Biology subjects

Yang, J.

Publications and source records attributed to Yang, J..

At least 181 records · Page 10Linked to original sources

bioRxiv2025

Unlocking karst biodiversity with eDNA: a validated qPCR toolkit for discovery and monitoring the worlds largest cavefish radiation (Cyprinidae: Sinocyclocheilus)

Environmental DNA (eDNA) enables non-invasive detection of aquatic species through trace genetic material, but uncertainties regarding DNA persistence, detection accuracy, and the limited taxonomic resolution of stygian fauna have constrained its use in subterranean aquatic environments. The species-rich cavefish genus Sinocyclocheilus, endemic to the karst landscapes of southwestern China, offers an excellent model for understanding these issues. With over eighty species, many of which are endemics, and many still being described, this lineage is taxonomically rich, poorly understood, and highly threatened. However, access to these deep habitats remains challenging, making eDNA detection one of the most practical methods for documenting and monitoring them. We developed and validated a qPCR assay targeting a hypervariable region of the mitochondrial 16S rRNA gene, allowing for genus-specific detection and species-level identification. Laboratory and field evaluations involving 47 representative species confirmed high specificity and a detection limit of around 20 DNA copies per reaction (R{superscript 2} = 0.995). Controlled degradation studies indicated that temperature and pH strongly affect DNA persistence. Field surveys across 47 karst sites (37 caves and 10 surface sites) detected Sinocyclocheilus eDNA in only 33 caves. Dideoxy sequencing of qPCR amplicons provided species-level identification and phylogenetic validation, including detection of a new species. At three cave sites yielding multiple qPCR detections, 16S metabarcoding enabled species discrimination. By integrating qPCR assay validation, eDNA degradation modelling, hierarchical genetic identification via dideoxy sequencing, and selective NGS metabarcoding, this study presents a strategic framework for applying eDNA methods in subterranean environments.

evolutionary biology↗

bioRxiv2025

Rare longevity-associated variants, including a reduced-function mutation in cGAS, identified in multigenerational long-lived families

Life expectancy has steadily increased in the last two centuries, while healthspan has been lagging behind. Survival into extreme ages strongly clusters within families which often exhibit a delayed onset of (multi)morbidity, yet the underlying protective genetic mechanisms are still largely undefined. We performed affected sib-pair linkage analysis in 212 sibships enriched for ancestral longevity and identified four genomic regions (LODmax [≥]3.0) at 1q21.1, 6p24.3, 6q14.3, and 19p13.3. Within these regions, we prioritized 12 rare protein-altering variants in seven candidate genes (NUP210L, SLC27A3, CD1A, CGAS, IBTK, RARS2, and SH2D3A) located in longevity-associated loci. Notably, a missense variant in CGAS (rs200818241), was present in two sibships. Using human- and mouse-based cell models, we showed that rs200818241 reduced protein stability and attenuated activation of the canonical cGAS-STING pathway in a cell-type specific manner. This dampened signalling mitigated inflammation and delayed cellular senescence, mechanisms that may contribute to the survival advantage of CGAS variant carriers. Our findings indicate novel rare variants and candidate genes linked to familial longevity and highlight the cGAS-STING pathway as a potential contributor to the protective mechanisms underlying human longevity.

genetics↗

bioRxiv2025

Domestication and Improvement Reshaped the Genomic Architecture of Abiotic Stress Tolerance in Sunflower

Sunflower (Helianthus annuus) has undergone extensive domestication and modern improvement, yet the extent to which selection has shaped its stress-adaptive variation remains largely unclear. Using genomic data from diverse accessions, including the wild ancestor of sunflower, we identified widespread balancing selection that maintains historical polymorphisms in abiotic-stress-related genes. Additionally, we identified strong positive selection signals associated with domestication and improvement. Using a cutting-edge DNA language model to characterize deleterious alleles, we found that modern cultivars exhibited an elevated deleterious mutation load, particularly within positively selected regions, consistent with patterns observed in other major domesticates. To cross-validate the functional consequences of the selected regions on stress responses, we assessed salt tolerance in an independent association panel and evaluated ancestral allele effects on salt-responsive traits. Although only a small subset of the selected regions reached genome-wide significance, several ancestral alleles within positively selected sweeps tended to enhance salt tolerance. Together, these results indicate that recent selection may have unintentionally eliminated historically beneficial alleles, reshaping the genomic landscape of sunflower stress responses. This study highlights a breeding strategy to recover ancestral beneficial alleles for enhancing abiotic stress tolerance in modern cultivars.

genomics↗

bioRxiv2025

Therapeutic Potential of Neural Rosette Stem Cell-Derived Small Extracellular Vesicles in the Management of Brain Edema

Small extracellular vesicles (sEVs) derived from neural stem cells (NSCs) have gained significant interest for their potential roles in neuroprotection, brain edema management, and the preservation of blood-brain barrier (BBB) integrity. These nanoscale particles facilitate intercellular communication and carry a diverse array of bioactive molecules, including proteins and RNAs, which influence various neurobiological processes. The composition of their cargo depends on the parental cell type, affecting their interactions with recipient cells. In this study, we characterized the cargo of sEVs derived from neural rosette stem cells (NRSCs) at both transcriptomic and proteomic levels and compared it to sEVs derived from cardiomyocyte progenitor cells (CMPCs) and human pluripotent stem cells (hPSCs). Furthermore, we assessed the therapeutic effects of NRSC-derived sEVs in a rat model of transient middle cerebral artery occlusion (tMCAO) to investigate neuronal injury and the progression of brain edema. Our proteomic and miRNA analyses revealed that the sEVs from NRSCs, CMPCs, and hPSCs possess distinct cargo compositions. In an in vitro neuronal ischemia model, NRSC-derived sEVs significantly enhanced cell viability, while in an endothelial model, they expedited the restoration of cell barrier integrity. Furthermore, the in vivo studies utilizing the rat model of tMCAO showed that intravenous administration of NRSC-derived sEVs at 3, 24 and 48 hours after reperfusion resulted in a significantly faster resolution of brain edema. These findings underscore the influence of the parental cell type on sEV composition and highlight the therapeutic potential of NRSC-derived sEVs in treating brain edema, paving the way for novel therapeutic strategies.

neuroscience↗

bioRxiv2025

{Phi}X174 bacteriophage viability predicted by protein biophysical modeling

The relationship between genotype and phenotype underlies our ability to understand and predict evolution. Efforts to build genotype-phenotype (GP) maps have revealed several unifying rules: epistasis is pervasive, fitness effects are not normally distributed, and the GP map is non-linear and complicated for high-level phenotypes. A critical step in developing GP maps is evaluating how well predictive models do in explaining observed phenotypes. We utilize the simplicity of a bacteriophage ({Phi}X174) study system to test if an intermediate phenotype (predicted stability of the G capsid protein) explains more complex phenotypes. In doing so, we compare the predictive performance of free energies of folding and binding obtained using numerous molecular modeling methods as well as phylogenetic and basic biochemical/biophysical properties of amino acid substitutions. By creating a large mutational library, we find that {Phi}X174 tolerates about 50% of the amino acid substitutions we inserted into the G protein and that molecular modeling compliments other substitution models for predicting viability. Mutations predicted to have large destabilizing effects are especially informative and are almost universally detrimental. These large-effect substitutions often coincide with the most conserved residues in the G protein. Apart from large-effect mutations, our ability to predict {Phi}X174 phenotypes is fairly poor and we explore various potential confounding factors (e.g., codon bias) that could be considered to improve viability predictions.

molecular biology↗

bioRxiv2025

DiCoLo: Integration-free and cluster-free detection of localized differential gene co-expression in single-cell data

Detecting changes in gene coordination patterns between biological conditions and identifying the cell populations in which these changes occur are key challenges in single-cell analysis. Existing approaches often compare gene co-expression between predefined cell clusters or rely on aligning cells across conditions. These strategies can be suboptimal when changes occur within small subpopulations or when batch effects obscure the underlying biological signal. To address these challenges, we introduce DiCoLo, a framework that identifies genes exhibiting differential co-localization, defined as changes in coordinated expression within localized cell neighborhoods-subsets of highly similar cells in the transcriptomic space. Importantly, DiCoLo does not rely on cell clustering or cross-condition alignment. For each condition, DiCoLo constructs a gene graph using Optimal Transport distances that reflect gene co-localization patterns across the cell manifold. Then, it identifies differential gene programs by detecting changes in connectivity patterns between the gene graphs. We show that DiCoLo robustly identifies differential gene co-localization even under weak signals or complex batch effects, outperforming existing methods across multiple benchmark datasets. When applied to mouse hair follicle development data, DiCoLo reveals coordinated gene programs and emerging cell populations driven by perturbations in morphogen signaling that underlie dermal condensate differentiation. Overall, these results establish DiCoLo as a powerful framework for uncovering localized differential transcriptional coordinated patterns in single-cell data.

bioinformatics↗

bioRxiv2025

Precision at Every Scale: Efficiency in AI-Driven De Novo Antibody Design

The precise de novo design of antibodies remains a therapeutic challenge. The AI platform, GaluxDesign, was evaluated in a high-efficiency Precision-Scale Workflow by synthesizing and testing only 50 full-length IgG candidates per epitope across eight distinct epitopes from six therapeutic targets. This campaign yielded a 10.5% binder rate (estimated EC50 < 100 nM), identifying target-specific binders for seven of eight epitopes, with multiple candidates exhibiting sub-nanomolar to single-digit nanomolar dissociation constants (Kd). We further assessed the same workflow on nine shared benchmark targets selected for external comparison, where GaluxDesign identified target-specific binders for eight of nine targets, demonstrating strong target-level performance relative to previously reported de novo antibody design approaches. Together, these results establish a high-efficiency, precision-scale workflow for generating novel, high-affinity therapeutic antibodies.

bioengineering↗

bioRxiv2025

CDH3 as a Novel Therapeutic Target in Basal-like Double-Negative Prostate Cancer

PurposeBasal-like (also known as double-negative) prostate cancers are aggressive tumors that lack effective targeted therapies. We aimed to delineate the role of CDH3 (P-cadherin) in basal-like prostate cancer and evaluate CDH3-directed therapeutic strategies. MethodsWe integrated genetically engineered mouse models (GEMMs) of prostate cancer, bulk and single-cell transcriptomic analyses, and a suite of in vitro and in vivo experiments. CDH3 expression and associated signaling pathways were examined in Pten/Apc double-knockout mouse prostates and human datasets. Functional studies included antibody-drug conjugate (ADC) cytotoxicity assays and the development of chimeric antigen receptor (CAR) T cells targeting CDH3, tested in prostate cancer cell lines and xenograft models. ResultsPten/Apc double deletion in prostate GEMMs led to highly aggressive tumors with markedly elevated CDH3 expression and enrichment of non-canonical WNT signaling components. Transcriptomic analyses of patient-derived prostate tumors confirmed that CDH3 is significantly upregulated in basal-like prostate cancer subtypes relative to luminal subtypes. Single-cell RNA sequencing revealed CDH3 expression predominantly in basal epithelial cells. Mechanistically, we found that active YAP1 signaling and a WNT5A-ROR2 non-canonical WNT axis drive CDH3 expression. Targeting CDH3 with a CDH3-specific ADC induced potent, antigen-dependent killing of CDH3 prostate cancer cells in vitro and significantly suppressed tumor growth in in vivo metastatic prostate cancer models. Likewise, CDH3-targeted CAR T cells specifically recognized and lysed CDH3-expressing prostate tumor cells while sparing CDH3-negative cells, leading to tumor regression and improved survival in mouse models, especially when combined with PD-1 checkpoint blockade. ConclusionsCDH3 is a key marker and functional driver of basal-like prostate cancer. Therapeutic strategies leveraging CDH3, including ADCs and CAR T cells, demonstrate strong preclinical efficacy, supporting the development of CDH3-targeted treatments to overcome resistance in aggressive prostate cancer.

cancer biology↗

bioRxiv2025

Evo2HiC: a multimodal foundation model for integrative analysis of genome sequence and architecture

Understanding how genomic sequences shape three-dimensional (3D) genome architecture is funda-mental to interpreting diverse biological processes. Although previous studies have shown that sequence information can predict 3D genome architecture, they fall short in capturing cell type-specific structures because they are trained solely on sequence inputs. The widely available Hi-C data, which contain rich structural information across biosamples, can provide complementary features to sequence data for study-ing cell type-specific architectures. Recently, DNA foundation models have demonstrated encouraging performance in capturing long-range genomic dependencies, holding promise for modeling chromatin interactions. However, the extremely high computational cost of running these models limits their applicability to Hi-C analysis, which requires genome-wide sequence embeddings. Here, we present Evo2HiC, a multimodal foundation model that jointly models genomic sequences and structures to study cell type-specific chromatin structure. The key idea of Evo2HiC is to distill a large-scale DNA foundation model, Evo 2 (7B), into a compact encoder, while guiding the distillation with Hi-C data to preserve genomic features critical for 3D genome analysis. The model supports two types of encoders, one that operates directly on DNA sequences, and a second that additionally takes as input corresponding Hi-C data. Using the DNA-only encoder and predicting Hi-C contact matrices, Evo2HiC improved Spearman correlation by 10.9% over Orca. Moreover, by jointly embedding Hi-C and sequence information Evo2HiC achieved the best overall Pearson correlation when predicting five representative epigenomic assays. Interpretation analysis of Evo2HiC revealed its ability to identify cell type-specific sequence motifs that explain changes in epigenomic signals. Finally, we demonstrated the cross-species generalizability of Evo2HiC on 177 species from the DNA Zoo dataset for Hi-C resolution enhancement. In summary, Evo2HiC is a foundation model that integrates genome sequences and 3D chromatin structure information, substantially reduces computational cost while maintaining state-of-the-art accuracy on predicting various epigenomic signals and genome architecture, enables the identification of cell type-specific motifs, and demonstrates robust generalizability across species.

bioinformatics↗

bioRxiv2025

Aging alters tumor cell - T cell crosstalk to promote breast cancer progression

Age is a dominant risk factor for all major breast cancer subtypes. However, the mechanisms by which aging influences tumor development remain unclear. Using a novel mouse model whereby breast cancer is induced in situ in young and old wild-type mice via intraductal delivery of a lentivirus encoding the HER2/neu oncogene, we found that old mice exhibited a higher oncogene-induced tumor burden than young mice. Old tumor cells showed reduced expression of interferon-related genes, particularly the T cell-recruiting chemokines Cxcl9 and Cxcl10, linked to their altered chromatin accessibility. CXCL9/10 expression also declined with age in human HER2+ tumors. Correspondingly, old tumors exhibited fewer T cells within tumor lesions. Targeted interventions showed that decreased expression of Cxcl9/10 is responsible for reduced T cell infiltration and weakened anti-tumor immunity. These results show how aged tumor cells are impaired in their recruitment of immune cells, leading to a defective anti-tumor immune response.

cancer biology↗

bioRxiv2025

Shape, Strain, and Stability: Epithelia Under High Strain

Mechanical forces play crucial roles in epithelia tissue morphogenesis and function, yet individual cells often respond differently to the same external force. Strain heterogeneity, the non-uniform deformation of cells under uniform external mechanical stimuli, may underlie tissue-level robustness and guide morphogenetic outcomes. In this study, we applied a defined uniaxial strain to Xenopus laevis epithelial explants and investigated how strain was distributed at the cellular level. Live imaging and quantitative analysis revealed that despite a uniform tissue-level strain, cellular strain was heterogeneous, suggesting variable responses to a uniform mechanical stimulus. To understand the source of this variability, we used an image analysis pipeline to explore multiple aspects of cell morphology. We found that cell intrinsic material properties, represented by Poisson index, had the strongest correlation with strain heterogeneity, suggesting a dominant role in variable mechanical response. We further analyzed how force was distributed at the cellular level using a vinculin force sensor and laser ablation. These experiments demonstrated that forces are primarily transmitted through the medio-apical actomyosin cortex whereas junctional actomyosin facilitates dissipation and remodeling. These findings provide new insights into the physical principles that underlie epithelial resilience and adaptive remodeling, highlighting the importance of distinct functions of junctional and medio-apical actomyosin networks in mechanical adaptation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/687918v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1d27474org.highwire.dtl.DTLVardef@1db811forg.highwire.dtl.DTLVardef@1fa36dforg.highwire.dtl.DTLVardef@288107_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Activation of IRF7/ISG15 axis in microglia inhibits NLRP3 Expression and Improves the Prognosis of Ischemia/Reperfusion in Mice

Ischemic stroke is a leading cause of disability and mortality, with neuroinflammation playing a key role in post-stroke injury. The molecular mechanisms remain incompletely defined. This study explored the functions of IRF7 and its downstream target ISG15 in stroke-associated neuroinflammation and prognosis of ischemic stroke. Bioinformatic analysis of transcriptomic datasets from microglia and ischemic brain tissues identified both molecules as hub genes. Their expression was validated in a mouse transient middle cerebral artery occlusion (tMCAO) model and in microglial cultures exposed to oxygen-glucose deprivation/reoxygenation (OGD/R). The effects of these molecules were assessed using siRNA knockdown, conditioned media assays with SY5Y neuronal cells, in vivo overexpression of ISG15, histological and functional assessments. Both IRF7 and ISG15 were significantly upregulated after stroke. IRF7 knockdown reduced ISG15 expression, whereas ISG15 knockdown did not affect IRF7, suggesting a unidirectional regulatory relationship. Conditioned media from microglia treated with siIRF7 or siISG15 increased SY5Y cell mortality, with a stronger effect in the siISG15 group, highlighting neuroprotective role of ISG15. ISG15 knockdown also enhanced microglial migration. Conversely, microglial ISG15 overexpression in vivo promoted a shift toward reduced neuroinflammation, improved neuronal survival, and enhanced functional recovery. Mechanistically, ISG15 stabilized NLRP3 protein but more strongly decreased its mRNA stability through accelerated degradation. These findings demonstrate that the activation of IRF7/ISG15 axis in microglia inhibits NLRP3 Expression and improve the prognosis of ischemia/reperfusion in mice, with ISG15 exerting stronger neuroprotective effects. Targeting microglial ISG15 may offer a promising therapeutic strategy for ischemic stroke. MAIN POINTSO_LIISG15 shows a stronger neuroprotective effect than IRF7 in ischemic stroke. C_LIO_LIISG15 stabilizes NLRP3 protein but more strongly decreases its mRNA stability. C_LIO_LIMicroglial ISG15 overexpression in vivo improves the prognosis of ischemic stroke in mice. C_LI

neuroscience↗

bioRxiv2025

An extensive forward genetic screen identifies the HWS-AGO1 axis as pivotal for target mimicry-mediated miRNA degradation in Arabidopsis

Target RNAs have emerged as key regulators of miRNA stability, thereby influencing development and physiological processes in both plants and animals. The F-box protein HAWAIIAN SKIRT (HWS) has been characterized as a crucial factor in targeted miRNA degradation in plants (pTMD), a process triggered by a specific type of target RNA called target mimicry. However, the precise mechanism by which HWS functions is still poorly understood. We previously established a genetic reporter system based on Short Tandem Target Mimicry of miR160 (STTM160) triggered miR160 degradation, resulting in pleiotropic developmental defects that are easy to monitor. Here, through an extensive forward genetic screen, we identified fourteen additional hws alleles that near-completely restored STTM160-induced developmental defects, highlighting a central role for HWS in pTMD. Intriguingly, we discovered two adjacent amino acid substitutions (R421K and G422D) in the PAZ domain of AGO1 that significantly suppressed the STTM160 phenotype. Similar to HWS dysfunction, the AGO1 R421K substitution caused a significant retention of target mimicry RNAs in AGO1 immunoprecipitates, suggesting that the R421 residue of AGO1 may cooperate with HWS for the clearance of AGO1-miRNA-target mimicry complexes. Although the R/G mutations only impacted a small set of endogenous miRNA levels, they were sufficient to rescue the developmental defects caused by HWS overexpression (HWS-OE), suggesting that the developmental abnormalities caused by HWS-OE largely depend on the AGO1-miRNA pathway. Taken together, our findings provide a solid genetic evidence for the coordination of HWS and AGO1 in pTMD.

plant biology↗

bioRxiv2025

PPAR-delta rather than PPAR-gamma is likely to be the key modulator of central carbon metabolism in human adipocytes

Adipogenesis involves adipocyte differentiation and synthesis and storage of fats. PPAR-{gamma} is the master regulator of adipogenesis and regulates genes for adipocyte differentiation, lipogenesis, adipocyte survival and adipokine secretion. Central carbon metabolism (CCM) comprises of three key pathways, glycolysis, tricarboxylic acid cycle and pentose phosphate pathway. CCM utilizes carbon sources to provide energy and building blocks for lipogenesis. Although several targets of PPAR-{gamma} have been identified in the CCM pathways, the exact process of how PPAR-{gamma} modulates adipogenesis via CCM remains elusive. In this study, we used real time-qPCR and metabolic arrays for CCM to understand the effect of PPAR activation by PPAR-{gamma} agonist 15d-PGJ2 on CCM and its role in adipogenesis. Our data show that PPAR-{delta} is likely the target of 15d-PGJ2 and the key modulator of adipogenesis in human SGBS adipocytes at least under current experimental conditions. Further studies are warranted to fully understand the regulation of CCM in adipocytes.

molecular biology↗

bioRxiv2025

A multi-tissue atlas of genetic regulatory effects in sheep

Characterizing the impact of genomic variants on genome function and ultimately complex traits in livestock is essential for the development of sustainable precision agriculture and comparative genomics. Here, as part of the Farm animal Genotype-Tissue Expression (FarmGTEx) project, we present the pilot phase of the SheepGTEx resource through analyzing 6,761 RNA-sequencing samples of 51 primary tissues in a multi-breed population of sheep. We identify millions of regulatory variants associated with seven types of molecular phenotypes, and fine-map 322,468 primary and 114,017 non-primary effects, revealing a high degree of regulatory allelic heterogeneity. We systematically characterize the pleiotropic effects of these variants on molecular phenotypes, assess their context-specific regulatory patterns across tissues, breeds, sexes, and developmental stages, as well as explore their evolutionary constraints across mammals. Finally, we demonstrate the substantial potential of the SheepGTEx resource (https://sheepgtex.farmgtex.org), by providing examples of regulatory mechanisms underpinning 34 complex traits, population divergence between European and Asian breeds, and adaptive evolution in sheep over the past ten millennia.

genomics↗

bioRxiv2025

The dominance of gene expression controlled by trans-eQTL hotspots contributes to phenotypic heterosis in maize

Heterosis, or hybrid vigor, is a key phenomenon in genetics research and agricultural production, and has been primarily attributed to non-additive genetic effects such as dominance -- a prevailing consensus shaped by decades of empirical research and theoretical debate. Although dominance may arguably arise from distal modifiers, their selective advantage is debated due to presumably small individual effects. To address this long-standing question, particularly how genetic dominance manifests at the transcriptomic level and contributes to phenotypic heterosis, we integrated transcriptomic and phenotypic data from a large population of maize hybrids and their inbred parents. We found that [~] 30% of the expressed seedling genes in a significant proportion of hybrids exhibited expression patterns deviating from the average of the two parents, indicative of non-additivity. Further analysis suggests that while hybrid gene expression per se is primarily regulated by cis-eQTLs, expression dominance (or non-additivity) is disproportionately controlled by trans-eQTLs. These trans-eQTLs cluster into hotspots that regulate the non-additivity of hundreds of target genes, mostly within co-expression networks, and are notably enriched for transcription factors (TFs). Focusing on one such hotspot, we functionally validated a classical maize gene ZmR1, a basic helix-loop-helix (bHLH) TF associated with multiple seedling trait heterosis, as a candidate regulator of expression dominance across hundreds of genes. Overexpression of ZmR1 enhances expression dominance of downstream genes and increases phenotypic heterosis in both seedling and adult traits. Further experiments confirmed its direct regulatory role in modulating genes involved in anthocyanin biosynthesis and lignin metabolism, driving transcriptome-level dominance. These results provide empirical support for the modifier hypothesis under an omnigenic model, suggesting that heterosis arises not from the modification of a single genes inheritance but through the coordinated regulation of hundreds of phenotype-associated genes, thereby helping to reconcile the long-standing debate over the genetic basis of dominance in heterosis.

genetics↗

bioRxiv2025

Models for the architecture of the human inner kinetochore CCAN complex on centromeric α-satellite CENP-A nucleosome arrays

Human kinetochores assemble onto centromeric DNA defined by tandem copies of thousands of the 171 bp -satellite repeat sequence. The centromere-specific CENP-A nucleosome (CENP-ANuc) recruits the inner kinetochore constitutive centromere associated network (CCAN) complex. A previous cryo-EM structure of CCAN bound to a CENP-ANuc reconstituted with a 171 bp -satellite repeat sequence showed how extranucleosomal linker DNA threads through an internal tunnel in the CCAN complex. To understand the higher-order architecture of the inner kinetochore assembled onto -satellite repeat arrays, we have determined cryo-EM structures of CCAN with longer DNA sequences. These include free DNA and single and dimeric -satellite repeats with CENP-A nucleosomes. We show from the structures of CCAN bound to both free DNA and monomeric CENP-ANuc that CCAN engages 65-70 bp of DNA comprising 30-35 bp of an upstream -satellite repeat. This upstream DNA interacts with the histone-fold domain subunits of the CENP-TWSX module in a manner resembling how DNA is wrapped in nucleosomes. A complex of CCAN assembled onto a dimeric -satellite repeat with two CENP-A nucleosomes showed that CCAN can only be accommodated on the linker DNA by unwrapping DNA from the CENP-TWSX module together with 25 bp of DNA from the upstream nucleosome. We discuss the implications of these results for models of CCAN assembly on arrays of -satellite chromatin containing CENP-ANuc.

molecular biology↗

bioRxiv2025

Extracellular matrix rigidity controls breast cancer metastasis via TYK2-mediated mechanotransduction

Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes. In breast cancer, increased tumor stiffness is associated with elevated metastasis risk and poor survival. We identify a unique role of the JAK family kinase TYK2 in suppressing breast cancer metastasis under low ECM stiffness. Genetic or pharmacological inhibition of TYK2 in mammary acini and patient-derived organoids leads to invasion at low ECM stiffness by promoting Epithelial-Mesenchymal Transition, which is independent of cytokine-induced JAK/STAT signaling. TYK2 blockade promotes metastasis in breast tumor cell- and patient-derived xenografts. TYK2 localizes at the plasma membrane via IFNAR1 association under low stiffness, but it becomes cytoplasmic and inactivated at high stiffness. Normal human breast epithelium displays membrane-localized TYK2, whereas invasive breast tumors exhibit cytoplasmic TYK2. These findings uncover a TYK2-dependent mechanism by which ECM rigidity suppresses breast cancer metastasis and underscore the need for vigilant breast cancer screening in patients receiving TYK2 inhibitors.

cancer biology↗