Biology subjects

Zhao, F.

Publications and source records attributed to Zhao, F..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

Mechanosensitive interactions between Jag1 and Myo1c control Jag1 trafficking in endothelial cells

Morphogenesis of the cardiovascular system is responsive to hemodynamic cues. In endothelial cells the organization of morphogenic signaling proteins can be regulated by membrane presentation and internalization of proteins. How these signaling proteins are regulated by hemodynamics is unclear. One of the signaling proteins that is regulated by hemodynamics is Jagged1, a ligand in the Notch pathway. Here we set out to identify factors that differentially interact with Jagged1 in response to shear stress exposure, by combining an orbital shaker as a shear stress platform with endothelial cells expressing Jagged1 coupled to an APEX2-tag for proximity labeling. Myo1c was identified and confirmed through coimmunoprecipitation as a Jag1 interacting factor under static conditions, with reduced interaction after exposure to shear in endothelial cells. We showed that Jagged1 polarized downstream of shear followed by nucleograde transport of Jagged1. Myo1c knockout inhibited shear-induced Jagged1 polarization and consequent nucleograde transport. Further, Myo1c knockdown reduced membrane levels of Jagged1 under static conditions, but not under shear conditions. Together, our data reveal a role for Myo1c in the hemodynamic control of Jagged1 localization in endothelial cells.

cell biology↗

bioRxiv2025

Contrasting responses of control and fibrotic lung fibroblasts to fibrotic stimuli: the role of osteoprotegerin in extracellular matrix remodeling

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease characterized by dysregulated wound healing, leading to excessive production of extracellular matrix (ECM) proteins, particularly collagen, by activated fibroblasts. Osteoprotegerin (OPG), a soluble protein associated with ECM remodeling in bone, has been found in higher levels in serum and lung tissue of IPF patients compared to controls. However, whether OPG is associated with ECM remodeling by fibroblasts in lung fibrosis remains poorly understood. In this study we assessed differences in OPG levels in ECM-derived hydrogels from non-disease controls and patients with IPF encapsulated with primary human lung fibroblasts from control and patients with IPF (7 days). OPG levels were assessed in a collected culture medium and stained lung tissue sections, and related to biomechanical properties. Fibrotic human lung fibroblasts secreted and deposited more OPG compared to controls, which was observed in both non-diseased and IPF ECM hydrogels. OPG levels correlated significantly with matrix stiffness and collagen fiber structural properties, but only in IPF hydrogels, independent of fibroblast source. Strikingly, we also observed opposite correlations for control and fibrotic fibroblasts in fibrotic hydrogels. Control fibroblasts within a fibrotic microenvironment, secreted less OPG when hydrogel stiffness increased and deposited more OPG when collagen fibers were more aligned. Interestingly, fibrotic fibroblasts secreted more OPG when collagen fiber length increased and when collagen fiber curvature increased. Overall, our findings suggest that control and fibrotic lung fibroblasts exhibited opposite behavior with respect to OPG and ECM remodeling under fibrotic conditions and this may have consequences for matrix stiffness

cell biology↗

bioRxiv2025

Super-omics and PCAS reveal rheumatoid arthritis as a tumor-like disease

Rheumatoid arthritis (RA) is a debilitating systemic autoimmune disorder that significantly impairs quality of life. To elucidate the molecular alterations in RA progression, we performed comprehensive super-omics analyses on synovial tissues from patients with joint trauma, arthritis, and RA. These analyses included transcriptomics, proteomics, metabolomics, microbiomics, and fourteen PTMs (phosphorylation, acetylation, lactylation, O-GlcNAc glycosylation, arginine monomethylation, lysine monomethylation, lysine dimethylation, lysine trimethylation, succinylation, malonylation, glutarylation, tyrosine nitration, N-glycosylation, and O-glycosylation). Additionally, we developed a protein-centered association study (PCAS) method to integrate these complex datasets. Using this approach, we identified key proteins, such as STK17B, and its interactors, which may be crucial in RA pathogenesis. Tumor-like features, including aberrant angiogenesis and epithelial-mesenchymal transition, were observed in RA, alongside the potential involvement of oncogenes and tumor suppressor genes. Finally, we constructed a molecular interaction blueprint of RA, providing a comprehensive framework for advancing RA pathogenesis, diagnosis, and therapy.

immunology↗

bioRxiv2024

Identification of QTL-by-Environment Interaction by Controlling Polygenic Background Effect

The QTL by environment interaction (QxE) effect is hard to detect because there are no effective ways to control the genomic background. In this study, we propose a novel linear mixed model that simultaneously analyzes data from multiple environments to detect QxE interactions. This model incorporates two different kinship matrices derived from the genome-wide markers to control both main and interaction polygenic background effects. Simulation studies demonstrated that our approach was more powerful than the meta-analysis and inclusive composite interval mapping methods. We further analyzed four agronomic traits of rice across four environments. A main effect QTL was identified for 1000-grain weight (KGW), while no QTLs were found for tiller number. Additionally, a large QTL with a significant QxE interaction was detected on chromosome 7 affecting grain number, yield and KGW. This region harbored two important genes, PROG1 and Ghd7. Furthermore, we applied our mixed model to analyze lodging in barley across six environments. The six regions exhibiting QxE interaction effects identified by our approach overlapped with the SNPs previously identified using EM and MCMC-based Bayesian methods, further validating the robustness of our approach. Both simulation studies and empirical data analyses showed that our method outperformed all other methods compared.

genetics↗

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

Jagged1 is a Notch-independent mechanotransducer in endothelial cells

The Notch signaling pathway plays a crucial role in regulating endothelial biology. Notch signaling is sensitive to hemodynamic forces and governs mechanically-driven cardiovascular development, physiology, and remodeling. However, the mechanisms by which mechanical forces integrate with the Notch pathway remain largely unknown. Here, we uncover a non-canonical role for the Notch ligand Jagged1 in regulating the activity of mechanosensitive kinases in endothelial cells. We show that stress induces expression and relocalization of Jagged1 to cell junctions downstream of flow. Jagged1 expression under stress demonstrates magnitude dependence and peaks at 0.8-1Pa without impacting Jagged1s Notch-activation potential. On the contrary Jagged1 regulates the activity of mechanosensitive kinases. Deletion of Jagged1 reduces the activity of VEGFR2 and ERK in vitro and diminished ERK activity in zebrafish embryos without affecting canonical Notch signaling. Furthermore, the direct physical stimulation of Jagged1 using antibody-conjugated beads triggers the activation of VEGFR2 and ERK, mediated by Jagged1-induces Src activation. Taken together, we demonstrate a novel non-canonical role for Jagged1 as a regulator of the activity of pathways involved in endothelial mechanotransduction.

cell biology↗

bioRxiv2024

In vivo genome editing of human hematopoietic stem cells for treatment of blood disorders by mRNA delivery

Ex vivo autologous hematopoietic stem cells (HSCs) gene therapy provides promising new treatments for hematological disorders. However, current methods involve complex processes and chemotherapeutic conditioning, leading to limited accessibility for treatment and significant side effects. Here, we developed an antibody-free targeted lipid nanoparticles (LNPs) for mRNA delivery to HSCs in vivo, enabling efficient base editing of the HBG target in human HSCs to reactivate fetal hemoglobin in derived erythroid cells. Delivery of ABE8e/sgRNA mRNA with optimized structure LNPs achieves efficient in vivo base editing of HBG in transfusion-dependent {beta}-thalassemia (TDT) patients derived HSCs engrafted in immunodeficient NCG-X mice, showing restored globin chain balance in erythroid cells. Our research indicated that utilizing LNPs for delivery of genome editor achieves efficient editing of endogenous genes of human HSCs. Notably, this non-viral delivery system eliminates the need for harvesting or mobilizing HSCs, providing a potent and one-time treatment potential for blood disorders like sickle cell disease (SCD) and TDT. HighlightsO_LIAntibody-free targeted LNPs system achieves efficient mRNA delivery and base editing in human bone marrow cells including HSCs via one-time intravenous injection. C_LIO_LIDelivery of ABE8e/sgRNA mRNA with optimized LNPs achieves efficient in vivo base editing of HBG in TDT patients derived HSCs engrafted in immunodeficient NCG-X mice, showing restored globin chain balance in erythroid cells. C_LIO_LILNPs efficiently deliver gene editing system to the BM for in vivo editing of human HSCs, providing preclinical evidence for the next generation non-viral gene therapy for blood disorders. C_LI

bioengineering↗

bioRxiv2024

Mechanical control of germ cell specification in Arabidopsis anthers

A central question in developmental biology is how the germline is established. We have studied the specification of the male germ cells (GCs) within the anther1-3. Hereby, we have focused on the potential role of mechanics, an aspect of anther development which has been very poorly characterized2,4. Using a combination of live imaging and mechanical measurements, we provide evidence that GCs originate in a special micro-mechanical niche, where inner tissues exert push on outer cell layers, placing them under compression. Mechanical perturbations significantly disrupted the GC specification and patterning. Moreover, we found that the master genetic regulator SPOROCYTELESS/NOZZLE (SPL/NZZ)5,6 is central in establishing this micro-mechanical environment by softening the cell wall. The mechanical cues, in turn, stabilize the transcription of SPL/NZZ. We propose here an intrinsic growth-derived mechano-chemical feedback loop that drives germ-cell fate acquisition.

developmental biology↗

bioRxiv2024

SeuratExtend: Streamlining Single-Cell RNA-Seq Analysis Through an Integrated and Intuitive Framework

Single-cell RNA sequencing (scRNA-seq) has revolutionized the study of cellular heterogeneity, but the rapid expansion of analytical tools has proven to be both a blessing and a curse, presenting researchers with significant challenges. Here, we present SeuratExtend, a comprehensive R package built upon the widely adopted Seurat framework, which streamlines scRNA-seq data analysis by integrating essential tools and databases. SeuratExtend offers a user-friendly and intuitive interface for performing a wide range of analyses, including functional enrichment, trajectory inference, gene regulatory network reconstruction, and denoising. The package seamlessly integrates multiple databases, such as Gene Ontology and Reactome, and incorporates popular Python tools like scVelo, Palantir, and SCENIC through a unified R interface. SeuratExtend enhances data visualization with optimized plotting functions and carefully curated color schemes, ensuring both aesthetic appeal and scientific rigor. We demonstrate SeuratExtends performance through case studies investigating tumor-associated high-endothelial venules and autoinflammatory diseases, and showcase its novel applications in pathway-Level analysis and cluster annotation. SeuratExtend empowers researchers to harness the full potential of scRNA-seq data, making complex analyses accessible to a wider audience. The package, along with comprehensive documentation and tutorials, is freely available at GitHub, providing a valuable resource for the single-cell genomics community. Practitioner PointsO_LISeuratExtend streamlines scRNA-seq workflows by integrating R and Python tools, multiple databases (e.g., GO, Reactome), and comprehensive functional analysis capabilities within the Seurat framework, enabling efficient, multi-faceted analysis in a single environment. C_LIO_LIAdvanced visualization features, including optimized plotting functions and professional color schemes, enhance the clarity and impact of scRNA-seq data presentation. C_LIO_LIA novel clustering approach using pathway enrichment score-cell matrices offers new insights into cellular heterogeneity and functional characteristics, complementing traditional gene expression-based analyses. C_LI

bioinformatics↗

bioRxiv2024

Rtf1 HMD domain facilitates global histone H2B monoubiquitination and regulates morphogenesis and virulence in the meningitis-causing pathogen Cryptococcus neoformans

Rtf1 is generally considered to be a subunit of the Paf1 complex (Paf1C), which is a multifunctional protein complex involved in histone modification and RNA biosynthesis at multiple stages. Rtf1 is stably associated with the Paf1C in Saccharomyces cerevisiae, but not in other species including humans. Little is known about its function in human fungal pathogens. Here, we show that Rtf1 is required for facilitating H2B monoubiquitination (H2Bub1), and regulates fungal morphogenesis and pathogenicity in the meningitis-causing fungal pathogen Cryptococcus neoformans. Rtf1 is not tightly associated with the Paf1C, and its histone modification domain (HMD) is sufficient to promote H2Bub1 and the expression of genes related to fungal mating and filamentation. Moreover, Rtf1 HMD fully restores fungal morphogenesis and pathogenicity; however, it fails to restore defects of thermal tolerance and melanin production in the rtf1{Delta} strain background. The present study establishes a role for cryptococcal Rtf1 as a Paf1C-independent regulator in regulating fungal morphogenesis and pathogenicity, and highlights the function of HMD in facilitating global H2Bub1 in C. neoformans.

molecular biology↗

bioRxiv2024

Androgen receptor activity inversely correlates with immune cell infiltration and immunotherapy response across multiple cancer lineages

There is now increasing recognition of the important role of androgen receptor (AR) in modulating immune function. To gain a comprehensive understanding of the effects of AR activity on cancer immunity, we employed a computational approach to profile AR activity in 33 human tumor types using RNA-Seq datasets from The Cancer Genome Atlas. Our pan-cancer analysis revealed that the genes most negatively correlated with AR activity across cancers are involved in active immune system processes. Importantly, we observed a significant negative correlation between AR activity and IFN{gamma} pathway activity at the pan-cancer level. Indeed, using a matched biopsy dataset from subjects with prostate cancer before and after AR-targeted treatment, we verified that inhibiting AR enriches immune cell abundances and is associated with higher IFN{gamma} pathway activity. Furthermore, by analyzing immunotherapy datasets in multiple cancers, our results demonstrate that low AR activity was significantly associated with a favorable response to immunotherapy. Together, our data provide a comprehensive assessment of the relationship between AR signaling and tumor immunity.

cancer biology↗

bioRxiv2024

The dual resistance mechanism of CYP325G4 and CYP6AA9 in Culex pipiens pallens legs according to transcriptome and proteome analysis

Mosquitoes within the Culex pipiens complex play a crucial role in human disease transmission. Insecticides, especially pyrethroids, are used to control these vectors. Mosquito legs are the main entry point and barrier for insecticides to gain their neuronal targets. However, the resistance mechanism in mosquito legs is unclear. Herein, we employed transcriptomic analyses and isobaric tags for relative and absolute quantitation techniques to investigate the resistance mechanism, focusing on Cx. pipiens legs. We discovered 2346 differentially expressed genes (DEGs) between deltamethrin-resistant (DR) and deltamethrin-sensitive (DS) mosquito legs, including 41 cytochrome P450 genes. In the same comparison, we identified 228 differentially expressed proteins (DEPs), including six cytochrome P450 proteins). Combined transcriptome and proteome analysis revealed only two upregulated P450 genes, CYP325G4 and CYP6AA9. The main clusters of DEGs and DEPs were associated with metabolic processes, such as cytochrome P450-mediated metabolism of drugs and xenobiotics. Transcription analysis revealed high CYP325G4 and CYP6AA9 expression in the DR strain at 72 hours post-eclosion compared with that in the DS strain, particularly in the legs. Mosquitoes knocked down for CYP325G4 were more sensitive to deltamethrin than the controls. CYP325G4 knockdown reduced the expression of several chlorinated hydrocarbon (CHC)-related genes, which altered the cuticle thickness and structure. Conversely, CYP6AA9 knockdown increased CHC gene expression without altering cuticle thickness and structure. P450 activity analysis demonstrated that CYP325G4 and CYP6AA9 contributed to metabolic resistance in the midgut and legs. This study identified CYP325G4 as a novel mosquito deltamethrin resistance factor, being involved in both metabolic and cuticular resistance mechanisms. The previously identified CYP6AA9 was investigated for its involvement in metabolic resistance and potential cuticular resistance in mosquito legs. These findings enhance our comprehension of resistance mechanisms, identifying P450s as promising targets for the future management of mosquito vector resistance, and laying a theoretical groundwork for mosquito resistance management. Author SummaryCulex pipiens mosquitoes are the primary vector of the filamentous nematode, Wuchereria bancrofti and also involved in the transmission of other pathogens, such as West Nile virus (WNV), avian malarias, and avian pox virus. Insecticides, particularly pyrethroids, continue to be the primary method to control these significant vectors. Worryingly, resistance to insecticides has become widespread and is rapidly intensifying in Culex mosquitoes throughout China, posing a threat to the efficacy of insecticides. Legs are the main sites of contact with ITNs and sprayed insecticides, and the insecticides have to penetrate the leg cuticle to reach their targets.Therefore, the resistance mechanisms in mosquito legs deserve further investigation. Several reports have found a certain amount of P450 in insect legs. Unfortunately, none of the above reports have conducted further functional studies on P450s in the legs. Here, we have identified two P450 enzymes, CYP325G4 and CYP6AA9, through the integrated analysis of transcriptomics and proteomics. CYP325G4 enriched in the cuticle of resistant mosquitoes might possess a dual resistance mechanism involving metabolic resistance and cuticle resistance. CYP6AA9 was slightly different, possibly exerting metabolic resistance as its main function and also being involved in cuticle synthesis. Understanding the dual resistance mechanism of P450s in the metabolism of pyrethroid insecticides will have an important role in optimizing vector control strategies.

physiology↗

bioRxiv2024

Comparative genomics of three non-hematophagous leeches (Whitmania spp.): focusing on antithrombotic genes

Leeches are well known for their blood-feeding habits and are widely used for medicinal purposes as they secrete various antithrombotic substances. However, some leeches such as Whitmania spp. exhibit non-hematophagous feeding habits and their significance for medicinal use is debated. In this study, we provide chromosome-level genomes of two non-hematophagous leeches Whitmania acranulata and Whitmania laevis, and combined with our previous results of Whitmania pigra, we systematically analyzed the similarities and differences on the genomes and especially their antithrombotic genes among the three non-hematophagous Whitmania leeches. For W. acranulata, W. laevis, and W. pigra, the genome size (181.72 Mb, 173.87 Mb, and 173.56), the percentage of repeat sites (29.55%, 28.28%, and 27.02%), and the number of protein-coding genes (27,068, 23,805, and 24,156) were close to each other, respectively. In contrast, both the total number of the antithrombotic genes (100, 63, and 79), and the detailed constitutes of different antithrombotic gene families were obviously different among the three leeches. There were also massive genetic variations among the members within each antithrombotic gene/protein family. RNA-Seq-based gene expression estimation showed that the expression profiles of the antithrombotic gene families were apparently different among the three leeches. This is the most comprehensive comparison of the genomes and antithrombic biomacromolecules for the Whitmania leeches to date. Our results will greatly facilitate the evolutionary research and application of leech derivatives for medical and pharmaceutical purposes of thrombosis.

genomics↗

bioRxiv2024

Flotillin-2 dampens T cell antigen-sensitivity and functionality

T cell receptor (TCR) engagement triggers T cell responses, yet how TCR-mediated activation is regulated at the plasma membrane remains unclear. Here, we report that deleting the membrane scaffolding protein Flotillin-2 (Flot2) increases T cell antigen sensitivity, resulting in enhanced TCR signaling and effector function to weak TCR stimulation. T cell-specific Flot2-deficient mice exhibited reduced tumor growth and enhanced immunity to infection. Flot2-null CD4+ T cells exhibited increased T helper 1 polarization, proliferation, Nur77 induction, and phosphorylation of ZAP70 and LCK upon weak TCR stimulation, indicating a sensitized TCR-triggering threshold. Single cell-RNA sequencing suggested that Flot2-null CD4+ T cells follow a similar route of activation as wild-type CD4+ T cells but exhibit higher occupancy of a discrete activation state under weak TCR stimulation. Given prior reports that TCR clustering influences sensitivity of T cells to stimuli, we evaluated TCR distribution with super-resolution microscopy. Flot2 ablation increased the number of surface TCR nanoclusters on naive CD4+ T cells. Collectively, we posit that Flot2 modulates T cell functionality to weak TCR stimulation, at least in part, by regulating surface TCR clustering. Our findings have implications for improving T cell reactivity in diseases with poor antigenicity, such as cancer and chronic infections.

immunology↗

bioRxiv2024

Extracellular electron transfer drives efficient H2-independent methylotrophic methanogenesis by Methanomassiliicoccus, a seventh order methanogen

Methylotrophic methanogenesis is achieved via methyl group dismutation or H2 reduction. This study reports extracellular electron droving efficient methylotrophic methanogenesis. The 7th order methanogen Methanomassiliicoccus luminyensis exclusively implements H2-dependent methylotrophic methanogenesis, but strain CZDD1 isolated from paddy soil possessed a higher methane-producing rate in coculture with Clostridium malenominatum CZB5 or the electrogenic Geobacter metallireducens. Chronoamperometry detected current production from CZB5, and current consumption accompanied CH4 production in a methanol-containing electrochemical culture of CZDD1. This demonstrated that M. luminyensis was capable of both direct species electron transfer (DIET) and extracellular electron transfer (EET) in methylotrophic methanogenesis. EET and DIET also enabled CZDD1 to produce methane from dimethyl arsenate. Differential transcriptomic analysis on H2-versus EET- and DIET-cocultures suggested that a membrane-bound Fpo-like complex and archaella of M. luminyensis CZDD1 could accept extracellular electrons. Given the ubiquitous environmental distribution of Methanomassiliicoccus strains, EET driven methylotrophic methanogenesis may contribute significantly to methane emission.

microbiology↗

bioRxiv2024

Chemoproteomics reveals immunogenic and tumor-associated cell surface substrates of ectokinase CK2α

New epitopes for immune recognition provide the basis of anticancer immunity. Due to the high concentration of extracellular adenosine triphosphate in the tumor microenvironment, we hypothesized that extracellular kinases (ectokinases) could have dysregulated activity and introduce aberrant phosphorylation sites on cell surface proteins. We engineered a cell-tethered version of the extracellular kinase CK2, demonstrated it was active on cells under tumor-relevant conditions, and profiled its substrate scope using a chemoproteomic workflow. We then demonstrated that mice developed polyreactive antisera in response to syngeneic tumor cells that had been subjected to surface hyperphosphorylation with CK2. Interestingly, these mice developed B cell and CD4+ T cell responses in response to these antigens but failed to develop a CD8+ T cell response. This work provides a workflow for probing the extracellular phosphoproteome and demonstrates that extracellular phosphoproteins are immunogenic even in a syngeneic system.

biochemistry↗

bioRxiv2024

Deep5hmC: Predicting genome-wide 5-Hydroxymethylcytosine landscape via a multimodal deep learning model

5-hydroxymethylcytosine (5hmC), a critical epigenetic mark with a significant role in regulating tissue-specific gene expression, is essential for understanding the dynamic functions of the human genome. Using tissue-specific 5hmC sequencing data, we introduce Deep5hmC, a multimodal deep learning framework that integrates both the DNA sequence and the histone modification information to predict genome-wide 5hmC modification. The multimodal design of Deep5hmC demonstrates remarkable improvement in predicting both qualitative and quantitative 5hmC modification compared to unimodal versions of Deep5hmC and state-of-the-art machine learning methods. This improvement is demonstrated through benchmarking on a comprehensive set of 5hmC sequencing data collected at four time points during forebrain organoid development and across 17 human tissues. Notably, Deep5hmC showcases its practical utility by accurately predicting gene expression and identifying differentially hydroxymethylated regions in a case-control study of Alzheimers disease.

bioinformatics↗

bioRxiv2024

scaDA: A Novel Statistical Method for Differential Analysis of Single-Cell Chromatin Accessibility Sequencing Data

Single-cell ATAC-seq sequencing data (scATAC-seq) has been widely used to investigate chromatin accessibility on the single-cell level. One important application of scATAC-seq data analysis is differential chromatin accessibility analysis. However, the data characteristics of scATAC-seq such as excessive zeros and large variability of chromatin accessibility across cells impose a unique challenge for DA analysis. Existing statistical methods focus on detecting the mean difference of the chromatin accessible regions while overlooking the distribution difference. Motivated by real data exploration that distribution difference exists among cell types, we introduce a novel composite statistical test named "scaDA", which is based on zero-inflated negative binomial model (ZINB), for performing differential distribution analysis of chromatin accessibility by jointly testing the abundance, prevalence and dispersion simultaneously. Benefiting from both dispersion shrinkage and iterative refinement of mean and prevalence parameter estimates, scaDA demonstrates its superiority to both ZINB-based likelihood ratio tests and published methods by achieving the highest power and best FDR control in a comprehensive simulation study. In addition to demonstrating the highest power in three real sc-multiome data analyses, scaDA successfully identifies differentially accessible regions in microglia from sc-multiome data for an Alzheimer s disease (AD) study, regions which are most enriched in GO terms related to neurogenesis, the clinical phenotype of AD, and SNPs identified in AD-associated GWAS. Author summaryUnderstanding the cis-regulatory elements that control the fundamental gene regulatory process is important to basic biology. scATAC-seq data offers an unprecedented opportunity to investigate chromatin accessibility on the single-cell level and explore cell heterogeneity to reveal the dynamic changes of cis-regulatory elements among different cell types. To understand the dynamic change of gene regulation using scATAC-seq data, differential chromatin (DA) analysis, which is one of the most fundamental analyses for scATAC-seq data, can enable the identification of differentially accessible regions between cell types or between multiple conditions. Subsequently, DA analysis has many applications such as identifying cell type-specific chromatin accessible regions to reveal the cell type-specific gene regulatory program, assessing disease-associated changes in chromatin accessibility to detect potential biomarkers, and linking differentially accessible regions to differentially expressed genes for building a comprehensive gene regulatory map. This paper proposes a novel statistical method named "scaDA" to improve the detection of differentially accessible regions by performing differential distribution analysis. scaDA is believed to benefit the research community of single-cell genomics.

bioinformatics↗