Biology subjects

Chen, X.

Publications and source records attributed to Chen, X..

At least 343 records · Page 19Linked to original sources

bioRxiv2025

Maternal CENP-C restores centromere symmetry in mammalian zygotes to ensure proper chromosome segregation

Across metazoan species, the centromere-specific histone variant CENP-A is essential for accurate chromosome segregation, yet its regulation at the parental-to-zygote transition in mammals is poorly understood. To address this, we developed a CENP-A-mScarlet knock-in mouse model, which revealed sex-specific dynamics: mature sperm retains 10% of the CENP-A levels present in MII-oocytes. However, in zygotes prior to the first mitosis, this difference is resolved, using maternally inherited cytoplasmic-CENP-A. Notably, the increase in CENP-A at paternal centromeres is independent of sensing CENP-A asymmetry or the presence of maternal chromosomes. Instead, CENP-A equalization relies on asymmetric recruitment of maternal CENP-C to paternal centromeres. Depletion of maternal CENP-A decreases total CENP-A in pronuclei without disrupting equalization. In contrast, reducing maternal CENP-C or disruption of its dimerization domains impairs CENP-A equalization and chromosome segregation. Therefore, maternal CENP-C acts a key epigenetic regulator that resets centromeric symmetry at fertilization to preserve genome integrity. Highlights[bullet] CENP-A asymmetry between sperm and oocyte centromeres is a conserved feature from flies to mammals including mice and humans. [bullet]CENP-A asymmetry between parental centromeres is resolved prior to the first zygotic division via maternally inherited, cytoplasmic CENP-A. [bullet]Zygotic CENP-A levels in zygotes are regulated in a pronucleus-autonomous manner. [bullet]CENP-A equalization relies on asymmetric CENP-C recruitment to the paternal pronucleus and requires CENP-C dimerization. Key TermsCentromere; CENP-A; CENP-C; sperm; oocyte; zygote; intergenerational; epigenetics; mouse

genetics↗

bioRxiv2025

Methionine γ-lyase Traps Cofactor Pyridoxal-5'-phosphate through Specific Serine-Mediated Affinity

Pyridoxal-5'-phosphate (PLP), the ubiquitous and ancient cofactor, plays important roles in enzymatic elimination, transamination and other reactions. The catalytic efficiency of PLP-dependent enzymes is significantly higher than that of free PLP. The recruitment of PLP from the environment by the enzymes, particularly through interactions outside the active site, is the key step determining the occurrence of PLP-mediated catalysis. However, the precise mechanism by which enzymes recruit PLP remains elusive. Methionine {gamma}-lyase (MGL), a PLP-dependent enzyme, catalyzes the degradation of L-methionine, thereby suppressing cancer cell proliferation through serum or dietary methionine depletion. Here, we report the crystal structure of yMGL, which belongs to a newly identified subgroup of cystathionine {gamma}-lyases, in complex with L-methionine and PLP. Through truncating the C-terminal domain of yMGL both in vitro and in vivo, we demonstrated that this domain, outside the canonical PLP-binding domain, is essential for the specific interaction between yMGL and PLP, as well as for efficient L-methionine catabolism. A conserved Ser in the C-terminal domain, positioned at the active-site entrance of MGLs, confers specificity for PLP. These findings elucidate a previously uncharacterized mechanism of PLP recruitment by MGLs and promote rational design of MGLs with tailored cofactor selectivity and catalytic performance.

biochemistry↗

bioRxiv2025

AutoMorphoTrack: An Automated Python Package for Organelle Morphology, Motility, and Colocalization Analysis in Live-Cell Imaging

Quantitative imaging of organelle dynamics provides crucial insights into cellular function, state, and organization; however, existing analysis workflows often require advanced coding expertise and multiple software tools. AutoMorphoTrack is an open-source Python toolkit that automates organelle detection, morphology classification, motility tracking, and colocalization from multichannel fluorescence microscopy image stacks. The platform includes adaptive segmentation, organelle trajectory reconstruction, and pixel-level overlap quantification within a unified, reproducible framework that can be executed as an interactive Jupyter notebook, a modular Python package, or through AI-assisted natural-language commands. Each analysis step outputs publication-ready images, time-lapse videos, and standardized quantitative data tables. To complement the main pipeline, an accompanying script--AMTComparison.py--is provided to demonstrate how AutoMorphoTracks outputs can be extended for comparative analysis across individual neurons or experimental conditions. Together, these tools provide an accessible and framework for high-content, reproducible quantification of subcellular morphology, motility, and interactions at single-cell resolution. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/665650v5_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@103ecb0org.highwire.dtl.DTLVardef@a81b6forg.highwire.dtl.DTLVardef@a145fcorg.highwire.dtl.DTLVardef@1f9c27c_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Construction of a genome-wide pooled CRISPRi library as a resource for exploring the acid tolerance mechanism in Streptococcus mutans

Streptococcus mutans is recognized as the primary etiological agent of dental caries, one of the most prevalent infectious diseases globally. Its remarkable acid tolerance enables survival and proliferation in the low-pH biofilm microenvironment, establishing S. mutans as the dominant species in dental plaque and a key contributor to cariogenesis. Although numerous studies have identified genes linked to acid tolerance mechanisms, the full set of essential acid tolerance genes within its genome remains incompletely characterized, largely due to the lack of systematic, genome-scale investigations. To address this knowledge gap, we constructed a genome-wide pooled CRISPR interference (CRISPRi) library targeting 95% of the predicted S. mutans genes and employed next-generation sequencing to identify acid tolerance determinants systematically. Our screen revealed 95 acid tolerance-associated genes, a subset of which were functionally validated through gene knockout studies. Functional enrichment analysis demonstrated significant associations with metabolic pathways (including cofactor biosynthesis and amino/nucleotide sugar metabolism), tRNA modification, and transcriptional regulation. Protein-protein interaction (PPI) network analysis identified critical interactors (ComYC, SMU_1979c, DeoC, AcpP, NadD, and SMU_1988c) and two functionally cohesive modules. These findings provide novel mechanistic insights into the acid adaptation strategies of S. mutans and highlight potential therapeutic targets for caries prevention.

microbiology↗

bioRxiv2025

Multifunctional Injectable GelMA Hydrogels with MSN-Mediated Spatiotemporal Icariin Release for Enhanced Osseointegration in Bone Defects

The repair of bone defects is faced with the dual challenges of limited autograft donors and single function of artificial materials. The development of new materials with mechanical adaptability, drug-controlled release and osteogenic induction has become a research hotspot in bone tissue engineering. Here, we developed an injectable photocrosslinkable hydrogel by integrating icariin (ICA)-loaded mesoporous silica nanoparticles (MSN) into gelatin methacryloyl (GelMA). The amino-functionalized MSN enhanced compressive modulus by 1.5-fold (p < 0.05) and enabled pH-responsive ICA release (91.2{+/-}4.2% cumulative release over 15 days). Our in vitro findings revealed the composite hydrogel promoted BMSC proliferation was 1.43{+/-}0.04 times that of GelMA group. And express excellent osteogenic differentiation ability (ALP activity: 3.25-fold; Mineralization: 5.01-fold). In vivo study, Micro-CT revealed significantly higher bone volume fraction (BV/TV) in rat calvarial defects at 12 weeks, with histology confirming mature trabecular bone formation. This MSN-mediated spatiotemporal delivery system synchronizes immunomodulation and osteogenesis, offering a promising strategy for non-load-bearing osseointegration.

bioengineering↗

bioRxiv2025

Real-time visualization of ligand-specific conformational dynamics of GPCR C-terminal domain in living cells

The cytoplasmic tails (C-tails) of membrane receptors are fundamental hubs for signal transductions. Yet directly investigating their dynamics in living cells is challenging. We developed a single-molecule technique to sense C-tail's conformational transitions. It was applied to investigate the ligand-induced activation of {beta}2AR, a prototypical G protein-coupled receptor (GPCR). Our data elucidate how the receptor transduces ligand binding to cell-level outcomes, with the ligand efficacy being interpreted in terms of the association and disassociation rates of the G protein. Moreover, upon G protein binding, not only the C-tail is displaced from the intracellular surface of the receptor but also the eighth helix H8 is dissociated from the plasma membrane. This work establishes a robust framework to directly decipher the activation of membrane receptors without interferences from overexpressed downstream transducers.

biophysics↗

bioRxiv2025

Small molecule intervention of actin-binding protein profilin1 reduces tumor angiogenesis in renal cell carcinoma

Angiogenesis plays a key role in the development and progression of renal cell carcinoma (RCC). Actin-binding protein profilin-1 (Pfn1) is overexpressed in clear cell RCC predominantly in tumor-associated vascular endothelial cells (ECs). We previously demonstrated that that EC-selective (over)expression of Pfn1 accelerates RCC progression, and conversely, genetic loss of EC-Pfn1 dramatically inhibits tumor angiogenesis impeding tumor initiation and/or progression in RCC, suggesting that Pfn1 could be an actionable therapeutic target in RCC. In this study, we demonstrate that 4,4-((4-bromophenyl)methylene)bis(3,5-dimethyl-1H-pyrazole), a small molecule that we had previously identified as an inhibitor of Pfn1-actin interaction, directly binds to Pfn1 and attenuates tumor angiogenesis when directly administered into subcutaneous RCC tumors. Next, we undertook a chemical optimization approach to design and synthesize 4,4-((4-(trifluoromethyl)phenyl)methylene)bis(3,5-dimethyl-1H-pyrazole), a structural analog of our originally identified inhibitor, that exhibits improved anti-angiogenic efficacy in vitro and in vivo. Finally, we demonstrate that Pfn1 inhibitor is amenable to lipid microbubble encapsulation and release in the tumor microenvironment (TME) by ultrasound-mediated disruption of circulating microbubbles to achieve anti-angiogenic and anti-tumor benefit. In summary, our findings suggest that tumor-localized release of Pfn1 inhibitor could be a potential therapeutic strategy in RCC.

cell biology↗

bioRxiv2025

Systematic investigation of Epstein-Barr virus transcriptional regulator interactions with the human genome

We systematically investigate interactions between Epstein-Barr virus (EBV) transcriptional regulators (vTRs) and the human genome. Starting with 16 known and candidate vTRs, we identify nine whose introduction into human cells results in substantial alterations to host gene expression. Genome-scale determination of vTR genomic binding events and alterations to chromatin accessibility reveals a detailed map of EBVs functional interactions with the human genome, including >100,000 vTR binding events impacting almost a quarter of all human genes. BMRF1 emerges as a potent regulator, impacting >7,000 genes and altering >37,000 chromatin regions. Our results provide new evidence that EBV RTA interacts with and stabilizes the binding of human RBPJ. Network analysis reveals that many human genes are targeted by multiple EBV vTRs, highlighting the vast coordinated impact of EBV on human gene expression. This study provides a valuable, extensive resource for examining EBV-induced alterations to human gene regulation, with data available on multiple platforms.

genomics↗

bioRxiv2025

Dynamic remodeling of the pancreas immune landscape in obesity.

Obesity is a known risk factor for diseases of the pancreas, including diabetes, pancreatic cancer and pancreatitis, but mechanisms remain unclear. To elucidate how obesity impacts pancreatic immune homeostasis, we performed spatial, transcriptomic and functional profiling of human pancreatic immune cells from obese and non-obese organ donors. Obesity was associated with higher density of tissue resident memory T-cells (TRM) in the exocrine pancreas which display high cytotoxic functions and aggregated around macrophages. Single cell sequencing of pancreatic macrophages revealed two main subsets - FOLR2+ CD11c- fetal-derived macrophages with pro-repair and immunoregulatory function and a FOLR2- CD11c+ monocyte-derived macrophages with greater T-cell interactions and pro-inflammatory function. In obesity, the pancreatic macrophage landscape shifts to lower predominance of FOLR2+ CD11c- macrophages and higher FOLR2- CD11c+ macrophages which interact selectively with the TRM and inflamed exocrine epithelium. Together, these results identify macrophage-T cell circuits and immune epithelial interactions that fuel chronic pancreatic inflammation in obesity - a potential unifying mechanism for obesity-related pancreatic diseases.

immunology↗

bioRxiv2025

Genome-Wide Investigation of Transcription Factor Occupancy and Dynamics Using cFOOT-seq

Gene regulation relies on the precise binding of transcription factors (TFs) at regulatory elements, but simultaneously detecting hundreds of TFs on chromatin is challenging. We developed cFOOT-seq, a cytosine deaminase-based TF footprinting assay, for high-resolution, quantitative genome-wide assessment of TF binding in both open and closed chromatin, even with small cell numbers. By utilizing the dsDNA deaminase SsdAtox, cFOOT-seq converts accessible cytosines to uracil while preserving genomic integrity, making it compatible with techniques like ATAC-seq for sensitive and cost-effective detection of TF occupancy at single-molecule and single-cell level. Our approach enables the delineation of TF footprints, quantification of occupancy, and examination of chromatin influences. Notably, cFOOT-seq, combined with FootTrack analysis, predicts TF occupancy dynamics. We demonstrate its application in capturing cell type-specific TFs, analyzing TF dynamics during reprogramming, and revealing TF dependencies on chromatin remodelers. Overall, cFOOT-seq represents a robust approach for investigating the genome-wide dynamics of TF occupancy and elucidating the cis-regulatory architecture underlying gene regulation.

molecular biology↗

bioRxiv2025

Non-invasive Free-breathing Gating-free Extracellular Cellular Volume Quantification for Repetitive Myocardial Fibrosis Evaluation in Rodents

BackgroundInterstitial myocardial fibrosis is a crucial pathological feature of many cardiovascular disorders. Myocardial fibrosis resulting in extracellular volume (ECV) expansion can be non-invasively quantified by cardiac MRI (CMR) with T1 mapping before and after gadolinium (Gd) contrast agent administration. However, longitudinal repetitive ECV measurements are challenging in rodents due to the prolonged scan time with cardiac and respiratory gating that is required for conventional T1 mapping and the invasive nature of the rodent intravenous lines. MethodsTo address these challenges, the objective of this study is to establish a fast, free- breathing, and gating-free ECV procedure with a non-invasive subcutaneous catheter for in- scanner Gd administration that can allow longitudinal repetitive ECV evaluations in rodent models. This is achieved by (1) IntraGate sequence for free-breathing gating-free cardiac imaging, (2) non-invasive subcutaneous in-scanner Gd administration, and (3) fast T1 mapping with varied flip angle (VFA) in conjunction with (4) triple jugular vein blood T1 normalization. Additionally, full cine CMR (multi-slice short-axis, long-axis 2-chamber, and long-axis 4- chamber) was acquired during the waiting period for comprehensive systolic cardiac functional and strain analysis. ResultsWe have successfully established a non-invasive fast ECV quantification protocol to enable longitudinal repetitive ECV quantifications in rodents. Non-invasive subcutaneous Gd bolus administration induced reasonable dynamic contrast enhancement (DCE) time course reaching a steady state in [~] 20 min for stable T1 quantification. The free-breathing gating-free VFA T1 quantification scheme allows for rapid cardiac ([~]2.5 min) and jugular vein (49 sec) T1 quantification with no motion artifacts. The triple jugular vein T1 acquisitions (1 pre-contrast and 2 post-contrast) immediately flanking the heart T1 acquisitions enable accurate myocardial ECV quantification. Our data demonstrated that left-ventricular myocardial ECV quantifications were highly reproducible with repeated scans, and the ECV values (0.25) are comparable to reported ranges among humans and rodents. This protocol was successfully applied to ischemic reperfusion injury model to detect myocardial fibrosis that was validated with histopathology. ConclusionWe have established a simple, fast, non-invasive, and robust CMR protocol in rodents that can enable longitudinal repetitive ECV quantifications for cardiovascular disease progression. It can be used to monitor disease regression with interventions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/664411v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@14b44e5org.highwire.dtl.DTLVardef@985392org.highwire.dtl.DTLVardef@96cef7org.highwire.dtl.DTLVardef@ede47d_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Genomic Surveillance Reveals Global Spread of Macrolide-Resistant Bordetella pertussis Linked to Vaccine Changes

The resurgence of whooping cough in regions utilizing acellular pertussis vaccines underscopes emerging public health challenges. Here, we characterized 178 Bordetella pertussis isolates collected from patients across all age groups in Shanghai (2018-2024) to assess genomic evolution and antibiotic susceptibility. Macrolide resistance to erythromycin, azithromycin, clarithromycin and clindamycin escalated from [≤]50% (pre-2020) to nearly 100% (post-2020), mechanistically linked to the 23S rRNA A2047G mutation. Genome-based analysis identified a genotype MT28-ptxP3-MRBP rapidlly dominated post-2020, exhibiting significantly higher prevalence in adults versus than age groups. Phylogenetic analysis of 178 Shanghai and 1596 global genomes revealed two major lineages corresponding to ptxP1 and ptxP3 alleles. MT28-ptxP3-MRBP cluster was identified in France, Japan and the United States in 2024, indicating potential cross-border dissemination. These findings advocate for intergrated surveillance spanning all ages and international borders to contain the global spread of macrolide-resistant Bordetella pertussis. HighlightsO_LIAfter 2020, MT28-ptxP3-MRBP lineage rapidly dominated, comprising 61.7% of isolates. C_LIO_LIMT28-ptxP3-MRBP exhibits a significant transmission advantage among older individuals. C_LIO_LIThe primary affected group shifted from [≤]36 months (pre-2020) to 37 months-18 years (post-2020). C_LIO_LIMacrolide resistance rose from [≤]50% pre-2020 to nearly 100% post-2020, with all resistant isolates carrying the A2047G mutation. C_LI

microbiology↗

bioRxiv2025

Vascularized Bioengineered Kidney Using Decellularized Scaffold Recellularized with human Placenta-Derived Angiogenic stem Cells and Kidney Organoids

A bioengineered kidney using a decellularized kidney scaffold (DKS) offers a promising solution to the kidney shortage. However, the transplantation of bioengineered kidneys using DKS recellularized with human endothelial, renal cells or others has not yet successfully achieved the vascular and renal reconstruction in vivo. In this study, we identified another type of stem cells, designated as human placenta-derived angiogenic stem cells (hPASCs), which serve as seeding cells for the vascularization of DKS. These hPASCs encompass angiogenic subpopulations, exhibit both stem cell properties and the capacity for vascular differentiation. Human fetal kidney organoids (KIO) were established as a source of renal parenchymal cells, comprising renal, immune, and vascular cell populations. We developed a vascularized bioengineered kidney by recellularizing DKS with hPASCs and KIO using a circulation perfusion culture system. The hPASCs bioengineered kidney demonstrated enhanced angiogenesis and reconstructed renal architecture in vivo, by transplantation into rat models of renal subcapsular and partial nephrectomy. Furthermore, immunofluorescence and single-cell analyses revealed that the hPASCs revascularized bioengineered kidney regenerated both vascular and renal parenchymal cells within the host. This study offers another strategy for kidney bioengineering and regeneration.

bioengineering↗

bioRxiv2025

Cell-resolved high-dimensional imaging analysis, therapeutic modeling, and a Phase Ib clinical study validate BCL-2 as a target across heterogeneous CRPC subtypes

BCL-2 has been implicated in prostate cancer (PCa) progression and development of castration-resistant disease (CRPC). However, it remains unclear how the BCL-2- and AR-expressing PCa cell populations evolve across the PCa continuum, how AR molecularly regulates BCL-2 and whether BCL-2 represents a common therapeutic target in heterogeneous CRPC. Importantly, BCL-2 inhibitors have yet to be approved for treating PCa patients. Here we first show the selective induction of BCL-2 by AR pathway inhibitors (ARPIs) in both patient specimens and xenograft models. Vectra-based quantitative multiplex immunofluorescence (qmIF) and image mass cytometry (IMC) analyses with single-cell resolution reveal markedly expanded BCL-2+ (AR+ or AR-) PCa cell populations in CRPC. Mechanistically, AR represses BCL-2 transcription through genomic binding via several AR binding sites and ARPIs relieve this repression, leading to BCL-2 upregulation. Comprehensive therapeutic studies in cells, organoids and xenografts establish that castration-induced BCL-2 is not merely associated with resistance but represents a shared and actionable vulnerability as the BCL-2 inhibitor ABT-199 potently suppressed the growth of multiple subtypes of CRPC. A Phase Ib clinical trial (NCT03751436) combining enzalutamide and BCL-2 inhibitor venetoclax demonstrated reduced circulating tumor cells in responding patients. Together, our findings elucidate the AR+/-BCL-2+/- PCa cell subpopulation dynamics during PCa progression, reveal a direct mechanistic link between AR inhibition and BCL-2-mediated resistance, and provide a strong rationale for targeting BCL-2 from the outset to eliminate emerging resistant subpopulations, inhibit treatment-induced cellular heterogeneity and plasticity, and improve therapeutic outcomes in CRPC.

cancer biology↗

bioRxiv2025

Convergent DNA Methylation Abnormalities at Bivalent Chromatin in Human Growth Disorders

Loss-of-function mutations in DNMT3A, a DNA methyltransferase, or NSD1, a histone methyltransferase, cause overgrowth syndromes. Conversely, disruption of the DNMT3A domain that binds NSD1-deposited H3K36 dimethylation (H3K36me2) results in growth restriction. To investigate the molecular basis of these opposing growth outcomes, we generated isogenic human embryonic stem cells carrying growth syndrome-associated mutations in DNMT3A and NSD1. Unexpectedly, both overgrowth- and growth restriction- associated DNMT3A mutations led to DNA hypomethylation in a shared subset of active enhancers, implicating H3K36me2 in directing enhancer methylation maintenance. In contrast, bivalent chromatin--marked by both active and repressive chromatin modifications--showed divergent DNA methylation changes: hypermethylation in growth restriction-associated DNMT3A mutants and hypomethylation in overgrowth-associated DNMT3A or NSD1 loss-of-function mutants. These findings identify locus-specific DNA methylation defects as a common molecular feature and nominate dysregulated DNA methylation at bivalent chromatin as a potential driver of abnormal growth phenotypes.

genomics↗

bioRxiv2025

Adaptive cytoskeletal responses to extracellular environment viscosity modulate cell migration

Cell migration is a pivotal process in metastasis, allowing cancer cells to invade surrounding tissues and disseminate to distant organs. While extracellular environment (ECE) viscosity serves as a critical modulator of cell motility, its regulatory mechanisms remain unclear. This study presents a mechanobiological model to investigate how ECE viscosity modulates cancer cell migration by regulating some key processes, including actin polymerization, retrograde flow, and adhesion adaptations. Our results reveal a biphasic response: a moderate increase in ECE viscosity enhances actin filament network density and adhesion strength, thereby accelerating migration, whereas excessively high viscosity hinders movement due to too large mechanical resistance. Furthermore, we identify a short-term migration memory phenomenon, where cancer cells exposed to high viscosity environments retain elevated migration speeds after transitioning to low viscosity conditions. This memory effect is sustained by the continued assembly of cytoskeletal proteins such as actin monomers and Arp2/3. These analyses reveal an adaptive mechano-chemo-biological mechanism by which cancer cells integrate and respond to mechanical cues from their viscous environment to optimize migration, and advance the understanding of cancer cell migration in various tissue environments.

biophysics↗

bioRxiv2025

FBL as a carrier mediate H2Aub degradation in the lysosome-like structures

Under glucose starvation, mammalian cells form lysosome-like structures within the nucleoli to mediate the metabolic shift of carbon sources from glucose to amino acids generated through H2Aub degradation for energy supply, but the mechanism by which lysosome-like structures mediate the degradation of monoubiquitinated histones during glucose starvation remains unclear. Through screening, we discovered that the fibrillarin (FBL) protein in the nucleolus acts as a carrier for mono-ubquitinated histone H2A (H2Aub), regulating the balance between BMI1-mediated mono-ubiquitination of H2A and USP36-mediated deubiquitination of H2Aub. Under glucose starvation, FBL undergoes aggregation, leading to a significant reduction in its interaction with BMI1, accompanied by markedly decreased binding to histone H2A and H2Aub. Concurrently, Midnolin, which serves as a receptor for monoubiquitinated histones, exhibits increased interaction with FBL during glucose starvation. USP36, FBL, Midnolin, and BMI1 collectively form a complex responsible for degrading monoubiquitinated histones. Furthermore, knockdown of FBL, BMI1, or USP36 resulted in cell cycle arrest at S phase and severely compromised cell viability. In summary, we identified a lysosome-like structure in the nucleolus that mediates the degradation of H2Aub. This structure plays a critical role in regulating cell cycle progression and survival, and may offer novel potential therapeutic targets for cancer.

cell biology↗

bioRxiv2025

A genome-wide MAGIC kit for recombinase-independent mosaic analysis in Drosophila

Mosaic analysis has been instrumental in advancing developmental and cell biology. Most current mosaic techniques rely on exogenous site-specific recombination sequences that need to be introduced into the genome, limiting their application. Mosaic analysis by gRNA-induced crossing-over (MAGIC) was recently developed in Drosophila to eliminate this requirement by inducing somatic recombination through CRISPR/Cas9-generated DNA double-strand breaks. However, MAGIC has not been widely adopted because gRNA-markers, a required component for this technique, are not yet available for most chromosomes. Here, we present a complete, genome-wide gRNA-marker kit that incorporates optimized designs for enhanced clone induction and more effective clone labeling in both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). With this kit, we demonstrate clonal analysis in a broad range of Drosophila tissues, including cell types that have been difficult to analyze using recombinase-based systems. Notably, MAGIC enables clonal analysis of pericentromeric genes and deficiency chromosomes and in interspecific hybrid animals, opening new avenues for gene function study, rapid gene discovery, and understanding cellular basis of speciation. This MAGIC kit complements existing systems and makes mosaic analysis accessible to address a wider range of biological questions. IMPACT STATEMENTA comprehensive toolkit enables genome-wide, recombinase-independent mosaic analysis in Drosophila, permitting clonal analysis of pericentromeric genes, deficiency chromosomes, and interspecific hybrids previously inaccessible to standard methods.

genetics↗