Biology subjects

Zeng, J.

Publications and source records attributed to Zeng, J..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

DNAH14 deficiency impairs sperm motility by reducing flagellar beat amplitude

Male infertility affects approximately 15-20% of couples worldwide, with asthenozoospermia accounting for 19% of cases. Nevertheless, the genetic basis of many asthenozoospermia cases remains poorly understood. In this study, we generated Dnah14 knockout mice, which displayed subfertility due to impaired sperm motility despite normal sperm morphology. Comprehensive kinematic analysis revealed that Dnah14-deficient spermatozoa exhibited reduced flagellar beat amplitude but increased beat frequency, while other dynein components remained unaffected. Notably, increasing sperm concentration during in vitro fertilization (IVF) experiments partially rescued the fertilization defects caused by Dnah14 deletion, suggesting a compensatory mechanism for this specific form of asthenozoospermia. Our results establish DNAH14 as a critical regulator of sperm motility and propose a potential therapeutic strategy for affected individuals.

developmental biology↗

bioRxiv2025

Why are so many fusogens rod-shaped?

Molecular fusogens catalyze membrane fusion for many basic biological processes. In eukaryotic cells, SNARE proteins drive membrane fusion for trafficking and for exocytic release in many contexts, from neurotransmission to enzymatic digestion, while other fusogens mediate cell-cell fusion for organ formation, placental development and gamete fusion. Enveloped viruses use glycoprotein fusogens for host cell entry and delivery of the viral genome. Despite this breadth of roles, a structural feature shared by many of these fusogens is their rod shape, conserved across the SNARE superfamily, the class I and II fusogen superfamilies and the class III fusogen family. Here we used highly coarse-grained molecular dynamics (MD) simulations to examine the collective behavior of rod-like fusogens on the microscopically long timescales of physiological membrane fusion. Rod-generated entropic forces maintained a cleared fusion site, squeezed and hemifused the membranes, and then expanded and ruptured the hemifused connection to yield fusion. More fusogens generated higher entropic forces and faster fusion, consistent with electrophysiological measurements at neuronal synapses. The required fusogenic feature was the rod shape, since simulated SNARE complexes, class II EFF-1 fusogens, and model rod-shaped complexes entropically drove fusion along similar pathways, whereas globular complexes failed. Thus, rod-like fusogens are optimally shaped generators of entropic forces that drive membrane fusion. These results suggest a universal rod-based fusion mechanism may have been the evolutionary driver of structural convergence among major classes of eukaryotic and viral fusogens. SignificanceLarge classes of eukaryotic and viral membrane fusion complexes are rod-shaped, a common structural feature despite a multiplicity of functions, from neurotransmission to organogenesis to cell entry. Here we used coarse-grained molecular dynamics to access the millisecond timescales of physiological fusion. Simulations revealed a universal mechanism whereby, regardless of structural details, rod-shaped fusogens generate entropic forces that drive fusion. This suggests the mechanism was a major evolutionary driver of structural convergence and multiple transmissions between eukaryotic hosts and viruses. Rod-mediated fusion is faster with more fusogens, explaining why release at presynaptic nerve terminals is enhanced or suppressed depending on the number of active SNARE fusogens. Thus, modulation of SNARE numbers and fusogenicities may represent a significant mechanism of synaptic plasticity.

biophysics↗

bioRxiv2025

ZIPcnv: accurate and efficient inference of copy number variations from shallow whole-genome sequencing

Shallow whole-genome sequencing (sWGS), a rapid and cost-effective sequencing technology, has gradually been widely adopted for CNV analyses. However, with genome-wide coverage of only 0.1-5x, sWGS data exhibit a pronounced zero-inflation phenomenon, whereby a large fraction of loci contains zero sequencing reads. Zero-inflation causes read counts to fluctuate by several-fold between adjacent windows. As a result, random upward blips in coverage can be misinterpreted as copy-number gains (false positives), and true deletions often become indistinguishable from pervasive zero-coverage noise. In addition, existing CNV detection tools developed for sWGS data often struggle to adapt across different CNV sizes. These combined effects severely constrain the accuracy of CNV inference. To address above challenges, we propose ZIPcnv, a novel CNV detection tool specifically designed for sWGS data. First, we apply a large sliding window to smooth the raw read depth signal, which transforms the original zero-inflated statistical characteristics into approximately normal distribution characteristics. We then design a statistical process model that robustly detects persistent shifts under high background noise using a cumulative sum strategy, classifying genomic regions into candidate and non-candidate CNV regions. Finally, dynamic sliding windows are used for one-pass detection of CNVs of varying lengths, with window size adapting to the CNV region size. We evaluated the performance of ZIPcnv on simulated data and 190 real whole-genome sequencing samples. Experimental results show that ZIPcnv consistently outperforms currently popular CNV detection tools. The ZIPcnv source code is freely available at https://github.com/Nevermore233/ZIPcnv.

bioinformatics↗

bioRxiv2025

The anaerobic WOR-3 Bacteria: Hydrogenotrophic metabolism and unique carbon fixation via Archaeal form III RuBisCO

The uncultured WOR-3 phylum is widely distributed in anaerobic environments, including hot springs, marine ecosystems, and hydrothermal vents, yet its ecological roles and metabolic capabilities remain poorly understood. In this study, we analyzed 180 medium- to high-quality metagenome-assembled genomes (MAGs), including 59 newly reconstructed from environmental samples and 121 retrieved from the Genome Taxonomy Database (GTDB). Phylogenetic analyses resolved the WOR-3 lineage into four subgroups (subgroup 1-4). Metabolic reconstruction revealed significant divergence of the carbon, sulfur, nitrogen and hydrogen metabolism pathways among the different subgroups. Subgroup 1 was characterized by fermentative metabolism involving formate and ethanol, and uniquely exhibited potential for carbon fixation via Calvin cycle, as indicated by the presence of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) gene. Notably, WOR-3 RuBisCO is phylogenetically affiliated with archaeal form III, although the carbon fixation pathway follows the canonical bacterial Calvin cycle--a feature of potential evolutionary significance. Subgroup 3 exhibits metabolic versatility, including genes for dissimilatory sulfate reduction, sulfur oxidation, partial denitrification, and fatty acid degradation. In addition, all subgroups harbored key components of hydrogen metabolism, including widespread NiFe hydrogenases and Rnf complexes, supporting H2-dependent electron transfer and energy conservation under anaerobic conditions. Featuring near-universal presence of complexes I and II and frequent occurrence of terminal oxidases (cydAB and coxABC)--suggests a facultative anaerobic lifestyle. Collectively, this study expands the genomic framework for the WOR-3 phylum and provides novel insights into the metabolic versatility and ecological functions of this previously uncharacterized lineage in biogeochemical cycles of carbon, nitrogen, and sulfur. IMPORTANCEThe WOR-3 phylum represents a widespread but poorly understood bacterial lineage inhabiting diverse anaerobic/microaerobic environments. By integrating 180 metagenome-assembled genomes, including 59 newly reconstructed, this study provides the most comprehensive genomic framework to date for WOR-3. Phylogenomic and metabolic analyses revealed four distinct subgroups with divergent capacities for carbon, sulfur, and nitrogen metabolism. Notably, subgroup 1 encodes a complete Calvin-Benson-Bassham (CBB) cycle featuring an archaeal-type form III RuBisCO, suggesting an unusual evolutionary trajectory for carbon fixation in this lineage. Subgroup 3 exhibits versatile metabolic potential, including dissimilatory sulfur metabolism, partial denitrification, and fatty acid degradation, highlighting its possible roles in multiple biogeochemical processes. The widespread presence of hydrogenases and respiratory complexes across all subgroups supports energy conservation under anaerobic or microaerobic conditions. These findings not only expand the taxonomic and functional landscape of the WOR-3 phylum but also offer key insights into its ecological roles in global element cycling.

microbiology↗

bioRxiv2025

Glucagon receptor signaling is indispensable for the healthspan effects of caloric restriction in aging male mice

Obesity and type 2 diabetes mellitus accelerate aging, shortening the duration of healthspan. Conversely, chronic calorie restriction (CR) extends healthspan. Research aimed at understanding the mechanism by which CR slows aging has focused heavily on insulin and downstream signaling cascades. Glucagon, a hormone that counter-regulates insulin, is commonly affected by these same interventions. To investigate the role of glucagon in aging we used dietary manipulation, global and liver-specific glucagon receptor knockout, and pharmacological glucagon receptor activation. We found that globally eliminating glucagon receptor signaling (Gcgr KO) decreases median lifespan by 35% in lean mice. These lifespan shortening effects are more robust in diet-induced obese mice (54%). Extending these findings to metabolic health, we found that glucagon receptor signaling is indispensable to the metabolic response to chronic CR in young and aged mice. While CR decreased liver fat, serum triglyceride, and serum cholesterol in WT mice, these metabolic benefits were absent in Gcgr KO mice. In line with these observations, we found that critical nutrient sensing pathways known to improve aging are dysregulated in mice lacking glucagon receptor signaling at the liver (Gcgrhep-/-). Liver-specific deletion of the glucagon receptor decreases hepatic AMP Kinase activation in aging mice, regardless of diet. Further, CR decreases hepatic mTOR activity in WT mice, but not in Gcgrhep-/- mice. Together, these findings propose that glucagon signaling plays a critical role in both normal aging and the lifespan and healthspan extension driven by caloric restriction.

physiology↗

bioRxiv2025

Chromosome-Specific Aneuploidy Engineering via dCas9-Induced Centromeric Chromatin Relaxation

Aneuploidy, the gain or loss of chromosomes, is prevalent in both normal and disease conditions, however, experimental approaches to engineer and study aneuploidy remain limited, leaving its functional significance under-characterized. Here, we present CRISPR-Taiji (CRISPRt), an efficient method for inducing chromosome-specific mis-segregation and aneuploidy generation across all 24 human chromosomes via dead Cas9 (dCas9)-induced centromeric chromatin relaxation. Using CRISPRt with scRNA-seq, we generated the first comprehensive transcriptomic alteration landscape of nearly all autosomal aneuploidies at chromosome-arm resolution. This genotype-phenotype map provides causal evidence linking recurrent aneuploidies in clear cell renal cell carcinoma (ccRCC) to molecular and clinical phenotypes observed in patient tumors. Notably, chromosome 3(p) loss, the ccRCC initiating event, specifically drives strong interferon signaling activation, offering novel insights into ccRCC tumorigenesis and immune modulation. Overall, we establish CRISPRt as a simple, efficient and scalable approach for chromosome-specific aneuploidy engineering and characterization in preclinical models to advance aneuploidy research across diverse biological contexts.

genomics↗

bioRxiv2025

Dysregulated Proline Metabolism Contributes to Subretinal Fibrosis in Neovascular AMD: Therapeutic Potential of Prolyl-4-Hydroxylase Inhibition

Subretinal fibrosis, a major cause of irreversible vision loss in neovascular age-related macular degeneration (nAMD), is driven by excessive deposition of extracellular matrix such as collagens. While proline metabolism is known to play a critical role in collagen biosynthesis and fibrosis, its involvement in subretinal fibrosis remains unclear. Here, we characterized the progression of fibrovascular lesions in JR5558 mice, observing significant molecular alterations as early as 4 weeks of age and phenotypic changes by 8 weeks. Transcriptomic and metabolomic analyses revealed elevated levels of 4-hydroxyproline, an essential component of collagen, alongside significant alterations of other fibrosis-related pathways. P4HA1, a catalytic subunit of prolyl-4-hydroxylase essential for 4-hydroxyproline biosynthesis, was prominently expressed in fibrotic lesions in retinas of JR5558 and two-stage laser-induced murine models, as well as human eyes with nAMD. Targeting P4HA1 with the small-molecule inhibitor diethyl pythiDC significantly attenuated fibrovascular lesion growth in the JR5558 murine models and reduced collagen turnover in human retinal pigment epithelium cells. Combining diethyl pythiDC with aflibercept had a stronger antifibrotic effect than monotherapies in JR5558 mice. These findings suggest a key contribution of proline metabolism, particularly proline hydroxylation, in subretinal fibrosis. Inhibiting P4HA1 with diethyl pythiDC inhibited fibrosis in the models we studied, offering a novel therapeutic strategy. Further research is warranted to explore the potential benefits of combining existing anti-angiogenic therapies with drugs that inhibit proline metabolism for the management of nAMD-associated fibrosis.

neuroscience↗

bioRxiv2025

High-accuracy protein complex structure modeling based on sequence-derived structure complementarity

In living organisms, proteins perform key functions required for life activities by interacting to form complexes. Determining the protein complex structure is crucial for understanding and mastering biological functions. Although AlphaFold2 has made a revolutionary breakthrough in predicting protein monomeric structures, accurately capturing inter-chain interaction signals and modeling the structures of protein complexes remain a formidable challenge. In this work, we report DeepSCFold, a pipeline for improving protein complex structure modeling. DeepSCFold uses sequence-based deep learning models to predict protein-protein structural similarity and interaction probability, providing a foundation for identifying interaction partners and constructing deep paired multiple-sequence alignments (MSAs) for protein complex structure prediction. Benchmark results show that DeepSCFold significantly increases the accuracy of protein complex structure prediction compared with state-of-the-art methods. For multimer targets from CASP15, DeepSCFold achieves an improvement of 11.6% and 10.3% in TM-score compared to AlphaFold-Multimer and AlphaFold3, respectively. Furthermore, when applied to antibody-antigen complexes from the SAbDab database, DeepSCFold enhances the prediction success rate for antibody-antigen binding interfaces by 24.7% and 12.4% over AlphaFold-Multimer and AlphaFold3, respectively. These results demonstrate that DeepSCFold effectively captures intrinsic and conserved protein-protein interaction patterns through sequence-derived structure-aware information, rather than relying solely on sequence-level co-evolutionary signals.

bioinformatics↗

bioRxiv2025

Homoharringtonine Promotes FTO Degradation to Suppress LILRB4-Mediated Immune Evasion in Acute Monocytic Leukemia

Acute monocytic leukemia (AML-M5), a subtype of acute myeloid leukemia, is a highly aggressive malignancy characterized by a poor prognosis, primarily due to the ability of leukemic cells to evade immune surveillance. In this study, we demonstrate that homoharringtonine (HHT), an FDA-approved therapeutic agent for chronic myeloid leukemia (CML), inhibits this immune evasion by targeting the FTO/m6A/LILRB4 signaling pathway in monocytic AML. Utilizing RNA sequencing (RNA-seq) and various functional assays, we reveal that HHT treatment significantly reduces LILRB4 expression at both the RNA and protein levels, suggesting that the effects of HHT on LILRB4 are distinct from its well-established role as a protein synthesis inhibitor. Mechanistically, HHT treatment markedly increases global levels of RNA m6A in THP-1 cells by promoting the degradation of FTO, which subsequently diminishes the expression of its downstream targets, MLL1 and LILRB4. Furthermore, in vitro and in vivo analyses employing monocytic AML cell lines, mouse-derived AML xenograft models, and patient samples collectively support the conclusion that HHT suppresses immune evasion in monocytic AML by reducing LILRB4 expression. Importantly, the downregulation of LILRB4 resulting from HHT treatment enhances the susceptibility of THP-1 cells to CD8+ T cell cytotoxicity, accompanied by increased markers of immune activation. Overall, our findings position HHT as a promising clinical agent for enhancing CD8+ T cell-based cancer immunotherapy by mitigating immune evasion in monocytic AML. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/643466v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@e72e5dorg.highwire.dtl.DTLVardef@19519aeorg.highwire.dtl.DTLVardef@1c8f786org.highwire.dtl.DTLVardef@f2a963_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Deciphering single-cell heterogeneity and cellular ecosystem dynamics during prostate cancer progression

Prostate cancer (PC) progresses from benign epithelium through pre-malignant lesions, localized tumors, metastatic dissemination, and castration-resistant stages, with some cases exhibiting phenotype plasticity under therapeutic pressure. However, high-resolution insights into how cell phenotypes evolve across successive stages of PC remain limited. Here, we present the Prostate Cancer Cell Atlas (PCCAT) by integrating [~]710,000 single cells from 197 human samples covering a spectrum of tumor stages. This comprehensive analysis dissects the cellular landscape and characterizes key cell types and molecular features that associate with PC progression and prognosis. In malignant cells, we highlight a distinctive profile denoted by high Major Histocompatibility Complex (MHC) expression, low Androgen Receptor (AR) activity, and enhanced stemness programs associated with enzalutamide resistance. Moreover, we reveal several cell states strongly correlated with PC progression and adverse prognosis, including lineage plasticity-like malignant cells (LPCs), neuroendocrine tumor cells, pericytes, and matrix cancer-associated fibroblasts (mCAFs). Furthermore, we uncover shared cell states that underpin the immune suppressive tumor microenvironment in advanced PC, including activated regulatory T cells, exhausted CD8+ T cells, and SPP1-expressing macrophages. Lastly, we pinpoint a spatial niche composed of mCAFs and SPP1-expressing macrophages localized near the tumor boundary in aggressive PC, which correlates with poor prognosis. Overall, our work provides a valuable resource and offers deeper insights into the diverse cell states, dynamics, and functional characteristics involved in PC progression at single-cell resolution.

cancer biology↗

bioRxiv2024

Regulation of Fungal Morphology, Conidiogenesis and Violet Pigment Synthesis by the Betalain Biosynthesis Pathway Genes in Aspergillus sydowii H-1

The biosynthesis of antioxidant pigments, namely, betalains, has predominantly been found in Caryophyllales. The potential betalains biosynthesis was firstly explored in Aspergillus sydowii H-1 under controlled culture conditions. This study identified, knocked out, and overexpressed genes involved in the betanin biosynthesis and assessed the activities of tyrosinase, 45-DOPA dioxygenase and LigB. The results indicated these betanin biosynthesis pathway was crucial for colony morphology, conidiogenesis, stress response, and violet pigment synthesis. Moreover, AsDODA and AsLigB catalyzed the conversion of L-DOPA into 45-seco-DOPA, a key intermediate in the formation of betalamic acid in vitro. Additionally, transcription factors such as AsbHLH, AsMYB1R, and AsWD40 positively regulated the expression of betalain biosynthesis genes. This research provides new insights into the evolutionary origins of betalain-producing species, expanding the scope of betalain biosynthesis to include Aspergillus species. ImportanceTo date, betalains were restricted to plants of the order Caryophyllales, fungi of Basidiomycota and several types of bacteria. This study is the first to demonstrate the potential of Ascomycetes A. sydowii H-1 to synthesize betalains under controlled culture conditions, providing a detailed genetic and biological characterization of the associated genes and metabolic pathways. This finding demonstrates that betalain biosynthesis can be expanded to other Aspergillus.

microbiology↗

bioRxiv2024

Oligomerization of PRMT1 Regulated RNA-Binding Protein Cascade Promotes Pancreatic Ductal Adenocarcinoma

PRMT1 is the predominant type I protein arginine methyltransferase responsible for generating monomethylarginine and asymmetric dimethylarginine (MMA and ADMA) in various protein substrates. It regulates numerous biological processes, including RNA metabolism, mRNA splicing, DNA damage repair, and chromatin dynamics. The crystal structure of rat PRMT1 has been previously reported as a homodimer; however, higher-order oligomeric species of human PRMT1 have been observed in vivo, and their structural basis remains elusive. In this study, we present the cryo-EM structure of human PRMT1 in its oligomeric form, revealing novel interfaces crucial for PRMT1 self-assembly and normal function. PRMT1 shows a strong preference for intrinsically disordered RGG/RG motifs, which are commonly found in RNA-binding proteins (RBPs), highlighting its critical role in regulating RNA metabolism. In vitro studies indicate that disrupting PRMT1 oligomerization impairs its binding affinity for RGG motif substrates, thereby reducing arginine methylation levels on these substrates. This finding emphasizes the essential role of the oligomeric state of PRMT1 in its function with RGG motif-containing RBPs. In vivo, the loss of PRMT1 oligomerization leads to decreased global ADMA levels in pancreatic ductal adenocarcinoma (PDAC) cells and inhibits PDAC tumor growth. Collectively, we propose that PRMT1 oligomerization, rather than mere dimerization, is sufficient for PRMT1-driven PDAC tumor growth, offering novel insights into the potential therapeutic targeting of PRMT1 oligomeric forms in PDAC. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/627716v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1ed6232org.highwire.dtl.DTLVardef@1c9e688org.highwire.dtl.DTLVardef@3e2c43org.highwire.dtl.DTLVardef@1cbf33e_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Aramchol attenuates fibrosis in mouse models of biliary fibrosis and blocks the TGFβ-induced fibroinflammatory mediators in cholangiocytes

BackgroundFibroinflammatory cholangiopathies, such as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC), are characterized by inflammation and biliary fibrosis, driving disease-related complications. In biliary fibrosis, cholangiocytes activated by transforming growth factor-{beta} (TGF{beta}) release signals that recruit immune cells to drive inflammation and activate hepatic myofibroblasts to deposit the extracellular matrix (ECM). TGF{beta} regulates stearoyl-CoA desaturase (SCD), an enzyme that catalyzes the synthesis of monounsaturated fatty acids, in stimulating fibroinflammatory lipid signaling. However, the role of SCD or its inhibitor, Aramchol, has not been investigated in biliary fibrosis or TGF{beta}-mediated cholangiocyte activation. Method10-16-week-old multi-drug resistance 2 knockout (Mdr2-/-) and 3,5-diethoxycarboncyl-1,4-dihydrocollidine (DDC) diet-fed mice were orally gavaged daily with Aramchol at 12.5 mg/kg/day for 4 and 3 weeks, respectively. Liver and serum were harvested for the assessment of fibrosis and inflammation. Transformed human cholangiocyte cells (H69) and mouse large biliary epithelial cells (MLEs) were used to test the effects of the SCD inhibitor, Aramchol, at varying doses on TGF{beta}-mediated expression of fibroinflammatory signals and were confirmed in PSC-derived cholangiocytes (PSC-Cs) using ELISA, qPCR, and Western blot analyses. ResultsAramchol treatment of Mdr2-/- mice with established biliary fibrosis (treatment) and DDC diet-induced (prevention) models of cholestatic injury and fibrosis demonstrated significant reductions in both measures of ECM synthesis (mRNA expression of ECM components in the liver), collagen content of the liver (picrosirius red staining and hydroxyproline content) and myofibroblast activation (SMA staining). Il6 and Tnfa were also reduced with Aramchol in the liver. RNA-seq analysis of H69 cells showed that Aramchol co-treatment led to significant inhibition of TGF{beta}-induced hepatic fibrosis pathways while upregulating peroxisome proliferator-activated receptor (PPAR) signaling. SCD expression was significantly increased in TGF{beta}-treated H69 cells (2-fold, p<0.05). Aramchol in a dose-dependent manner significantly attenuated the increased expression of the fibrotic marker, plasminogen activator inhibitor-1 (PAI-1/SERPINE1), and hepatic stellate cell-activating genes (VEGFA and PDGFB) in TGF{beta}-activated H69 and MLEs. Aramchol also markedly reduced the expression of the inflammatory cytokine, interleukin 6 (IL6). SCD siRNA knockdown produced similar results in H69 cells. Furthermore, in PSC-Cs, the expressions of SCD, VEGFA and IL6 were significantly reduced with Aramchol. The expression of the anti-fibroinflammatory factors PPAR and -{gamma} were modestly increased in cholangiocyte cell lines with increased expression of PPAR-responsive genes and increased nuclear binding of DNA PPAR response elements with Aramchol co-treatment compared to TGF{beta} only. ConclusionAramchol, an SCD inhibitor, both attenuates and prevents biliary fibrosis in mouse models of cholestatic injury and fibrosis. This effect is partially due to Aramchol inhibiting TGF{beta}-induced fibroinflammatory mediators in cholangiocytes by upregulating PPAR and -{gamma} expression and activity. These findings, along with Aramchols excellent safety profile in clinical trials, provide the rationale for assessing Aramchol in further clinical studies in patients with biliary fibrosis, particularly PSC, where a treatment is desperately needed.

cell biology↗

bioRxiv2024

The mechanism of checkpoint-dependent DNA replication fork stabilization in human cells

The DNA damage checkpoint is crucial for maintaining genome stability after genotoxic stress; without it, excess DNA replication origins are activated, stalled DNA replication forks cannot restart normally, high levels of DNA damage and single-stranded DNA (ssDNA) accumulate and cells cannot complete S phase. Preventing excess origin firing suppresses all these effects. Here we show that when replication is not restrained by a functional checkpoint, excess DNA synthesis sequesters the processivity factor PCNA and its loader RFC, preventing normal fork restart. Nascent DNA ends unprotected by RFC/PCNA are attacked by the Helicase-Like Transcription Factor (HLTF), causing irreversible replication fork collapse and hyperaccumulation of single-stranded DNA. This explains how the checkpoint stabilizes stalled replication forks and has implications for how origin firing is normally coordinated with fork progression. Loss of HLTF suppresses fork collapse and cell lethality in checkpoint-deficient cells, which has implications for how resistance to anti-checkpoint therapies may arise.

cell biology↗

bioRxiv2024

Epigenetic constraint of cellular genomes evolutionarily links genetic variation to function

Cellular diversity is a product of evolution acting to drive divergent regulatory programs from a common genome. Here, we use cross-cell-type epigenetic conservation to gain insight into the impact of selective constraints on genome function and phenotypic variation. By comparing chromatin accessibility across hundreds of diverse cell-types, we identify 1.4% of the human genome safeguarded by conserved domains of facultative heterochromatin, which we term regions under "cellular constraint". We calculate single-base resolution cellular constraint scores and demonstrate robust prediction of functionally important coding and non-coding loci in a cell-type-, trait-, and disease-agnostic manner. Cellular constraint annotation enhances causal variant identification, drug discovery, and clinical diagnostic predictions. Furthermore, cell-constrained sequences share paradoxical evolutionary signals of positive and negative selection, suggesting a dynamic role in driving human adaptation. Overall, this study demonstrates that evolutionary chromatin dynamics can be leveraged to inform the translation of genetic discoveries into effective biological, therapeutic, and clinical outcomes.

genetics↗

bioRxiv2024

Reduction in ETFDH expression optimizes cancer cell bioenergetics

Mitochondrial electron transport flavoprotein (ETF) insufficiency causes metabolic diseases known as a multiple acyl-CoA dehydrogenase deficiency (MADD). In contrast to muscle, ETFDH is a non-essential gene in acute lymphoblastic leukemia NALM-6 cells, and its expression is reduced across human cancers. ETF insufficiency caused by decreased ETFDH expression limits flexibility of OXPHOS fuel utilization but paradoxically increases cancer cell bioenergetics and accelerates neoplastic growth by activation of the mTORC1/BCL-6/4E-BP1 axis. Collectively, these findings reveal that while ETF insufficiency is rare and has detrimental effects in non-malignant tissues, it is common in neoplasia, where ETFDH downregulation leads to bioenergetic and signaling reprogramming that accelerate neoplastic growth.

cancer biology↗

bioRxiv2024

AutoMorFi: Automated Whole-image Morphometry in Fiji/ImageJ for Diverse Image Analysis Needs

Running on the highly popular and accessible ImageJ/Fiji platform for biological image analysis, we have established AutoMorFi as a streamlined interface for automated whole-image morphometric analysis that generates at least 47 measurements per cell or object in under 1 minute. We performed multiple validated cluster and principal component analyses on nonredundant morphometric parameters derived from AutoMorFi for various cell types, objects, and organisms. We used images of rodent macrophages, human postmortem brain tissues from multiple sclerosis (MS) and Alzheimers disease (AD) patients, iPSC/animal models for Downs syndrome and autism spectrum disorder (ASD), and organisms such as sea anemone and corals. AutoMorFis adaptability extends across diverse imaging modalities including brightfield, confocal, or widefield fluorescence microscopy as well as underwater photography. Due to its unlimited and unbiased sampling across any image and high potential for modification and customization, using AutoMorFi has led to the discovery of new distinguishing features in previously studied cell types and organisms as well as the development of rapid diagnostic approaches. AutoMorFi represents a transformative tool that will accelerate morphometric analysis and offer broad relevance in biological studies.

cell biology↗

bioRxiv2024

Scalable assessment of genome editing off-targets associated with genetic variants

Genome editing with RNA-guided DNA binding factors carries risk of off-target editing at homologous sequences. Genetic variants may introduce sequence changes that increase homology to a genome editing target, thereby increasing risk of off-target editing. Conventional methods to verify candidate off-targets rely on access to cells with genomic DNA carrying these sequences. However, for candidate off-targets associated with genetic variants, appropriate cells for experimental verification may not be available. Here we develop a method, Assessment By Stand-in Off-target LentiViral Ensemble with sequencing (ABSOLVE-seq), to integrate a set of candidate off-target sequences along with unique molecular identifiers (UMIs) in genomes of primary cells followed by clinically relevant gene editor delivery. Gene editing of dozens of candidate off-target sequences may be evaluated in a single experiment with high sensitivity, precision, and power. We provide an open-source pipeline to analyze sequencing data. This approach enables experimental assessment of the influence of human genetic diversity on specificity evaluation during gene editing therapy development.

genomics↗