genome integrity. How this activity is spatially controlled to trigger mitotic onset remains unclear. Here, we show that chromosome condensation triggers an increase in nuclear envelope (NE) tension. This increased tension is required for translocation of cyclin B1 into the nucleus and dynein loading on nuclear pore complexes (NPCs), ensuring timely mitotic entry. Micromanipulation experiments further indicate this tension-dependent mechanism requires SUN proteins on the NE. Impairment of chromosome condensation leads to the nuclear accumulation of the G2 checkpoint kinase Wee1 and an inhibition of CDK1 activity, which result in a temporary delay in mitotic entry. This delay can be overridden by increasing tension on the NE, which accelerates the nuclear translocation of cyclin B1 and dynein loading. We propose that mitotic onset is controlled by a chromosome-dependent NE tension mechanism that enables robust spatiotemporal coupling between chromosome condensation and the NE structural changes required for an efficient mitosis.
Neuronal remodeling is crucial for proper nervous system development and function. Despite significant advances, the underlying mechanisms that govern this process remain poorly understood. Here, we adapted C. elegans IL...Neuronal remodeling is crucial for proper nervous system development and function. Despite significant advances, the underlying mechanisms that govern this process remain poorly understood. Here, we adapted C. elegans IL2 sensory neurons as a model system to study developmental and organismal stress-associated dendrite remodeling. Upon entering developmental diapause, IL2 dendrites grow a complex dendritic arbor, which is later pruned when reproductive development resumes. We identified unexpected specificity in the pruning process, with distinct genetic requirements to direct branch-specific elimination of secondary, tertiary, and quaternary branches. The serine/threonine kinase SAX-1/NDR promotes elimination of secondary and tertiary, but not quaternary, dendrites. SAX-1 functions with its conserved interactors SAX-2/Furry and MOB-2 in the removal of both dendritic branches. The guanine-nucleotide exchange factor RABI-1/Rabin8 and the small GTPase RAB-11.2 mediate the elimination of secondary branches with SAX-1, but their effect on tertiary branches is minimal. Consistent with the known roles of RABI-1 and RAB-11.2 in regulating membrane dynamics, we find that SAX-1 promotes endocytosis during remodeling. Together, our findings reveal distinct mechanisms for dendrite branch-specific elimination during neuronal remodeling.
Human ATP synthase is a molecular rotary machine bound in inner mitochondrial membranes, built from twenty-eight subunits of seventeen kinds, two encoded in mitochondrial DNA, the remainder in nuclear genes. The machine...Human ATP synthase is a molecular rotary machine bound in inner mitochondrial membranes, built from twenty-eight subunits of seventeen kinds, two encoded in mitochondrial DNA, the remainder in nuclear genes. The machine consists of a rotor and an interacting stator. Turning of the rotor driven by a transmembrane proton motive force effects a cycle of structural changes in the catalytic part of the stator, producing three ATP molecules per rotation. Here, to establish how the stator and rotor are assembled, we deleted subunits and known assembly factors from human cells, purified and accumulated assembly intermediate complexes, and characterized them by gel analysis and mass spectrometry, allowing us to propose pathways of assembly of the rotor and the catalytic F-module of the stator. These observations provide opportunities for further development by structural analysis of the accumulated intermediates. The compositions of the various assembly intermediates support the view that ATP synthase arose via independent evolution of its three constituent structural components, the catalytic F-module, the peripheral stalk module, and the membrane-associated F-module.
The Sec translocon and the YidC/Oxa1-type insertases universally mediate biogenesis of α-helical membrane proteins, but the molecular basis of their cooperation has remained disputed. Recent discovery of multi-subunit in...The Sec translocon and the YidC/Oxa1-type insertases universally mediate biogenesis of α-helical membrane proteins, but the molecular basis of their cooperation has remained disputed. Recent discovery of multi-subunit insertases assembled at the back of the translocon in fungi and higher eukaryotes has raised questions about the architecture and mechanism of the putative bacterial ortholog SecYEG-YidC. Here, we combine cryogenic electron microscopy with cell-free protein synthesis to visualize biogenesis of the SecYEG/YidC-dependent multipass membrane protein NuoK. The nascent chain of NuoK does not enter the lateral gate of SecYEG but instead crosses the translocon towards its back side, where YidC is recruited in the nascent substrate-dependent manner. The SecY-YidC interface promotes folding of the transmembrane helices before insertion, consistent with thermodynamic principles of membrane protein folding. YidC forms extensive contacts with the nascent chain, suggesting its key role in the insertion event. These findings provide mechanistic insight into membrane protein insertases, support evolutionary conservation of a gate-independent insertion route, and expand current models of membrane protein biogenesis.
Phages with contractile tails employ elaborate mechanisms to penetrate bacterial cell walls and deliver their genomes into the host cytoplasm. Here, we used cryo-EM to show that the baseplate of phage 812, a member of th...Phages with contractile tails employ elaborate mechanisms to penetrate bacterial cell walls and deliver their genomes into the host cytoplasm. Here, we used cryo-EM to show that the baseplate of phage 812, a member of the Kayvirus genus, which infects Gram-positive Staphylococcus strains, is formed of a core, wedge modules, and baseplate arms carrying receptor-binding proteins 1 and 2 and tripod complexes. Upon binding to a host cell, the receptor-binding proteins of phage 812 baseplate reorient and undergo conformational changes. The changes to the tripod complexes trigger the release of the central spike and weld proteins, which expose peptidoglycan-degrading domains of the hub proteins. Changes in the positions of baseplate arms are transmitted through wedge modules to tail sheath initiator proteins. The ring of the tail sheath initiator proteins expands and triggers the contraction of the tail sheath, which shortens to 50% and pushes the tail tube 10-30 nm into the bacterial cytoplasm. Homologous molecular mechanisms are probably shared by phages of the Herelleviridae family with contractile tails to infect Gram-positive bacteria.
Navalayeu T, Beer N, Bebjaková M
… +11 more, Kalis RW, Hohmann U, Stejskal K, Krššáková G, Fasching N, Herzog VA, Popitsch N, Roitinger E, Plaschka C, Zuber J, Ameres SL
Most eukaryotic proteins assemble into multisubunit complexes that coordinate essential cellular functions, yet the principles governing their assembly and proteostatic control remain largely undefined. Here, we systemat...Most eukaryotic proteins assemble into multisubunit complexes that coordinate essential cellular functions, yet the principles governing their assembly and proteostatic control remain largely undefined. Here, we systematically dissect the cellular assembly and functional organization of the RNA exosome, an essential ribonucleolytic complex, using an inducible dual-guide CRISPR/Cas9 system in mouse embryonic stem cells. We reveal a sequential assembly pathway where Exosc2, Exosc4, and Exosc7 initiate complex formation, facilitating the incorporation of barrel and cap subunits in a defined hierarchy. Unlike other structural subunits, the terminally incorporated cap subunit Exosc1 is dispensable for cell viability, revealing a modular, functionally resilient architecture. We demonstrate that orphan subunits are selectively degraded via the ubiquitin-proteasome system, enforcing stringent quality control over RNA exosome biogenesis. These findings define an assembly logic of of the mammalian exosome and uncover previously unrecognized plasticity in the composition and function of this essential ribonucleolytic complex.
Martí-Mateos Y, Muñoz-Hernández MDM, Gómez de Las Heras MM
… +17 more, Escrig-Larena JI, Cabrera-Alarcón JL, Acín-Pérez R, Calvo E, Jaroszewicz SN, de Prado-Rivas L, Martínez-Jiménez ER, De Andrés-Laguillo M, García-Domínguez E, Gómez-Cabrera MC, Viña J, Benedito R, Efeyan A, Vázquez J, Mittelbrunn M, Jiménez-Gómez MC, Enríquez JA
Hepatic inflammation and immunosurveillance play major roles in the progression of liver cancer. A common trigger for hepatic inflammation is oxidative stress, which stems from mitochondrial dysfunction. Here, we demonst...Hepatic inflammation and immunosurveillance play major roles in the progression of liver cancer. A common trigger for hepatic inflammation is oxidative stress, which stems from mitochondrial dysfunction. Here, we demonstrate that deletion of the mitochondrial stress integrator OMA1 increases hepatic primary tumor incidence and impairs survival in mice. Persistent activation of the KEAP1-Nrf2 oxidative stress pathway in the absence of OMA1 promotes early liver injury, which progresses into chronic hepatic inflammation and fibrosis during aging. Exhausted CD8 and CD4 T cells gradually accumulate in Oma1 livers, facilitating hepatic tumor progression. Adoptive transfer and bone marrow-transplant experiments indicate that hepatic immunogenicity increases in the absence of parenchymal OMA1. Furthermore, hepatocyte-specific Oma1 deletion is sufficient to trigger NRF2 signaling, hepatocyte death, and immune exhaustion, suggesting that immunosurveillance during liver aging relies on the hepatic expression of OMA1. Given the therapeutic interest of OMA1 in several pathologies, these data are crucial to guide the generation of safe OMA1-targeted therapies.
Acidification of lysosomes, endosomes and the Golgi underpins organelle-specific functions within the endomembrane system. This process is driven by vacuolar-type H -ATPases (V-ATPases), proton pumps that reversibly asse...Acidification of lysosomes, endosomes and the Golgi underpins organelle-specific functions within the endomembrane system. This process is driven by vacuolar-type H -ATPases (V-ATPases), proton pumps that reversibly assemble from peripheral V and membrane-integral V domains to regulate organelle pH. In yeast, V-V assembly at the vacuole is mediated by the RAVE complex, but V-ATPase assembly in mammalian cells remains less well understood. Here, we systematically characterize physiological roles of the mammalian RAVE complex, composed of the subunits Dmxl1 or Dmxl2, Wdr7 and Rogdi. Under basal conditions, mRAVE broadly promotes V-ATPase assembly and luminal acidification of endomembrane organelles. Upon mTORC1 inactivation, mRAVE is recruited to lysosomes and required for the resulting increase in V-ATPase assembly and catabolic activity. Loss of mRAVE disrupts organelle acidification, leading to suppression of lysosomal catabolism, accumulation of dysfunctional lysosomes and lysosomal exocytosis. Restoring lysosomal pH rescues basal function in mRAVE-deficient cells but not the mTORC1-regulated increase in catabolic activity. Thus, mRAVE is an essential V-ATPase assembly factor that couples acidification to organelle function and nutrient signaling.
From a historical perspective, long non-coding RNAs (lncRNAs) represent a relatively short story. However, this story has many plot lines, thrills, twists, and turns that altogether form quite a long saga of its own. lnc...From a historical perspective, long non-coding RNAs (lncRNAs) represent a relatively short story. However, this story has many plot lines, thrills, twists, and turns that altogether form quite a long saga of its own. lncRNAs stay at the forefront of a recent paradigm shift from a protein-only world to the mysterious RNA world, the enormous complexity of which we are only beginning to appreciate. Here, we review the most enigmatic aspect of lncRNAs, their coding ability in the context of whole-cell regulation. What peptides do lncRNAs encode as true translons? What is the mechanism of their translation? What roles do these ncRNA-encoded peptides (ncPEPs) play during differentiation and development and in distinct pathologies? Do these ncPEPs contribute to the already known regulatory roles of lncRNAs? Shouldn't we coin the coding subset of lncRNAs its apt name: cryptic long non-coding RNAs (crpt-lncRNAs) for their cryptic coding capacity? These and other questions concerning these current winners of the spotlight in molecular biology, which await resolution, are discussed so that molecular history can be rewritten once again.
Xu Y, Zhang L, Liao X
… +19 more, Fan Y, Shao R, Wu M, Zhang L, Zhang M, Wang G, Zhang H, Li E, Wu Q, Zhao J, Zhang J, Wang W, Cai L, Zhong J, Hu L, Wang J, Mao J, Yang B, Zhang J
Whether metabolites enriched at early developmental stages affect cellular and organismal aging remains unclear. In this study, we comprehensively profiled the metabolic landscape of mouse oocytes in comparison to cleava...Whether metabolites enriched at early developmental stages affect cellular and organismal aging remains unclear. In this study, we comprehensively profiled the metabolic landscape of mouse oocytes in comparison to cleavage-stage embryos. Our analysis revealed that oocytes display accumulation of reductive metabolites that diminish following fertilization. Notably, we identified serotonin (5-hydroxytryptamine, 5-HT) as an oocyte-enriched metabolite with protective roles in aging. The underlying mechanisms operate through dual pathways: (i) in a canonical pathway serotonin acts via its receptor 5HTR1B to modulate mitochondrial function, and (ii) in a non-canonical pathway serotonin promotes serotonylation of HSP90β, which effectively reduces endoplasmic reticulum stress. Overall, our study demonstrates that oocyte-enriched metabolites including serotonin can alleviate aging-related cellular and systemic phenotypes, suggesting new avenues for anti-aging strategies.
The manchette is a transient microtubule (MT)-based structure that is vital for the correct shaping of sperm during spermiogenesis. Throughout spermiogenesis, the manchette retains structural integrity for several days,...The manchette is a transient microtubule (MT)-based structure that is vital for the correct shaping of sperm during spermiogenesis. Throughout spermiogenesis, the manchette retains structural integrity for several days, raising the question of how its MTs are regulated. Here, using cryo-electron tomography of manchettes isolated from rat testes, we find that manchette MT ends are structurally diverse. We show that the MT-binding protein CLASP2 is present throughout the manchette and likely regulates both MT ends. Using cryo-electron microscopy single particle analysis and super-resolution microscopy, we reveal that SPACA9 and MNMIP1 (SH3D21) bind to the seam of manchette MTs from the luminal side. SPACA9 binds to both α- and β-tubulin of protofilament 1 but does not interact directly with protofilament 13, while MNMIP1 binds directly to protofilament 13. MNMIP1 further extends and threads through the MT lattice at the seam. Our study reveals a novel seam MT inner protein complex with a unique binding mode, providing a plausible explanation for MT regulation that maintains manchette integrity over an extended period.
Bennison DJ, Chaudhary I, Chaudhuri D
… +18 more, Wong JCN, Punwatkar A, Biswas P, Stephenson M, Zhong Q, Kallemeijn WW, Guenot M, Koigi S, Papp D, Thomas JP, Dixit D, Korcsmaros T, Talman AM, Frickel EM, Tate EW, Visweswariah SS, Frankel G, Shenoy AR
The IFNγ-induced GTPase guanylate-binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial ou...The IFNγ-induced GTPase guanylate-binding protein 1 (GBP1) binds to lipopolysaccharide (LPS) on cytosolic gram-negative bacteria and promotes pyroptosis via the recruitment and activation of caspase-4 on the bacterial outer membrane. Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) are extracellular pathogens that adhere to host cells and stimulate dense actin polymerisation underneath their attachment sites, generating structures described as actin-rich pedestals. Here, we show that GBP1 traffics to actin-rich pedestals in human cells infected with EPEC or EHEC in vitro and mouse colonocytes infected with the EPEC-like murine pathogen Citrobacter rodentium in vivo. GBP1 promotes caspase-4 recruitment to actin-rich pedestals, leading to pyroptosis and IL-18 release. GBP1 mutants defective in LPS coatomer formation also localise to EPEC pedestals. A novel assay that mimics pathogenic effector activity reveals GBP1 recruitment to sterile actin polymerisation sites. We conclude that cytosolic GBP1 is mobilised to sites of pathogen-induced actin remodelling independently of LPS. Our study establishes that GBP1 not only operates as a pattern-recognition receptor but also orchestrates effector-triggered immunity against pathogens that hijack the actin cytoskeleton.
Bacteria encode numerous stress-response pathways that protect their hosts against both internal and external threats. A key question is how these pathways are regulated, especially anti-phage immune pathways that mediat...Bacteria encode numerous stress-response pathways that protect their hosts against both internal and external threats. A key question is how these pathways are regulated, especially anti-phage immune pathways that mediate host-cell killing. Here, we identify two proteins termed CapK and CapS that are encoded upstream of diverse immune operons, and regulate these operons' expression in response to DNA damage. CapK resembles bacterial anti-sigma factor kinases, and CapS resembles STAS-domain antagonists of these proteins. CapS is a DNA-binding transcriptional repressor, and phosphorylation of CapS by CapK results in dissociation of a CapS homodimer and de-repression of transcription. The CapK kinase is directly activated by single-stranded DNA generated as a byproduct of DNA repair. Finally, we show that CapK and CapS-like proteins have been co-opted into an anti-phage toxin-antitoxin system with a VapC-like protein, where they similarly respond to DNA damage to activate VapC nuclease activity. Overall, our results reveal how a kinase-substrate pair can regulate expression of an adjacent operon in response to DNA damage, and highlight the modularity of immune and other stress-response pathways.
The Golgi apparatus expands during differentiation and increased secretory demand, yet the transcriptional mechanisms governing its biogenesis remain poorly defined. To investigate this process, we developed an enzymatic...The Golgi apparatus expands during differentiation and increased secretory demand, yet the transcriptional mechanisms governing its biogenesis remain poorly defined. To investigate this process, we developed an enzymatic approach to ablate preexisting Golgi and induce large-scale de novo organelle formation. Transcriptional profiling of cells rebuilding the Golgi revealed a coordinated induction of a broad gene network, coinciding with structural and functional maturation of the organelle. This network spans all Golgi subcompartments and activities, supporting the existence of a unified "Golgi regulon" that synchronizes expression of components required for organelle integrity. Failure to activate this regulon impaired Golgi reassembly. Promoter analysis and RNAi screening identified CREB3L1 as a central transcription factor for Golgi gene activation. Following Golgi loss, CREB3L1 translocates to the nucleus and activates its target genes, thereby driving biosynthesis of the components that are essential for organelle assembly. These findings demonstrate that Golgi biogenesis is governed by a coordinated transcriptional program that promotes organelle regeneration and enables secretory-pathway plasticity. This mechanism may support physiological remodeling by coupling Golgi biogenesis to cellular demand.
Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) represent the most investigated type of "immunosuppressive" neutrophils in humans. However, a detailed understanding of human PMN-MDSCs has been hindered for...Polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) represent the most investigated type of "immunosuppressive" neutrophils in humans. However, a detailed understanding of human PMN-MDSCs has been hindered for years by limited knowledge of their specific markers for sorting and purification, as well as by their very low numbers in patient circulation. Recently, the use of advanced technologies has shown that human PMN-MDSCs are generated during emergency granulopoiesis and comprise a heterogeneous population of both immature and mature neutrophils. It has also been shown that human mature PMN-MDSCs (mPMN-MDSCs) perform immunosuppressive functions through transcriptional reprogramming during maturation. Furthermore, single-cell RNA sequencing experiments have shown that human mPMN-MDSCs and tumor-associated neutrophils (TANs) share transcriptomic similarities, therefore uncovering a potential relationship between them. In this Perspective, we discuss: i) how our knowledge on human PMN-MDSCs has advanced; ii) the criteria for discriminating human PMN-MDSCs from normal neutrophils or other immunosuppressive neutrophil populations; iii) recent findings revealing that human mPMN-MDSCs exhibit transcriptomic features suggestive of functions beyond immunosuppression.
The antiviral protein MORC3 is frequently inhibited by viruses. To counteract viral antagonism, MORC3 represses a noncanonical pathway of type-I-interferon (IFN) such that viral inhibition of MORC3 triggers ( > 10,000-fo...The antiviral protein MORC3 is frequently inhibited by viruses. To counteract viral antagonism, MORC3 represses a noncanonical pathway of type-I-interferon (IFN) such that viral inhibition of MORC3 triggers ( > 10,000-fold) IFN induction. How MORC3 represses this pathway, and why IFN induction upon MORC3 loss is so potent without canonical IRF3/7 transcription factors, is unknown. Here, we show that MORC3 restricts chromatin accessibility at tandem repeat elements harboring up to 61 homotypic transcription factor motifs. One such element becomes a potent enhancer of IFNB1 upon MORC3 loss. Its motif cluster contains 45 PU.1 binding sites and is necessary and sufficient for MORC3-mediated repression and enhancer activity upon MORC3 loss. PU.1 recruits MORC3 to repress this enhancer by recruiting DAXX and enabling H3.3 incorporation. Upon MORC3 loss, PU.1 drives IRF3/7-independent IFN induction. Other restricted tandem repeats contain homotypic motif clusters of SPI, AP-1, and SP/KLF transcription factors. Our findings uncover a TF motif cluster-driven repression mechanism by MORC3 at tandem repeats, enabling specific repression of an IFNB1 enhancer such that viral antagonism of MORC3 induces interferon.