The AAA ATPase VCP/p97 has emerged as a critical regulator of ubiquitin and chromatin-associated processes but progress in understanding has been hampered by the complexity of p97 functions and the various p97 cofactors involved. Here, we combined ubiquitin profiling with acutely induced degradation of the Ufd1 subunit of the p97 ubiquitin adapter, Ufd1-Npl4, in human cells. We identified a set of chromatin regulators, HUS1, XRCC1, MORF4L1, and the cohesin subunit RAD21 as targets of p97 Ufd1-Npl4 . We find that RAD21 is ubiquitylated and targeted by p97 Ufd1-Npl4 specifically in S phase to remove a subpopulation of cohesin from chromatin. Acute degradation of Ufd1 in S phase, after replication licensing is completed, impedes replication and leads to replication-associated DNA damage. Our findings suggest that a fraction of cohesin rings need to be removed by p97 Ufd1-Npl4 from DNA to allow unhindered replication and reveal a critical function of p97 that ensures genome stability.
In periods of sustained hyper-nutrition, pancreatic β-cells undergo functional compensation through transcriptional upregulation of gene programs driving insulin secretion. This adaptation is essential for maintaining systemic glucose homeostasis and metabolic health. Using single nuclei multiomics, we have mapped the early transcriptional adaptive mechanisms in murine islets of Langerhans exposed to high-fat diet (HFD) for 1 and 3 wk. We show that β-cells exhibit the largest transcriptional response to HFD, characterized by early activation of pro-inflammatory eRegulons and down-regulation of β-cell identity genes, particularly in a distinct subset of β-cells. These observations extend to humans, where the prevalence of an β-cells with a high inflammatory signature is increased in diabetes. Collectively, these observations point to cellular crosstalk through pro-inflammatory signaling as a central and early driver of β-cell dysfunction that limits the compensatory capacity of β-cells, which is closely linked to the development of diabetes.
Retrotransposons are emerging as novel regulators of embryonic and brain development. We recently demonstrated that the LINE-1-encoded protein ORF1p is abundantly expressed in adult mouse and human neurons, although its function remains unclear. Here, we characterize the ORF1p interactome in differentiated mouse and human neurons using mass spectrometry and identify novel partners implicated in gene regulation and neuron-specific processes. ORF1p localizes not only to neuronal nuclei, where it associates with chromatin under steady-state conditions, but also to neurites, supporting a role in neuronal physiology. To further explore its nuclear functions, we sorted human post-mortem neurons with high or low nuclear ORF1p levels and performed ORF1p knockdown in cultured human neurons, followed by chromatin accessibility assays. Both approaches revealed consistent patterns of differential chromatin accessibility dependent on ORF1p. Loss of ORF1p also led to the down-regulation of long, neuron-specific genes and altered neurite morphology. Together, these findings point to a physiological role of ORF1p in post-mitotic neurons, mediated through converging interactions with proteins and chromatin.
Legionella pneumophila infects and replicates within protozoa and macrophages in a specialized compartment termed the Legionella-containing vacuole. To establish this niche, the bacterium delivers more than 350 effector proteins through its type IV secretion system to modulate host pathways and prevent phagolysosome fusion. Here, we identify the effector Lpg0733/RavH as a tandem WH2 domain-containing actin nucleator. RavH localizes to endosomal membranes via its N-terminal lipid-binding domain and promotes actin polymerization when expressed in yeast or mammalian cells. We further demonstrate that RavH functions as a membrane-dependent pointed-end actin nucleator, requiring both its lipid-binding domain and multiple C-terminal WH2-like domains to drive robust actin assembly on PI(3)P-containing membranes. Although dispensable for Legionella intracellular replication in Acanthamoeba castellanii amoeba cells, RavH is recruited to the Legionella-containing vacuole during the early stages of infection in macrophages, where it may contribute to vacuole positioning and motility.
Protein folding by the bacterial chaperonin GroEL/ES relies on ATP-driven conformational cycles that promote substrate encapsulation and folding. Under oxidative stress, the redox-active co-chaperone CnoX protects oxidized proteins and associates with GroEL, yet the structural basis of its interaction with the GroEL/ES remains incompletely understood. Using single-particle cryo-electron microscopy, we resolved four distinct nucleotide-bound conformational states of CnoX-associated GroEL/ES complexes. CnoX remains tethered to GroEL through its C-terminal TPR domain despite substantial rearrangements of the GroEL apical domains. We further captured a GroEL/ES-CnoX ternary assembly in which CnoX and GroES simultaneously occupy the same GroEL ring, demonstrating that their binding sites are structurally distinct and non-overlapping. Comparison of two GroES-bound states reveals how apical-domain compaction occludes the CnoX-binding surface and coincides with loss of CnoX from the cis-ring. Together, these structures define how CnoX is accommodated and excluded across distinct GroEL/ES conformations and provide a structural framework for understanding the interplay between redox co-chaperones and chaperonin assemblies.
LIMD1, a tumour suppressor located at chromosome 3p21.3, is frequently lost in non-small-cell lung cancer, yet its role in tumour-immune interactions remains unclear. Here, we show LIMD1 loss increases PD-L1 protein abundance across multiple lung cancer models and primary airway epithelial cells. Mechanistically, LIMD1 restrains PD-L1 through post-transcriptional and post-translational mechanisms. LIMD1 loss can relieve microRNA-mediated repression of the CD274 3'UTR, and LIMD1 loss can also disrupt ARIH1-PD-L1 association, reduce PD-L1 polyubiquitination, and stabilise PD-L1 protein without a commensurate increase in CD274 transcript levels in isogenic models. Functionally, LIMD1-deficient tumour cells suppress CD8+ T-cell activation in vitro and show enhanced sensitivity to PD-1/PD-L1 blockade in tumour-PBMC co-culture assays. Analysis of TRACERx non-small-cell lung cancer samples revealed clonal LIMD1 loss of heterozygosity in ∼40% of lung adenocarcinomas, where it is associated with increased tumour PD-L1 expression. Across independent patient cohorts receiving immune checkpoint blockade, low LIMD1 expression was enriched among responders. We identify LIMD1 as a tumour-intrinsic regulator of PD-L1 turnover and suggest that tumour suppressor loss can shape immune checkpoint biology and influence immunotherapy response.
P-bodies are cytoplasmic membraneless organelles involved in mRNA storage, yet their role in cellular stress responses remains unresolved. Here, we demonstrate that P-bodies are remodeled during the early response to ER stress throughout Drosophila melanogaster oogenesis. Notably, this remodeling occurs within minutes of stress induction and precedes stress granule formation. This early remodeling is characterized by changes in P-body morphology and internal organization and promotes selective mRNA storage. Mechanistically, we find that this process is driven by transcriptional up-regulation of the RNA-binding protein, Bruno 1, downstream of ATF4-dependent stress signaling, thereby establishing a connection between the unfolded protein response and condensate regulation. Consistent with this model, loss of Bruno 1 abolishes, whereas its overexpression enhances, P-body remodeling, demonstrating that stress-induced changes in RNA-binding protein levels can reprogram condensate properties. Together, our findings reveal that P-bodies function as stress-responsive hubs enabling selective preservation of essential mRNAs during ER stress. More broadly, this work uncovers a previously unrecognized mechanism by which stress signaling pathways reorganize cytoplasmic architecture to shape mRNA fate.
The Jagged1 (JAG1) gene is essential for cardiac development, yet its tissue-specific transcriptional regulation remains poorly understood. In this study we used an integrative screening approach to identify 19 candidate enhancers within the ±100 kb region flanking the JAG1 locus, among which R7 exhibited the highest activity in dual-luciferase assays. CRISPR/Cas9-mediated deletion of R7 in AC16 cells significantly reduced JAG1 expression, decreased proliferative and migratory capacities, and increased apoptosis. Mechanistically, R7 deletion altered local chromatin contacts and reduced accessibility at CTCF-bound regions near the JAG1 promoter, accompanied by decreased H3K27ac, H3K4me3, RNA polymerase II, and SRF occupancy. These findings identify R7 as a cardiac-associated promoter-proximal regulatory element with enhancer-like activity that contributes to local chromatin organization and transcriptional activity at the JAG1 locus.
Cell-cycle dynamics of gene expression are fundamental to life, yet their mechanistic basis remains unclear. A prevailing model, derived from cancer cells, posits that RNAs transcribed in one cell-cycle phase peak in abundance in subsequent phases, suggesting temporal segregation of transcriptional regulation and RNA maturation during the cell cycle. However, whether such extensive transcription-to-maturation lags occur in normal cells remains unclear. Here, we report a lack of evidence for systematic transcription-to-maturation lags extending across the G1-to-G2-phase boundary in normal fibroblasts. Using FUCCI cell-cycle reporters coupled with RNA-seq and GRO-seq, we defined the cell-cycle dynamics of nascent transcription and mature RNA abundance in BJ-5ta human fibroblasts. Transcription and mature RNA accumulation were strongly temporally concordant. Genes with peak transcription in G1, early S, or late S/G2/M showed peak mature RNA abundance in the corresponding phases, without extensive lags. Minor lags within the G1 phase were detected, confirming the sensitivity of our approach. Thus, our data do not support the generality of the prevailing model of extensive transcription-to-accumulation lags in normal human cells.
Oxygen-sensing and the hypoxia stress response play vital roles in physiological homeostasis. Subterranean species that are naturally adapted to hypoxia provide powerful tools for understanding the mechanisms of hypoxia tolerance. The plateau zokor Eospalax baileyi and the Gansu zokor Eospalax cansus, which inhabit the Qinghai-Tibet Plateau of China, and the blind mole rat Spalax galili, which lives in Israel, are all subterranean rodents adapted to hypoxic environments. Here, we showed that the T480S variant of the hypoxia-inducible factor-2ɑ (encoded by the endothelial PAS domain protein 1 gene, Epas1) in Chinese zokors stabilized EPAS1 by reducing phosphorylation, thereby regulating fibrosis and contributing to hypoxia adaptation on the Qinghai-Tibet Plateau. In Israeli blind mole rats, variants at positions -2023/-1810 in the Epas1 regulatory region distinguish two abutting populations undergoing incipient sympatric speciation by altering the binding of cMYB and hepatocyte nuclear factor 4 gamma, linking transcriptional activity to the interferon mRNA transcription. This finding reveals an adaptive regulation of metabolism and O2 homeostasis by the Epas1 gene in adaptation to diverse habitats, providing evidence for both divergent and convergent functional molecular evolution.
Increased sympathetic tone and hypertension are hallmarks of metabolic syndrome and contribute to chronic kidney disease. Although renal sympathetic denervation transiently lowers blood pressure, its role in the development of metabolic and renal alterations remains unclear. Here, we evaluated the contribution of renal sympathetic input to the onset and progression of high-fat diet-induced alterations. Male Wistar rats underwent bilateral renal denervation before metabolic challenge and were fed a standard or high-fat diet for 8 or 12 wk. High-fat feeding induced hypertension, proteinuria, increased angiotensin II, and reduced creatinine clearance, urinary flow, and potassium excretion, independently of denervation. Renal norepinephrine content confirmed effective denervation and was not affected by diet. Denervation attenuated ketonuria in high-fat diet-fed rats. The phosphorylation of AKT, PI3K, and ERK1/2 in the kidney was modulated by interactions among diet, renal sympathetic input, and time. These findings indicate that renal sympathetic nerves contribute to early stages of metabolic dysregulation, whereas prolonged hypercaloric exposure overrides autonomic control and promotes cardiovascular and renal complications.
The genome organizer special AT-rich sequence binding protein 1 (SATB1) is critical for the development of T lymphocyte subsets by regulating Foxp3 and Pdcd1 expression. However, its physiological role in mature peripheral T cells has remained obscured by the severe developmental defects caused by early-stage gene knockout models. Here, we used a Thpok-cre driver to specifically delete Satb1 after CD4 lineage commitment. We demonstrate that SATB1 is continuously required to suppress Foxp3 expression in conventional CD4+ T cells; however, this aberrant derepression does not confer Treg suppressive activity. In contrast, FoxP3+CD25+ Tregs isolated from SATB1-deficient mice exhibit functional defects in immune suppressive function. Although SATB1 deletion does not cause severe global defects in the Treg transcriptome, it disrupts essential FoxP3-mediated gene regulation, resulting in a targeted failure to silence transcripts detrimental to Treg fitness. Physiologically, this Treg-specific functional defect significantly enhances antitumor immunity in a melanoma model. Our findings reveal a dual requirement for SATB1, preserving lineage fidelity by repressing Foxp3 in conventional T cells, while enforcing foundational programming required for Treg suppressive function.
Spinal muscular atrophy is a neuromuscular disorder primarily caused by mutations in the SMN1 (Survival of Motor Neuron 1) gene. SMN1 is ubiquitously expressed and encodes a protein essential for the assembly of small nuclear ribonucleoproteins, key components of pre-mRNA splicing. The SMN protein also participates in several other fundamental cellular processes, including RNA transport, regulation of actin dynamics, transcription, and translation. While multiple hypotheses have been put forward to explain the selective motor neurons (MNs) vulnerability to SMN deficiency, the precise mechanisms involved remain incompletely understood. In this study, we used neuron-specific smn-1 RNAi silencing in D-type MNs or in touch receptor neurons in C. elegans. In touch receptor neurons, smn-1 silencing caused distinct defects in neuronal process morphology. Our results reveal pronounced neuron-specific differences in sensitivity within the neurons of C. elegans, providing a robust framework to dissect the mechanisms underlying selective neuronal vulnerability of spinal cord MNs in spinal muscular atrophy.
Mutations in the human SPTLC1 gene have recently been linked to early-onset amyotrophic lateral sclerosis (ALS), characterized by global atrophy, motor impairments, and symptoms such as tongue fasciculations. All known ALS-linked SPTLC1 mutations cluster within exon 2, and a specific variant, c.58G>T, results in exon 2 skipping. However, it is unclear how the exon 2 deletion affects SPTLC1 function in vivo and contributes to ALS pathogenesis. Leveraging the high genomic sequence similarity between mouse and human SPTLC1, we created a novel knock-in mouse model with a CRISPR/Cas9-mediated deletion of exon 2 in the endogenous murine Sptlc1 locus. Although heterozygous mice did not develop motor defects or ALS-like neuropathology, homozygous mutants died prematurely. These findings provide valuable insights into SPTLC1 exon 2 biology and serve as a useful resource for future mechanistic studies.
Peroxisomal dysfunction contributes to a broad spectrum of multisystem disorders, yet mechanistic understanding and therapeutic options remain limited, posing significant challenges for clinical management. Network-based computational strategies support hypothesis generation, biomarker discovery, and drug repurposing, but their usage is constrained by incomplete human interactome coverage-especially by scarcity of high-confidence protein-protein interaction (PPI) data for peroxisomal proteins. We present the first comprehensive map of the peroxisomal interactome, generated using an automated, informatics-guided bioluminescence resonance energy transfer strategy. We profiled PPIs for 92 peroxisomal proteins and six isoforms, validating 68% of known interactions and identifying 333 novel ones. Integration with curated PPIs yielded an expanded peroxisomal interactome, enriched for drug targets and disease-associated proteins. A disease-linked subnetwork enabled prioritization of drug repurposing candidates. Tissue-specific expanded peroxisomal interactome variants, derived from transcriptomic data, revealed distinct functional submodules across nine tissues. Gene ontology analysis of 1,272 non-peroxisomal interactors suggested pathways contributing to tissue-specific vulnerability. Our approach provides a systems-level framework for mechanistic insight in peroxisomal disease, the identification of treatment targets, and application to other organelle systems.
Epithelial tissues undergo rapid expansion during development, repair, and morphogenesis, yet how tissue-scale growth is coordinated to re-establish homeostasis remains unclear. Here, we show that large epithelial monolayers confined at a wide range of initial densities and mechanochemical states robustly converge to the same final size and density upon release, despite differences in initial cell size, YAP activity, and cell number dynamics. To investigate the underlying mechanism, we combined quantitative experiments with a mechanochemical agent-based model in which mechanical pressure arising from confinement acts as a tissue-scale signal that modulates intracellular cell-cycle activity over time. Using this framework, we show that transient mechanical relaxation during confinement selectively elevates cell-cycle activity in higher-density tissues at the time of release, accelerating early expansion without disrupting final homeostatic outcomes. Together, these results reveal how epithelial tissues coordinate collective growth and robustly restore homeostasis during expansion.
Myopia is a global cause of vision impairment, yet its molecular mechanisms remain unclear. We applied a multi-omics approach to the retina, choroid, and sclera of guinea pigs (GPs), a model of spontaneous myopia. Integrated transcriptomic and proteomic analyses suggest a two-state molecular framework. In a myopia-prone state, the choroid and sclera exhibited widespread transcript-protein discordance, with RNA and protein changes in remodeling pathways not aligned in direction, alongside metabolic down-regulation and suppressed immune transcription, a primed yet quiescent state preceding elongation. In a spontaneous myopic state, molecular regulation becomes more concordant, with RNA and protein changing in the same direction, indicating active remodeling, whereas ribosome/translation emerged as a consistent cross-tissue signal. Integration with human myopia GWAS loci, followed by validation of CYP26A1 expression in GP ocular tissues at both transcript and protein levels, highlighted post-transcriptional regulation in the sclera and underscored translational relevance. These findings provide a molecular framework for myopia development and nominate targets in neurogenesis, synaptic remodeling, and translational control for therapeutic intervention.
Single-cell proteomics (SCP) reveals cellular heterogeneity and biological insights inaccessible to bulk analysis. Existing limitations are cost, sample loss during processing, and accessibility to state-of-the-art instrumentation. We describe a label-free SCP methodology in human tissue, combining FACS, oil-immersion cell handling, mass spectrometry, and neural-network-derived spectral libraries, which address these issues. We tested this methodology in a skin tumor syndrome, CYLD cutaneous syndrome (CCS), assessing tumor heterogeneity. Using a Bruker timsTOF HT platform, we quantified >4,000 proteins, averaging ∼700 per cell, through a cost-effective pipeline without specialised liquid handling infrastructure. By using preexisting bioinformatic tools from the scRNA-seq field, we implemented a robust analysis methodology, discriminating between macrophages, dendritic cells, and tumor keratinocytes, in an unbiased analysis of 419 CCS tumor cells. We validated the biological accuracy of cell annotations by cross referencing with each cell's FACS markers. Furthermore, we identified a novel CCS tumor-associated macrophage population, which carried a tumor microenvironment remodelling signature. Our findings demonstrate an accessible SCP technology capable of yielding novel biological discoveries in clinical tissue.
The switch from fetal to adult hemoglobin is tightly regulated during erythropoiesis, and its dysregulation can contribute to β-hemoglobinopathies. Although transcriptional repression of γ-globin by BCL11A is well established, the posttranscriptional mechanisms that sustain BCL11A expression in adult erythroid cells remain incompletely understood. Here, we report that the long noncoding RNA UCA1/miR-148b-mediated regulatory axis is critical for globin gene switching. We identified miR-148b as a direct posttranscriptional regulator of BCL11A. lncRNA UCA1, which is abundantly expressed in adult erythroid cells, functions as a molecular decoy for miR-148b, thereby attenuating miR-148b-mediated repression of BCL11A. Depletion of UCA1 increases miR-148b availability and reduces BCL11A expression, which can lead to robust induction of γ-globin. Conversely, the ectopic expression of UCA1 restores BCL11A levels by antagonizing miR-148b, thereby promoting γ-globin silencing in adult erythroid cells. Mechanistically, UCA1 orchestrates a posttranscriptional regulatory axis that reinforces γ-globin silencing by stabilizing BCL11A levels in adult erythroid cells. Taken together, our studies uncover a previously unrecognized lncRNA-mediated mechanism that integrates miRNA activity with transcriptional control to fine-tune hemoglobin switching during adult erythropoiesis.
Heat shock proteins safeguard proteostasis under stress. We examined mitochondrial chaperonin HSP60 in three cnidarians to assess stress responses. We evaluated HSP60 expression in Pocillopora acuta (hard coral), Exaiptasia diaphana (sea anemone), and Cassiopea xamachana (upside-down jellyfish) using immunoblotting. In P. acuta , HSP60 was not detected at the fragment level under either control (25°C) or heat-stress (30°C). In contrast, isolated cells showed transient HSP60 expression under both temperature conditions, indicating context-dependent regulation in coral. E. diaphana and C. xamachana showed elevated HSP60 expression over 24 h when stressed (+5°C above laboratory optima). These patterns indicate lineage-specific regulatory mechanisms underlying chaperone-mediated stress response pathways. Thus, thermal sensitivity varies among species and across biological contexts. Consistent antibody cross-reactivity prompted evolutionary analysis. Phylogenetic analyses confirmed cnidarian HSP60 proteins are orthologous to vertebrate HSP60, demonstrating deep conservation across Metazoa. Although HSP60 is ancient and highly conserved, its role in regulating mitochondrial proteostasis varies across early-diverging metazoans. This study underlines the role of chaperone plasticity in cnidarian thermotolerance and diverging bleaching susceptibility of symbiotic cnidarians.
