EMBO reports

The master kinases of the DNA damage response (DDR), ATR, ATM and DNA-PK, become active in response to DNA damage and orchestrate a downstream wave of phosphorylations contributing to DNA damage repair and preservation of cellular homeostasis. Of them, we recently demonstrated that ATM binds the pool of the lipid phosphatidyl-inositol-4-phosphate (PI4P) situated at the Golgi membrane. Depending on PI4P availability at Golgi membranes, ATM is more or less titrated away from the nucleus, which translates into responses to nuclear DNA damage of matching intensity. Building on this knowledge, in this work we asked if, beyond the Golgi merely serving as a docking platform that retains ATM away from the nucleus, ATM does exert any role important for Golgi biology. We found that ATM maintains Golgi morphology by counteracting its excessive deployment. This occurs both by its mere presence (likely antagonizing the Golgi-stretching action of the protein GOLPH3) and by phosphorylating Golgi-resident substrates. Of relevance, we also report that the morphological alterations caused to the Golgi without ATM affect the biology of a model Golgi cargo. Our findings nourish the growing evidence that kinases of ATMs family display functional interactions with membranes and highlights an underappreciated crosstalk between the Golgi and the nucleus.

Oxidative phosphorylation is the most efficient way of generating ATP in respiring cells. As high energy electrons are the major source of reactive oxygen species their production needs to be carefully calibrated. In most organisms, NADH dehydrogenase serves as the primary source and gateway of electrons. This complex is responsible for oxidizing NADH to NAD+, which liberates two electrons that are then fed into the respiratory chain. In the Gram-positive model bacterium, Bacillus subtilis, a transcription factor (Rex) is utilized to monitor the rise in NADH level and subsequently increase the production of the NADH dehydrogenase Ndh. Thus, the generation of electrons through this pathway is tightly regulated. In this report, we reveal the presence of another independent mechanism to moderate Ndh activity involving a previously uncharacterized protein, YfhS. Additionally, we present the first experimental evidence showing that the functional NADH dehydrogenase is a two-protein complex comprised of a membrane-associated YjlC and the enzyme Ndh. We find that absence of YfhS leads to cell morphology and growth defects that are corrected by spontaneous mutations in ndh. We note that increased production of NADH dehydrogenase complex proteins by itself is not detrimental. However, strikingly, it is lethal in a strain lacking yfhS. These results reveal that YfhS is an important moderator of NADH dehydrogenase activity. We also demonstrate that YfhS and YjlC are interaction partners. A model developed based on our data indicates that YfhS is an important regulator of intracellular NADH concentration. Compounds that target specific microbial (Type II) NADH dehydrogenase, which is absent in human mitochondria, are considered promising drug candidates to help address the threat posed by antibiotic-resistant bacteria. Overall, our data unveiling the importance of YfhS and YjlC in controlling Ndh activity could be harnessed for the development of new therapeutics.

Epidermal Growth factor (EGF) signaling is associated with (oncogenic) proliferation. Conversely, EGF-family ligands are able to trigger a differentiation program in cultured cells, an effect attributed to ligand affinity and EGFR phosphorylation. How EGF/EGFR driven proliferation-differentiation dynamics underlie tissue self-renewal has not been addressed. We show that culturing mouse small intestinal organoids (mSIOs) without EGF enhanced EGFR expression and base phosphorylation while maintaining a balanced development of proliferative crypts and differentiated villi. Addition of EGF or EREG triggers receptor endocytosis, reducing cell-surface and expression levels. While EGF promoted crypt proliferation, EREG promoted both proliferation and villus differentiation compared to untreated controls. Removal or re-introduction of EGF or EREG proved sufficient to induce development comparable to constant presence of ligands over 96h. Sub-saturating concentrations of EGF led to increased villus differentiation, resembling EREG treatments, suggesting that control over EGFR endocytic cycle ultimately regulates the balance of proliferation and differentiation in mSIOs SummaryExpression and signaling competency at the plasma membrane of EGFR drives crypt proliferation vs villus differentiation by medium ligand-composition, aiding mouse intestinal organoids self-renewal and regeneration.

The suppression of type I interferon (IFN) signalling by the ISG15-USP18-STAT2 inhibitory complex (ISG15 IC) is an established regulatory mechanism of the antiviral response. However, a molecular understanding of how the ISG15 IC forms to suppress IFN signalling is still emerging. Here, we use AlphaFold modelling in conjunction with biochemical and biophysical approaches to elucidate the interactions of this multiprotein assembly. Our analysis identified a unique STAT2 binding loop (SBL) in USP18, which is critical for the USP18-STAT2 association. Further biochemical characterisation through site-directed mutagenesis confirmed the importance of residues within and surrounding the SBL, enabling the design of mutants with both increased and decreased binding affinities. Moreover, several USP18 and STAT2 patient mutations severely disrupted this interaction. Lastly, using influenza B virus (IBV) and Zika virus (ZIKV) proteins, we investigated the influence of these viral effector proteins on these interactions. Taken together, these results provide much-needed insights into a key aspect of IFN signalling control.

Crossing over during meiosis drives genetic diversity and ensures the accurate segregation of homologous chromosomes. Variation in the rate of crossing over has been linked to evolutionary divergence and environmental adaptability, shaping fitness and responses to selective pressures. Despite its significance, the molecular mechanisms underlying this variation remain poorly understood. Crossover sites are selected from a large pool of potential sites initiated by programmed DNA double-strand breaks. Post-translational modification by SUMO (Small Ubiquitin-like Modifier) has been implicated in this process. Here, we show that crossover rate, chromosome length, and abundance of chromosome-associated SUMO are positively correlated across a range of vertebrate species, including mouse, chicken, pig, cattle, sheep, and goat. Crossover variation between goat breeds across the Indian subcontinent was also positively correlated with chromosomal SUMO level. Furthermore, modulating SUMO levels in cultured goat spermatocytes altered crossover frequency. Cumulatively, these observations point to a central role for SUMO in mediating crossover variation both between and within vertebrate species.

Following their engagement towards differentiation, tissue stem cells often transit through a precursor state that is difficult to define because of its transient nature; similarly, the precise role of lineage precursors in implementation of tissue architecture and function is unknown. In the present work, we used two mouse models of deficient feedback regulation to characterize precursors of the pituitary corticotrope lineage that regulates the stress response. Both the POMC knockout and adrenalectomized mouse models develop glucocorticoid deficiency and compensatory accumulation of corticotrope precursors that have so far eluded characterization. We found that pre-corticotrope differentiation depends on the lineage-specific factor Tpit and is repressed by glucocorticoids. We identified brain-derived neurotrophic factor (BDNF) as the signal that engages pituitary stem cells towards differentiation in these models as well as in normal pituitary development. A glucocorticoid-sensitive BDNF autocrine loop active in pre-corticotropes turns these cells into signaling hubs for maintenance of pituitary-adrenal homeostasis. HighlightsO_LIPituitary lineage precursors expand in conditions of deficient feedback regulation C_LIO_LIBDNF mobilizes pituitary stem cells during establishment of tissue size and architecture C_LIO_LICorticotrope precursors are a signaling hub for tissue homeostasis C_LI

Persistence of memory T lymphocytes, in the apparent absence of antigen, is a hallmark of immune memory and key to adaptive immunity to recurrent infections. The signaling pathways ensuring survival and quiescence of the memory T cells are largely enigmatic. Here we show, by inhibition in vivo, that persistence of surface CD69+KLF2-tissue-resident memory T cells of murine bone marrow and spleen is blocked by antibodies to the integrins VLA-4 and LFA-1, connecting the memory T cells to VCAM1 and ICAM1 of stromal cells. Persistence requires the PI3K/AKT signaling pathway, since it is blocked by Wortmannin, and it involves PI3K-dependent survival genes. Surface CD69-KLF2+ memory T cells of the bone marrow are also dependent on integrin-mediated contact to stromal cells. Their persistence critically depends on the NF-kB pathway, their PI3K signaling pathway is not relevant. Blocking Jak1 and 3 of the interleukin-7 and -15 signaling pathways does affect memory T cells of the spleen, but not those of the bone marrow. Thus, tissue-resident KLF2+ and KLF2-memory T cells, and memory T cells of spleen and bone marrow, use different signaling pathways, adapting them to their respective tissues and reflecting an unexpected heterogeneity in the molecular mechanisms of persistence.

Pancreatic beta cells have the unique function of synthesizing and secreting high amounts of the inhibitory neurotransmitter {gamma}-aminobutyric acid (GABA). The mechanism of GABA secretion, whether vesicular or channel-mediated, is debated. Our study reveals surprising temporal complexity in the pattern of islet GABA secretion. We used insulin secretion modulators to demonstrate that GABA release is not directly correlated with insulin secretion. VGAT reporter mice also showed that beta cells do not express the requisite vesicular GABA transporter (VGAT) for vesicular GABA release. Instead, GABA is secreted from the cytosol in pulses by the LRRC8A/D isoform of the volume regulatory anion channel (VRAC). We further demonstrate the dynamic coordination of GABA release with calcium influx in beta cells and dependence on beta cell depolarization. These results suggest a model where GABA is released during the peaks of beta cell calcium oscillations to provide feedback which strengthens and reinforces the oscillation waveform.

The restart of replication forks that have become stalled or disintegrated during the replication cycle is vital for all organisms, and in many bacterial species involves the conserved and essential DNA helicase PriA. PriA has been shown to physically interact with the C-terminus of SSB, which also binds to several other proteins involved in DNA repair and restart. It has been proposed that PriA is enriched at all replication forks in Bacillus subtilis via SSB interaction, such that it is instantly present to respond to a requirement for restart. Using single molecule tracking, we show that SSB and PriA are comprised of populations having very different diffusion constants, ruling out that PriA is co-migrating with fork-bound SSB. Indeed, PriA was only enriched at a subset of cells in exponentially growing cells, dependent on the C-terminus of SSB, but largely showed confined motion through the entire genome, searching for target sites in a transcription factor-like manner. Upon stalling of forks, SSB became highly enriched in all cells, suggesting a first line of response. PriA was also visibly enriched at forks following replication stress, in contrast to primosome proteins DnaD and DnaI, who showed only moderate changes in localization or in single molecule motion. PriA dwell times were affected by the lack of the SSB C-terminus, and also by the absence of RecG helicase, which is involved in recombination events. Heterogeneity of restart proteins at replication forks also extends to translesion DNA polymerases PolY1 and PolY2. Both proteins are low-abundant such that a considerable fraction of cells is devoid of any molecule. Our findings show that SSB accumulation is an initial response to replication stress, and that translesion synthesis and lesion skipping are less frequent events than fork remodelling.

AO_SCPLOWBSTRACTC_SCPLOWMeiotic DNA double-strand break (DSB) formation and repair by homologous recombination is crucial for ensuring proper chromosome segregation. In mice, the mini-chromosome maintenance family protein, MCM8, has been proposed to function in meiotic recombination and its loss leads to infertility, but the underlying mechanisms are poorly understood. Here we used cytological and genomic assays to infer the role of MCM8 during meiotic recombination in mouse spermatocytes. We show that MCM8-deficient spermatocytes exhibit increased levels of SPO11-dependent DSBs at recombination hotspots during early prophase. DSBs are resected normally and accumulate strand-exchange proteins. However, downstream recombination intermediates are barely detected and recombination intermediate-associated MutSgamma foci do not form efficiently. Consistent with a role in early recombination intermediate processing, MCM8 binds to displacement loop (D-loop) structures in vitro. We propose that MCM8 controls meiotic recombination in at least two ways. MCM8 participates in regulating meiotic DSB number. Further, MCM8 plays a role in the formation and/or stability of post-resection recombination intermediates, steps that are critical for DSB repair via recombination and for efficient synapsis of homologous chromosomes during mouse meiosis.

Mitochondrial permeabilization by the apoptotic executioners BAK and BAX represents a critical stage of mitochondrial apoptosis and facilitates the release of pro-inflammatory mitochondrial DNA via herniation of the inner mitochondrial membrane. This study utilises TurboID proximity labelling to investigate the temporal changes of the BAK proximal proteome during mitochondrial herniation and apoptosis. In doing so, we detail a comprehensive BAK proximal proteome, both at steady state and during apoptosis and observe the loss of MICOS complex stability and proximity to the BAK pore as apoptosis proceeds. In addition, we identify the mitochondrial microprotein SLC35A4-MP proximal to the BAK pore and reveal a SLC35A4-MP dependent modulation of OPA1 processing. Furthermore, loss of SLC35A4-MP delays mitochondrial fragmentation in response to a variety of stressors, uncovering a previously unrecognised role for SLC35A4-MP in fine-tuning mitochondrial rearrangement during apoptotic stress.

Germ cells are the only cells of an organism that pass onto the next generation and, hence perpetuate the species. To ensure this, germ cells need dedicated transcriptional repertoire, that ensure specification, proliferation, differentiation and fate maintenance. We previously characterized LSL-1, a conserved zinc-finger transcription factor that acts as a major direct transcriptional activator of genes involved in germ cell development, fate specification, meiosis and genome stability. Here, we show that LSL-1 interacts with the transcription factor HIM-17, the chromatin proteins BRA-2 and XND-1. These proteins are functionally related to LSL-1 and they colocalize at germline gene promoters, forming most likely a transcription-promoting complex. Furthermore, LSL-1 lies in close proximity to members of the COMPASS and the MOF complexes, corroborating the observation that HIM-17 and LSL-1 are required to maintain normal level of H3K4 methylation in the gonad. Finally, we show that LSL-1 interacting partners are necessary to maintain germ cell fate. Altogether, we propose that LSL-1 interacts with transcription regulators and chromatin modifiers to ensure the establishment of the transcriptional repertoire appropriate for germline function as well as for cell fate maintenance.

Fibrosis and pathological stiffening of tissue are driven by mechanical and biochemical signaling pathways. Here, we find that Sun2, an integral inner nuclear membrane component of Linker of Nucleoskeleton and Cytoskeleton (LINC) complexes, is up-regulated in the lung of patients suffering from fibrotic conditions and in fibroblasts during an injury-induced mouse model of lung fibrosis. Sun2 protein levels also increase in primary lung fibroblasts in a substrate stiffness-dependent manner. Sun2-/- primary lung fibroblasts respond to TGF{beta}, become contractile, and express a key marker of extracellular matrix-producing fibroblasts, Cthrc1. Consistent with this, Sun2 is dispensable for myofibroblast formation and repairing the alveolar barrier after bleomycin injury. Remarkably, however, fibrosis does not develop in bleomycin-treated Sun2-/- mouse lungs. This is explained by the requirement for Sun2 to up-regulate genes encoding extracellular matrix proteins. We therefore suggest that Sun2-containing LINC complexes contribute to a mechanical coincidence detection mechanism that acts in concert with canonical TGF{beta} signaling necessary for pathologic extracellular matrix protein production, representing a nuclear mechanosensing node for intervention in fibrotic diseases of the lung.

ABSTRACT/SUMMARYIonotropic GABAA receptors (GABAARs) mediate fast inhibitory neurotransmission in mammalian brains. While recent structural studies have identified that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], a well-established regulator of numerous ion channels, binds to the 1 subunits of GABAARs, the functional relevance of this binding has remained elusive. Here, we combine electrophysiology, molecular dynamics simulations, and a recently developed caged lysine technology to define the role of PI(4,5)P2 in GABAARs. We show that GABAARs are insensitive to acute PI(4,5)P2 depletions by voltage-sensing phosphatase, but sensitivity is conferred by neutralizing the K311 binding site, indicating high-affinity binding. Caging of K311 by use of genetic code expansion recapitulated phenotypes of K311 mutant, conferring sensitivity to PI(4,5)P2 depletion, whereas uncaging restored insensitivity. Furthermore, caging K311 revealed decelerated activation, which then can be accelerated by uncaging. Additionally, PI(4,5)P2-dependence extends to glycine receptors, suggesting PI(4,5)P2 is an important endogenous phospholipid modulator of inhibitory receptor channels.

Ewing sarcoma (EwS) is an aggressive, human-exclusive tumor typically driven by the EWS::FLI1 fusion protein. To assess whether the neomorphic functions of EWS::FLI1 are fundamentally dependent on evolutionarily recent cofactors such as ETS transcription factors (ETS-TFs), Plycomb group (PcG) proteins, CBP/p300, or specific subunits of the BAF complex, we expressed EWS::FLI1 in the model organism Saccharomyces cerevisiae. This minimal system was chosen because several key EWS::FLI 's cofactors possess greatly reduced sequence homology (e.g., BAF) or are lacking altogether (e.g., ETS-TFs, PcG, or CBP/p300). We used co-IP/MS to map the yeast interactome, Chip-Seq to identify gDNA binding sequences, RNA-Seq for global gene expression, and engineered reporters to test conversion of (GGAA) tandem repeats (GGAASat) into neoenhancers. We found that the yeast EWS::FLI1 interactome was more limited and qualitatively distinct from its human counterpart, sharing core machinery (e.g. RNA Polymerase II, FACT) but lacking the BAF/SWI-SNF and spliceosome complexes, and showing strong enrichment for the SAGA chromatin remodeling complex. We also found that EWS::FLI1 binds to hundreds of sites in the yeast genome with a clear preference for putative ETS-TF consensus sequences and (CA) dinucleotide repeats. Yet, EWS::FLI1 expressing cells presented only minimal transcriptional dysregulation, a stark contrast to the extensive changes observed in humans and Drosophila cells. Finally, we found that EWS::FLI1 successfully converted silent GGAASat sequences into active enhancers in yeast. This remarkable result occurs despite the absence of homologs for key human activators, such as CBP/p300, strongly suggesting that EWS::FLI1 can mobilize functionally related, non-homologous pathways to establish neoenhancers at GGAASat sites. Altogether, our results indicate that EWS::FLI1's core ability to drive GGAASat-dependent gene expression is a conserved, ancient property, while GGAASat-independent extensive transcriptome reprogramming is dependent on co-factors and pathways specific to animal cells.

TNFR superfamily members such as 4-1BB sustain T cell responses to control virus infections or tumors. However, the precise role of 4-1BB during an acute infection remains incompletely understood. Here we used mixed bone marrow chimeras and transcriptome analysis to show that intrinsic 4-1BB signaling in lung T cells during influenza A virus (IAV) infection induces the transcriptional coregulator PR domain containing 16 (Prdm16), known for its role in regulating mitochondrial biology in other cell types. T cell-specific deletion of Prdm16 reduced the number of Ag-specific CD8 T cells, with a larger effect on T cells in the lung parenchyma compared to the vasculature or lymphoid tissues. Conversely, Prdm16 overexpression in T cells increased effector and memory CD8 T cell accumulation during IAV infection. Single nuclei transcriptomics suggested that Prdm16 allows the accumulation of T cells with high protein translation and mitochondrial activity. Prdm16 increased genes associated with oxidative phosphorylation and mitophagy. Consistently, Prdm16 overexpressing cells had more compact mitochondrial cristae, which has been associated with more efficient electron transport. Prdm16 also repressed some genes, including Herpes virus entry mediator, which can inhibit T cell responses through B and T lymphocyte attenuator. These findings reveal a 4-1BB-Prdm16 axis that is induced in T cells during viral infection to support T cell accumulation and memory formation.

Signalling via the epidermal growth factor receptor (EGFR) is indispensable for morphogenesis and tissue homeostasis. It is activated by extracellular ligands, typically released from transmembrane precursors by proteolysis. Ligand shedding activity is provided by the conserved rhomboid intramembrane serine proteases in Drosophila, but by the unrelated ADAM family metalloproteases in mammals, leaving the functions of mammalian non-mitochondrial rhomboids underexplored. Using quantitative proteomics, we show that EGFR is the main endogenous substrate of the human rhomboid protease RHBDL2 in keratinocytes. By shedding the EGFR ectodomain, thus producing a decoy receptor, RHBDL2 suppresses EGFR signalling, limiting cell migration and invasion. Conspicuously, RHBDL2 activity is upregulated by elevated intracellular calcium concentration, a condition typical for keratinocyte differentiation. These effects are recapitulated in primary human keratinocytes, and human skin equivalents deficient in RHBDL2 display incomplete differentiation and are morphologically disordered compared to wild type cells. We propose that context-specific fine-tuning of EGFR signalling and sensitivity to cross-talk from other signalling pathways could be important and hitherto overlooked roles of rhomboid proteases in mammals.

The PBAF is one of three biochemically distinct BAF chromatin remodelers in humans. We previously proposed the role of ARID2, a PBAF component, as a bonafide tumor suppressor in colorectal cancer (CRC). Here, we validated loss of tumor suppression under conditions of ARID2 deficiency emanating from a marked reduction in PBAF complex assembly resulting from destabilization of PBAF-specific components BRD7, PHF10, and PBRM1. Transcriptome profiling of ARID2 deficient CRC cells revealed perturbation of disease processes, including CRC and neurodegenerative disorders, as well as CRC relevant pathways including Wnt/{beta}-catenin signalling, but transcript levels of PBAF-specific components remained unchanged, confirmed by RT-qPCR and TCGA data analysis. Our study establishes ARID2 as a critical stabilizer of the PBAF complex of relevance to CRC.

A powerful way to enhance heat tolerance is to prime organisms with a moderate heat treatment to establish a molecular stress memory permitting the survival of the organism when exposed to subsequent heat shocks. While this has been extensively studied in multicellular organisms, we demonstrate that the unicellular red alga Cyanidioschyzon merolae exhibits heat stress memory. We show that, similarly to more complex organisms, thermomemory in this alga is underpinned by transcriptomic reprogramming, with the chloroplast emerging as the main site of gene trainability. Additionally, we find a conserved small heat shock protein (sHSP)-encoding locus in the nuclear genome to be heat-trainable, likely by histone depletion and sustained removal of the repressive mark histone H3 Lysine 27 trimethylation (H3K27me3). Of C. merolaes two sHSPs, only the nuclear-localizing CmsHSP2 is necessary for proper HS memory establishment. Finally, we reveal a role for the H3K27me3-transferase CmE(z) (Enhancer of zeste) in heat stress memory which shapes the transcriptome to recurring heat exposures, beyond regulating the trainable sHSP locus. Overall, our work provides a molecular framework for the regulation of heat stress memory in a unicellular eukaryote.

In vertebrate early embryos and embryonic stem cells, the interferon (IFN)-centered double-stranded RNA (dsRNA) sensing and signaling pathway is markedly suppressed, implying the existence of an alternative repertoire of dsRNA sensors in these stages. We recently reported that dsRNA treatment triggers translation inhibition using Prkra as a sensor in zebrafish and mouse early embryos. Independently, here we show that dsRNA stimulates the expression of a subset of interferon-stimulated genes (ISGs) in the absence of IFN production, establishing a defensive state in mouse embryonic stem cells (mESCs). Upon dsRNA stimulation, the multifunctional DExD/H-box RNA helicase Dhx9 is recruited into dsRNA-induced condensates, where it promotes the recruitment and functional suppression of the Mdm2/Cul4A ubiquitin ligase machinery by excluding their substrate adaptor Ddb1, thereby stabilizing p53 and Stat1. Dhx9, p53 and Stat1 then cooperate to stimulate an ISG response. This signaling is important for the defense against ZIKV infection in mESCs as well as other dsRNA stresses. Such a cascade is conserved in human ESCs, while in zebrafish embryos, p53 but not Stat1 is required for the transcriptional response. Our findings define an immediate, cell-autonomous innate immune pathway operating in ESCs and vertebrate embryos.