Biology subjects

Hagai, T.

Publications and source records attributed to Hagai, T..

16 recordsLinked to original sources

bioRxiv2026

KLHL9 acts as a KSHV vBcl-2-interacting host factor that supports lytic replication

Kaposis sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that causes Kaposis sarcoma and several lymphoproliferative disorders. The KSHV open reading frame (ORF) 16 encodes viral Bcl-2 (vBcl-2), which inhibits apoptosis and autophagy. vBcl-2 is required for efficient lytic replication and production of infectious progeny; however, this essential role is independent of its established functions regulating cell death. To identify host factors that may contribute to vBcl-2 function, we employed proteomic analysis of HA-vBcl-2 immunoprecipitates from lytically reactivated KSHV-infected cells. This analysis identified the BTB-Kelch protein KLHL9, a substrate-specific adaptor for Cullin 3-RING ligase (CRL3) complex, as a candidate vBcl-2-associated factor. We validated the vBcl-2-KLHL9 association by reciprocal co-immunoprecipitation from infected cells, and by ectopic expression in uninfected cells. During lytic reactivation, KLHL9 redistributed from a predominantly cytoplasmic/perinuclear pattern to a mitochondrial pattern that overlapped with HA-vBcl-2. Alanine-scanning mutagenesis mapped a KLHL9-interaction determinant to the N-terminal region of vBcl-2, and vBcl-2 mutants defective in binding failed to colocalize with KLHL9. Conversely, KLHL9 deletion analysis implicated the C-terminal Kelch-repeat region in efficient association with vBcl-2. AlphaFold modeling supported the interface regions. However, vBcl-2 did not show detectable KLHL9-dependent ubiquitination, suggesting that it is unlikely to be a substrate of a KLHL9-containing complex. Functionally, CRISPR/Cas9-mediated KLHL9 disruption reduced lytic viral protein accumulation and infectious progeny production, while re-expression of sgRNA-resistant KLHL9 partially restored these phenotypes. Together, these findings suggest that vBcl-2 may engage host CRL3 complexes during productive infection, and identify KLHL9 as a vBcl-2-associated host factor that supports efficient KSHV lytic replication. Author SummaryKaposis sarcoma-associated herpesvirus (KSHV) is a cancer-associated virus that alternates between a latent cycle and a productive lytic cycle, during which new infectious virus particles are made. The viral protein vBcl-2 is best known for regulating cell-death and autophagy pathways, but earlier studies showed that it is required for efficient virus production through additional, non-canonical functions. In this study, we identify the cellular protein KLHL9 as a host factor that associates with KSHV vBcl-2. KLHL9 normally functions as a specific adaptor in protein ubiquitination pathways that regulate the turnover, localization, or activity of target proteins through ubiquitination. We show that vBcl-2 and KLHL9 associate with one another, and that KLHL9 redistributes toward the mitochondria in cells expressing vBcl-2. Importantly, loss of KLHL9 impairs the expression of viral lytic proteins and reduces the production of new infectious viruses. This defect resembles the phenotype caused by loss of vBcl-2, suggesting that KSHV employs vBcl-2 to engage KLHL9-dependent host machinery during productive infection. Understanding this interaction may help reveal how herpesviruses reprogram infected cells to create an environment that supports virus production.

microbiology↗

bioRxiv2026

From bats to humans: uncovering ISG15 as a new resistance factor in type 1 diabetes

Viral infections are one of the main environmental factors triggering type 1 diabetes (T1D). Pancreatic alpha cells are more resistant than beta cells to diabetogenic viruses, partially explaining their survival in T1D. Similarly, bats have enhanced viral resistance, suggesting putative convergent evolution in antiviral mechanisms. Herein, we compared global gene expression in bat fibroblasts under basal conditions or exposed to double-stranded RNA to human alpha and beta cells and found that alpha cells exhibit greater similarity than beta cells to the antiviral responses of bat cells, as well as stronger intrinsic resistance to viral infection. Interferon-stimulated gene 15 (ISG15), a key regulator of antiviral responses in humans and bats, has higher expression in alpha compared to beta cells in five single-cell RNASeq datasets from human islet cells and in human induced pluripotent stem cell (hiPSC)-derived alpha-like cells. ISG15 knockdown in human insulin-producing EndoC-{beta}H1 cells and human islets increases apoptosis under basal conditions and after IFN exposure, exacerbates IFN responses and increases cell death and viral production after infection with the diabetogenic virus coxsackievirus B1, while its overexpression protects EndoC-{beta}H1 cells from the virus. Collectively, the present results demonstrate that alpha cells but not beta cells have similarities with the virus resistance gene program present in bats and identify ISG15 as an important factor for islet cells to cope with viral and diabetogenic stresses.

immunology↗

bioRxiv2025

Decoy Antibodies Block Extracellular HSP70, Prevent Self Signaling and Inhibit Melanoma Cell Survival

Melanoma cells actively secrete melanosomes-large, extracellular vesicles (EVs) enriched with oncogenic factors that reprogram the tumor microenvironment, enhance self-signaling, and promote tumor growth. Despite their abundance and immunogenic potential, humoral responses to melanoma-derived melanosomes remain unexplored. Here, we identify a novel immune surveillance mechanism in which melanosome-elicited decoy antibodies target melanoma-derived melanosomes by binding to the extracellular form of heat shock protein 70 (HSP70), a chaperone broadly implicated in cancer cell survival and stress adaptation. Anti-HSP70 decoy antibodies potently and effector-independently inhibit growth and survival of both murine and human melanoma cells and suppress key transcriptional programs involved in proliferation, cytoskeletal dynamics, and metabolism. In a preclinical B16 melanoma model, prophylactic administration of decoy monoclonal antibodies Mel322-34 and Mel321-35 conferred significant survival benefits of 27% and 48%, respectively. Strikingly, anti-HSP70 antibodies were enriched in the sera of melanoma patients achieving complete responses to immune checkpoint blockade, in contrast to non-responders with progressive disease. Collectively, our findings uncover a novel EV-antibody axis as a promising avenue to block cancer-promoting signaling pathways. Decoy autoantibodies targeting the extracellular form of HSP70 advance the understanding of tumor-intrinsic vulnerability and promote biomarker-driven immunotherapy in melanoma.

immunology↗

bioRxiv2024

Evolutionary divergence of induced versus constitutive antiviral gene expression between primates and rodents

BackgroundHundreds of genes are upregulated in response to viral infection. These genes sequences often diverge across mammals, to counteract rapid virus evolution. However, the transcriptional divergence of these genes, their relative levels before and after infection in different host species, remains poorly understood. ResultsWe studied this divergence by comparing gene expression before and after viral stimulation in cells from primates and rodents. We developed a method to identify orthologs upregulated in one species that are unchanged in response to stimulus in another species. Using human and mouse data, we detected 578 transcriptionally divergent orthologous genes. While most divergent genes do not belong to the same cellular process, several pathways and protein complexes are enriched in this set, suggesting that divergence in immune responses between closely related mammals is limited to specific modules rather than involving entire pathways. Transcriptional divergence between human and mouse orthologs was also observed when ortholog expression from different primates and rodents were compared, when responses were studied in several cell types, and was recapitulated at the chromatin level, using histone mark patterns that denote active promoter regions. ConclusionsIn summary, we found genes whose orthologs diverge between primates and rodents in response to immune stimulation. Some of these genes are constitutively expressed in one species even before infection, potentially facilitating rapid antiviral activity, and suggesting clade-specific adaptation to confer greater resistance against viruses. Further comparative studies on diverse infections can point to additional species-specific responses and how they enable different species to overcome infection.

bioinformatics↗

bioRxiv2023

Spatial and single-cell transcriptomics illuminate bat immunity and barrier tissue evolution

The Egyptian fruit bat displays tolerance to lethal viruses and unique dietary adaptations, but the molecular basis for this is poorly understood. To this end, we generated detailed maps of bat gut, lung and blood cells using spatial and single-cell transcriptomics. We compared bat with mouse and human cells to reveal divergence in genetic programs associated with environmental interactions and immune responses. Complement system genes are transcriptionally divergent, uniquely expressed in bat lung and gut epithelium, and undergo rapid coding-sequence evolution. Specifically in the tip of the gut villus, bat enterocytes express evolutionarily young genes while lacking expression of genes related to specific nutrient absorption. Profiling immune stimulation of PBMCs revealed a monocyte subset with conserved cross-species interferon expression, suggesting strong constraints to avoid an excessive immune response. Our study thus uncovers conserved and divergent immune pathways in bat tissues, providing a unique resource to study bat immunity and evolution.

genomics↗

bioRxiv2023

A comparative analysis of the antiviral response in two bat species reveals conserved and divergent innate immune pathways

Bats host a range of viruses that cause severe disease in humans without displaying clinical symptoms to these infections. The mechanisms of bat adaptation to these viruses are a continuous source of interest but remain largely unknown. To understand the landscape of bat antiviral response in a comprehensive and comparative manner, we studied this response in two bat species - the Egyptian fruit bat and the insectivore Kuhls pipistrelle, representing the two major bat subordinal clades. We profiled the transcriptional response to dsRNA - that triggers a rapid innate immune response - in skin fibroblasts from a large cohort of replicates from each bat species, using RNA-sequencing, and compared bat response with responses in primates and rodents. Both bat species upregulate a similar set of genes, many of which are known to be involved in the antiviral response across mammals. However, a subset of these genes is transcriptionally divergent in response between the two bat species. These transcriptionally divergent genes also evolve rapidly in coding sequence across the bat clade and have particular regulatory and functional characteristics, including specific promoter architectures and association with expression programs thought to underlie tolerance and resistance in response to viral infection. In addition, using single-cell transcriptomics, we show that transcriptionally divergent genes display high expression variability between individual cells. A focused analysis of dsRNA-sensing pathways further points to significant differences between bat and human in basal expression of genes important for triggering antiviral responses. Finally, a survey of genes recently lost or duplicated in bats points to a limited set of antiviral genes that have undergone rapid gene loss or gain in bats, with the latter group resulting in paralogs displaying divergence in both coding sequence and expression in bat tissues. Our study reveals a largely conserved regulatory program of genes upregulated in response to viral infection across bats and other mammals, and points to a set of genes that evolved rapidly in bats through multiple evolutionary mechanisms. This divergence can contribute to bat adaptation to viral infection and provides directions to understanding the mechanisms behind it.

evolutionary biology↗

bioRxiv2023

Lifelong tissue memory relies on spatially organised dedicated progenitors located distally from the injury

It is believed epithelial cells that have participated in a wound repair elicit a more efficient but locally restricted response to future injuries. However here we show that the cell adaptation resulting from a localised tissue damage has a wide spatial impact at a scale not previously noticed. We demonstrate that away from injured site, after a first injury a specific epithelial stem cell population gives rise to long term wound-memory progenitors residing in their own niche of origin. Notably these progenitors have not taken part in the first wound healing but become pre-activated through priming. This adaptation differs from classical features of trained immunity previously shown to be adopted by other epithelial stem cells. Our newly identified wound-distal memory cells display a cell-autonomous transcriptional pre-activated state leading to an enhanced wound repair ability that can be partially recapitulated through epigenetic perturbation even in absence of an injury. Importantly, the harmful consequences of wound repair, such as exacerbated tumorigenesis, occur within these primed cells and follow their spatial distribution. Overall, we show that sub-organ scale adaptation of an injury relies on spatially organised and memory-dedicated progenitors, characterised by an epigenetic actionable cell state, that predisposes to tumour onset.

cell biology↗

bioRxiv2022

Distinct gene programs underpinning 'disease tolerance' and 'resistance' in influenza virus infection

When challenged with an invading pathogen, the host defense response is engaged to eliminate the pathogen (resistance) and to maintain health in the presence of the pathogen (disease tolerance). However, the identification of distinct molecular programs underpinning disease tolerance and resistance remained obscure. We exploited transcriptional and physiological monitoring across 33 mouse strains, during in vivo influenza virus infection, to identify two host-defense gene programs - one is associated with hallmarks of disease tolerance and the other with hallmarks of resistance. Both programs constitute generic responses in multiple mouse and human cell types. Our study describes the organizational principles of these programs and validates Arhgdia as a regulator of disease-tolerance states in epithelial cells. We further reveal that the baseline disease-tolerance state in macrophages is associated with the pathophysiological response to injury and infection. Our framework provides a paradigm for the understanding of disease tolerance and resistance at the molecular level.

systems biology↗

bioRxiv2022

Rapidly evolving viral motifs target biophysically constrained binding pockets of host proteins

Evolutionary changes in the host-virus interactome can alter the course of infection, but the biophysical and regulatory constraints that shape interface evolution remain largely unexplored. Here, we focus on viral mimicry of short host-like peptide motifs that allow binding to host domains and modulation of cellular pathways. We observe that motifs from unrelated viruses preferentially target conserved, widely expressed and highly connected host proteins, enriched with regulatory and essential functions. The interface residues within these host domains are more conserved and bind a larger number of cellular proteins than similar motif-binding domains that are not known to interact with viruses. In stark contrast, rapidly evolving viral-binding human proteins form few interactions with other cellular proteins, display high tissue specificity and their interface residues have few inter-residue contacts. Our results distinguish between highly conserved and rapidly evolving host-virus interfaces, and show how regulatory, functional and biophysical factors limit host capacity to evolve, allowing for efficient viral subversion of host machineries.

microbiology↗

bioRxiv2021

Mapping interindividual dynamics of innate immune response at single-cell resolution

Common genetic variants modulate the cellular response to viruses and are implicated in a range of immune pathologies, including infectious and autoimmune diseases. The transcriptional antiviral response is known to vary between infected cells from a single individual, yet how genetic variants across individuals modulate the antiviral response (and its cell-to-cell variability) is not well understood. Here, we triggered the antiviral response in human fibroblasts from 68 healthy donors, and profiled tens of thousands of cells using single-cell RNA-seq. We developed GASPACHO (GAuSsian Processes for Association mapping leveraging Cell HeterOgeneity), the first statistical approach designed to identify dynamic eQTLs across a transcriptional trajectory of cell populations, without aggregating single-cell data into pseudo-bulk. This allows us to uncover the underlying architecture and variability of antiviral response across responding cells, and to identify more than two thousands eQTLs modulating the dynamic changes during this response. Many of these eQTLs colocalise with risk loci identified in GWAS of infectious and autoimmune diseases. As a case study, we focus on a COVID-19 susceptibility locus, colocalised with the antiviral OAS1 splicing QTL. We validated it in blood cells from a patient cohort and in the infected nasal cells of a patient with the risk allele, demonstrating the utility of GASPACHO to fine-map and functionally characterise a genetic locus. In summary, our novel analytical approach provides a new framework for delineation of the genetic variants that shape a wide spectrum of transcriptional responses at single-cell resolution.

genetics↗

bioRxiv2021

Promoter architecture links gene duplication with transcriptional divergence

Gene duplication is thought to be a central mechanism in evolution to gain new functions, but gene families vary greatly in their rates of gene duplication and long-term retention. Here, we discover a link between the promoter architecture of vertebrate genes and their rate of duplication: Genes that harbor CpG Islands in their promoters (CGI genes) - nearly 60% of our genes - have rarely duplicated in recent evolutionary times, and most CGI gene duplication events predate the emergence of CGI as a major regulatory element of vertebrate genes. In contrast, CGI-less genes predominate duplications that have occurred since the divergence of vertebrates. Furthermore, CGI-less paralogs are transcriptionally more divergent than CGI paralogs, even when comparing CGI and CGI-less paralogs that have duplicated at similar evolutionary times - suggesting greater capacity of CGI-less promoters to enable divergence in expression. This higher divergence between CGI-less paralogs is also reflected in lower similarity of transcription factors that bind to the promoters of CGI-less paralog pairs in comparison with CGI paralogs. Finally, CGI-less paralogs have a greater tendency to sub- and neo-functionalize, and they transcriptionally diversify faster following duplication. Our results highlight the links between promoter architecture, gene expression plasticity and their impact on gene expansion, and unravel an unappreciated role of CGI elements in shaping genome evolution. Significance statementGene duplication generates extra gene copies, providing material for evolution of new functions. However, many duplicated genes are eliminated due to functional and regulatory constraints. The evolutionary processes that govern the elimination and persistence of duplicated genes are not well understood. Here, we focus on CpG Islands (CGIs) - important elements that occur in the majority of gene promoters. We show that genes with CGIs in their promoters have duplicated almost exclusively in ancient times, and nearly all recent duplications involve CGI-less genes. Furthermore, duplicated CGI-less genes diverge more in expression and display more distinctive transcription and cis-regulation compared to duplicated CGI-genes. Our results demonstrate how promoter structure influences transcriptional evolvability and, in turn, the retention of new genes.

evolutionary biology↗

bioRxiv2020

Poised cell circuits in human skin are activated in disease

The human skin confers biophysical and immunological protection through a complex cellular network that is established early in development. We profiled ~500,000 single cells using RNA-sequencing from healthy adult and developing skin, and skin from patients with atopic dermatitis and psoriasis. Our findings reveal a predominance of innate lymphoid cells and macrophages in developing skin in contrast to T cells and migratory dendritic cells in adult skin. We demonstrate dual keratinocyte differentiation trajectories and activated cellular circuits comprising vascular endothelial cells mediating immune cell trafficking, disease-specific clonally expanded IL13/IL22 and IL17A/F-expressing lymphocytes, epidermal IL23-expressing dendritic cells and inflammatory keratinocytes in disease. Our findings provide key insights into the dynamic cellular landscape of human skin in health and disease. One Sentence SummarySingle cell atlas of human skin reveals cell circuits which are quantitatively and qualitatively reconfigured in inflammatory skin disease.

immunology↗

bioRxiv2018

Single-cell RNA sequencing reveals a dynamic stromal niche within the evolving tumour microenvironment

Non-cancerous stromal cells represent a highly diverse compartment of the tumour, yet their role across tumour evolution remains unclear. We employed single-cell RNA sequencing to determine stromal adaptations in murine melanoma at different points of tumour development. Naive lymphocytes recruited from lymph nodes underwent activation and clonal expansion within the tumour, prior to PD1 and Lag3 expression, while tumour-associated myeloid cells promoted the formation of a suppressive niche through cytokine secretion and inhibitory T cell interactions. We identified three temporally distinct cancer-associated fibroblast (CAF) populations displaying unique signatures, and verified these in human datasets. In early tumours, immune CXCL12/CSF1 and complement-expressing CAFs supported recruitment of macrophages, whereas contractile CAFs became more prevalent in later tumours. This study highlights the complex interplay and increasing diversity among cells that co-evolve with the tumour, indicating that from early stages of development, stromal cells acquire the capacity to modulate the immune landscape towards suppression.

cancer biology↗

bioRxiv2018

Cardelino: Integrating whole exomes and single-cell transcriptomes to reveal phenotypic impact of somatic variants

Decoding the clonal substructures of somatic tissues sheds light on cell growth, development and differentiation in health, ageing and disease. DNA-sequencing, either using bulk or using single-cell assays, has enabled the reconstruction of clonal trees from frequency and co-occurrence patterns of somatic variants. However, approaches to systematically characterize phenotypic and functional variations between individual clones are not established. Here we present cardelino (https://github.com/PMBio/cardelino), a computational method for inferring the clone of origin of individual cells that have been assayed using single-cell RNA-seq (scRNA-seq). After validating our model using simulations, we apply cardelino to matched scRNA-seq and exome sequencing data from 32 human dermal fibroblast lines, identifying hundreds of differentially expressed genes between cells from different somatic clones. These genes are frequently enriched for cell cycle and proliferation pathways, indicating a key role for cell division genes in non-neutral somatic evolution.\n\nKey findingsO_LIA novel approach for integrating DNA-seq and single-cell RNA-seq data to reconstruct clonal substructure for single-cell transcriptomes.\nC_LIO_LIEvidence for non-neutral evolution of clonal populations in human fibroblasts.\nC_LIO_LIProliferation and cell cycle pathways are commonly distorted in mutated clonal populations.\nC_LI

genomics↗

bioRxiv2018

Neutralizing Gatad2a-Chd4-Mbd3 Axis within the NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency

The Nucleosome Remodeling and Deacytelase (NuRD) complex is a co-repressive complex involved in many pathological and physiological processes in the cell. Previous studies have identified one of its components, Mbd3, as a potent inhibitor for reprogramming of somatic cells to pluripotency. Following OSKM induction, early and partial depletion of Mbd3 protein followed by applying naive ground-state pluripotency conditions, results in a highly efficient and near-deterministic generation of mouse iPS cells. Increasing evidence indicates that the NuRD complex assumes multiple mutually exclusive protein complexes, and it remains unclear whether the deterministic iPSC phenotype is the result of a specific NuRD sub complex. Since complete ablation of Mbd3 blocks somatic cell proliferation, here we aimed to identify alternative ways to block Mbd3-dependent NuRD activity by identifying additional functionally relevant components of the Mbd3/NuRD complex during early stages of reprogramming. We identified Gatad2a (also known as P66), a relatively uncharacterized NuRD-specific subunit, whose complete deletion does not impact somatic cell proliferation, yet specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuit during both stem cell differentiation and reprogramming to pluripotency. Complete ablation of Gatad2a in somatic cells, but not Gatad2b, results in a deterministic naive iPSC reprogramming where up to 100% of donor somatic cells successfully complete the process within 8 days. Genetic and biochemical analysis established a distinct sub-complex within the NuRD complex (Gatad2a-Chd4-Mbd3) as the functional and biochemical axis blocking reestablishment of murine naive pluripotency. Disassembly of this axis by depletion of Gatad2a, results in resistance to conditions promoting exit of naive pluripotency and delays differentiation. We further highlight context- and posttranslational dependent modifications of the NuRD complex affecting its interactions and assembly in different cell states. Collectively, our work unveils the distinct functionality, composition and interactions of Gatad2a-Chd4-Mbd3/NuRD subcomplex during the resolution and establishment of mouse naive pluripotency.

developmental biology↗

bioRxiv2017

A Balance Between Regulatory Constraints And Pathogen Pressure Shapes The Evolution Of Innate Immunity

As the first line of defence against pathogens, cells mount an innate immune response, which is highly variable from cell to cell. The response must be potent yet carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here, we characterise this programmes transcriptional divergence between species and expression variability across cells. Using bulk and single-cell transcriptomics in primate and rodent fibroblasts challenged with an immune stimulus, we reveal a striking architecture of the innate immune response. Rapidly diverging genes, including cytokines and chemokines, also vary across cells and have distinct promoter structures. Conversely, genes involved in response regulation, such as transcription factors and kinases, are conserved between species and display low cell-to-cell variability. We suggest that this unique expression pattern, observed across species and conditions, has evolved as a mechanism for fine-tuned regulation, to achieve an effective but balanced response.

evolutionary biology↗