Cell reports最新文献

Quantifying enzymatic activity at the single-cell level remains challenging. Building on previous development of metal-tagged activity-based probes (time-of-flight [TOF]-probes) compatible with mass cytometry, we adapt this technology to quantify individual proteases in immune cells. We optimize and validate TOF-probes targeting catalytically active cysteine and serine proteases in two primary immune populations: neutrophils and monocyte-derived macrophages. Using this platform, we observe striking shifts in cathepsin activity during monocyte-to-macrophage differentiation, along with a functional rewiring from apoptotic to pyroptotic protease programs. In neutrophils, unexpectedly, we detect high levels of active serine proteases even in the resting state, highlighting their potential role as pre-armed effector cells poised for rapid inflammatory responses. These findings lay the foundation for using TOF-probes to quantify active proteases in situ at the single-cell level and provide new insights into protease activation states across immune cells.

While high-fiber diets (HfiDs) promote weight loss, their long-term efficacy is limited by rapid weight regain upon returning to high-fat diets (HFDs). Using C57BL/6J mice in diet-switching paradigms, we characterized tissue-specific responses to HfiD-to-HFD transitions through single-nucleus and spatial transcriptomics. HfiD pre-feeding enhanced mesenteric white adipose tissue progenitor/adipocyte sensitivity to subsequent HFD exposure. In the intestine, HfiD prevented HFD-induced immune-enterocyte expansion in the duodenum and reversed the enterocyte-to-goblet cell shift in the colon while maintaining persistent epigenetic reprogramming. Although HfiD-induced microbiome changes were largely reversed by HFD, we identified sexually dimorphic remodeling of adipose cell populations during diet transitions. Our findings demonstrate that prior HfiD feeding fundamentally reprograms adipose and intestinal responses to subsequent HFD challenge, providing mechanistic insights into dietary intervention outcomes. This work establishes a spatiotemporal resource for understanding tissue plasticity during dietary changes, offering new perspectives for obesity management strategies.

NLRP3 inflammasome activation requires both transcriptional priming and complex assembly, but how RNA m⁶A methylation coordinates these steps remains unclear. Here, we show that m⁶A levels increase during macrophage NLRP3 inflammasome activation and that METTL3 loss suppresses this activation. Myeloid-specific Mettl3 knockout mice display reduced inflammation and improved metabolic outcomes in lipopolysaccharide (LPS)-induced sepsis, monosodium urate (MSU)-induced arthritis, and diet-induced obesity. Integrated chromatin-associated RNA sequencing (chrRNA-seq), kethoxal-assisted single-stranded DNA sequencing (KAS-seq), and chrRNA-methylated RNA immunoprecipitation (MeRIP)-seq analyses show that METTL3 installs m⁶A co-transcriptionally on nascent Jak1, Nlrp3, and Il1β RNAs and that METTL3 regulates dynamic transcription and chromatin accessibility while selectively maintaining Nlrp3/Il1β transcription. YTHDF1-driven translation of Jak1 activates the JAK1-STAT3-C/EBPβ axis to initiate Nlrp3/Il1β transcription, and m⁶A-YTHDF1 translation of Nlrp3/Il1β amplifies protein output, forming a coupled transcriptional-translational circuit. Pharmacologic STAT3 inhibition and METTL3 catalytic rescue validate this pathway and identify METTL3-mediated m⁶A as a therapeutic target for inflammasome-driven diseases.

Ruminants thrive in diverse ecosystems by leveraging their rumen microbiome to ferment fibrous plants. However, the spatial biogeography of rumen microbiome and the genetic diversity of the ventral rumen epithelium remain unknown. Here, we present a multi-omics study in roe deer, sika deer, and sheep, integrating region-resolved microbiome and metabolome across 11 ruminal sacs, as well as single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin using sequencing (ATAC-seq), and bulk RNA sequencing (RNA-seq) of ventral epithelium. We reveal species-specific microbial compositions and metabolic capacities contributing to differences in short-chain fatty acid and vitamin B production. We uncover functional divergence, genomic specialization, and metabolic changes across the microbiome of distinct ruminal sacs. Single-cell profiling reveals changes of immune responses and structural remodeling of the ruminal ventral epithelium. We demonstrate that vitamin B₁₂ promotes epithelial growth and we identify genes enhancing stem cell differentiation. Our results highlight variation in microbial ecology and epithelial architecture among three ruminant species, offering insights to improve livestock productivity.

Interferon regulatory factor 3 (IRF3) functions as a key transcription factor in the innate antiviral immune response, which depends on its nuclear localization. However, its function in the cytoplasm during non-infection states remains largely unknown. In this study, we found that resting cytoplasmic IRF3 interacts with hypoxia-inducible factor (HIF)-1α and HIF-2α, two master regulators of hypoxia signaling. This interaction retains HIF-α in the cytoplasm under hypoxic conditions, preventing it from exerting its transcription factor function and attenuating hypoxia signaling. Disruption of IRF3 in both mice and zebrafish resulted in increased expression of hypoxia response genes and enhanced tolerance to hypoxia. These findings suggest that, in the absence of viral infection, cytoplasmic IRF3 modulates hypoxia signaling by retaining HIF-α in the cytoplasm under hypoxic conditions.

Promoter-proximal pausing of RNA polymerase II (Pol II) primes genes for rapid activation, yet how Pol II dynamics are temporally organized in adult stem cells to enable fast and flexible responses to environmental cues remain unknown. To address this, we developed sciCUT&Tag2in1 for joint profiling of Pol II and histone modifications in single cells. By profiling over 200,000 CD34⁺ hematopoietic stem cells (HSCs) and progenitors, we identify a Pol II regulatory cascade that directs the response to granulocyte colony-stimulating factor (G-CSF)-induced inflammatory stress. HSCs are activated by elevated Pol II occupancy and reduced Polycomb repression of immune response genes. Lineage commitment proceeds through sequential modes of Pol II activation, beginning with rapid pause-and-release genes, followed by slower initiate-and-release of Polycomb-repressed targets. sciCUT&Tag2in1 defines the temporal logic of how adult stem cells use paused Pol II to enable flexible lineage decisions, providing a powerful tool for studying the intersection of development, inflammation, and disease.

The levels of N6-methyladenosine (m6A)-modified RNAs are reduced within cells with high-risk human papillomaviruses (HPVs) in comparison to normal cells. These reduced m6A levels stabilize RNA:DNA hybrids (R-loops), whose regulation is critical for viral replication. HPV E6 induces the degradation of the m6A methyltransferases, Mettl3 and Mettl14, in an E6AP-dependent manner to reduce m6A levels, while the RNA helicase senataxin (SETX) counteracts this by activating the expression of m6A regulators. Maintenance of the residual m6A levels in undifferentiated cells is also critical for viral replication. A subset of early viral transcripts is m6A modified, and the inhibition of Mettl3 impairs viral gene expression. Upon differentiation, the levels of SETX and m6A are increased by over 8-fold, which is necessary for the productive replication of HPVs. These studies identify a role for SETX and E6 in regulating m6A deposition to control HPV pathogenesis in a differentiation-dependent manner.

Precise control of pre-mRNA splicing is vital for transcriptome integrity, and its disruption is an emerging cancer vulnerability. Here, we use indisulam to degrade RBM39 and show that the clinical ALK inhibitor alectinib can be repurposed to inhibit SRPK1. Co-treatment of indisulam and alectinib inhibited cell proliferation, induced apoptosis, and caused cell-cycle arrest in multiple cancer cell lines, including MYCN-amplified high-risk neuroblastoma. RNA sequencing revealed enhanced splicing defects preferentially in DNA repair-related genes following combination treatment, leading to R-loop accumulation and increased DNA damage. In the Th-MYCN/ALK^F1174L neuroblastoma mouse model, combination therapy induced complete tumor regression and significantly improved survival rates compared with monotherapies. These findings demonstrate that combining indisulam and alectinib is a promising approach to treating aggressive malignancies such as high-risk neuroblastoma, exploiting the untapped polypharmacology of alectinib as an RNA splicing inhibitor and supporting the therapeutic value of co-targeting interdependent splicing factors for synergistic benefit.

The identities of primary afferents transducing mechanical allodynia following nerve injury remain unclear. We genetically label brushing-activated (BA) trigeminal ganglia (TG) neurons using Fos^CreER mice with trigeminal nerve injury (TNI). BA TG neurons are largely medium-sized. Many express neurofilament200 and Ntrk3, markers for low-threshold mechanoreceptors, with lower co-localization with nociceptor markers such as Calca or Trpv1. Chemogenetic inhibition of BA TG neurons reduces mechanical allodynia, whereas their chemogenetic activation increases spontaneous face wiping after TNI. Brushing-induced conditional knockdown (bcKD) of Piezo2 from BA TG afferents reduces punctate and dynamic mechanical allodynia. In vivo TG GCaMP Ca²⁺ imaging shows that Piezo2 bcKD reduces not only hypersensitivity to low-force mechanical stimulation, mostly among medium-sized neurons, but also, unexpectedly, TNI-induced spontaneous activity. Therefore, Fos is useful for genetic labeling and manipulation of BA TG neurons. Furthermore, innocuous mechanical stimuli activate multiple TG afferent subtypes after TNI, possibly accounting for the complexity of resulting painful symptoms.

The FANCD2-FANCI heterodimer contributes to DNA repair at interstrand crosslinks and sites of replication stress. This complex has been physically and mechanistically linked to double-strand break (DSB) repair, but its role in that process remains undefined. Here, we show that the FANCD2-FANCI heterodimer dynamically interacts with open chromatin regions, including transient DSB-induced open chromatin, where it can be stabilized through co-activation by the DNA repair kinase ATM and the Fanconi anemia core ubiquitin ligase. The loaded FANCD2-FANCI heterodimer stabilizes open chromatin and promotes resection and loading of RPA through increased association of BRCA1 and BLM. Chromatin-loaded FANCD2-FANCI has a second, distinct function promoting a G2 cell cycle arrest that is dependent on the ATR-CHK1-WEE1 axis. Our results support a two-step genome surveillance model in which FANCD2-FANCI monitors open chromatin sites and is stably loaded to coordinate DNA repair activities in response to signaling from a DNA repair kinase.