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Summary Tumor-associated macrophages (TAMs) play an important role in tumor immunity and comprise of subsets that have distinct phenotype, function, and ontology. Transcriptomic analyses of human medulloblastoma, the most common malignant pediatric brain cancer, showed that medulloblastomas (MBs) with activated sonic hedgehog signaling (SHH-MB) have significantly more TAMs than other MB subtypes. Therefore, we examined MB-associated TAMs by single-cell RNA sequencing of autochthonous murine SHH-MB at steady state and under two distinct treatment modalities: molecular-targeted inhibitor and radiation. Our analyses reveal significant TAM heterogeneity, identify markers of ontologically distinct TAM subsets, and show the impact of brain microenvironment on the differentiation of tumor-infiltrating monocytes. TAM composition undergoes dramatic changes with treatment and differs significantly between molecular-targeted and radiation therapy. We identify an immunosuppressive monocyte-derived TAM subset that emerges with radiation therapy and demonstrate its role in regulating T cell and neutrophil infiltration in MB.

Summary: E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.

SUMMARY Vitamin B12 is known to play critical roles during the development and aging of the brain, and vitamin B12 deficiency has been linked to neurodevelopmental and degenerative disorders. However, the underlying molecular mechanisms of how vitamin B12 affects the development and maintenance of the nervous system are still unclear. Here, we report that vitamin B12 can regulate glial migration and synapse formation through control of isoform-specific expression of PTP-3/LAR PRTP (leukocyte-common antigen-related receptor-type tyrosine-protein phosphatase). We found the uptake of diet-supplied vitamin B12 in the intestine to be critical for the expression of a long isoform of PTP-3 (PTP-3A) in neuronal and glial cells. The expression of PTP-3A cell autonomously regulates glial migration and synapse formation through interaction with an extracellular matrix protein NID-1/nidogen 1. Together, our findings demonstrate that isoform-specific regulation of PTP-3/ LAR PRTP expression is a key molecular mechanism that mediates vitamin-B12-dependent neuronal and glial development., In Brief Zhang et al. describe glial migration and synapse formation defects stemming from vitamin B12 deficiency. Their study identifies a cell-autonomous mechanism regulating glial migration, where vitamin B12 regulates expression of the cell adhesion molecule PTP-3/LAR PRTP in an isoform-specific manner., Graphical Abstract

SUMMARY IQGAP1 is a master regulator of many cellular processes, including intracellular vesicle trafficking and endocytosis. We show that depletion of IQGAP1 in a variety of cell types increases the release of HIV-1 infectious virions and that overexpression diminishes virion production, with neither affecting the early stages of infection. IQGAP1 negatively regulates the steady-state levels of HIV-1 Gag at the plasma membrane, the site of assembly. We establish that IQGAP1 interacts with both the nucleocapsid and p6 domains of Gag, and interaction with either domain is sufficient for its regulatory function. Finally, we demonstrate that IQGAP1 regulation is independent of HIV-1 Gag “late-domains” sequences required by the virus to recruit the cellular ESCRT machinery. Thus, we provide evidence that IQGAP1 is a negative regulatory factor inhibiting efficient budding of HIV-1 by reducing Gag accumulation at the plasma membrane., Graphical Abstract, In Brief IQGAP1 is a ubiquitously expressed master regulator of many cellular processes, including intracellular trafficking. Sabo et al. demonstrate that in a variety of cell types, IQGAP1 acts as a negative regulator of HIV-1 viral particle release by reducing accumulation of the Gag viral structural protein at the plasma membrane.

Summary: Although intramuscular (i.m.) administration is the most commonly used route for licensed vaccines, subcutaneous (s.c.) delivery is being explored for several new vaccines under development. Here, we use rhesus macaques, physiologically relevant to humans, to identify the anatomical compartments and early immune processes engaged in the response to immunization via the two routes. Administration of fluorescently labeled HIV-1 envelope glycoprotein trimers displayed on liposomes enables visualization of targeted cells and tissues. Both s.c. and i.m. routes induce efficient immune cell infiltration, activation, and antigen uptake, functions that are tightly restricted to the skin and muscle, respectively. Antigen is also transported to different lymph nodes depending on route. However, these early differences do not translate into significant differences in the magnitude or quality of antigen-specific cellular and humoral responses over time. Thus, although some distinct immunological differences are noted, the choice of route may instead be motivated by clinical practicality. : Route of immunization, especially intramuscular versus subcutaneous administration, is often debated. Ols et al. use a rhesus macaque model to determine the tissues targeted by a nanoparticle vaccine administered by either route. The authors demonstrate that tissue dissemination is route dependent, but innate and adaptive immune responses develop comparably. Keywords: vaccination, intramuscular, subcutaneous, antigen transport, HIV envelope glycoprotein, dendritic cell, follicular dendritic cell, monocytes, lymph node, B cell follicle

Summary: Oxidation resistance gene 1 (OXR1) protects cells against oxidative stress. We find that male mice with brain-specific isoform A knockout (Oxr1A−/−) develop fatty liver. RNA sequencing of male Oxr1A−/− liver indicates decreased growth hormone (GH) signaling, which is known to affect liver metabolism. Indeed, Gh expression is reduced in male mice Oxr1A−/− pituitary gland and in rat Oxr1A−/− pituitary adenoma cell-line GH3. Oxr1A−/− male mice show reduced fasting-blood GH levels. Pull-down and proximity ligation assays reveal that OXR1A is associated with arginine methyl transferase PRMT5. OXR1A-depleted GH3 cells show reduced symmetrical dimethylation of histone H3 arginine 2 (H3R2me2s), a product of PRMT5 catalyzed methylation, and chromatin immunoprecipitation (ChIP) of H3R2me2s shows reduced Gh promoter enrichment. Finally, we demonstrate with purified proteins that OXR1A stimulates PRMT5/MEP50-catalyzed H3R2me2s. Our data suggest that OXR1A is a coactivator of PRMT5, regulating histone arginine methylation and thereby GH production within the pituitary gland. : Yang et al. show that OXR1A interacts with methyltransferase PRMT5 to promote arginine methylation of histone H3R2 and to regulate transcription of growth hormone in the pituitary gland. Male mice with OXR1A knockout display growth-hormone deficiency and develop fatty liver. Keywords: Oxidation resistance gene 1, OXR1, protein arginine methyltransferase, PRMT1, PRMT5, Arginine Methylation, H3R2me2s, pituitary gland, brain-liver axis, Growth hormone, Non-alcoholic fatty liver disease, NAFLD, neuroendocrine regulation, epigenetic regulation

Summary: CD137, a potent costimulatory receptor for CD8+ T cells, is expressed in various non-T cells, but little is known about its regulatory functions in these cells. In this study, we show that CD137 signaling, specifically in intestinal CD11b−CD103+ dendritic cells (DCs), restricts acute colitis progression. Mechanistically, CD137 engagement activates TAK1 and subsequently stimulates the AMPK-PGC-1α axis to enhance expression of the Aldh1a2 gene encoding the retinoic acid (RA) metabolizing enzyme RALDH2. RA can act on CD11b+CD103− DCs and induce SOCS3 expression, which, in turn, suppresses p38MAPK activation and interleukin-23 (IL-23) production. Administration of RA in DC-specific CD137−/− mice represses IL-23-producing CD11b+CD103− DCs and TH17 cells, indicating that RA is a major inhibitory effector molecule against intestinal CD11b+CD103− DCs. Additionally, the therapeutic effect of the anti-CD137 antibody is abrogated in DC-specific CD137−/− mice. Taken together, our results define a mechanism of paracrine immunoregulation operating between adjacent DC subsets in the intestine. : Jin et al. demonstrate that CD137 signaling functions as an immune checkpoint by controlling the survival and function of regulatory intestinal CD11b−CD103+ dendritic cells to coordinate the balance between regulatory T cells and pathogenic IL-23 during acute colitis. Keywords: CD137, regulatory CD11b−CD103+ DC, Foxp3+ Treg, acute colitis, retinoic acid, RALDH2, IL-23, TH17, immune checkpoint, immune tolerance

Summary: The nucleus accumbens (NAc) contributes to behavioral inhibition and compulsions, but circuit mechanisms are unclear. Recent evidence suggests that amygdala and thalamic inputs exert opposing control over behavior, much like direct and indirect pathway output neurons. Accordingly, opponent processes between these NAc inputs or cell types may underlie efficient reward seeking. We assess the contributions of these circuit elements to mouse operant behavior during recurring conditions when reward is and is not available. Although direct pathway stimulation is rewarding and indirect pathway stimulation aversive, the activity of both cell types is elevated during periods of behavioral suppression, and the inhibition of either cell-type selectively increases unproductive reward seeking. Amygdala and thalamic inputs are also necessary for behavioral suppression, even though they both support self-stimulation and innervate different NAc subregions. These data suggest that efficient reward seeking relies on complementary activity across NAc cell types and inputs rather than opponent processes between them. : Although previous work highlights antagonistic interactions between NAc cell types and inputs, Lafferty et al. show that the suppression of behavioral responding is distributed across D1 and D2 neurons and PVT and BLA inputs. Efficient reward seeking thus arises from complementary activity, rather than opponent processes between these circuit elements. Keywords: nucleus accumbens, basolateral amygdala, paraventricular nucleus of the thalamus, fiber photometry, optogenetics, behavioral inhibition, self-stimulation, place preference, direct pathway, indirect pathway

SUMMARY Brown adipose tissue (BAT) activity protects animals against hypothermia and represents a potential therapeutic target to combat obesity. The transcription factor early B cell factor-2 (EBF2) promotes brown adipocyte differentiation, but its roles in maintaining brown adipocyte fate and in stimulating BAT recruitment during cold exposure were unknown. We find that the deletion of Ebf2 in adipocytes of mice ablates BAT character and function, resulting in cold intolerance. Unexpectedly, prolonged exposure to cold restores the thermogenic profile and function of Ebf2 mutant BAT. Enhancer profiling and genetic assays identified EBF1 as a candidate regulator of the cold response in BAT. Adipocyte-specific deletion of both Ebf1 and Ebf2 abolishes BAT recruitment during chronic cold exposure. Mechanistically, EBF1 and EBF2 promote thermogenic gene transcription through increasing the expression and activity of ERRα and PGC1α. Together, these studies demonstrate that EBF proteins specify the developmental fate and control the adaptive cold response of brown adipocytes., Graphical Abstract, In Brief Angueira et al. show that early B cell factors (EBFs) control both basal and cold-induced thermogenic activity in brown adipocytes. EBF2 is required to maintain BAT fate under basal conditions, ensuring a capacity for thermogenesis upon cold challenge. BAT recruitment during chronic cold exposure requires EBF1 or EBF2 activity.

Summary: During the 2013–2016 Ebola virus (EBOV) epidemic, a significant number of patients admitted to Ebola treatment units were co-infected with Plasmodium falciparum, a predominant agent of malaria. However, there is no consensus on how malaria impacts EBOV infection. The effect of acute Plasmodium infection on EBOV challenge was investigated using mouse-adapted EBOV and a biosafety level 2 (BSL-2) model virus. We demonstrate that acute Plasmodium infection protects from lethal viral challenge, dependent upon interferon gamma (IFN-γ) elicited as a result of parasite infection. Plasmodium-infected mice lacking the IFN-γ receptor are not protected. Ex vivo incubation of naive human or mouse macrophages with sera from acutely parasitemic rodents or macaques programs a proinflammatory phenotype dependent on IFN-γ and renders cells resistant to EBOV infection. We conclude that acute Plasmodium infection can safeguard against EBOV by the production of protective IFN-γ. These findings have implications for anti-malaria therapies administered during episodic EBOV outbreaks in Africa. : Rogers et al. demonstrate that acute Plasmodium infection protects against lethal Ebola virus challenge. Protection is conferred by Plasmodium-elicited interferon gamma (IFN-γ) that causes M1 polarization of tissue macrophages. These studies provide insight into conflicting clinical data regarding whether malaria protects or sensitizes hosts to Ebola virus. Keywords: Plasmodium, Ebola virus, macrophage polarization, co-infection, interferon gamma, malaria, filovirus, innate immunity, macrophage

SUMMARY Defective cholesterol efflux pathways in mice promote the expansion of hematopoietic stem and progenitor cells and a bias toward the myeloid lineage, as observed in chronic myelomonocytic leukemia (CMML). Here, we identify 5 somatic missense mutations in ABCA1 in 26 patients with CMML. These mutations confer a proliferative advantage to monocytic leukemia cell lines in vitro. In vivo inactivation of ABCA1 or expression of ABCA1 mutants in hematopoietic cells in the setting of Tet2 loss demonstrates a myelosuppressive function of ABCA1. Mechanistically, ABCA1 mutations impair the tumor-suppressor functions of WT ABCA1 in myeloproliferative neoplasms by increasing the IL-3Rβ signaling via MAPK and JAK2 and subsequent metabolic reprogramming. Overexpression of a human apolipoprotein A-1 transgene dampens myeloproliferation. These findings identify somatic mutations in ABCA1 that subvert its anti-proliferative and cholesterol efflux functions and permit the progression of myeloid neoplasms. Therapeutic increases in HDL bypass these defects and restore normal hematopoiesis., In Brief Viaud et al. show that ABCA1 mutants identified in CMML patients diminish the tumor-suppressor functions of ABCA1 and cooperate with Tet2 loss to confer the hypersensitivity of myeloid progenitors to IL-3 receptor β canonical signaling, which can be prevented by raising HDL levels., Graphical Abstract

Despite extensive study, the morphogenetic mechanisms of heart looping remain controversial because of a lack of information concerning precise tissue-level deformation and the quantitative relationship between tissue and cellular dynamics; this lack of information causes difficulties in evaluating previously proposed models. To overcome these limitations, we perform four-dimensional (4D) high-resolution imaging to reconstruct a tissue deformation map, which reveals that, at the tissue scale, initial heart looping is achieved by left-right (LR) asymmetry in the direction of deformation within the myocardial tube. We further identify F-actin-dependent directional cell rearrangement in the right myocardium as a major contributor to LR asymmetric tissue deformation. Our findings demonstrate that heart looping involves dynamic and intrinsic cellular behaviors within the tubular tissue and provide a significantly different viewpoint from current models that are based on LR asymmetry of growth and/or stress at the tube boundaries. Finally, we propose a minimally sufficient model for initial heart looping that is also supported by mechanical simulations., 心臓が左右非対称になる仕組みを解明 --細胞集団運動によるダイナミックな形のリモデリング--. 京都大学プレスリリース. 2020-03-18.

Summary: Human naive pluripotent stem cells established from the epiblasts of preimplantation blastocysts provide a useful model for mechanistic studies of pluripotency regulation and lineage differentiation. Important advances have been made to optimize culture conditions and define molecular criteria for naive pluripotency. However, the identity of naive-specific surface markers and the underlying molecular mechanism of naive pluripotency regulation remain poorly understood. Here, we identify alkaline phosphatase placental-like 2 (ALPPL2) as a prominent naive-specific surface marker by systematic proteomic and transcriptomic analyses. Furthermore, we demonstrate that ALPPL2 is essential for both the establishment and maintenance of naive pluripotency. Moreover, we show that ALPPL2 can interact with the RNA-binding protein IGF2BP1 to stabilize the mRNA levels of the naive pluripotency transcription factors TFCP2L1 and STAT3 to regulate naive pluripotency. Overall, our study identifies a functional surface marker for human naive pluripotency, providing a powerful tool for human-naive-pluripotency-related mechanistic studies. : Bi et al. identify ALPPL2 as a functional surface marker specific for naive pluripotency that interacts with IGF2BP1 to regulate STAT3 and TFCP2L1 mRNA stability to affect naive pluripotency maintenance and establishment, providing a powerful tool for human-naive-pluripotency-related mechanistic studies. Keywords: naive pluripotency, ALPPL2, IGF2BP1, functional surface marker

SUMMARY Bacteria adapt to dynamic changes in the host during chronic and recurrent infections. Bacterial microevolution is one type of adaptation that imparts a selective advantage. We hypothesize that recurrent episodes of disease promote microevolution through genetic mutations that modulate disease severity. We use a pre-clinical model of otitis media (OM) to determine the potential role for microevolution of nontypeable Haemophilus influenzae (NTHI) during sequential episodes of disease. Whole genome sequencing reveals microevolution of hemoglobin binding and lipooligosaccharide (LOS) biosynthesis genes, suggesting that adaptation of these systems is critical for infection. These OM-adapted strains promote increased biofilm formation, inflammation, stromal fibrosis, and an increased propensity to form intracellular bacterial communities (IBCs). Remarkably, IBCs remain for at least one month following clinical resolution of infection, suggesting an intracellular reservoir as a nidus for recurrent OM. Additional approaches for therapeutic design tailored to combat this burdensome disease will arise from these studies., Graphical Abstract, In Brief Harrison et al. develop a sequential model of otitis media (OM) to investigate microevolution through genetic mutations that modulate disease severity. OM-adapted strains promote increased biofilm, inflammation, stromal fibrosis, and intracellular bacterial community (IBC) development. IBCs remain one month following clinical resolution of infection, suggesting a nidus for recurrent OM.

Summary: In the mammalian primary visual cortex, neural tuning to stimulus orientation is organized in either columnar or salt-and-pepper patterns across species. For decades, this sharp contrast has spawned fundamental questions about the origin of functional architectures in visual cortex. However, it is unknown whether these patterns reflect disparate developmental mechanisms across mammalian taxa or simply originate from variation of biological parameters under a universal development process. In this work, after the analysis of data from eight mammalian species, we show that cortical organization is predictable by a single factor, the retino-cortical mapping ratio. Groups of species with or without columnar clustering are distinguished by the feedforward sampling ratio, and model simulations with controlled mapping conditions reproduce both types of organization. Prediction from the Nyquist theorem explains this parametric division of the patterns with high accuracy. Our results imply that evolutionary variation of physical parameters may induce development of distinct functional circuitry. : Across mammalian species, orientation tuning in the primary visual cortex is arranged in different manners, such as columnar orientation map in primates or salt-and-pepper organization in rodents. Here, Jang et al. propose that the retina-to-cortex sampling ratio is the key factor in determining the organization of the orientation tuning. Keywords: primary visual cortex, orientation tuning, columnar orientation map, salt-and-pepper organization, retino-cortical sampling, retinal ganglion cell, statistical wiring model, magnification factor, Nyquist frequency, receptive field

SUMMARY Many chaperones promote nascent polypeptide folding followed by substrate release through ATP-dependent conformational changes. Here we show cryoEM structures of Gα subunit folding intermediates in complex with full-length Ric-8A, a unique chaperone-client system in which substrate release is facilitated by guanine nucleotide binding to the client G protein. The structures of Ric-8A-Gαi and Ric-8A-Gαq complexes reveal that the chaperone employs its extended C-terminal region to cradle the Ras-like domain of Gα, positioning the Ras core in contact with the Ric-8A core while engaging its switch2 nucleotide binding region. The C-terminal α5 helix of Gα is held away from the Ras-like domain through Ric-8A core domain interactions, which critically depend on recognition of the Gα C terminus by the chaperone. The structures, complemented with biochemical and cellular chaperoning data, support a folding quality control mechanism that ensures proper formation of the C-terminal α5 helix before allowing GTP-gated release of Gα from Ric-8A., Graphical Abstract, In Brief Seven et al. present cryoEM structures of Gα subunit folding intermediates in complex with their universal chaperone, Ric-8. Ric-8 forms key interactions with the Ras domain to prepare GTP-gated release of Gα subunits from their chaperone. The structures, complemented by biochemical and cellular experiments, reveal a folding quality control mechanism.

Summary: Pathogenic bacteria can alter host gene expression through post-translational modifications of histones. We show that a natural colonizer, Streptococcus pneumoniae, induces specific histone modifications, including robust dephosphorylation of histone H3 on serine 10 (H3S10), during infection of respiratory epithelial cells. The bacterial pore-forming toxin pneumolysin (PLY), along with the pyruvate oxidase SpxB responsible for H2O2 production, play important roles in the induction of this modification. The combined effects of PLY and H2O2 trigger host signaling that culminates in H3S10 dephosphorylation, which is mediated by the host cell phosphatase PP1. Strikingly, S. pneumoniae infection induces dephosphorylation and subsequent activation of PP1 catalytic activity. Colonization of PP1 catalytically deficient cells results in impaired intracellular S. pneumoniae survival and infection. Interestingly, PP1 activation and H3S10 dephosphorylation are not restricted to S. pneumoniae and appear to be general epigenomic mechanisms favoring intracellular survival of pathogenic bacteria. : Dong et al. show that Streptococcus pneumoniae infection induces histone H3 dephosphorylation in lung epithelial cells, through two bacterial factors, PLY and SpxB. Infection-triggered activation of the host phosphatase PP1 is required for this histone modification and efficient intracellular infection. Keywords: histone modification, Streptococcus pneumoniae, H3S10 dephosphorylation, protein phosphatase 1, bacterial infection, pneumolysin, SpxB

Summary: Timely completion of DNA replication is central to accurate cell division and to the maintenance of genomic stability. However, certain DNA-protein interactions can physically impede DNA replication fork progression. Cells remove or bypass these physical impediments by different mechanisms to preserve DNA macromolecule integrity and genome stability. In Saccharomyces cerevisiae, Wss1, the DNA-protein crosslink repair protease, allows cells to tolerate hydroxyurea-induced replication stress, but the underlying mechanism by which Wss1 promotes this function has remained unknown. Here, we report that Wss1 provides cells tolerance to replication stress by directly degrading core histone subunits that non-specifically and non-covalently bind to single-stranded DNA. Unlike Wss1-dependent proteolysis of covalent DNA-protein crosslinks, proteolysis of histones does not require Cdc48 nor SUMO-binding activities. Wss1 thus acts as a multi-functional protease capable of targeting a broad range of covalent and non-covalent DNA-binding proteins to preserve genome stability during adverse conditions. : Maddi et al. show that Wss1 degrades unincorporated histones to tolerate replication stress. Wss1 is a DNA-dependent metalloprotease that cleaves DNA-protein crosslinks to maintain genome stability. Unlike DNA-protein crosslink removal, Wss1 does not require interactions with Sumo or Cdc48 to act on histones that bind non-specifically to DNA. Keywords: Wss1, metalloprotease, hydroxyurea, HU, histone H3, histone H2A, histone H4, Ddi1, Rad51, replication stress, DNA-protein crosslinks

Summary: G3BP RNA-binding proteins are important components of stress granules (SGs). Here, we analyze the role of the Drosophila G3BP Rasputin (RIN) in unstressed cells, where RIN is not SG associated. Immunoprecipitation followed by microarray analysis identifies over 550 mRNAs that copurify with RIN. The mRNAs found in SGs are long and translationally silent. In contrast, we find that RIN-bound mRNAs, which encode core components of the transcription, splicing, and translation machinery, are short, stable, and highly translated. We show that RIN is associated with polysomes and provide evidence for a direct role for RIN and its human homologs in stabilizing and upregulating the translation of their target mRNAs. We propose that when cells are stressed, the resulting incorporation of RIN/G3BPs into SGs sequesters them away from their short target mRNAs. This would downregulate the expression of these transcripts, even though they are not incorporated into stress granules. : Laver et al. show that in early embryos, Rasputin, the Drosophila G3BP ortholog, binds short mRNAs and is associated with polysomes. They provide evidence for a direct role for Rasputin and its human homologs in stabilizing and upregulating the translation of their target mRNAs. Keywords: RNA-binding protein, post-transcriptional regulation, mRNA translation, mRNA stability, stress granule, Drosophila, embryo, G3BP, Rasputin

SUMMARY Cell-based therapies have shown promise for treating myriad chronic pulmonary diseases through direct application of epithelial progenitors or by way of engineered tissue grafts or whole organs. To elucidate environmental effects on epithelial regenerative outcomes in vitro, here, we isolate and culture a population of pharmacologically expanded basal cells (peBCs) from rat tracheas. At peak basal marker expression, we simultaneously split peBCs into four in vitro platforms: organoid, air-liquid interface (ALI), engineered trachea, and engineered lung. Following differentiation, these samples are evaluated using single-cell RNA sequencing (scRNA-seq) and computational pipelines are developed to compare samples both globally and at the population level. A sample of native rat tracheal epithelium is also evaluated by scRNA-seq as a control for engineered epithelium. Overall, this work identifies platform-specific effects that support the use of engineered models to achieve the most physiologic differential outcomes in pulmonary epithelial regenerative applications., Graphical Abstract, In Brief Greaney et al. compare pulmonary epithelial regeneration across multiple modalities in vitro, finding that decellularized scaffolds achieved the most physiologic differentiation over more artificial platforms. scRNA-seq enables high-resolution comparison between engineered and native cell populations, thereby better gauging progress toward the generation of a tissue that may function on implantation.

Summary: Genetic variations in TMEM106B, coding for a lysosomal membrane protein, affect frontotemporal lobar degeneration (FTLD) in GRN- (coding for progranulin) and C9orf72-expansion carriers and might play a role in aging. To determine the physiological function of TMEM106B, we generated TMEM106B-deficient mice. These mice develop proximal axonal swellings caused by drastically enlarged LAMP1-positive vacuoles, increased retrograde axonal transport of lysosomes, and accumulation of lipofuscin and autophagosomes. Giant vacuoles specifically accumulate at the distal end and within the axon initial segment, but not in peripheral nerves or at axon terminals, resulting in an impaired facial-nerve-dependent motor performance. These data implicate TMEM106B in mediating the axonal transport of LAMP1-positive organelles in motoneurons and axonal sorting at the initial segment. Our data provide mechanistic insight into how TMEM106B affects lysosomal proteolysis and degradative capacity in neurons. : Genetic variants in the TMEM106B gene, coding for a lysosomal transmembrane protein, are linked to various neurodegenerative diseases. The function of TMEM106B remains enigmatic. Lüningschrör et al. analyze Tmem106b-knockout mice and find drastically enlarged LAMP1-positive vacuoles in proximal axons of selected motoneuron nuclei. Vacuolization is caused by impaired axonal transport. Keywords: TMEM106B, frontotemporal lobar degeneration, lysosome, retrograde, axon, axon initial segment, motoneurons, FTLD

Summary The functional impact of single interneurons on neuronal output in vivo and how interneurons are recruited by physiological activity patterns remain poorly understood. In the cerebellar cortex, molecular layer interneurons and their targets, Purkinje cells, receive excitatory inputs from granule cells and climbing fibers. Using dual patch-clamp recordings from interneurons and Purkinje cells in vivo, we probe the spatiotemporal interactions between these circuit elements. We show that single interneuron spikes can potently inhibit Purkinje cell output, depending on interneuron location. Climbing fiber input activates many interneurons via glutamate spillover but results in inhibition of those interneurons that inhibit the same Purkinje cell receiving the climbing fiber input, forming a disinhibitory motif. These interneuron circuits are engaged during sensory processing, creating diverse pathway-specific response functions. These findings demonstrate how the powerful effect of single interneurons on Purkinje cell output can be sculpted by various interneuron circuit motifs to diversify cerebellar computations., Graphical Abstract, Highlights • Single spikes in single interneurons can inhibit Purkinje cell firing in vivo • Climbing fiber input excites or inhibits interneurons via glutamate spillover in vivo • Interneuron circuit motifs are spatially organized by depth in the molecular layer • These direct and disinhibitory motifs can explain the responses to sensory stimulation, Using dual two-photon guided patch-clamp recordings from molecular-layer interneurons and Purkinje cells in vivo, Arlt and Häusser show that single-interneuron spikes can potently inhibit Purkinje cell output and uncover the microcircuit logic by which interneurons and their targets are recruited by different excitatory afferents.

Summary: Schlafen 11 (SLFN11) was recently discovered as a cellular restriction factor against replication stress. Here, we show that SLFN11 increases chromatin accessibility genome wide, prominently at active promoters in response to replication stress induced by the checkpoint kinase 1 (CHK1) inhibitor prexasertib or the topoisomerase I (TOP1) inhibitor camptothecin. Concomitantly, SLFN11 selectively activates cellular stress response pathways by inducing the transcription of the immediate early genes (IEGs), including JUN, FOS, EGR1, NFKB2, and ATF3, together with the cell cycle arrest genes CDKN1A (p21WAF1) and GADD45. Both chromatin remodeling and IEG activation require the putative ATPase and helicase activity of SLFN11, whereas canonical extrinsic IEG activation is SLFN11 independent. SLFN11-dependent IEG activation by camptothecin is also observed across 55 non-isogenic NCI-60 cell lines. We conclude that SLFN11 acts as a global regulator of chromatin structure and an intrinsic IEG activator with the potential to engage the innate immune activation in response to replicative stress. : Schlafen 11 (SLFN11), a promising therapeutic biomarker, binds chromatin and sensitizes cancer cells to DNA-targeting agents by blocking DNA replication. Murai et al. show that, in response to replication stress, SLFN11 selectively increases chromatin accessibility at promoters and activates a subset of genes known as the immediate early genes (IEGs). Keywords: chromatin, replication stress, ATR, CHK1, cell cycle checkpoint, DNA damage, topoisomerase inhibitor, native immune response, JUN, FOS

SUMMARY The mammalian heart is incapable of regenerating a sufficient number of cardiomyocytes to ameliorate the loss of contractile muscle after acute myocardial injury. Several reports have demonstrated that mononucleated cardiomyocytes are more responsive than are binucleated cardiomyocytes to proproliferative stimuli. We have developed a strategy to isolate and characterize highly enriched populations of mononucleated and binucleated cardiomyocytes at various times of development. Our results suggest that an E2f/Rb transcriptional network is central to the divergence of these two populations and that remnants of the differences acquired during the neonatal period remain in adult cardiomyocytes. Moreover, inducing binucleation by genetically blocking the ability of cardiomyocytes to complete cytokinesis leads to a reduction in E2f target gene expression, directly linking the E2f pathway with nucleation. These data identify key molecular differences between mononucleated and binucleated mammalian cardiomyocytes that can be used to leverage cardiomyocyte proliferation for promoting injury repair in the heart., Graphical Abstract, In Brief Windmueller et al. develop a strategy to separate mononucleated and binucleated cardiomyocytes and examine transcriptional differences acquired as the two subsets diverge during the neonatal period. Binucleation is associated with silencing of proliferation genes and upregulation of maturation genes. Genetic loss of Ect2 links binucleation to the Rb/E2f pathway.

SUMMARY Netherton syndrome (NS) is a monogenic skin disease resulting from loss of function of lymphoepithelial Kazal-type-related protease inhibitor (LEKTI-1). In this study we examine if bacteria residing on the skin are influenced by the loss of LEKTI-1 and if interaction between this human gene and resident bacteria contributes to skin disease. Shotgun sequencing of the skin microbiome demonstrates that lesional skin of NS subjects is dominated by Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis). Isolates of either species from NS subjects are able to induce skin inflammation and barrier damage on mice. These microbes promote skin inflammation in the setting of LEKTI-1 deficiency due to excess proteolytic activity promoted by S. aureus phenol-soluble modulin α as well as increased bacterial proteases staphopain A and B from S. aureus or EcpA from S. epidermidis. These findings demonstrate the critical need for maintaining homeostasis of host and microbial proteases to prevent a human skin disease., Graphical Abstract, In Brief Williams et al. show how an abnormal skin microbiome promotes inflammation associated with Netherton syndrome, a monogenic disorder in the human protease inhibitor SPINK5. Subjects with Netherton syndrome have excess colonization by S. aureus or S. epidermidis that then produce and promote increased protease production in the epidermis.

SUMMARY Our knowledge of transcriptional heterogeneities in epithelial stem and progenitor cell compartments is limited. Epidermal basal cells sustain cutaneous tissue maintenance and drive wound healing. Previous studies have probed basal cell heterogeneity in stem and progenitor potential, but a comprehensive dissection of basal cell dynamics during differentiation is lacking. Using single-cell RNA sequencing coupled with RNAScope and fluorescence lifetime imaging, we identify three non-proliferative and one proliferative basal cell state in homeostatic skin that differ in metabolic preference and become spatially partitioned during wound re-epithelialization. Pseudotemporal trajectory and RNA velocity analyses predict a quasi-linear differentiation hierarchy where basal cells progress from Col17a1Hi/Trp63Hi state to early-response state, proliferate at the juncture of these two states, or become growth arrested before differentiating into spinous cells. Wound healing induces plasticity manifested by dynamic basal-spinous interconversions at multiple basal transcriptional states. Our study provides a systematic view of epidermal cellular dynamics, supporting a revised “hierarchical-lineage” model of homeostasis., Graphical Abstract, In Brief Haensel et al. performed a comprehensive dissection of the cellular makeup of skin during homeostasis and wound healing and the molecular heterogeneity and cellular dynamics within its stem-cell-containing epidermal basal layer. Their work provides insights and stimulates further investigation into the mechanism of skin maintenance and repair.

Summary: Understanding the mechanisms of activity-dependent gene transcription underlying adaptive behaviors is challenging at neuronal-subtype resolution. Using cell-type specific molecular analysis in agouti-related peptide (AgRP) neurons, we reveal that the profound hunger-induced transcriptional changes greatly depend on plant homeodomain finger protein 6 (PHF6), a transcriptional repressor enriched in AgRP neurons. Loss of PHF6 in the satiated mice results in a hunger-state-shifting transcriptional profile, while hunger fails to further induce a rapid and robust activity-dependent gene transcription in PHF6-deficient AgRP neurons. We reveal that PHF6 binds to the promoters of a subset of immediate-early genes (IEGs) and that this chromatin binding is dynamically regulated by hunger state. Depletion of PHF6 decreases hunger-driven feeding motivation and makes the mice resistant to body weight gain under repetitive fasting-refeeding conditions. Our work identifies a neuronal subtype-specific transcriptional repressor that modulates transcriptional profiles in different nutritional states and enables adaptive eating behavior. : Gan et al. show that PHF6 is a transcriptional repressor enriched in AgRP neurons and regulates immediate-early gene (IEG) expression. Depletion of PHF6 in AgRP neurons decreases hunger-driven feeding motivation and makes the mice resistant to body weight gain under repetitive fasting/refeeding conditions. Keywords: PHF6, hunger-driven feeding behavior, AgRP neuron, activity-dependent gene transcription, immediate-early genes, IEGs, Börjeson-Forssman-Lehmann syndrome, BFLS

SUMMARY Nucleotide deprivation and imbalance present detrimental conditions for animals and are thus expected to trigger cellular responses that direct protective changes in metabolic, developmental, and behavioral programs, albeit such mechanisms are vastly underexplored. Following our previous finding that Caenorhabditis elegans shut down germ cell proliferation in response to pyrimidine deprivation, we find in this study that endonuclease ENDU-2 regulates nucleotide metabolism and germ cell proliferation in response to nucleotide imbalance and other genotoxic stress, and that it affects mitotic chromosomal segregation in the intestine and lifespan. ENDU-2 expression is induced by nucleotide imbalance and genotoxic stress, and ENDU-2 exerts its function in the intestine, mostly by inhibiting the phosphorylation of CTPS-1 through repressing the PKA pathway and histone deacetylase HDA-1. Human EndoU also affects the response to genotoxic drugs. Our work reveals an unknown role of ENDU-2 in regulating nucleotide metabolism and animals’ response to genotoxic stress, which may link EndoU function to cancer treatment., In Brief Jia et al. show that an endonuclease plays a critical role in a nucleotide response system in nematodes and possibly mammalian cells. It affects nucleotide metabolism and reproductive development in response to nucleotide imbalance or other stresses, and it does so by inhibiting the phosphorylation of CTP synthase in the gut., Graphical Abstract

Summary: HIRA is a histone chaperone that deposits the histone variant H3.3 in transcriptionally active genes. In DiGeorge syndromes, a DNA stretch encompassing HIRA is deleted. The syndromes manifest varied abnormalities, including immunodeficiency and thrombocytopenia. HIRA is essential in mice, as total knockout (KO) results in early embryonic death. However, the role of HIRA in hematopoiesis is poorly understood. We investigate hematopoietic cell-specific Hira deletion in mice and show that it dramatically reduces bone marrow hematopoietic stem cells (HSCs), resulting in anemia, thrombocytopenia, and lymphocytopenia. In contrast, fetal hematopoiesis is normal in Hira-KO mice, although fetal HSCs lack the reconstitution capacity. Transcriptome analysis reveals that HIRA is required for expression of many transcription factors and signaling molecules critical for HSCs. ATAC-seq analysis demonstrates that HIRA establishes HSC-specific DNA accessibility, including the SPIB/PU.1 sites. Together, HIRA provides a chromatin environment essential for HSCs, thereby steering their development and survival. : Chen et al. show that the histone H3.3 chaperone HIRA directs development and survival of adult hematopoietic stem cells (HSCs). Hira deletion impairs development of all lineages of hematopoiesis. Keywords: HIRA, histone chaperone, H3.3, hematopoietic stem cells, DiGeorge syndrome

Summary: To understand the conditions necessary to initiate and terminate seizures, we investigate optogenetically induced hippocampal seizures with LFP, fMRI, and optogenetic inhibition. During afterdischarge induction using optogenetics, LFP recordings show that stimulations with earlier ictal onset times are more likely to result in afterdischarges and are more difficult to curtail with optogenetic inhibition. These results are generalizable across two initiation sites, the dorsal and ventral hippocampus. fMRI shows that afterdischarges initiated from the dorsal or ventral hippocampus exhibit distinct networks. Short-duration seizures initiated in the dorsal and ventral hippocampus are unilateral and bilateral, respectively, while longer-duration afterdischarges recruit broader, bilateral networks. When optogenetic inhibition is ineffective at stopping seizures, the network activity spreads more extensively but largely overlaps with the network activity associated with seizures that could be curtailed. These results provide insights into how seizures can be inhibited, which has implications for targeted seizure interventions. : Duffy et al. show that success in generating or curtailing seizures is related to their onset time, with inhibition proving less effective on earlier-onset afterdischarges. To identify downstream targets for intervention, fMRI is used to map the seizure network of afterdischarges initiated from the dorsal and ventral hippocampus. Keywords: seizure, epilepsy, hippocampus, fMRI, optogenetrics, generation, inhibition, stopping, network, afterdischarge

Summary: ATP-powered unfoldases containing D1 and D2 AAA+ rings play important roles in protein homeostasis, but uncertainty about the function of each ring remains. Here we use single-molecule optical tweezers to assay mechanical unfolding and translocation by a variant of the ClpAP protease containing an ATPase-inactive D1 ring. This variant displays substantial mechanical defects in both unfolding and translocation of protein substrates. Notably, when D1 is hydrolytically inactive, ClpAP often stalls for times as long as minutes, and the substrate can back-slip through the enzyme when ATP concentrations are low. The inactive D1 variant also has more difficulty traveling in the N-to-C direction on a polypeptide track than it does moving in a C-to-N direction. These results indicate that D1 normally functions as an auxiliary/regulatory motor to promote uninterrupted enzyme advancement that is fueled largely by the D2 ring. : Kotamarthi et al. employ optical tweezers to examine the individual rings in a double-ring AAA+ unfoldase. ClpAP inactivated in the D1 ring is partially defective in unfolding and pauses frequently during translocation. Thus, D1 functions as a booster motor, thereby assisting the more powerful unfoldase motor of the D2 ring. Keywords: AAA+ proteases, ATP motors, single-molecule force spectroscopy, protein unfolding, protein translocation, translocation pauses, Hsp104/ClpB, ClpAP, optical tweezers, protein degradation

SUMMARY HIV-1-infected infants develop broadly neutralizing antibodies (bnAbs) more rapidly than adults, suggesting differences in the neonatal versus adult responses to the HIV-1 envelope (Env). Here, trimeric forms of HIV-1 Env immunogens elicit increased gp120-and gp41-specific antibodies more rapidly in neonatal macaques than adult macaques. Transcriptome analyses of neonatal versus adult immune cells after Env vaccination reveal that neonatal macaques have higher levels of the apoptosis regulator BCL2 in T cells and lower levels of the immunosuppressive interleukin-10 (IL-10) receptor alpha (IL10RA) mRNA transcripts in T cells, B cells, natural killer (NK) cells, and monocytes. In addition, immunized neonatal macaques exhibit increased frequencies of activated blood T follicular helper-like (Tfh) cells compared to adults. Thus, neonatal macaques have transcriptome signatures of decreased immunosuppression and apoptosis compared with adult macaques, providing an immune landscape conducive to early-life immunization prior to sexual debut., In Brief Han et al. demonstrate that infant macaques are capable of responding to HIV vaccines currently studied in human clinical trials. The nature of the neonatal immune system following immunization also suggests a state permissive for generating HIV-1 antibodies. Thus, infant HIV-1 immunization remains a viable strategy to end this pandemic., Graphical abstract

Summary Compensatory endocytosis of released synaptic vesicles (SVs) relies on coordinated signaling at the lipid-protein interface. Here, we address the synaptic function of C-terminal binding protein 1 (CtBP1), a ubiquitous regulator of gene expression and membrane trafficking in cultured hippocampal neurons. In the absence of CtBP1, synapses form in greater density and show changes in SV distribution and size. The increased basal neurotransmission and enhanced synaptic depression could be attributed to a higher vesicular release probability and a smaller fraction of release-competent SVs, respectively. Rescue experiments with specifically targeted constructs indicate that, while synaptogenesis and release probability are controlled by nuclear CtBP1, the efficient recycling of SVs relies on its synaptic expression. The ability of presynaptic CtBP1 to facilitate compensatory endocytosis depends on its membrane-fission activity and the activation of the lipid-metabolizing enzyme PLD1. Thus, CtBP1 regulates SV recycling by promoting a permissive lipid environment for compensatory endocytosis., Graphical Abstract, Highlights • Nuclear CtBP1 restricts synaptogenesis and vesicular release probability • CtBP1 is required for efficient compensatory endocytosis at the presynapse • CtBP1 facilitates synaptic vesicle retrieval via activation of the lipid enzyme PLD1 • Phosphorylation by Pak1 shifts CtBP1 from the active zone to endocytic sites, Ivanova et al. demonstrate a dual role of CtBP1 in synaptic transmission. Nuclear CtBP1 restricts synaptogenesis and vesicular release probability, whereas presynaptic CtBP1 promotes compensatory endocytosis via activation of the lipid enzyme PLD1. Phosphorylation by Pak1 controls the redistribution of CtBP1 from active zones toward endocytic sites linking presynaptic exo- and endocytosis.

Summary: Bone metabolism depends on the balance between osteoclast-driven bone resorption and osteoblast-mediated bone formation. Diseases like osteoporosis are characterized by increased bone destruction due to partially enhanced osteoclastogenesis. Here, we report that the post-translational SUMO modification is critical for regulating osteoclastogenesis. The expression of the SUMO-specific protease SENP3 is downregulated in osteoclast precursors during osteoclast differentiation. Mice with SENP3 deficiency in bone marrow-derived monocytes (BMDMs) exhibit more severe bone loss due to over-activation of osteoclasts after ovariectomy. Deleting SENP3 in BMDMs promotes osteoclast differentiation. Mechanistically, loss of SENP3 increases interferon regulatory factor 8 (IRF8) SUMO3 modification at the K310 amino acid site, which upregulates expression of the nuclear factor of activated T cell c1 (NFATc1) and osteoclastogenesis. In summary, IRF8 de-SUMO modification mediated by SENP3 suppresses osteoclast differentiation and suggests strategies to treat bone loss diseases. : Osteoclast over-activation leads to osteoporosis, but the role of post-translational modification in osteoporosis is not fully understood. Zhang et al. show that the SUMO protease SENP3 can alter the SUMO modification status on the critical osteoclast suppressor IRF8 in osteoclast precursor cells, which affects osteoclastogenesis and osteoporosis. Keywords: SENP3, osteoclastogenesis, SUMOylation, osteoporosis

Summary: Cancer cell-derived secretomes have been documented to play critical roles in cancer progression. Intriguingly, alternative extracellular roles of intracellular proteins are involved in various steps of tumor progression, which can offer strategies to fight cancer. Herein, we identify lung cancer progression-associated secretome signatures using mass spectrometry analysis. Among them, PKM2 is verified to be highly expressed and secreted in lung cancer cells and clinical samples. Functional analyses demonstrates that secreted PKM2 facilitates tumor metastasis. Furthermore, mass spectrometry analysis and functional validation identify integrin β1 as a receptor of secreted PKM2. Mechanistically, secreted PKM2 directly bound to integrin β1 and subsequently activated the FAK/SRC/ERK axis to promote tumor metastasis. Collectively, our findings suggest that PKM2 is a potential serum biomarker for diagnosing lung cancer and that targeting the secreted PKM2-integrin β1 axis can inhibit lung cancer development, which provides evidence of a potential therapeutic strategy in lung cancer. : Wang et al. find that PKM2 is highly expressed and secreted in lung cancer. Secreted PKM2 facilitates metastasis by interacting with its receptor integrin β1, which activates the FAK/SRC/ERK pathway and subsequently elevates MMP-9 expression. These results suggest that secreted PKM2 and integrin β1 are potential targets in lung cancer patients. Keywords: integrin β1, lung cancer, metastasis, PKM2, secretome

Summary Condensin is a multi-subunit protein complex regulating chromosome condensation and segregation during cell division. In Plasmodium spp., the causative agent of malaria, cell division is atypical and the role of condensin is unclear. Here we examine the role of SMC2 and SMC4, the core subunits of condensin, during endomitosis in schizogony and endoreduplication in male gametogenesis. During early schizogony, SMC2/SMC4 localize to a distinct focus, identified as the centromeres by NDC80 fluorescence and chromatin immunoprecipitation sequencing (ChIP-seq) analyses, but do not form condensin I or II complexes. In mature schizonts and during male gametogenesis, there is a diffuse SMC2/SMC4 distribution on chromosomes and in the nucleus, and both condensin I and condensin II complexes form at these stages. Knockdown of smc2 and smc4 gene expression reveals essential roles in parasite proliferation and transmission. The condensin core subunits (SMC2/SMC4) form different complexes and may have distinct functions at various stages of the parasite life cycle., Graphical Abstract, Highlights • SMC2/SMC4 and condensin complex I and II proteins are present in Plasmodium • SMC2/SMC4GFP has discrete centromeric localization in nuclei during early schizogony • SMC2/SMC4GFP are dispersed in the nucleus in male gametogenesis and late schizogony • SMC2/SMC4 gene knockdown (KD) impairs oocyst development and parasite transmission, Pandey et al. use live cell imaging, proteomics, and genetic approaches to show that Plasmodium condensin core subunits (SMC2/SMC4) have differential cellular location and condensin complex formation during parasite development and have essential roles in parasite proliferation and transmission.

The innate immune system safeguards the organism from both pathogenic and environmental stressors. Also, physiologic levels of nutrients affect organismal and intra-cellular metabolism and challenge the immune system. In the long term, over-nutrition leads to low-grade systemic inflammation. Here, we investigate tissue-resident components of the innate immune system (macrophages) and their response to short- and long-term nutritional challenges. We analyze the transcriptomes of six tissue-resident macrophage populations upon acute feeding and identify adipose tissue macrophages and the IL-1 pathway as early sensors of metabolic changes. Furthermore, by comparing functional responses between macrophage subtypes, we propose a regulatory, anti-inflammatory role of heat shock proteins of the HSP70 family in response to long- and short-term metabolic challenges. Our data provide a resource for assessing the impact of nutrition and over-nutrition on the spectrum of macrophages across tissues with a potential for identification of systemic responses., Cell Reports, 30 (5), ISSN:2666-3864, ISSN:2211-1247

Summary: Heterochromatin functions as a scaffold for factors responsible for gene silencing and chromosome segregation. Heterochromatin can be assembled by multiple pathways, including RNAi and RNA surveillance. We identified factors that form heterochromatin using dense profiles of transposable element integration in Schizosaccharomyces pombe. The candidates include a large number of essential proteins such as four canonical mRNA cleavage and polyadenylation factors. We find that Iss1, a subunit of the poly(A) polymerase module, plays a role in forming heterochromatin in centromere repeats that is independent of RNAi. Genome-wide maps reveal that Iss1 accumulates at genes regulated by RNA surveillance. Iss1 interacts with RNA surveillance factors Mmi1 and Rrp6, and importantly, Iss1 contributes to RNA elimination that forms heterochromatin at meiosis genes. Our profile of transposable element integration supports the model that a network of mRNA cleavage and polyadenylation factors coordinates RNA surveillance, including the mechanism that forms heterochromatin at meiotic genes. : Lee et al. use dense profiles of transposon integration to identify genes important for the formation of heterochromatin. Among many candidates, Iss1 is a canonical mRNA cleavage and polyadenylation factor found to be important for heterochromatin at meiotic genes by recruiting the nuclear exosome. Keywords: Tn-seq, heterochromatin, polyadenylation, Schizosaccharomyces pombe, Iss1, RNA elimination, Rrp6, ssm4, Mmi1, mei4

Summary: Upon fertilization, the ovary increases in size and undergoes a complex developmental process to become a fruit. We show that cytokinins (CKs), which are required to determine ovary size before fertilization, have to be degraded to facilitate fruit growth. The expression of CKX7, which encodes a cytosolic CK-degrading enzyme, is directly positively regulated post-fertilization by the MADS-box transcription factor STK. Similar to stk, two ckx7 mutants possess shorter fruits than wild type. Quantification of CKs reveals that stk and ckx7 mutants have high CK levels, which negatively control cell expansion during fruit development, compromising fruit growth. Overexpression of CKX7 partially complements the stk fruit phenotype, confirming a role for CK degradation in fruit development. Finally, we show that STK is required for the expression of FUL, which is essential for valve elongation. Overall, we provide insights into the link between CKs and molecular pathways that control fruit growth. : Di Marzo et al. establish that defects in Arabidopsis fruit size are accompanied by alterations in the metabolism of the plant hormone cytokinin, which is critical for cell elongation. SEEDSTICK influences fruit elongation by directly regulating cytokinin degradation via CKX7 and indirectly modulating the expression of the MADS-box gene FRUITFULL. Keywords: Arabidopsis, cell elongation, CKX7, cytokinins, FRUITFULL, fruit size, MADS-box, SEEDSTICK

SUMMARY Type I interferons (IFNs) play critical roles in anti-viral and anti-tumor immunity. However, they also suppress protective immune responses in some infectious diseases. Here, we identify type I IFNs as major upstream regulators of CD4+ T cells from visceral leishmaniasis (VL) patients. Furthermore, we report that mice deficient in type I IFN signaling have significantly improved control of Leishmania donovani, a causative agent of human VL, associated with enhanced IFNγ but reduced IL-10 production by parasite-specific CD4+ T cells. Importantly, we identify a small-molecule inhibitor that can be used to block type I IFN signaling during established infection and acts synergistically with conventional anti-parasitic drugs to improve parasite clearance and enhance anti-parasitic CD4+ T cell responses in mice and humans. Thus, manipulation of type I IFN signaling is a promising strategy for improving disease outcome in VL patients., Graphical Abstract, In Brief CD4+ T cells are critical for control of intracellular parasites such as Leishmania donovani. Kumar et al. show that type I interferons (IFNs) suppress Th1 cells and promote IL-10-producing CD4+ T cells during visceral leishmaniasis (VL). Thus, manipulation of type I IFN signaling may improve disease outcome in VL patients.

SUMMARY Retinal degeneration is a form of neurodegenerative disease and is the leading cause of vision loss globally. The Toll-like receptors (TLRs) are primary components of the innate immune system involved in signal transduction. Here we show that TLR2 induces complement factors C3 and CFB, the common and rate-limiting factors of the alternative pathway in both retinal pigment epithelial (RPE) cells and mononuclear phagocytes. Neutralization of TLR2 reduces opsonizing fragments of C3 in the outer retina and protects photoreceptor neurons from oxidative stress-induced degeneration. TLR2 deficiency also preserves tight junction expression and promotes RPE resistance to fragmentation. Finally, oxidative stress-induced formation of the terminal complement membrane attack complex and Iba1+ cell infiltration are strikingly inhibited in the TLR2-deficient retina. Our data directly implicate TLR2 as a mediator of retinal degeneration in response to oxidative stress and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology., Graphical Abstract, In Brief Oxidative stress and complement deposition are common to many retinal degenerative diseases. Mulfaul et al. demonstrate that TLR2 blockade protects against photoreceptor neuronal cell death and RPE fragmentation in experimental models of oxidative stress-induced retinal degeneration and present TLR2 as a bridge between oxidative damage and complement-mediated retinal pathology.

SUMMARY Human brain organoid systems offer unprecedented opportunities to investigate both neurodevelopmental and neurological disease. Single-cell-based transcriptomics or epigenomics have dissected the cellular and molecular heterogeneity in the brain organoids, revealing a complex organization. Similar but distinct protocols from different labs have been applied to generate brain organoids, providing a large resource to perform a comparative analysis of brain developmental processes. Here, we take a systematic approach to compare the single-cell transcriptomes of various human cortical brain organoids together with fetal brain to define the identity of specific cell types and differentiation routes in each method. Importantly, we identify unique developmental programs in each protocol compared to fetal brain, which will be a critical benchmark for the utility of human brain organoids in the future., In Brief Tanaka et al. report integrative analyses of single-cell RNA-seq for human brain organoids derived from different protocols. They find a unique preference of cell differentiation routes across protocols and provide a benchmark for the use and the improvement of human brain organoids., Graphical Abstract

SUMMARY Pathological activation of TGF-β signaling is universal in fibrosis. Aberrant TGF-β signaling in conjunction with transdifferentiation of hepatic stellate cells (HSCs) into fibrogenic myofibroblasts plays a central role in liver fibrosis. Here we report that the DNA demethylase TET3 is anomalously upregulated in fibrotic livers in both humans and mice. We demonstrate that in human HSCs, TET3 promotes profibrotic gene expression by upregulation of multiple key TGF-β pathway genes, including TGFB1. TET3 binds to target gene promoters, inducing demethylation, which in turn facilitates chromatin remodeling and transcription. We also reveal a positive feedback loop between TGF-β1 and TET3 in both HSCs and hepatocytes. Furthermore, TET3 knockdown ameliorates liver fibrosis in mice. Our results uncover a TET3/TGF-β1 positive feedback loop as a crucial determinant of liver fibrosis and suggest that inhibiting TET3 may represent a therapeutic strategy for liver fibrosis and perhaps other fibrotic diseases., In Brief Xu et al. unmask a positive feedback loop between chromatin demethylase TET3 and TGF-β1 in stressed hepatocytes and stellate cells in humans and mice. Activation of this loop stimulates expression of fibrotic genes, whereas knockdown of TET3 reduces liver fibrosis in mice, suggesting a strategy for treating fibrosis., Graphical Abstract

Summary Transcription factor binding to enhancer and promoter regions critical for homeostatic adult gene activation is established during development. To understand how cell-specific gene expression patterns are generated, we study the developmental timing of association of two prominent hepatic transcription factors with gene regulatory regions. Most individual binding events display extraordinarily high temporal variations during liver development. Early and persistent binding is necessary, but not sufficient, for gene activation. Stable gene expression patterns are the result of combinatorial activity of multiple transcription factors, which mark regulatory regions long before activation and promote progressive broadening of active chromatin domains. Both temporally stable and dynamic, short-lived binding events contribute to the developmental maturation of active promoter configurations. The results reveal a developmental bookmarking function of master regulators and illuminate remarkable parallels between the principles employed for gene activation during development, during evolution, and upon mitotic exit., Graphical Abstract, Highlights • HNF4α and C/EBPα binding are highly dynamic during development • HNF4α and C/EBPα bookmark target genes for future activation • Stable and transient binding of multiple factors is required for transcription • Dynamic binding events facilitate the formation of broad open chromatin domains, Karagianni et al. show that during liver development, master transcription factors bind to their targets in a temporally stable or dynamic manner. Transcription factors mark regulatory regions long before gene activation. Commonalities among the mechanisms of gene activation during development, evolution, and mitotic exit are described.

SUMMARY In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are bound by the Rhino-Deadlock-Cutoff complex. The HP1 homolog Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription from both genomic strands. Cuff function is less well understood, but this Rai1 homolog shows hallmarks of adaptive evolution, which can remodel functional interactions within host defense systems. Supporting this hypothesis, Drosophila simulans Cutoff is a dominant-negative allele when expressed in Drosophila melanogaster, in which it traps Deadlock, TRF2, and the conserved transcriptional co-repressor CtBP in stable complexes. Cutoff functions with Rhino and Deadlock to drive non-canonical transcription. In contrast, CtBP suppresses canonical transcription of transposons and promoters flanking the major germline clusters, and canonical transcription interferes with downstream non-canonical transcription and piRNA production. Adaptive evolution thus targets interactions among Cutoff, TRF2, and CtBP that balance canonical and non-canonical piRNA precursor transcription., Graphical Abstract, In Brief Parhad et al. use cross-species complementation to determine the functional impact of adaptive evolution. These studies show that adaptive evolution of the piRNA pathway protein Cutoff, which is required for transposon silencing and genome maintenance, targets interactions with conserved canonical and non-canonical transcription factors that regulate piRNA precursor expression.

Summary: Reactive oxygen species (ROS) play critical roles in metabolism and disease, yet a comprehensive analysis of the cellular response to oxidative stress is lacking. To systematically identify regulators of oxidative stress, we conducted genome-wide Cas9/CRISPR and shRNA screens. This revealed a detailed picture of diverse pathways that control oxidative stress response, ranging from the TCA cycle and DNA repair machineries to iron transport, trafficking, and metabolism. Paradoxically, disrupting the pentose phosphate pathway (PPP) at the level of phosphogluconate dehydrogenase (PGD) protects cells against ROS. This dramatically alters metabolites in the PPP, consistent with rewiring of upper glycolysis to promote antioxidant production. In addition, disruption of peroxisomal import unexpectedly increases resistance to oxidative stress by altering the localization of catalase. Together, these studies provide insights into the roles of peroxisomal matrix import and the PPP in redox biology and represent a rich resource for understanding the cellular response to oxidative stress. : Despite its importance in metabolism and disease, a comprehensive analysis of the cellular response to oxidative stress is lacking. Here, Dubreuil et al. use genome-wide screens to identify cellular regulators of oxidative stress. They investigate paradoxical mechanisms by which disruption of the pentose phosphate and peroxisomal import pathways protect cells. Keywords: CRISPR, genome-wide screen, shRNA, pentose phosphate pathway, peroxisomal import pathway, catalase, phosphogluconate dehydrogenase, oxidative stress, reactive oxygen species

Summary: Synapses are fundamental to the normal function of the nervous system. Glia play a pivotal role in regulating synaptic formation. However, how presynaptic neurons assemble synaptic structure in response to the glial signals remains largely unexplored. To address this question, we use cima-1 mutant C. elegans as an in vivo model, in which the astrocyte-like VCSC glial processes ectopically reach an asynaptic neurite region and promote presynaptic formation there. Through an RNAi screen, we find that the Rho GTPase CDC-42 and IQGAP PES-7 are required in presynaptic neurons for VCSC glia-induced presynaptic formation. In addition, we find that cdc-42 and pes-7 are also required for normal synaptogenesis during postembryonic developmental stages. PES-7 activated by CDC-42 promotes presynaptic formation, most likely through regulating F-actin assembly. Given the evolutionary conservation of CDC-42 and IQGAPs, we speculate that our findings in C. elegans apply to vertebrates. : Dong et al. reveal a role of the conserved CDC-42 and IQGAP/PES-7 in glia-mediated synaptogenesis during postembryonic development. At the glia-neurite contact sites, PES-7 activated by CDC-42 promotes presynaptic formation, possibly through regulating the F-actin assembly, providing insight into the neuronal responses to pro-synaptic signaling from glia. Keywords: synaptogenesis, glia, glia-neuron interaction, IQGAP/PES-7, CDC-42, synaptic spatial specificity, synaptic specificity, F-actin, Caenorhabditis elegans

Summary: Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, systematic analyses of DUBs that regulate this pathway have not been performed. Using a ubiquitin C-terminal electrophile, we profile DUBs that function during influenza A virus (IAV) infection and isolate OTUB1 as a key regulator of RIG-I-dependent antiviral responses. Upon infection, OTUB1 relocalizes from the nucleus to mitochondrial membranes together with RIG-I, viral PB2, and NS1. Its expression depends on competing effects of interferon stimulation and IAV-triggered degradation. OTUB1 activates RIG-I via a dual mechanism of K48 polyubiquitin hydrolysis and formation of an E2-repressive complex with UBCH5c. We reconstitute this mechanism in a cell-free system comprising [35S]IRF3, purified RIG-I, mitochondrial membranes, and cytosol expressing OTUB1 variants. A range of IAV NS1 proteins trigger proteasomal degradation of OTUB1, antagonizing the RIG-I signaling cascade and antiviral responses. : Jahan et al. describe the role of Otub1, a deubiquitylase that is induced during virus infection in an interferon-dependent manner to regulate optimal signaling via the RIG-I pathway. Influenza A virus NS1 is able to bind and degrade newly synthesized Otub1 as a means to evade the innate immune response. Keywords: deubiquitylases, RIG-I signaling, ubiquitylation, innate immune response, RNA virus, influenza A, viral subversion strategies

SUMMARY Repeat-mediated deletions (RMDs) often involve repetitive elements (e.g., short interspersed elements) with sequence divergence that is separated by several kilobase pairs (kbps). We have examined RMDs induced by DNA double-strand breaks (DSBs) under varying conditions of repeat sequence divergence (identical versus 1% and 3% divergent) and DSB/repeat distance (16 bp–28.4 kbp). We find that the BLM helicase promotes RMDs with long DSB/repeat distances (e.g., 28.4 kbp), which is consistent with a role in extensive DSB end resection, because the resection nucleases EXO1 and DNA2 affect RMDs similarly to BLM. In contrast, BLM suppresses RMDs with sequence divergence and intermediate (e.g., 3.3 kbp) DSB/repeat distances, which supports a role in heteroduplex rejection. The role of BLM in heteroduplex rejection is not epistatic with MSH2 and is independent of the annealing factor RAD52. Accordingly, the role of BLM on RMDs is substantially affected by DSB/repeat distance and repeat sequence divergence., In Brief Mendez-Dorantes et al. identify the BLM helicase as a key regulator of repeat-mediated deletions (RMDs). BLM, EXO1, and DNA2 mediate RMDs with remarkably long DNA break/repeat distances. BLM suppresses RMDs with sequence divergence that is optimal with a long non-homologous tail and is independent of MSH2 and RAD52., Graphical Abstract

Summary The transcriptional corepressor complex CoREST is one of seven histone deacetylase complexes that regulate the genome through controlling chromatin acetylation. The CoREST complex is unique in containing both histone demethylase and deacetylase enzymes, LSD1 and HDAC1, held together by the RCOR1 scaffold protein. To date, it has been assumed that the enzymes function independently within the complex. Now, we report the assembly of the ternary complex. Using both structural and functional studies, we show that the activity of the two enzymes is closely coupled and that the complex can exist in at least two distinct states with different kinetics. Electron microscopy of the complex reveals a bi-lobed structure with LSD1 and HDAC1 enzymes at opposite ends of the complex. The structure of CoREST in complex with a nucleosome reveals a mode of chromatin engagement that contrasts with previous models., Graphical Abstract, Highlights • The activities of LSD1 and HDAC1 are closely coupled in the CoREST complex • Both LSD1 and HDAC1 exist in two different kinetic states • CoREST has a bi-lobed, flexible structure with the two enzymes located at opposite ends • CoREST interacts with methylated nucleosomes via LSD1, but not HDAC1 or RCOR1, Using a real-time NMR assay, Song et al. characterize crosstalk between LSD1 and HDAC1 in the CoREST complex. Activation or inhibition of one enzyme strongly affects activity of the other. Electron microscopy studies of the complex reveal a bi-lobed structure with implications for the mode of interaction with nucleosomes.