Biology subjects

Yu, Y.

Publications and source records attributed to Yu, Y..

At least 109 records · Page 6Linked to original sources

bioRxiv2026

Structural insights into the recognition and catalysis of tRNA by human NSUN2

The human RNA m5C methyltransferase NSUN2 catalyzes site-specific cytosine methylation across diverse RNA substrates and thereby regulates a wide range of biological and physiological processes. However, the molecular basis by which NSUN2 achieves broad substrate recognition while maintaining catalytic specificity has remained unclear. Here we determine structures of human NSUN2 in both substrate-free and substrate-bound states using X-ray crystallography and cryo-electron microscopy. Structures of NSUN2 in complex with multiple tRNA substrates reveal a structure-first, sequence-tolerant strategy in which NSUN2 actively remodels tRNA architecture, exposing the buried target cytosine and positioning it within the catalytic pocket for methyl transfer. This recognition strategy enables NSUN2 to accommodate diverse tRNA substrates through a largely conserved interaction interface. Guided by these structural insights, we identify a small-molecule inhibitor that suppresses NSUN2 activity and cancer cell proliferation. Together, our findings define the molecular principles underlying NSUN2-mediated RNA m5C modification and provide a structural foundation for targeting NSUN2 in cancers.

molecular biology↗

bioRxiv2026

Donor Macrophage Depletion Permits Post-Transplant Tolerance Induction in a Murine Islet Transplant Model

Current strategies for experimental tolerance induction for allogeneic transplantation typically require recipient preparation days to weeks prior to transplantation, making them not applicable to deceased donor transplantation. Developing tolerance strategies feasible for deceased donor transplantation would greatly increase the pool of eligible patients for tolerance induction. Here, we aimed to induce tolerance with post-transplant only interventions in a murine pancreatic islet transplant model. We demonstrated that transplant tolerance induction by recipient infusions of ethylcarbodiimide-treated donor splenocytes (ECDI-SPs) could be reliably delayed to the post-transplant timeframe provided that donor islets were depleted of intra-islet macrophages prior to transplantation. Mechanistically, islet production of CCL3, CCL4, and CCL5 (RANTES) was significantly reduced by intra-islet macrophage depletion. On POD+1, islet allograft depleted of donor intra-islet macrophages exhibited significantly reduced infiltration of recipient innate immune cells, including monocytes, macrophages, and neutrophils. Interestingly, perioperative inhibition of CCR5, the receptor for CCL3, CCL4 and CCL5, also reduced POD+1 innate immune cell infiltration, and similarly permitted tolerance induction by post-transplant donor ECDI-SP infusions. This study thus demonstrates the efficacy of a strategy that would allow transplant tolerance induction by post-transplant-only interventions, thereby expanding the applicability of tolerance induction regimens to additional clinically relevant settings.

immunology↗

bioRxiv2026

Delta PSA: A New Metric for Conformational Dynamics Underlying Macrocyclic Peptide Permeability

Development of oral cyclic drugs often suffers from low oral bioavailability resulting from limited passive permeability making accurate prediction a central challenge in drug development. Existing approaches generally fall into two categories: deep learning-based models and accelerated molecular dynamics (aMD) simulations. While deep learning models enable rapid, high-throughput predictions, they often suffer from dependence on training data, and sensitivity to dataset biases. On the other hand, aMD provide mechanistic interpretability by explicitly modeling peptide translocation across lipid bilayers, however, suffer from huge computational cost. Here, we present a conventional MD-based framework for predicting macrocyclic peptide permeability, designed to facilitate interpretation. Importantly, we show that Delta PSA ({Delta}PSA) directly quantifies a peptides chameleon propensity, providing a mechanistically meaningful measure. Furthermore, we identify two key structural indicators--the sidechain PSA ratio and the radius of gyration--as refined metrics for assessing conformational behavior. When applied to a benchmark dataset of macrocyclic peptides, our framework achieves an MSE of 0.22, surpassing the 0.25 reported for Multi_CycGT and demonstrating its superior performance. Our work also provides a large dataset of MD trajectories for macrocyclic peptides in both polar and nonpolar environments. This dataset offers access to a broader conformational space for cyclic peptide studies. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/697862v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@18fc9e4org.highwire.dtl.DTLVardef@10fbc70org.highwire.dtl.DTLVardef@1c763aorg.highwire.dtl.DTLVardef@95d9e4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Cryo-EM reveals the structural basis of subtype-specific, noncompetitive inhibition of the human P2X3 receptor

P2X receptors are ATP-gated cation channels, and the P2X3 subtype plays crucial roles in peripheral sensory neurons, including in chronic pain and chronic cough. Accordingly, P2X3 has attracted substantial interest as a therapeutic target. Gefapixant, a negative allosteric modulator (NAM) of P2X3, has been approved in some countries for the treatment of chronic cough; however, its limited selectivity for P2X3 homomers over P2X2/P2X3 heteromers is associated with taste disturbance as a prominent adverse effect. These limitations have motivated the development of next-generation NAMs with improved subtype selectivity, but their subtype-specific allosteric inhibition mechanisms are unclear. Here, we report the cryo-EM structure of the human P2X3 receptor in complex with ATP and the P2X3-selective next-generation NAM sivopixant, an investigational drug. Sivopixant binds to an allosteric site at the portal of the central pocket in the extracellular domain, and structure-based mutational analysis by electrophysiology identifies key residues required for sivopixant-dependent inhibition of human P2X3. Comparisons with P2X structures from other subtypes, together with gain-of-function mutants, define a structural basis for subtype-selective allosteric inhibition of the P2X3 receptor. Furthermore, structural comparisons with apo and ATP-bound open states of P2X3 receptors, together with molecular dynamics simulations, revealed that sivopixant expands the upper body domain to suppress the lower-body movements required for channel activation, thereby preventing channel opening even in the presence of ATP.

biophysics↗

bioRxiv2025

IGHV3-53 antibody abundance drives divergent SARS-CoV-2 immune imprinting

The mechanisms driving divergent SARS-CoV-2 immune imprinting in populations with distinct SARS-CoV-2 exposure histories remain unclear. However, conventional wild-type mouse models fail to recapitulate severe imprinting, even after potent ancestral-strain mRNA immunization, hindering imprinting-related mechanistic investigation and vaccine evaluation. Here, we surprisingly found that V(D)J-humanized mice could faithfully recapitulate human severe imprinting phenotypes. Comprehensive antibody repertoire analysis and deep mutational scanning-based epitope mapping of 583 monoclonal antibodies from these models revealed that the pre-existing IGHV3-53/66 antibody abundance determine imprinting severity through antibody-mediated masking of Omicron-specific epitopes. Both passive IGHV3-53/66 antibody transfer and IGHV3-53 knock-in were sufficient to induce severe imprinting in wild-type mice. Together, these findings demonstrate that the V(D)J germline repertoire--even a single germline-encoded antibody response--can profoundly shape humoral imprinting severity. Accordingly, we established an IGHV3-53 knock-in mouse model that accurately recapitulates the human antibody landscape, providing a valuable tool for guiding future COVID-19 vaccine updates.

immunology↗

bioRxiv2025

Conformation-specific Antibody Deciphers K27-linked Ubiquitination in Chaperone-Mediated Proteostasis

Lysine 27 (K27)-linked polyubiquitination plays critical yet incompletely defined roles in proteostasis, innate immunity, and disease progression; however, investigations into this process have long been hindered by its extremely low abundance and the lack of conformation-specific enrichment tools. Herein, we describe the development of a long-sought conformation-specific antibody, K27-IgG, which can selectively recognize--among all ubiquitin chain types--the unique architecture of K27-linked polyubiquitin (K27-polyUb) characterized by a distinct buried K27-isopeptide bond, with high affinity (KD = 4.66 nM). This antibody was derived from synthetic antibodies initially generated via phage display, using chemically synthesized K27-linked diubiquitin (K27-diUb) as the antigen. High-resolution co-crystal structures uncovered the unique K27-diUb interface targeted by these sAbs. Subsequent reformatting of these sAbs into a full-length human immunoglobulin G (IgG) scaffold yielded K27-IgG, notably exhibiting markedly enhanced affinity without compromising selectivity. Using K27-IgG as a tool, we achieved sensitive detection and immunoprecipitation (IP) of endogenous K27-polyUb in cells, and delineated the intracellular interaction landscape of K27-polyUb through complementary proteomic approaches. Two key findings emerged: 1) The molecular chaperone DNAJB1 is a specific reader of K27-linked ubiquitin chains (but not other linkages) and that K27-polyUb chains themselves exhibit chaperone-like activity, suggesting a novel mechanism by which K27-polyUb regulates chaperone-mediated proteostasis; 2) The E2 enzyme UBE2Q1 assembles K27-diUb, identifying it as a potential writer for this ubiquitin chain topology. Collectively, this study establishes K27-IgG as a robust tool for deciphering the K27-linked ubiquitin code, thereby opening new avenues for investigating the biological functions of K27-linked polyubiquitination. HighlightsO_LIFirst K27-linkage conformation-specific antibody with nanomolar affinity overcomes a major barrier in the field. C_LIO_LIK27-IgG unlocks functional mapping of the K27 ubiquitin landscape under proteotoxic stress. C_LIO_LIMolecular chaperone DNAJB1 is a selective "reader" of K27-linked ubiquitin chains. C_LIO_LIK27 chains possess intrinsic chaperone activity, enabling protein refolding and suppressing aggregation. C_LIO_LIE2 enzyme UBE2Q1 is a "writer" that directly assembles K27-linked ubiquitin chains. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/695067v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@113b980org.highwire.dtl.DTLVardef@1f7c1a0org.highwire.dtl.DTLVardef@c91656org.highwire.dtl.DTLVardef@1d9a507_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Structural basis of Retron-Eco8-mediated anti-phage defense

Retrons represent a novel class of bacterial defense systems that employ reverse transcriptase (RT), non-coding RNA, and effector proteins to counteract phage infections. In this study, we elucidate the molecular mechanism of a retron system, Retron-Eco8. Biochemical experiments reveal that the Retron-Eco8 holocomplex, rather than the effector alone, exhibits double-stranded DNA cleavage activity in an ATP-independent manner, triggering abortive infection and effectively halting phage propagation. Cryo-electron microscopy (cryo-EM) analysis reveals a supramolecular assembly comprising four RT subunits, four msDNA (multicopy single-stranded DNA) molecules, and four OLD (overcoming lysogenization defect) nucleases--a configuration critical for anti-phage defense. Structural comparisons between apo and ATP-Mg2+-bound states demonstrate a local conformational change. Notably, we identify the phage SSB (single-stranded DNA-binding) protein as an activator of Retron-Eco8, and phylogenetic analysis of SSB proteins further elucidates the phage resistance specificity. Collectively, our findings delineate the structural architecture of the Retron-Eco8 defense complex and provide mechanistic insights into retron-mediated bacterial immunity.

biophysics↗

bioRxiv2025

Single-cell transcriptomic dynamics exposes hidden survival trajectory under antibiotic treatment

Heterogeneity plays a major role in bacterial resistance to antibiotic treatment while the mechanism at single-cell level is largely unknown. Here, we employed a robust integration of bulk and bacterial single-cell RNA-seq (scRNA-seq) to uncover how individual cells of Acinetobacter baumannii reorganise the heterogenous transcriptome in response to antibiotic. Using polymyxin as a representative, bulk RNA-seq showed canonical envelope- and efflux-centred responses but obscured underlying heterogeneity. Single-cell profiling resolved these averages into discrete subpopulations whose abundances shifted with concentration and time. Specifically, in early time an envelope-stress programme predominated survival at the low concentration, whereas an outer-membrane repair/efflux programme dominated survival at the high concentration. These patterns revealed structured and time-resolved heterogeneity, highlighting an ingenious bacterial stress responsive strategy. Through trajectory inference, we further revealed a concentration-dependent bifurcation of cell fates: low-concentration treated cells detoured through a transient stress state and rejoined growth, whereas high-concentration treated survivors diverted into a slow-growing, tolerant branch. Perturbing marker genes from these programmes altered fitness eventually and rapidly increased permeability, depolarisation and reactive-oxygen burden, linking state to survival capability. Collectively, we map a dynamic, concentration-structured landscape of antibiotic responses at the single-cell level, revealing diverse survival trajectories that are obscured in conventional population-averaged analyses. Our developed single-cell based framework revealed tolerant bacterial cells with rewiring of the transcriptional landscape, causing emergence of antibiotic resistance. Importantly, these findings urge precision antimicrobial therapy in patients to minimise emergence of antibiotic resistance.

microbiology↗

bioRxiv2025

Epitope and functional classification of human neutralizing antibodies against SFTSV Gn

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a lethal bunyavirus lacking approved countermeasures. From SFTS survivors, we isolate 84 human monoclonal antibodies (mAbs) against the viral glycoproteins Gn and Gc. Gn-specific mAbs demonstrate superior neutralization breadth and potency compared to the restricted neutralizing activity observed with Gc. Using a high-throughput yeast display deep mutational scanning (DMS) platform, we classify Gn-head mAbs into eight epitope groups, among which four groups (IA, ID, IIIA, IIIB) conferring neutralization. Notably, mAbs BD70-4003 (group IA) and BD70-4017 (group IIIA) exhibit broad neutralization and provide 100% protection in a lethal mouse model. Cryo-EM structural analysis of these mAbs in complex with the Gn-head reveal their binding interfaces, directly validating the epitope residues identified by DMS. Our study delineates the antigenic landscape of SFTSV Gn, identifies potent therapeutic candidates, and establishes DMS coupled with structural validation as a powerful framework for antibody discovery against bunyavirus.

immunology↗

bioRxiv2025

Systematic Discovery of Novel Phosphoinositide-Binding Effectors in Legionella Reveals Conserved α-Helical Folds

Intracellular bacterial pathogens deploy effector proteins to hijack host membranes by targeting specific lipids. Phosphoinositides are key eukaryotic signaling molecules that govern membrane identity and dynamics, making them powerful levers for microbial interference. However, the bacterial repertoire of phosphoinositide-binding effectors remains largely undefined. In this study, we systematically identified such proteins using an expression library of 241 Legionella pneumophila effectors. We applied a combination of lipid bead pulldowns, mass spectrometry, colocalization with phosphoinositide biosensors, and protein-lipid overlay assays, leading to the validation of 18 new phosphoinositide-binding effectors. Structural predictions revealed that these proteins share compact alpha-helical folds distinct from canonical eukaryotic lipid-binding domains. Guided by this structural signature, we performed a computational screen across L. pneumophila, Coxiella burnetii, and Burkholderia pseudomallei. This analysis identified 15 additional effectors, which were experimentally validated as phosphoinositide binders. Altogether, we report 33 previously unrecognized bacterial phosphoinositide-binding effectors. While their lipid-binding profiles were diverse, many effectors showed a preference for compartments enriched in phosphatidylinositol 3-phosphate. These findings expand the known diversity of lipid-binding effectors produced by intracellular pathogens, identify new structural modules bacteria use for phosphoinositide recognition, and broaden our understanding of how pathogens exploit phosphoinositide signaling to manipulate host membranes. ImportanceIntracellular bacterial pathogens hijack host membrane trafficking by targeting phosphoinositides (PIPs), essential eukaryotic signaling lipids. Despite their importance in virulence, bacterial PIP-binding effectors remain largely uncharted because they lack recognizable sequence motifs. Using Legionella pneumophila as a model system, we systematically identified 30 novel PIP-binding effectors, doubling the known repertoire, and discovered that they employ compact -helical folds structurally distinct from canonical eukaryotic PIP-binding domains. Remarkably, these -helical modules are conserved in Coxiella burnetii and Burkholderia pseudomallei, revealing a convergent evolutionary strategy for PIP recognition across phylogenetically diverse pathogens. This work expands our understanding of bacterial lipid-targeting mechanisms and provides a structural blueprint for identifying virulence factors in emerging pathogens.

microbiology↗

bioRxiv2025

Rapid and accurate quantification of viable Listeria monocytogenes with clonal specificity using microfluidic droplet digital PCR based technology

To overcome the technical bottlenecks in the precise quantification and molecular typing of viable foodborne pathogens, this study establishes a microfluidic droplet digital PCR (ddPCR) based method for rapid and accurate detection and quantification of viable Listeria monocytogenes with clonal specificity. In contrast to the time-consuming plate culture methods and unspecific rapid detection methods, the method in this study employs clonal complex (CC) specific primers and probe for strain-specificity and integrate the nucleic acid dye propidium monoazide (PMA) to effectively distinguish viable from dead bacteria. The rapid and precise quantification of viable bacteria is achieved through microdroplet counting. This method does not require DNA extraction, and the entire detection process takes only about 3 hours, with a quantitative detection limit of 3.3x102 CFU/mL, providing strong technical support for the risk monitoring of highly pathogenic specific types of L. monocytogenes.

microbiology↗

bioRxiv2025

Spontaneous differentiation across cell lineages of separate germ layer origin during progenitor cell-mediated regeneration of the central nervous system

During CNS regeneration, neuroepithelial-derived oligodendrocyte progenitor cells (OPCs) can cross germ-layer boundaries to generate neural-crest-derived Schwann cells (SCs). However, the underlying mechanism and disease relevance of this unique phenomenon of cellular plasticity is unclear. Here, we combine single cell genomics in rodent models of CNS injury and samples from multiple sclerosis patients to characterise OPC-derived SCs. We discover that integrin signalling and bone morphogenetic protein (BMP) activation activate a core SOX10/OLIG2 transcriptional circuit to drive OPC-to-SC differentiation. We show that OPC-derived SCs myelinate in vitro and in vivo, and unlike peripheral SCs, can integrate into astrocyte-rich territories. Together, these findings define a conserved molecular mechanism of adult plasticity, enabling the control of OPC fate choices beyond their germ layer origin for CNS repair.

neuroscience↗

bioRxiv2025

Deep learning reveals a microprotein atlas in maize and uncovers novel regulators of seed amino acid metabolism

Microproteins represent a vast and functionally important class of genes that remain largely unexplored in plant genomes. Here, we developed DeepMp, a deep learning framework that integrated multi-omics evidence and built the most comprehensive plant microprotein atlas to date, identifying 18,338 high-confidence candidates in maize. The majority of these appear to have originated de novo from regions previously annotated as noncoding, and they show hallmarks of rapid, lineage-specific evolution and pronounced tissue specificity. These novel microproteins were found integrated into core regulatory networks, particularly in organ development and nutrient storage. Focusing on the maize kernel, population-scale analyses linked microprotein expression to natural variation in amino acid content. We functionally validated three grain-filling-specific candidates originating from noncoding regions by CRISPR-Cas9 knockouts, which confirmed their roles as precise modulators of arginine, aspartate, and methionine levels, without pleiotropic effects on kernel morphology. Our study provides a foundational resource and analytical framework, establishes microproteins as a new and functionally important coding layer in maize, and uncovers a previously untapped source of targets for crop improvement.

genomics↗

bioRxiv2025

Dissociable neural substrates of integration and segregation in exogenous attention

Impact StatementThis study offers the first direct neuroimaging evidence for the integration-segregation theory of exogenous attention by identifying dissociable neural signatures of the competing integration and segregation processes, strengthening the understanding of attentional orienting mechanisms. The integration-segregation theory proposes that early facilitation and later inhibition (i.e., inhibition of return, IOR) in exogenous attention arise from the competition between cue-target event integration and segregation. Although widely supported behaviorally, the theory lacked direct neural evidence. Here, we used event-related fMRI with an optimized cue-target paradigm to test this account. Cued targets elicited stronger activation in the frontoparietal attention networks, including the bilateral frontal eye field (FEF) and intraparietal sulcus (IPS), right temporoparietal junction (TPJ), and left dorsal anterior cingulate cortex (dACC), consistent with the notion of attentional demand of reactivating the cue-initiated representations for integration. In contrast, uncued targets engaged the medial temporal cortex, particularly the bilateral parahippocampal gyrus (PHG) and superior temporal gyrus (STG), reflecting the segregation processes associated with new object-file creation and novelty encoding. These dissociable activations provide the first direct neuroimaging evidence for the integration-segregation theory. Moreover, we observed neural interactions between IOR and cognitive conflict, suggesting a potential modulation of conflict processing by attentional orienting. Taken together, these findings provide new insights into exogenous attention by clarifying the neural underpinnings of integration and segregation and uncovering the interaction between spatial orienting and conflict processing.

neuroscience↗

bioRxiv2025

Lessons learned from a Kaggle challenge for particle picking in cryo-electron tomography

The difficulty of particle picking in cryo-electron tomography remains a barrier to routine in situ structure determination. Machine learning is well-suited to overcome this bottleneck with efficient algorithms that generalize across molecular species. To spur new algorithm development, we held a three-month Kaggle challenge that tasked contestants with annotating five molecular species across hundreds of experimental tomograms. This competition successfully engaged >1000 participants from diverse fields and delivered particle pickers that outperformed existing state-of-the-art. Systematic comparisons of the contestants submissions revealed the tolerance of subtomogram averaging to moderate but not severe over-picking and underscored the need for more robust measures of annotation quality. The winning models also highlighted the importance of data augmentation to overcome limited training data. All competition tomograms along with the ground truth and winning teams annotations have been released on the CryoET Data Portal as a resource to benchmark current and future tools for particle picking.

cell biology↗

bioRxiv2025

Oxidative Peptide Backbone Cleavage by a HEXXH Enzyme During RiPP Biosynthesis

Ribosomally synthesized and post-translationally modified peptides (RiPPs) rely on a diverse array of enzymes to tailor peptide backbones and side chains. In this study, we characterized enzymes from two different biosynthetic gene clusters (BGCs) from Pseudomonas strains (pfl and pos) that catalyze new transformations in RiPP biosynthesis. Two -ketoglutarate-dependent HEXXH enzymes, PflC and PosC, perform hydroxylation of multiple consecutive glutamine residues and selectively recognize a C-terminal ARMD tetrapeptide to trigger oxidative backbone cleavage that generates an amide terminus. Mutational analysis pinpoints the first position of this motif as a critical determinant. Notably, PflC displays proteolytic activity in the absence of the leader peptide, indicating that leader peptide-enzyme interactions modulate the observed reaction selectivity. The biosynthetic gene clusters also encode a unique MNIO-nitroreductase fusion enzyme that installs a rare Z-dehydrophenylalanine and hydroxylates an Asp residue. Collectively, this work expands both the catalytic repertoire and structural diversity accessible through bacterial RiPP biosynthesis.

biochemistry↗

bioRxiv2025

Toward simple, rapid, and deep plant proteome analysis with an in-cell proteomics strategy

Although modern mass spectrometry (MS) based proteomics has been broadly employed by the plant science community, proteomics analysis of plant tissues remains highly challenging to general plant biology laboratories, mainly due to the lack of effective and easily adaptable strategies that can process plant samples for MS analysis rapidly and reliably. Existing methods often involves lengthy mechanical disruption and protein extraction, which not only require large starting materials but also can cause significant variations, compromising quantitation accuracy. Here, we present an "in-cell proteomics" strategy and benchmark its performance against conventional processing methods using several model plants, including Arabidopsis thaliana, Nicotiana benthamiana, Zea mays and Sorghum bicolor. The comprehensive datasets from this study indicate that the on-filter in-cell (OFIC) processing-based "in-cell proteomics" strategy can eliminate cell lysis of plant leaves and pollen grains, and process proteins directly, enabling the identification of 9,000 to 12,000 proteins from leaves, and 7,000 to 9,000 proteins from pollen grains using one-shot LCMS. The OFIC strategy demonstrated broadly equivalent or better performance over conventional approaches in the context of simplicity, speed, digestion effectiveness, and quantitation accuracy. Applying the strategy to investigate plant leaf infection of N. benthamiana leaves revealed interesting signature proteins that respond differentially to host-adapted Colletotrichum destructivum and nonhost-adapted Colletotrichum sublineola fungus. The single-vessel approach bypasses cell disruption, substantially simplifying plant sample preparation while reducing the required sample input and enhancing proteomic sensitivity. It offered a generalizable approach to study plant leaf infection and other significant questions in plant biology and beyond.

biochemistry↗

bioRxiv2025

Distinct and compensatory roles of Stag1 and Stag2 in post-mitotic genome refolding

The three-dimensional architecture of the eukaryotic genome is largely shaped by the cohesin complex, which contains either Stag1 or Stag2 subunits. Although both subunits contribute to chromatin organization, their specific functions in de novo loop formation during post-mitotic genome refolding remain elusive. Here, we leverage the mitosis-to-G1 transition to dissect their individual roles. We found that Stag1 depletion has a negligible impact on post-mitotic genome restructuring or transcription reactivation. In contrast, Stag2 orchestrates chromatin remodeling in a manner that is both cell cycle stage-specific and chromatin context-dependent. During early-G1, Stag2 preferentially associates with euchromatin, where it drives the rapid formation of small chromatin loops. This facilitates prompt promoter-enhancer (P-E) contact formation and enables efficient transcription activation. As the nuclear concentration of Stag2 increases by late-G1, it progressively suppresses large loops, likely due to its shorter chromatin residence time and its potential to competitively displace the more extrusion-capable Stag1-associated cohesin. Mechanistically, Stag2-mediated loop extrusion is constrained by CTCF-bound barriers, rather than by genomic travel distance. Although Stag2 associates rapidly with euchromatin in early-G1, its recruitment to heterochromatin is delayed until late-G1. Simultaneous depletion of both Stag proteins results in a synergistic loss of virtually all structural loops and a more severe disruption of transcription than that caused by individual deletions. Together, these results establish Stag2 as the principal regulator of post-mitotic genome reorganization among Stag paralogs, mediating spatiotemporal control of chromatin architecture, while Stag1 provides compensatory support to ensure functional robustness.

molecular biology↗