Cell Reports

Efficient myelin regeneration requires coordinated signaling between oligodendrocyte progenitor cells (OPCs) and the extracellular matrix, yet how specific heparan sulfate sulfation patterns regulate this process remains poorly understood. The extracellular endosulfatases Sulf1 and Sulf2 selectively remove 6-O-sulfate groups from heparan sulfate proteoglycans (HSPGs). We previously demonstrated that these enzymes are highly expressed by OPCs following demyelination and inhibit OPC recruitment, differentiation, and remyelination through activation of BMP and WNT signaling. We investigated whether sulfotransferases that establish 2-O and 6-O sulfation during HSPG biosynthesis regulate remyelination. Using OPC-specific conditional knockout models, deletion of the 2-O sulfotransferase Hs2st1 enhanced OPC recruitment, oligodendrocyte differentiation, and remyelination through suppression of BMP and WNT signaling. Loss of 2-O sulfation induced compensatory remodeling of the heparan sulfate glycome, with a marked enrichment of 6-O-sulfated HS species. Conversely, deletion of 6-O sulfotransferases impaired OPC recruitment and differentiation and reversed the Hs2st1 phenotype, identifying 6-O sulfation as the dominant functional determinant of remyelination. Together, these findings establish hierarchical control of remyelination by heparan sulfate sulfation, demonstrating that compensatory remodeling of the heparan sulfate glycome governs regenerative signaling following demyelination.

The tumor suppressor p53 and the transcription factors estrogen receptor and androgen receptor regulate growth in a sex-dependent hormone-responsive manner, as in breast and prostate tumors and muscle tissue. Using a model of transcriptional regulation, we investigated co-regulation between p53 and hormone receptors in different cellular contexts and highlight an enrichment in mammary and muscle cell types. Sequence, structural and epigenomic evidence demonstrate a broadly distributed functional 73-bp p53-estrogen receptor grammar spanning nucleosome entry/exit to dyad, while p53-androgen receptor grammar spans 146 bp. At a functional breast cancer enhancer regulating Cyclin D, varied spacing led to lower predicted p53 binding. Genome-wide analysis found a major role of Alu sequences in distributing this grammar, in particular in mTORC1 pathway genes, with an evolutionary association of primate body-size sexual dimorphism and muscle aging.

The digestive tract is a major site of nutrient absorption. It also harbors the densest microbial communities in our body. It is well known that the brain perceives ingested nutrient information through the intestinal epithelium enteroendocrine cell (EEC)-vagal pathway. Little is known about how the brain senses gut bacteria that reside in the intestinal lumen. In this study, we combined novel in vivo EEC calcium imaging, EEC optochemical stimulation, and vagal sensory neuron calcium imaging in the zebrafish model, and revealed that segregated pathways mediated by different EEC and vagal sensory neuron subtypes differentially perceive beneficial nutrient information and harmful irritant pathogenic bacterial information. Our study revealed that a group of EECs that express the nociceptive receptor Transient receptor potential ankyrin A1 (Trpa1) can be activated by the pathogenic bacterium Edwardsiella tarda (E. tarda) or E. tarda-produced irritant metabolites. These Trpa1+EECs do not sense nutrients. Trpa1+EECs, but not other EECs, uniquely and highly express both Serotonin and the neuronal peptide Pituitary adenylate cyclase-activating polypeptide (Pacap). Using pharmaceutical and genetic approaches, our data showed that Pacap synergizes with Serotonin and is required for Trpa1+EEC-induced vagal activation. Stimulating the Trpa1+EEC-vagal sensory pathway potently inhibits a group of vagal motor neurons that innervate visceral organs in the caudal dorsal motor nucleus of the vagus (cDMV). However, the same group of cDMV neurons is activated by nutrients. Microbially induced cDMV inhibition enhances liver gluconeogenesis and promotes intestinal neutrophil recruitment. Inhibiting glucose metabolism, disrupting Trpa1+EEC-vagal sensory pathway, or optogenetically activating cDMV neurons in E. tarda colonized zebrafish induces intestinal E. tarda to switch from an aggregated phenotype to a diffused phenotype and hinders E. tarda clearance. Furthermore, our data show that disrupting the Trpa1+EEC-vagal sensory pathway impairs intestinal neutrophil recruitment and hinders survival, suggesting a protective role of the Trpa1+EEC-vagal microbial sensing pathway in pathogen defense. Together, our data revealed that a distinct non-nutrient-sensing Trpa1+EEC-vagal circuit senses gut microbial irritants, coordinating neuronal, metabolic, and immune responses to enhance host defense and pathogen clearance.

The mammalian BRG-/BRM-associated factor (BAF) chromatin remodeling complexes are crucial regulators of hematopoietic stem and progenitor cell (HSPC) fate. Expression of the ncBAF subcomponent, BRD9, is altered in both normal aging and myeloid malignancies. We find that in adult human HSPC, BRD9 regulates the differentiation of myeloid progenitors. Upon degradation of BRD9 in these cells, ncBAF is displaced from the chromatin, leading in turn to enhanced recruitment of cBAF to bona fide CEBPA target sites. With the shift from ncBAF to cBAF, HSPC differentiation results in increased generation of myeloid-derived suppressor cells (MDSC) that secrete elevated levels of IL-8. Soluble factors from these myeloid cells, which have lost BRD9, enhance the stemness of breast cancer cells in vitro. In contrast, bromodomain inhibition of BRD9 results in minimal alterations of ncBAF binding and myeloid differentiation, indicating that BRD9 acts as a barrier to human MDSC differentiation in a bromodomain-independent manner.

Aging and tobacco exposure are major determinants of risk and outcome in HPV-negative oral cavity squamous cell carcinoma (OSCC), yet the cellular programs linking host factors to tumor immunity remain poorly defined. We reprocessed and reanalyzed previously published single-cell RNA-seq data from 19 HPV-negative OSCC tumors and we identified a robust age- and smoking-associated expansion of SPP1high tumor-associated macrophages (TAMs), accompanied by a shift toward a myeloid-dominant and lymphocyte-reduced tumor microenvironment. SPP1high TAMs exhibit a distinct transcriptional state enriched for extracellular-matrix remodeling and metabolic programs and reduced antigen-presentation pathways. They also express inflammatory chemokines such as CXCL2 and CXCL3 and down-regulate the cytotoxic mediator TNFSF10, suggesting a broadly protumor TAM phenotype. Ligand-receptor modeling suggests that SPP1high TAMs act as key communication hubs, with dominant SPP1-CD44 signaling to T-cell subsets. Transcription-factor analysis further implicates a POU2F1/POU2F2/SP1 module in maintaining this state. In the TCGA HNSCC cohort, the association between SPP1 expression and overall survival did not reach statistical significance after adjustment for age and tumor purity. These findings demonstrate that aging and tobacco exposure converge to expand an SPP1high TAM population that remodels the extracellular matrix and suppresses antitumor immunity, highlighting the SPP1-CD44 axis and its upstream regulators as potential therapeutic targets in high-risk OSCC.

Infected cells rapidly adapt their transcriptional profile to upregulate genes that limit pathogen replication and promote inflammation. Viruses also induce widespread changes in host gene expression, and both host and virus further remodel the transcriptome through targeted mRNA degradation, modulation of alternative splicing, and alterations at transcript 5 and 3 prime ends. The complexity of the response to infection makes it difficult to understand which transcriptional changes are driven by the virus and which reflect the host antiviral program. Short-read RNA sequencing has been the primary tool for studying these changes, but its inability to resolve full-length transcript isoforms limits isoform switching-based analyses, narrowing our understanding of how transcript usage shapes immunity. Here we profiled the host transcriptome in response to interferon-{beta} treatment or influenza A virus infection using both short- and long-read RNA sequencing analyzed with the SQANTI3 pipeline. Because isoform switching and transcript abundance change are distinct regulatory events, we focused our analysis on differential transcript usage to uncover a layer of transcriptome remodeling that standard expression analysis cannot detect. Pathway analysis revealed distinct regulons for genes undergoing transcriptional upregulation compared with those undergoing isoform switching. These data provide a rich resource for querying the isoform changes underlying shared and distinct transcriptional programs triggered by viral infection or innate immune stimulation. To identify putative mechanisms coordinating these stimulation-dependent isoform changes, we integrated 232 eCLIP datasets and found a small set of RNA-binding proteins localized at these isoform switches, including DDX3X. Comparing the responses to interferon and to virus highlighted RNA-binding proteins associated with infection-dependent immune remodeling. Altogether, these results demonstrate the value of hybrid sequencing for deciphering the post-transcriptional regulatory code active during the antiviral response.

Natural Killer (NK) cells eliminate virally infected and cancerous cells by secreting cytotoxic granules and pro-inflammatory cytokines. However, the epigenetic mechanisms that coordinate these processes in human NK cells remain poorly understood. We identified two NK deficiency (NKD) patients with mutations in the histone methyltransferase KMT2D that demonstrated impaired cytotoxicity and degranulation. CRISPR-mediated KMT2D deletion or introduction of a patient-specific mutation in healthy human NK cells was sufficient to inhibit effector functions. NK cell-specific KMT2D deletion in mice resulted in defective NK cell degranulation and IFN-{gamma} production, increasing mortality following MCMV infection. KMT2D loss reduced H3K4me1 deposition, associated with decreased levels of RAB3D. RAB3D-deficient human NK cells reduced the release of GZMB and IFN-{gamma}, without impacting intracellular levels. Thus, KMT2D acts as a conserved epigenetic regulator of mature NK cell functions, promoting rapid effector molecule release.

Definitive hematopoietic stem cells (HSCs) emerge predominantly along the ventral surface of the dorsal aorta in the embryonic aorta-gonad-mesonephros (AGM) region, yet the spatial organization of the niche underlying this developmental asymmetry remains poorly understood. Defining the native niche of pre-HSCs has also been hindered by their rarity, transient nature and lack of a single definitive marker for their identification in situ. Here, we integrated single-cell and spatial transcriptomics to construct a spatiotemporal atlas of the niche in the AGM. Neural cells and chondrogenic mesenchyme predominated dorsally, whereas N-cadherin mesenchymal stromal cells (N-cad+ MSCs) formed a ventral layer between the aortic endothelium and mesonephric cells. BMP and SHH signaling showed overall dorsal enrichment, whereas WNT, NOTCH and KIT signaling were enriched ventrally, with these cellular and signaling patterns dynamically remodeled across HSC ontogeny. Imaging-based spatial transcriptomics validated the findings and further enabled the identification of individual pre-HSCs in situ and revealed a multicellular niche comprising endothelial cells, N-cad+ MSCs, intra-aortic hematopoietic cluster cells and macrophages. TGF{beta} and NOTCH signals were primarily supplied locally, whereas major sources of BMP and WNT were more distant. Functional perturbation identified niche-derived CD200 as an immune-regulatory signal promoting HSC development, validating a spatially predicted niche interaction. Together, our findings link tissue-scale spatial organization with the local multicellular niche supporting the preferential development of HSCs along the ventral aorta.

Antibodies engage both activating Fc Receptors and the inhibitory receptor Fc{gamma}RIIB. Why macrophages need a dedicated inhibitory receptor rather than simply tuning activating receptor signaling is unclear. Using DNA-based chimeric receptors and in silico modeling, we independently controlled activating and inhibitory Fc Receptors. We found that Fc{gamma}RIIB imposed a time limit on macrophage phagocytosis and ERK signaling. The time limit is due to activating Fc Receptors converting PI(4,5)P2 to PI(3,4,5)P3, which is subsequently converted to PI(3,4)P2 by Fc{gamma}RIIB. This leads to a pulse of active signaling, which is sufficient for phagocytosis of small bacteria-sized targets but not phagocytosis of large targets and TNF secretion. Unlike engaging Fc{gamma}RIIB, reducing activating Fc Receptor signaling decreased initiation of phagocytosis, the speed of PI(3,4,5)P3 generation, and the amplitude of ERK signaling. Our results demonstrate that Fc{gamma}RIIB controls the duration of IgG signaling, while the activating Fc Receptors control sensitivity.

Functional ovarian plasticity depends on the granulosa cell (GC) fate determination, differentiation, oocyte support and steroid production. After birth, GC identity must be maintained in differentiating follicles, while from puberty, ovarian plasticity is cyclically challenged by gonadotropic hormones. TRIM28-FOXL2 interactions form a key chromatin component maintaining ovarian identity programs and repressing testis-specific genes. This TRIM28 role depends on its E3-SUMO-ligase activity, suggesting SUMOylation in GC can be regulated by transient hormonal cues, although hormonal involvement in modulating GC identity remains to be established. Here, we demonstrated the in vivo interplay between gonadotropic-PKA-signaling and hypo-SUMOylation waves underlying ovarian GC plasticity. In vitro TRIM28 mutagenesis and phospho-proteomics demonstrated direct PKA inhibitory control on TRIM28. This prompted us to highlight SUMOylation alterations in Carney Complex ovarian lesions resulting from chronic PKA activity from rare genetic mutations. Using mutant models, transcriptomics and TRIM28/FOXL2-ChIP-Seq analyses, we specified the in vivo PKA inhibitory mechanisms on TRIM28/FOXL2 chromatin binding and its consequences on adult ovarian somatic cell identity. Together, our data support an integrative model where gonadotrophin pulses create transient windows of PKA-mediated TRIM28 phosphorylation, hypo-SUMOylation, and FOXL2-TRIM28 destabilization. This positions SUMOylation dynamics as central integrator of hormonal signaling and epigenetic identity maintenance in the ovary.

Coronaviruses cause human infections with divergent clinical outcomes. Viral proteins likely play important roles in the severity of human diseases but are poorly defined. In this study, we discovered that viral nucleocapsid (N) proteins from various human coronaviruses have distinct abilities to antagonize human innate immune responses. N proteins from more pathogenic coronaviruses, such as SARS-CoV and SARS-CoV-2, have more potent activities specifically against human RIG-I-like receptor (RLR) activation than less pathogenic coronaviruses including OC43 and HKU1. Mechanistically, the SARS-CoV-2 N protein interacts with RLR pathway viral RNA sensors MDA5 and RIG-I and inhibits their interaction with MAVS. The CTD of SARS-CoV-2 N protein is an important region for RLR antagonism. Consequently, N proteins from more pathogenic coronaviruses promote viral replication more effectively than N proteins from less pathogenic coronaviruses. Blocking N protein function may be a promising antiviral strategy.

The endocytic pathway plays key roles in facilitating and regulating synaptic plasticity, but how its components are organized at spines undergoing plasticity is not fully understood. Here we use live-cell imaging and single-particle tracking to examine the localization of endosomes and lysosomes in response to plasticity-inducing stimuli. We find that endosomes and lysosomes are positioned and retained near the bases of stimulated spines after the induction of structural plasticity in a manner dependent on actin polymerization in the dendritic shaft. Spines with endocytic compartments near their bases exhibit greater glutamate receptor turnover and more sustained enlargement after stimulation than spines lacking them. These results suggest that actin polymerization in the dendritic shaft acts as a hub to organize critical cellular machinery at the bases of spines undergoing synaptic plasticity.

Human coronaviruses (HCoVs) cause pathogenic outcomes ranging from mild illness to severe respiratory disease. Determining how respiratory cells coordinate an early antiviral response is necessary to understand successful control of HCoV replication. The Type I Interferon (IFN) response is a major component of innate viral immunity. IFN signaling leads to the upregulation of hundreds of interferon-stimulated genes (ISGs) that can have antiviral activity, though the individual ISGs that are responsible for restricting mildly pathogenic HCoVs in the respiratory epithelium are not clear. Here, we developed a targeted CRISPR-Cas9 knockout sgRNA library (termed the respISG library), focusing on ISGs that are upregulated across a panel of respiratory cells, to screen for innate host factors that restrict the mildly pathogenic human coronavirus, HCoV-OC43. We executed cell death-based screens in immortalized human small airway epithelial cells and identified lymphocyte antigen 6E (LY6E) as a top hit. We confirmed LY6E activity in validation studies and found it regulates entry of various SARS-CoV-2 Spike-pseudotyped lentiviruses. In addition to demonstrating a key role for LY6E in blocking a variety of HCoVs in small airway epithelial cells, this study establishes a loss-of-function CRISPR-Cas9 screening platform for identification of HCoV antiviral ISGs in respiratory cells.

Acutely induced cellular senescence is increasingly recognized as vital for tissue repair but the precise roles of senescent cells in skeletal muscle regeneration remain paradoxical. Here, using single-cell RNA sequencing and a Unified Senescence Score (USS) methodology, we identify a distinct USSHigh (USSHi) muscle stem cell (MuSC) subpopulation emerging in early phase of acute injury induced muscle regeneration. Isolation via the surface marker MHCII enables characterization of these cells as exhibiting canonical senescent-like features. Further characterization through integrated transcriptomics, proteomics, and secretomics reveals their highly pro-inflammatory secretory capacity. Moreover, lipidomic profiling uncovers that the USSHi MuSCs undergo profound sphingolipid metabolic remodeling to generate upregulated bioactive lipid Sphingosine-1-phosphate (S1P). In vivo specific ablation of this subpopulation using a Pax7-CreERT; Ccl2-LSL-DTA genetic strategy significantly impaired muscle regeneration. Furthermore, spatial transcriptomics and cell-cell communication analyses revealed that USSHi MuSCs act as critical niche modulators, directing crosstalk with multiple cell types. Specifically, S1P is essential for mediating direct USSHi interaction with endothelial cells and USSLo cells to promote endothelial angiogenesis and MuSC expansion in the regenerating muscle. Crucially, exogenous S1P rescues the regenerative deficits caused by USSHi MuSC ablation. Altogether our findings define a senescent-like MuSC population emerging in acute injury induced muscle regeneration and provide definitive evidence to demonstrate the positive role of these cells in enhancing muscle repair via their robust secretory phenotypes and inter-cellular communicative capacity.

The SARS-CoV-2 nsp1 protein is a virulence factor that broadly inhibits cellular gene expression. Although cellular mRNAs are translationally inhibited by nsp1 and subsequently degraded, viral transcripts possess a 5' leader sequence (CoV2L) that enables them to escape nsp1-mediated repression. Both transcript targeting selectivity and mRNA decay require coordination by the nsp1 N-terminal domain (NTD) through an unknown mechanism. Here, we generated an alanine-scanning library of mutations encompassing all residues in the nsp1 NTD to gain a deeper understanding of how this domain coordinates nsp1 function. We screened this library for the ability to repress mRNA bearing a host- or CoV2L-derived 5' untranslated region, revealing two predominant clusters of residues required for target selectivity. These largely comprised adjacent surface-exposed beta-sheets on the NTD, whose deletion rendered CoV2L-containing mRNA susceptible to repression and prevented nsp1-induced mRNA decay. This work provides residue-level information on the role of the NTD in distinguishing among mRNA targets and further links nsp1 target selectivity to mRNA decay.

Despite decades of HIV-1 vaccine development, the clonal and cellular determinants of durable vaccine-induced T cell memory remain incompletely understood. Here, we combined antigen-specific T cell receptor (TCR) identification, longitudinal TCR sequencing, and single-cell multi-omics to characterize Env- and Gag-specific memory precursor T cells elicited by the HIV Vaccine Trials Network (HVTN) 505 DNA prime-recombinant adenovirus serotype 5 (rAd5) boost (DNA/rAd5) vaccine regimen. We developed a generalizable high-throughput approach to identify HIV-1 Env- and Gag-specific TCRs and found distinct patterns of clonal expansion, persistence, and contribution to memory between Env- and Gag-specific CD8 T cell responses. The DNA prime and rAd5 boost differentially shaped these repertoires, with rAd5-induced clones contributing proportionally more to the Gag-specific than to the Env-specific memory precursor compartment. Single-cell immune profiling further revealed distinct memory precursor states, with Env-specific responses enriched for GZMBPRF1 cytotoxic effector-memory (EM) CD8 T cells and Gag-specific responses containing a larger cycling/proliferative population. Together, these findings demonstrate that heterologous DNA/rAd5 vaccination generates antigen-specific CD8 T cell memory with distinct clonal trajectories and cellular programs, providing new insights into how vaccine platform and antigen-specificity shape the durability and functional properties of HIV-1 specific cellular immunity.

The human HSP70 chaperone network maintains cellular proteostasis through a diverse repertoire of HSP70s and co-chaperones. Here we examine alternative isoforms and co-chaperone hetero-complexes as additional sources of network complexity. To that end, we systematically mapped the isoform, tissue-expression, and interaction landscapes of the human HSP70 network, revealing a modular interactome containing known and novel DNAJ-DNAJ interactions. We found both tissue-expression isoform divergence as well as widespread alternative-canonical isoform co-expression, suggesting additional modes of functional diversification. Focusing on the uncharacterized DNAJA2-DNAJA4 hetero-complex, we identified the stress-inducible isoform DNAJA4-CTD-II. DNAJA4-CTD-II formed hetero-complexes with DNAJA2 and DNAJA4, with both interactions enhanced following sodium arsenite stress. Functionally, DNAJA4-CTD-II co-localized with TDP-43 aggregates and significantly suppressed their accumulation in a DNAJA2-dependent manner. Together, our data reveal extensive, uncharted isoform and interaction complexity within the HSP70 network, and uncover isoform-dependent hetero-complex remodeling as a new layer of chaperone network regulation.

The balance between spermatogonial proliferation and differentiation is essential for sustaining male fertility, yet the contribution of cellular metabolism remains poorly understood. Here, we showed that adenosine levels increased significantly during spermatogonial differentiation and promoted the formation of differentiating spermatogonia. Using a conditional knockout mouse model in which Ahcy, a gene encoding an enzyme in adenosine synthesis, is deleted from spermatogonia, we found that disruption of adenosine generation blocked spermatogonial differentiation, resulting in a transient delay in spermatogenesis. We further demonstrated that adenosine exerted its effects partially through ADORA2A- and ADORA2B-mediated signaling. Downregulating these receptor activities by specific inhibitors in spermatogonial culture or by genetic ablation in mice led to increased spermatogonial proliferation. Taken together, our study identified adenosine as a metabolic signal in refining the balance between spermatogonial proliferation and differentiation, linking cellular metabolism to critical developmental timing and stem cell fate decisions.

The coronavirus nucleocapsid (N) protein is indispensable for the viral lifecycle as part of the viral Replication-Transcription Complex together with Nsp3. Recent research demonstrated that phosphorylation of N by several host kinases intricately regulates its functions during infection. However, the mechanisms that control N dephosphorylation, and its physiological consequence, remain poorly understood. Here, we show that SARS-CoV-2 Nsp3 is a key orchestrator of N dephosphorylation by recruiting the phosphatase PP1/{gamma} via a conserved PP1-binding motif in the Ubl1 domain. Disruption of this motif abolishes N dephosphorylation and selectively impairs subgenomic RNA synthesis. Comparative interactome proteomics analysis of phospho-mimic vs. phospho-deficient N, together with functional validation, revealed that the host splicing factor SRSF1 cooperates with unphosphorylated N to promote subgenomic RNA transcription. These findings uncover an unrecognized mechanism in which Nsp3-guided N dephosphorylation by PP1/{gamma} enables coronavirus subgenomic RNA replication and highlight a potentially targetable axis for antiviral intervention.

Middle East respiratory syndrome coronavirus (MERS-CoV), first reported in 2012, belongs to the Betacoronavirus genus, including SARS-CoV and SARS-CoV-2. Human-to-human transmission of MERS-CoV appears inefficient, but repeated spillover events have been reported from at least 27 countries, raising concern for future emergent events. Coronaviruses (CoVs) rely extensively on host proteins to support their replication. Several studies have implicated the paraspeckle non-POU domain-containing octamer-binding protein (NONO) as an RNA-binding protein (RBP) that binds to CoV genomes, but the role of this protein in regulating replication is unknown. Here, we show that NONO is required for expression of dipeptidyl peptidase 4 (DPP4, also known as CD26), the major cellular receptor required for MERS-CoV attachment and entry. NONO did not impact DPP4 mRNA processing or stability but instead promoted DPP4 transcription through control of active H3K4me3 and repressive H3K27me3 histone modifications at the DPP4 locus. Together, these findings identify NONO as a key proviral host factor for MERS-CoV and reveal an epigenetic mechanism linking a host RBP to viral entry receptor expression that may represent a target for therapeutic strategies.