The activation of the embryonic genome is a crucial step in development. In addition to thousands of genes, many transposable elements (TEs) are robustly transcribed during early mammalian development. However, their tra...The activation of the embryonic genome is a crucial step in development. In addition to thousands of genes, many transposable elements (TEs) are robustly transcribed during early mammalian development. However, their transcriptional regulators remain largely unexplored. Here, we set out to identify transcription factors regulating the expression of TEs from the LINE, SINE and ERVL families during mouse preimplantation development. In particular, the MaLR family are the most abundant ERVL in the mouse genome and are also the most abundant constituent of the transcriptome in early mouse embryos. We find that the general transcription factor TBP binds and activates MaLRs in mouse embryos. Loss-of-function of TBP leads to downregulation of MaLRs, specifically the ORR1A family, which is the youngest ORR subclass and contributes a significant portion of major zygotic genome activation transcripts. Our work identifies regulators of TE expression in vivo and highlights a previously unrecognised role for the general transcription factor TBP in regulating a highly specific TE transcriptional programme.
Ubiquilins are molecular chaperones that play multifaceted roles in proteostasis, with point mutations in UBQLN2 leading to altered phase-separation properties and amyotrophic lateral sclerosis (ALS). Our mechanistic und...Ubiquilins are molecular chaperones that play multifaceted roles in proteostasis, with point mutations in UBQLN2 leading to altered phase-separation properties and amyotrophic lateral sclerosis (ALS). Our mechanistic understanding of this essential process has been hindered by a lack of structural information on the STI1 domain, which is essential for ubiquilin chaperone activity and phase separation. Here, we present the first crystal structure of a ubiquilin-family STI1 domain bound to a transmembrane domain (TMD), and show that ALS mutations disrupt the STI1-TMD interaction. We further demonstrate that ubiquilins contain multiple conserved internal sequences that bind to the STI1 domain, including the PXX-repeat region that is a hotspot for ALS mutations. We propose that these placeholder sequences prevent solvent exposure of the STI1 hydrophobic groove and contribute to the multivalency that drives ubiquilin phase-separation. Together, this work provides a new paradigm for understanding how STI1 domains modulate ubiquilin chaperone activity and phase separation, and offers insights into the molecular basis of ALS pathogenesis.
Meiotic prophase I, characterized by homologous recombination and synapsis, is a critical step in spermatogenesis. This process entails extensive changes to chromatin and transcription. Prior to prophase I, accessible ch...Meiotic prophase I, characterized by homologous recombination and synapsis, is a critical step in spermatogenesis. This process entails extensive changes to chromatin and transcription. Prior to prophase I, accessible chromatin bound by paused Pol II at meiotic gene promoters is essential for their timely activation later during meiosis. However, the factors responsible for establishing accessible chromatin at meiotic gene promoters before entry into prophase I are unknown. Here, we discovered that NFYA, expressed in pre-meiotic germ cells, regulates accessible chromatin at meiotic gene promoters, including those activated by the STRA8/MEISON axis. Concordantly, conditional germline deletion of Nfya in male mice blocks meiotic entry. Single-cell ATAC-seq analysis shows that loss of NFYA in pre-meiotic cells disrupts accessible chromatin at poised meiotic gene promoters. These findings establish NFYA as a regulator of accessible chromatin at meiotic gene promoters and of the timely activation of the meiotic genetic program.
Bacteriophages have evolved diverse inhibitors targeting key bacterial processes, including virulence and anti-phage defense systems, which could inspire novel antimicrobial strategies and enhance phage therapy approache...Bacteriophages have evolved diverse inhibitors targeting key bacterial processes, including virulence and anti-phage defense systems, which could inspire novel antimicrobial strategies and enhance phage therapy approaches. In this study, we characterize Dap2, a protein encoded by a Pseudomonas aeruginosa phage PaoP5, which disrupts host virulence by sequestering the type III secretion system (T3SS) transcriptional activator ExsA, thus suppressing bacterial pathogenicity. Furthermore, Dap2 also directly binds the host Lon protease to prevent degradation of the phage-encoded HNH endonuclease. Deletion of dap2 in PaoP5 strongly impairs phage genome packaging due to insufficient levels of HNH. Finally, Dap2 synergizes with its genomically adjacent partner Dap1, a previously identified HNH-binding protein providing partial Lon resistance, to completely protect HNH against degradation. Together, these findings reveal a dual-function phage protein that simultaneously modulates bacterial virulence and anti-phage immunity, and showcase a synergistic mechanism for complete neutralization of bacterial defense system against which individual components provide only partial protection.
Animals activate regenerative processes to repair injuries and restore homeostasis following tissue damage. A central question in regeneration is how damage signals are sensed and translated into regenerative growth. Tis...Animals activate regenerative processes to repair injuries and restore homeostasis following tissue damage. A central question in regeneration is how damage signals are sensed and translated into regenerative growth. Tissue injuries lead to the release of intracellular contents and bodily fluids and disturb the osmotic balance. However, the role of osmolarity in regeneration remains largely unexplored. Using Drosophila and mouse intestine, as well as samples from inflammatory bowel disease (IBD) patients, we identify a key role for the osmolarity-sensing WNK-OXSR1 kinase cascade in intestinal regeneration. Mechanistically, OXSR1 phosphorylates the RhoB GTPase at threonine 37 upon intestinal injury, thereby disrupting its interaction with ARHGAP17 and increasing the levels of GTP-bound RhoB. RhoB activation in turn leads to enhanced F-actin polymerization and YAP activation, thus promoting tissue regeneration. We further show that pharmacological inhibition of WNK or OXSR1 reduces the oncogenic potential of intestinal regeneration. These findings reveal osmolarity as a critical damage signal in regeneration and position WNK-OXSR1 as a potential therapeutic target for stimulating intestinal repair.
The exceptional virulence of the human malaria parasite, Plasmodium falciparum, is attributed to the adhesive properties of infected red blood cells and the parasite's ability to avoid antibody recognition through antige...The exceptional virulence of the human malaria parasite, Plasmodium falciparum, is attributed to the adhesive properties of infected red blood cells and the parasite's ability to avoid antibody recognition through antigenic variation. Both properties are derived from the hypervariable surface protein PfEMP1, which is encoded by members of the multi-copy var gene family. Waves of parasitemia during an infection are thought to correspond to var transcriptional switching, enabling parasites to avoid elimination by antibodies targeting previously expressed forms of PfEMP1. The mechanisms underlying and regulating var transcriptional switching remain incompletely understood. Here, we show how transient activation of the var2csa locus mediates var switching, while the expression of non-coding RNAs from this locus contributes to repression of var2csa transcription and affects var switching frequencies. Furthermore, we find that an upstream open reading frame in the 5'-untranslated region of the var2csa transcript destabilizes the var2csa mRNA through the induction of the nonsense-mediated RNA decay pathway. This process promotes transcriptional activation of an alternative var gene. Our findings provide molecular insights into the coordinated transcriptional switching of the var gene family, which contributes to chronic infection.
The encephalomyocarditis virus (EMCV) internal ribosomal entry side (IRES) and other Type 2 IRESs favor translation of the viral genome during infection. The domains H-L of these IRESs specifically interact with the cell...The encephalomyocarditis virus (EMCV) internal ribosomal entry side (IRES) and other Type 2 IRESs favor translation of the viral genome during infection. The domains H-L of these IRESs specifically interact with the cellular translation initiation factors eIF4G/eIF4A through their essential JK domain. However, the JK domain is not sufficient for IRES activity, which also strictly requires the preceding domain I of unknown function. To identify interactions that drive ribosomal attachment to eIF4G/eIF4A-bound Type 2 IRESs, we determined the cryo-EM structure of 48S initiation complexes formed on the EMCV IRES. The apical cloverleaf of domain I contacts ribosomal proteins uS13 and uS19 via its subdomain Id, whereas the essential GNRA tetraloop in subdomain Ic interacts with the TψC domain of initiator tRNA. The IRES-tRNA interaction also provides a mechanism for release of the IRES after eIF2 is replaced by eIF5B during subunit joining to allow attachment of 60S subunits. Functional assays supported the exceptional role of these interactions for initiation on this IRES. The strong conservation of the apex of domain I amongst Type 2 IRESs suggests that the reported interactions provide a common general mechanism of ribosomal attachment on them all.
Pan L, Zhu S, Yang SL
… +12 more, Mellor N, Iacobini FR, Zhu T, Neyt P, van de Cotte B, Vandorpe M, Swarup R, Van Damme D, Geisler MM, Gevaert K, Band LR, De Smet I
Gravity-directed growth ensures that shoots and roots grow upwards and downwards, respectively. To achieve this, the organ's angle with respect to gravity drives the asymmetric redistribution of the plant hormone auxin a...Gravity-directed growth ensures that shoots and roots grow upwards and downwards, respectively. To achieve this, the organ's angle with respect to gravity drives the asymmetric redistribution of the plant hormone auxin and consequently differential elongation, thus creating a curvature of the organ. In addition to efflux transporters, the auxin influx transporter AUXIN RESISTANT 1 (AUX1) is essential for auxin redistribution during root gravitropism. Here, we show that the F-box protein SLOMO regulates AUX1 via two distinct mechanisms. First, SLOMO promotes proteolytic degradation of AUX1, possibly in an indirect manner. Second, SLOMO controls the ubiquitination of K261, K264, and K266, thus potentially affecting AUX1 auxin transport properties that are regulated by these residues. This reveals a dual mode of SLOMO-mediated regulation of AUX1, including a novel, non-proteolytic role for SLOMO-mediated ubiquitination in addition to SLOMO-mediated degradation of AUX1.
Advanced paternal age is associated with reduced fertility and increased health risks in offspring, but the molecular events that convert “chronological age” into “biological risk” remain incompletely defined. Recent wor...Advanced paternal age is associated with reduced fertility and increased health risks in offspring, but the molecular events that convert “chronological age” into “biological risk” remain incompletely defined. Recent work by Shi et al (2026) uncovers a conserved sharp transition, i.e., an “aging cliff”, in mouse and human sperm small non-coding RNA profiles, suggesting roles of age-dependent remodeling of the sperm epigenetic playload in early embryogenesis and transgenerational outcomes.
Targeting β-oxidation has been proposed as a strategy for shortening tuberculosis (TB) treatment by killing non-replicating Mycobacterium tuberculosis within granulomas where the pathogen relies on host-derived lipids. T...Targeting β-oxidation has been proposed as a strategy for shortening tuberculosis (TB) treatment by killing non-replicating Mycobacterium tuberculosis within granulomas where the pathogen relies on host-derived lipids. The protein EtfD is thought to couple β-oxidation of fatty acids with the respiratory chain in mycobacteria. However, the structure of EtfD is not known and, as the presumed link between two complex processes, its activity has been difficult to measure, impeding its exploitation as a drug target. Here we show that Mycobacterium smegmatis, a fast growing and nonpathogenic model for M. tuberculosis, relies on EtfD for extracting energy from β-oxidation. The electron cryomicroscopy structure of M. smegmatis EtfD reveals an unusual linear [3Fe-4S] cluster that has not been seen in other protein structures, and suggests how EtfD transfers electrons from β-oxidation to the respiratory chain. We devised an assay that couples EtfD activity to a fluorescent readout of proton pumping by the respiratory chain, which can be used to identify compounds that block mycobacteria from using β-oxidation to power oxidative phosphorylation.
Bacterial flagella are essential for motility, but their structure and how they generate movement vary greatly. Most motile bacteria use external helical flagella, whereas spirochetes have periplasmic flagella (PFs) that...Bacterial flagella are essential for motility, but their structure and how they generate movement vary greatly. Most motile bacteria use external helical flagella, whereas spirochetes have periplasmic flagella (PFs) that distort the cell body to drive forward movement. Here, we generated sheath protein knockout mutants and used high-resolution cryo-electron microscopy to elucidate the mechanisms underlying PF assembly, curvature, and rigidity in Leptospira biflexa. The PF consists of a FlaB1-based core filament surrounded asymmetrically by sheath proteins. Weak but essential binding of FlaA2 to the core enables asymmetric localization of the coiling protein FcpA. FcpA alone can induce curvature, whereas FcpB acts as a structural wedge that reinforces PF rigidity and enables efficient swimming in liquid. Specific glycosylation of FlaB1 mediates sheath-core interactions and may guide the assembly of sheath components. We propose that sheath proteins interact transiently with the core and may be anchored to the outer membrane, allowing core rotation beneath a static sheath. These findings reveal how cooperative interactions among sheath components confer structural and mechanical specialization to spirochete flagella.
How can expression of a specific gene be quantitatively regulated? In this review, we discuss two possible modalities. In one, the level of mRNA generated from each gene copy can be smoothly varied giving graded analogue...How can expression of a specific gene be quantitatively regulated? In this review, we discuss two possible modalities. In one, the level of mRNA generated from each gene copy can be smoothly varied giving graded analogue control. In a second, some gene copies generate high mRNA levels whilst others generate very low levels, giving ON/OFF digital control, with the fraction of copies with high or low expression being regulated. We focus on why in different contexts one modality would be preferred over the other, how these two modalities can be generated through transcriptional regulation, and discuss whether ON/OFF control is particularly linked to epigenetic memory. We argue that digital control arises for memory mediated by trans-factor feedback loops and histone modifications, but not necessarily for DNA methylation. We also examine how these expression modes can be established at one specific target, Arabidopsis FLOWERING LOCUS C (FLC). Graded expression and switching to ON/OFF control occur during early development and during long-term cold exposure and both are key to FLC regulation.
Plasmids are the workhorses of molecular biology: fast, flexible, and often taken for granted. We clone, overexpress, tag, and mutate freely, assuming they will faithfully produce RNA transcripts that match the intended...Plasmids are the workhorses of molecular biology: fast, flexible, and often taken for granted. We clone, overexpress, tag, and mutate freely, assuming they will faithfully produce RNA transcripts that match the intended DNA sequence. This assumption is rarely tested and often invalidated. Sequences in plasmid backbones, epitope tags, and codon-optimized regions may inadvertently harbor cryptic promoters or splice sites. The resulting unexpected transcripts and proteins, while often undetected, can distort results and propagate false conclusions through papers, grants, and even clinical trials. In this perspective, we highlight published cases where plasmids have distorted results and misled interpretation. We examine the mechanisms and consequences of plasmid-associated expression artifacts and offer practical strategies to minimize them. Finally, we call for a revision of community standards for experiments using transgenes: deposit complete plasmid sequences and verify the resulting transcripts using RNA-seq.
The mammalian genome is organised into large topologically associating domains (TADs) and smaller sub-TADs or enhancer-promoter loops, which may contribute to the regulation of gene expression. These dynamic structures a...The mammalian genome is organised into large topologically associating domains (TADs) and smaller sub-TADs or enhancer-promoter loops, which may contribute to the regulation of gene expression. These dynamic structures arise, at least partly, via cohesin-mediated loop extrusion delimited by insulator elements. By studying the structure and function of the alpha-globin locus during erythroid differentiation, we have previously shown that the juxtaposition of the enhancers and promoters during this process partly depends on cohesin-mediated loop extrusion, which appears to be delimited by 12 largely convergently orientated CTCF boundary elements. To define the downstream boundary of the sub-TAD, we removed four CTCF sites in informative combinations. This showed that rather than CTCF insulators, it is the transcriptionally active alpha-globin gene that defines the downstream boundary of the sub-TAD. Further, insertion of actively transcribed fragments of the α-globin gene between the enhancers and native genes leads to a reduction in native α-globin expression and accumulation of cohesin at the insertion site. This highlights an overlap in the functional role of the fundamental elements of the genome.
Current human pluripotency models are hindered by the need for co-culture with little-defined mouse feeder cells. In this issue, Rossignoli et al (2026) demonstrate that naïve human pluripotent stem cells (hPSCs) can be...Current human pluripotency models are hindered by the need for co-culture with little-defined mouse feeder cells. In this issue, Rossignoli et al (2026) demonstrate that naïve human pluripotent stem cells (hPSCs) can be stably maintained long-term on serum-coated substrates without feeder cells, retaining their transcriptional and functional properties, and offering a scalable, reproducible, and broadly accessible platform for studying early human development.
Rossignoli G, Oberhuemer M, Brun IS
… +21 more, Zorzan I, Osnato A, Wenzel A, van Genderen E, Drusin A, Panebianco G, Magri N, Becker M, Solis MA, Colantuono C, van Knippenberg SSFA, Pham TXA, Khodeer S, Grumati P, Cacchiarelli D, Martini P, Rivron N, Pasque V, Żylicz JJ, Leeb M, Martello G
Naive human pluripotent stem cells (hPSCs) represent a pre-implantation epiblast state able to efficiently differentiate into embryonic and extraembryonic pre-implantation lineages and to self-organise in vitro into blas...Naive human pluripotent stem cells (hPSCs) represent a pre-implantation epiblast state able to efficiently differentiate into embryonic and extraembryonic pre-implantation lineages and to self-organise in vitro into blastocyst-like structures called blastoids. Naive hPSC maintenance routinely relies on co-culture with mouse embryonic fibroblast (MEFs) as feeder cells, a method prone to variability and analytical confounders. Here, we describe a feeder-free culture system based on serum coating that supports long-term maintenance of naive hPSCs. Across five laboratories, 30 serum batches were evaluated for the expansion of eight naive hPSCs lines for up to 25 passages. Mass spectrometry analysis identified fibronectin and collagens as extracellular matrix proteins consistently present in serum coating. Cells cultured on serum coating displayed growth kinetics, clonogenic capacity, mutation rates, and global gene expression profiles comparable to MEF-based cultures. Importantly, serum-cultured naive hPSCs efficiently underwent germ layer specification, retained trophectoderm competence, and generated blastoids with efficiency similar to MEF-based cultures. Collectively, serum coating provides a scalable, cost-effective, and robust alternative to feeder-based systems, preserving genomic stability and developmental potential while eliminating MEF-associated disadvantages and variability. This platform facilitates large-scale applications of naive hPSCs and enables more reproducible mechanistic studies.
The endoplasmic reticulum (ER) is an important site for accurate folding and processing of secretory and membrane proteins. Signal peptides within such proteins are recognized by the signal recognition particle (SRP), wh...The endoplasmic reticulum (ER) is an important site for accurate folding and processing of secretory and membrane proteins. Signal peptides within such proteins are recognized by the signal recognition particle (SRP), which guides them to the ER. When this process is impaired, cells rely on quality control mechanisms to prevent the accumulation of misfolded or mislocalized proteins. One of these mechanisms, known as regulation of aberrant protein production (RAPP), detects nascent proteins with aberrant signal peptides and degrades their mRNA templates. Using functional genetic screens, we identify the zinc finger antiviral protein (ZAP) as a key component of the RAPP pathway. Proteomics and enhanced UV-crosslinking and immunoprecipitation (eCLIP) experiments reveal that the short isoform ZAP-S associates with SRP components and facilitates degradation of aberrant mRNAs. ZAP-S recognizes faulty proteins early in their biogenesis and targets their corresponding mRNAs for degradation. Loss of ZAP activates the unfolded protein response and the downstream integrated stress response, highlighting its central role in safeguarding protein targeting and maintaining cellular homeostasis.
N-methyladenosine (mA) and its binding proteins are critical regulators of gene expression and development in mammals. Despite its extent and importance, the regulatory mechanisms of mA-binding reader proteins Ythdf1, Yt...N-methyladenosine (mA) and its binding proteins are critical regulators of gene expression and development in mammals. Despite its extent and importance, the regulatory mechanisms of mA-binding reader proteins Ythdf1, Ythdf2, and Ythdf3 during the early stages of mammalian development remain incompletely understood. Here, we show that Ythdf2 and Ythdf3, but not Ythdf1, are required for early embryo development in mice. Mechanically, we demonstrate that all three Ythdf proteins mediate the decay of their target transcripts by binding to similar mA sites, including maternal mRNAs, mid-preimplantation-activated transcripts, and retrotransposon RNAs. Among these, retrotransposon B2 RNAs emerge as one of the primary targets of Ythdf proteins throughout early embryo development, and deficiency in Ythdf1-3 leads to the accumulation of SINE/B2 RNAs, which, in turn, attenuates RNA polymerase II (Pol II) transcription through trans-regulatory mechanisms. In parallel, Ythdf1-3 deficiency represses Pol III-driven B2 transcription, thereby modulating RNA polymerase II activity at genomic regions adjacent to B2 loci via cis-regulatory effects. Together, the coordinated regulatory axis of Ythdf-SINE/B2-gene expression governs a broad transcriptional network that is crucial for embryogenesis.
VE-cadherin controls endothelial junction integrity, and thereby inflammation-induced vascular permeability and leukocyte extravasation. The adhesive function of VE-cadherin is influenced by its binding to β-catenin, whi...VE-cadherin controls endothelial junction integrity, and thereby inflammation-induced vascular permeability and leukocyte extravasation. The adhesive function of VE-cadherin is influenced by its binding to β-catenin, which is linked by α-catenin to actin. Plakoglobin can replace β-catenin in such complexes, and both types of complexes co-exist in endothelial cells. Here, we have investigated whether β-catenin and plakoglobin differ in their relevance for controlling endothelial junctions. Based on gene silencing in vitro and conditional endothelium-specific gene inactivation in mice in vivo, we found that both leukocyte diapedesis through endothelium and induction of vascular permeability by inflammatory mediators depend on plakoglobin, but not β-catenin. Mechanistically, we demonstrated that plakoglobin is crucial for the generation of tension across VE-cadherin by transmigrating leukocytes and by inflammatory mediators, whereas β-catenin was dispensable in this context. Transgenic mice expressing a VE-cadherin tension sensor revealed that plakoglobin is essential in vivo for histamine-induced tension across VE-cadherin. Thus, plakoglobin, but not β-catenin, is needed for leukocyte diapedesis, the induction of vascular permeability, and the stimulation of mechanical tension across VE-cadherin.
Many organisms maintain generalized stress responses activated by adverse conditions. A common theme is the induction of stress-defense proteins with reduced production of growth-promoting proteins, including ribosomes....Many organisms maintain generalized stress responses activated by adverse conditions. A common theme is the induction of stress-defense proteins with reduced production of growth-promoting proteins, including ribosomes. Yet the precise roles of these coupled programs are difficult to dissect. Here, we investigated Saccharomyces cerevisiae responding to salt as a model stressor. We used molecular, genomic, and single-cell microfluidic methods to examine the interplay between transient induction of stress-defense genes and coordinated repression of growth-promoting genes in the yeast environmental stress response (ESR). Loss of transcriptional inducers Msn2/4 accelerates growth during multiple mild stress doses, at the expense of acquired tolerance to subsequent severe stresses. In contrast, loss of Dot6/Tod6 repressors of growth-promoting genes delays stress acclimation, showing that gene repression accommodates the cost of the Msn2/4 response. Msn2/4 bind the DOT6 promoter, influence Dot6 abundance and activation dynamics, and are required for full repression of Dot6 targets and other growth-promoting genes. Thus, Msn2/4 participate in regulating resource reallocation needed to induce their transcripts, underscoring a common theme in stress responses utilized in other organisms.