Biology subjects

Zheng, L.

Publications and source records attributed to Zheng, L..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

DNA replication errors drive genome-wide small inverted triplication dynamics

Structural variants (SVs) have a profound impact on phenotype and diversity and are associated with human diseases. To explore the origination of SVs, we have analyzed 1,340 cancer genomes with annotation of 4,608 novel small inverted triplication (SIT) events and found that FEN1 is strongly associated with SIT incidence. Then, we performed long-read sequencing and developed PacBioR to annotate SITs in yeast FEN1 mutant cells. We found that SIT structures mimic classic inverted triplications but with a smaller DUP/IN/DUP structure of 184/160/184 bp on average, with a spacer sequence of 30 bp and breakpoint junction of 6 bp. We further showed that breakpoints of SITs preferentially occurred at nucleosome midpoints, aligned with Okazaki fragment termini, and those harbored in plasmids were precisely eliminated via DNA polymerase slippage over SIT-derived hairpin structures. This study provides mechanistic insight into SIT origination and offers practical tools for future studies on genome rearrangements.

genetics↗

bioRxiv2025

Pan-cancer tumor classification by a holistic tumor microenvironment atlas

The tumor microenvironment (TME) heterogeneity presents a major bottleneck to effective cancer immunotherapies. We address this by establishing a pan-cancer tumor classification system based on the holistic TME cellular components. First, using single-cell transcriptomes from 1,271 patients across 26 cancer types, we compile a TME cellular atlas, leading to the identification of recurrent cell states and multicellular modules across cancer types. Among these, we highlight a type I interferon (IFN-I)-related multicellular module, including IFIT1+ tumor-associated macrophages, as a critical contributor to immune-activation TMEs. From a holistic perspective, pan-cancer TMEs exhibit trichotomous patterns predominantly driven by T, myeloid, and stromal compartments. Leveraging fine-grained TME cellular composition, we further stratify all tumors into 10 stable groups. These groups display varying responses to immune checkpoint blockade (ICB), and each rationally selected therapeutic strategy specifically perturbs the corresponding expression signature. Our pan-cancer tumor classification scheme reveals the underlying patterns of TME heterogeneity and provides a framework for stratifying patients, guiding treatment selection, and developing novel therapeutic options.

cancer biology↗

bioRxiv2025

Adaptive regulation of dNTP homeostasis confers osimertinib resistance in EGFR mutant non-small cell lung carcinoma

Maintaining sustained deoxyribonucleotide triphosphate (dNTP) pools is essential for DNA replication fidelity and genome stability. In EGFR-mutant non-small cell lung carcinoma (NSCLC), we find that disruption of dNTP homeostasis plays a critical role in determining sensitivity to the EGFR inhibitor osimertinib and in shaping mechanisms of acquired resistance. Transcriptomic and biochemical analyses revealed that osimertinib suppresses RRM2 expression, a key regulator of dNTP synthesis, through the downregulation of the transcription factor MYBL2. In response to osimertinib-mediated replication stress and dNTP depletion, cells activated a compensatory pathway involving the stress-inducible ribonucleotide reductase subunit RRM2B via a novel transcriptional regulator, TNNT3. We further identified CHK2 signaling as essential for TNNT3 nuclear translocation and RRM2B transcriptional activation. Inhibition of CHK2 or combined CHK1/2 blockade impaired RRM2B induction, exacerbated replication stress, and delayed the development of osimertinib resistance both in vitro and in xenograft models. Collectively, our findings reveal that EGFR-mutant NSCLC cells rely on a dynamic EGFR-MYBL2-RRM2 and CHK2-TNNT3-RRM2B regulatory axis to maintain dNTP pool balance under therapeutic pressure. Disruption of this axis sensitizes tumors to osimertinib and impairs the acquisition of resistance, highlighting dNTP metabolism as a critical vulnerability and actionable target in EGFR-driven lung cancer. STATEMENT OF SIGNIFICANCETargeting dNTP metabolism via the EGFR-MYBL2-RRM2 and CHK2-TNNT3-RRM2B regulatory axis sensitizes EGFR-mutant NSCLC to osimertinib treatment and delays drug resistance, revealing a novel and actionable therapeutic vulnerability in EGFR-driven lung cancer.

cancer biology↗

bioRxiv2025

Oncometabolite fumarate impairs ATR-CHK1 signaling by succinating RPA1 in Fumarate Hydratase-deficient renal cell carcinoma cells

Abnormal accumulation of oncometabolite fumarate drives susceptibility in fumarate hydratase-deficient renal cell carcinoma (FH-dRCC), but the precise mechanisms remain not fully understood. In this study, we demonstrate that high fumarate levels impair activation of ATR-CHK1 signaling in response to replication stress and DNA damage. Mechanistically, fumarate modifies RPA1, an essential factor for ATR-CHK1 activation through succination, a post-translational modification. Succination of RPA1 occurs mainly at cysteine residues 481 and 486, which reduces its binding affinity for single-stranded DNA (ssDNA). RPA1 succination leads to deficient recruitment of TOPBP1 to ssDNA, resulting in attenuated CHK1 activation and defective cell cycle arrest in response to DNA damage. Succinated RPA1 compromises homologous recombination-mediated DNA repair. Our findings establish that fumarate-induced succination of RPA1 impairs DNA repair and cell cycle control, promoting genomic instability in FH-dRCC. This work reveals a novel mechanism by which oncometabolites contribute to genomic instability.

cancer biology↗

bioRxiv2025

Selective induction by statins of FAM134B-mediated sarcoplasmic reticulum (SR)-phagy degrades the SR calcium pump SERCA1 and contributes to myopathy

Statins, HMG-CoA reductase inhibitors, are the most prescribed medication for lowering low-density lipoprotein cholesterol. Although statin intolerance due to statin-associated muscle symptoms (SAMS) has been a clinically important issue for decades, the molecular mechanisms of statin myopathy remain incompletely understood. To seek to clarify how statins induce myopathy, here we perform transcriptome analysis and identify FAM134B short isoform (FAM134B-S), an endoplasmic reticulum (ER)-phagy receptor, as a statin-inducible gene. FAM134B-S expression is regulated by the mevalonate pathway and sterol regulatory element-binding proteins. We further show that FAM134B plays a critical role in the regulation of skeletal muscle integrity both in the steady-state and statin-treated conditions in human iPS cell-derived myocytes and mice. Mechanistically, FAM134B-S interacts with the sarcoplasmic reticulum (SR) calcium pump SERCA1 and promotes its autophagic degradation. Collectively, our work reveals a hitherto undisclosed mechanism of statin myopathy; blocking the mevalonate pathway induces SERCA1 degradation through FAM134B-mediated SR-phagy, exerting myotoxicity.

pharmacology and toxicology↗

bioRxiv2025

Integrated multi-omic and functional evidence uncover substantial strain-specific divergence in zebrafish

The zebrafish (Danio rerio) is a foundational vertebrate model in biomedical and developmental research, yet genetic divergence among laboratory strains remains an underappreciated source of biological and experimental variation. Among these, the AB and Tubingen (TU) strains are used globally, forming the basis for most zebrafish-based discoveries. Here, we integrated long- and short-read sequencing, ATAC-seq, Hi-C, transcriptomics, and functional assays to generate a comprehensive multi-omic comparison of these two strains. Our analyses revealed substantial genomic and regulatory divergence, including a 62-bp insertion in the asmt promoter that elevates transcription and alters melatonin biosynthesis, driving strain-specific circadian phenotypes. We also identify a non-synonymous variant (mybbp1aG2612C) associated with heightened immune responsiveness. Together, these findings construct the first genome-wide atlas of functional variation between zebrafish strains, illuminating how hidden genetic differences can shape phenotype, regulatory architecture, and experimental reproducibility in this essential vertebrate model.

genomics↗

bioRxiv2025

XPF mediates 3' flap processing for FEN1-independent Okazaki fragment maturation

Okazaki fragment maturation (OFM), the process that removes RNA-DNA primers, is a major source of DNA replication stress and mutations. It involves Pol{delta}-mediated DNA strand displacement synthesis that produces 5 flaps, FEN1-mediated 5 flap cleavage, and LIG1-catalyzed nick ligation. Recently, we discovered that under stress conditions, yeast cells convert 5 flaps into 3 flaps, which are degraded by 3 flap nucleases to produce ligatable DNA nicks. However, little is known about this stress-induced, 3 flap-based OFM in human cells. Here, we report that 3 flaps frequently form in various human cancer cells, and that FEN1 deficiency significantly enhances 3 flap levels. XPF1 is recruited to the replication forks in FEN1 mutant or FEN1-chemically inhibited cells. Notably, XPF deficiency or inhibition in those defective cells leads to accumulation of 3 flaps, replication-related DNA strand breaks, and unique mutation signatures. Furthermore, XPF and FEN1 inhibitors show synergistic effects in killing human cancer cells. In summary, we demonstrate that 3 flap-based OFM is an important alternative of 5 flap-based OFM in mammalian cells. XPF is a key nuclease to degrade 3 flaps and complete OFM for survival. Targeting this compensatory mechanism could provide new therapeutic strategies to selectively impair cancer cell survival under pre-existed replication stress. HighlightsO_LIXPF is recruited to DNA replication forks in mouse or human cells of functional deficiency of FEN1 due to genetic mutations or chemical inhibition. C_LIO_LIXPF gene deficiency or chemical inhibition results in accumulation of 3 flaps and replicative DNA single-stranded breaks. C_LIO_LIFunctional deficiency of XPF and FEN1 displays a synergy in inducing genome instability and cell death in human cancer cells. C_LI

cell biology↗

bioRxiv2025

DNASE1L3 surveils mitochondrial DNA on the surface of distinct mammalian cells

The extracellular space is a critical environment for discriminating self versus non-self nucleic acids and initiating the appropriate immune responses through signaling cascades to relay information about extracellular nucleic acids. Here, we provide evidence that oxidized mitochondrial DNA is tethered to the surface of select mammalian cells through cell surface proteins and heparan sulfate proteoglycans. We demonstrate that cell surface DNA accumulates in large clusters that partially overlap with domains enriched in RNA binding proteins. Finally, we show that human and murine B cell surfaces contain DNA that can be cleared by the secreted nuclease DNASE1L3, and that patients with a DNASE1L3 missense variant associated with increased risk for autoimmune disease harbor increased levels of surface DNA on B and T cells. Taken together, this work expands the scope of cell surface nucleic acid biology and provides a mechanistic link between cell surface molecules and DNA targeting in autoimmune disease.

cell biology↗

bioRxiv2025

JMJD1B-mediated FEN1 demethylation allows timely switching of Okazaki fragment maturation core enzymes to avoid mutagenic flap ligation by PARP1-LIG3

Efficient and faithful Okazaki fragment maturation (OFM) depends on the PCNA-coordinated actions of core enzymes Pol{delta}, FEN1, and LIG1. We demonstrate that Pol{delta}, FEN1, and LIG1 sequentially but not simultaneously bind to PCNA in mammalian cells. The association of FEN1 with PCNA, which lies at the center of this orderly program, is mediated by FEN1 R192 methylation, which is also crucial for preventing premature loading of LIG1. Conversely, FEN1 demethylation by the recently identified arginine demethylase JMJD1B promotes FEN1 dissociation from PCNA and LIG1 recruitment. Disruption of the sequential PCNA binding program in R192Q or Jmjd1b-/- cells results in unprocessed 5 flaps that prevent OFM and induction of PARP1-dependent recruitment of LIG3, which has flap ligation activity to join incompletely processed OFs. This alternative OFM process supports cell survival but causes duplications and other DNA mutations. Our findings define fundamental and alternative DNA replication processes underlying mutagenesis and cell survival. HighlightsO_LIThe core enzymes involved in Okazaki fragment maturation (OFM), DNA polymerase {delta} (Pol{delta}), flap endonuclease 1 (FEN1), and ligase 1 (LIG1), sequentially bind to the scaffold protein PCNA for their concerted actions C_LIO_LIFEN1 R192 methylation enables FEN1 binding to PCNA; JMJD1B-mediated FEN1 arginine demethylation provokes FEN1 exit from PCNA and LIG1 recruitment C_LIO_LIFEN1 R192Q mutation and JMJD1B deficiency oppositely alter dynamic PCNA interactions with FEN1 and LIG1 and result in distinct OFM DNA intermediate structures C_LIO_LIActivation of PARP1 at replication forks due to disruption of the sequential PCNA binding program induces LIG3-mediated alternative mutagenic 5 flap ligation for survival. C_LI

cell biology↗

bioRxiv2025

The Pdgfd-Pdgfrb axis orchestrates tumor-nerve crosstalk in pancreatic cancer

Nerves are an integral component of the tumor microenvironment, contributing to cancer progression, metastasis, morbidity, and mortality. In pancreatic ductal adenocarcinoma (PDAC), worse clinical outcomes are associated with perineural invasion (PNI), a process by which cancer cells surround and invade nerves. Here, we employed whole-transcriptome and single-cell spatial transcriptomics to identify candidate tumor-nerve interactions that promote PNI. We discovered that Pdgfd signaling promotes key features of nerve invasion. Mechanistically, Pdgfd stimulated cancer cell invasiveness, neurite outgrowth, and direct physical engagement with glia. Pharmacological blockade of this axis reduced each of these processes in vitro as well as PNI in vivo. Thus, Pdgfd-Pdgfrb signaling mediates PNI by coordinating multifaceted cancer-neuron-glia interactions and represents a promising therapeutic strategy aimed at disrupting harmful cancer-nerve crosstalk.

cancer biology↗

bioRxiv2025

Overcoming breeding constraints in polyploid oat from evolutionary insights

Polyploidy provides adaptive advantages in plants by buffering deleterious mutations1,2. While polyploidization can enhance agronomic traits such as increased biomass, known as the gigas effect1-4, increasing genetic gain in polyploid crops remains a critical but difficult goal due to the difficulty in dissecting complex trait inheritance5,6. Here we present chromosome-scale genome assemblies for 26 Avena taxa, spanning diploid, tetraploid, and hexaploid lineages. We traced four independent polyploidization events across the genus, including the formation of A. agadiriana as an allotetraploid (AgAgAg'Ag'), and revealed a reticulate evolutionary history shaped by gene flow involving four subgenomes (A, B, C, D), for example, the hybrid speciation of A. hirtula. Transcriptomic analysis of 286 samples across 11 tissues, combined with deleterious mutation analysis from a cultivated population of 112 accessions, showed that polyploidization led to widespread functional redundancy among homoeologs, supporting a genome-wide buffering effect. However, derived allele frequency analysis revealed that, while disrupting functional genes may yield desirable traits, the buffering effect impedes the fixation of beneficial loss-of-function mutations and thereby limits breeding efficiency. Based on these integrative analyses, we propose breeding strategies to circumvent these limitations by targeting beneficial loss-of-function alleles within the complex polyploid background of oat. Our study highlights broader challenges in the improvement of polyploid crops and provides a foundation for future breeding strategies.

evolutionary biology↗

bioRxiv2025

Learning the native-like codons with a 5'UTR and secondary RNA structure aided species-informed transformer model

Efficient protein expression across heterologous hosts remains a major challenge in synthetic biology, largely due to species-specific differences in codon usage and regulatory sequence context. A key difficulty lies in reconstructing the codon landscape of the target expression system within a foreign host with a native-like codon preference. To address this, we present TransCodon, a Transformer-based deep learning model that leverages both 5 untranslated regions (5UTRs) and coding sequences (CDS), along with explicit species identifiers and RNA secondary structure information, to learn nuanced codon usage patterns across diverse organisms. By incorporating multisource genomic data and modeling sequence dependencies in a masked language modeling paradigm, TransCodon effectively captures both local and global determinants of codon preference. Our experiments demonstrate that integrating species-level information during training significantly improves the models ability to predict optimal synonymous codons when considering different evaluation metrics. More importantly it identifies native-like codons with less divergence from natural sequences compared to other methods. Besides, TransCodon could capture more low-frequency codons which are often omitted by other deep learning-based methods. The results thus indicate that TransCodon as a robust codon language model has the potential for generating native-like CDS with high translational efficiency in target hosts.

bioinformatics↗

bioRxiv2025

DNA2 and FANCM function in two distinctive pathways in disrupting TERRA R-loops and suppressing replication stress at ALT telomeres

Cancers maintain their telomeres through two main telomere maintenance mechanisms (TMMs): 85-90% of cancers rely on telomerase, while 10-15% of cancers adopt the Alternative Lengthening of Telomere (ALT) pathway. Previously, we and others reported that FANCM, one of the Fanconi Anemia proteins, plays a critical role in suppressing replication stress and DNA damage at ALT telomeres by actively disrupting TERRA R-loops [1-4]. Here, we showed that inactivation of DNA2 in ALT-positive (ALT+) cells, but not in telomerase-positive (TEL+) cells, induces a robust increase of replication stress and DNA damage at telomeres, which leads to a pronounced increase of many ALT properties, including telomere dysfunction-induced foci (TIFs), ALT-associated PML bodies (APBs), and C-circles. We further demonstrated that depletion of DNA2 induces a pronounced increase of TERRA R-loops and a decrease in replication efficiency at ALT telomeres. Most importantly, we uncovered a strong additive genetic interaction between DNA2 and FANCM in the ALT pathway. Furthermore, co-depletion of DNA2 and FANCM causes synthetic lethality in ALT+ cells, but not in TEL+ cells, suggesting that targeting DNA2 and FANCM could be a viable strategy to treat ALT+ cancers. Finally, utilizing the single-molecule telomere assay via optical mapping (SMTA-OM) technology, we thoroughly characterized genome-wide changes in DNA2 deficient cells and FANCM deficient cells and found that most chromosome arms manifested increased telomere length. Unexpectedly, we uncovered many chromosome arm-specific telomere changes in those cells, suggesting that telomeres at different chromosome arms may regulate and respond to replication stress differently. Collectively, our study not only shed new light on the molecular mechanisms of the ALT pathway, but also discovered a new strategy for targeting ALT+ cancer.

cancer biology↗

bioRxiv2025

Multiplexed, scalable analog recording of gene regulation dynamics over weeks using intracellular protein tapes

Gene expression is constantly regulated by gene regulatory networks that consist of multiple regulatory components to mediate cellular functions. An ideal tool for analyzing gene regulation processes would provide simultaneous measurements of the dynamics of many components in the gene regulatory network, but existing methodologies fall short of simultaneously tracking the dynamics of components over long periods of time. Here, we present CytoTape--a genetically encoded, modular, and scalable analog recorder for continuous, multiplexed in situ recording of gene regulation dynamics over multiple days and weeks at single-cell resolution. CytoTape consists of a flexible, thread-like, elongating intracellular protein self-assembly engineered via AI-guided rational design. Gene regulation dynamics, together with timestamps for reconstruction of the continuous time axis, are directly encoded via distinct molecular tags distributed along single CytoTape assemblies in live cells, to be readout at scale after fixation via standard immunofluorescence imaging. CytoTape recorders are modularly designed to record gene expression driven by a variety of activity-dependent promoters. We demonstrated the utility of CytoTape in mammalian embryonic kidney cells, cancer cells, glial cells, and neurons, achieving simultaneous recording of five cell plasticity-associated transcription factor activities and immediate early gene expression levels, namely CREB, c-fos, Arc, Egr1, and Npas4 activities, within single cells in a spatiotemporally scalable manner. CytoTape revealed complex waveforms and nonlinear temporal couplings among these cellular activities, enabling investigations of how gene regulation histories and intrinsic signaling states shape transcriptional logics. We envision CytoTape to have broad applications in both basic and disease-related cell biology research.

synthetic biology↗

bioRxiv2025

NF2 loss malignantly transforms human pancreatic acinar cells and enhances cell fitness under environmental stress

Pancreatic ductal adenocarcinoma (PDAC) occurs as a complex, multifaceted event driven by the interplay of tumor permissive genetic mutations, nature of cellular origin and microenvironmental stress. In this study, using primary human pancreatic acinar 3D organoids, we performed CRISPR knockout screen targeting 199 previously underappreciated potential tumor suppressors curated from clinical PDAC samples. Our data revealed significant enrichment of a list of candidates, with NF2 emerging as the top target. Functional validation confirmed that loss of NF2 promotes the transition of PDAC to an invasive state, potentially through extracellular matrix modulation. NF2 inactivation was found to enhance PDAC cell fitness under nutrient starvation. This adaptation not only reinforces the oncogenic state but also confers therapeutical resistance. Additionally, we found that NF2 loss is associated with the fibroblast heterogeneity and cancer-stroma communications in tumor evolution. These findings establish NF2 as a critical tumor suppressor in PDAC and uncover its role in mediating nutrient adaptation and drug resistance. Importantly, this study provides new insights into drug resistance mechanisms and potential therapeutic targets in PDAC.

cancer biology↗

bioRxiv2025

ER fusogens maintain membrane reservoir to ensure brain function

How the morphological dynamics of the endoplasmic reticulum (ER) are linked to its functions is unclear. Atlastins (ATLs), a class of GTPases, mediate ER fusion, and human mutations in ATL1 cause hereditary spastic paraplegia (HSP). Here, we show that ATL2-knockout mice are embryonic lethal with compromised development, particularly of the cerebellum. ATL2 is highly expressed in neuroglia, though ATL1 is dominant in the brain. Lack of ATL2 disorganizes the positioning of Bergmann glia, which in turn interferes with granule cell migration. These cells have significant shrinkage in the intracellular membrane area, which is associated with decreased phosphatidylcholine and cholesterol synthesis. When tested in calyx-type synapses in ATL-deleted mice, a reduced membrane reservoir, represented by fewer presynaptic vesicles, leads to defective synaptic function and deafness. Collectively, these findings suggest that ER-shaping activity by ATL is essential for sustained lipid synthesis, and boosted lipid uptake is potentially beneficial for HSP patients.

neuroscience↗

bioRxiv2025

Newly trained navigation and verbal memory skills elicit changes in task-related networks but not brain structure

Training cognitive skills, such as remembering a list of words or navigating a new city, has important implications for everyday life. Yet, understanding what brain changes underlie the acquisition of complex cognitive skills remains unresolved. Here, we developed and validated intensive multiweek interventions in which participants were randomly assigned training in either navigation or verbal memory. Healthy young participants (N=75) underwent structural and functional imaging prior to and following the training. Based on pre-registered and exploratory analyses, we did not find any evidence for changes to gross hippocampal or hippocampal subfield volume, cortical brain volume, or white matter connectivity due to the training. In contrast, network-based analyses suggested changes in task-related informational connectivity, which occurred primarily between cortical areas and mostly involved putative cognitive control networks. These results suggest that cognitive interventions target more transient configurations in network connectivity rather than more durable structural changes.

neuroscience↗

bioRxiv2025

Structural basis for prohibitin-mediated regulation of mitochondrial m-AAA protease

Mitochondrial function critically dependents on protein quality control systems, with the m-AAA protease plays a key role at the inner mitochondrial membrane (IMM). The evolutionarily conserved prohibitins (PHBs) are essential modulators of this protease across species, yet the molecular mechanisms remain unclear. Here, we present the Cryo-EM structure of the Chaetomium thermophilum PHB (CtPHB) complex, revealing a cage-like assembly composed of 11 copies of PHB1/PHB2 heterodimers. Electron microscopic and biochemical analyses suggest that m-AAA proteases are enclosed within the PHB complex through interactions mediated by their SPFH-interacting motif (SIM) exposed in the intermembrane space. Further in situ cryo-ET directly visualizes these cage-protease assemblies in native mitochondria. Disruption of their interface leads to elevated m-AAA protease activity and diminished mitochondrial stress resistance. These data establish PHB complexes as spatial organizers that compartmentalize m-AAA proteases in membrane microdomains to fine tune proteolytic homeostasis. Our findings reveal the critical role of the PHB complex in maintaining mitochondrial proteostasis, providing a unified mechanistic model to explain and reconcile the pleiotropic, and often contradictive phenotypes of PHBs and m-AAA protease in mitochondrial physiology and various disease conditions.

molecular biology↗