Biology subjects

Grosveld, F.

Publications and source records attributed to Grosveld, F..

10 recordsLinked to original sources

bioRxiv2026

scRIPT: a rapid, high-coverage single-cell lineage-tracing method applied to the hematoendothelial differentiation of embryonic stem cells

Single-cell lineage tracing, which often involves CRISPR-Cas-based barcode insertion followed by novel barcode creation, links a living cell's transcriptome to its clonal history. It is often less suitable for short cell differentiation protocols: the barcoding time window does not always align well with that of differentiation, barcode dropout rates can be too high, library preparation remains laborious, and many studies lack appropriate lineage-specific quality control. We developed scRIPT (single-cell Rapid Integrated Platform for Tracing), which combines rapid ribonucleoprotein (RNP)-based barcoding with fluorescent reporter coding sequence insertion, reporter-based enrichment of informative cells by cell sorting, streamlined library preparation, and dual short-read sequencing, coupled to dedicated quality-control metrics for lineage data. scRIPT achieves a low cell dropout rate (<5% of cells presenting without a detectable lineage barcode) and a reliable assignment of barcodes (of >90% of cells). We applied scRIPT to document mouse embryonic stem cell (ESC) differentiation into early hematoendothelial cells and revealed clonal relationships and fate coupling among the emerging cell (sub)types. A subset of cell clones was biased towards single fates, and bifurcating progenitors already express mRNA of canonical regulators of their eventual fate. The results also suggest a heritable, fate-independent clonal memory enriched with imprinted genes. Taken together, scRIPT provides a convenient, rapid, reliable, high-coverage, and quality-controlled platform that combines cell lineage and transcriptome readouts.

developmental biology↗

bioRxiv2025

Single-Cell Roadmap of Early Hemato-Endothelial Development: Functions of Atf3, Zfp711 and Bcl6b

Hematopoiesis is the process of producing blood cells. In mammalian embryos, hematopoiesis occurs in three consecutive overlapping waves (Neo et al. 2021; Dzierzak and Bigas 2018) that are regulated by transcription factors (TFs) and signaling proteins. We investigated the functions of three relatively poorly studied TFs in early embryonic hematopoietic development at single-cell resolution: Activating transcription factor 3 (Atf3), Zinc finger protein 711 (Zfp711), and B cell CLL/lymphoma 6, member B (Bcl6b). These TFs are upregulated early in development when hematopoietic and endothelial lineages separate from cardiac and other mesodermal lineages. We combined multiplexed single-cell RNA sequencing (scRNA-seq) and cell identity analysis using Flow Cytometric Analysis (FCA) with TF knockouts (KO) in in-vitro differentiating mouse embryonic stem cells (mESCs) to dissect the function of these TFs in lineage induction, specification, and separation. The Atf3-KO showed an increase of distinct mesodermal subpopulations, but a decrease of endothelial and erythro-myeloid progenitors (EMPs) by downregulation of important genes (i.e. Runx1, Mafb, Egr1, Jun, Jund, Fos, Batf3, and Zf608) that likely explains the effects on EMPs and the increased expression of interferon-related genes. In Zfp711-KO cells, the number of blood progenitor cells and erythroid cells increased, while the number of endothelial cells decreased. Furthermore, Hoxa expressing mesoderm decreased, while Hoxb expressing mesoderm increased. In contrast, the Bcl6b-KO had no observable effects on early hematopoiesis. In conclusion, we report the function of these three TFs function at different stages of hemato-endothelial lineage specification. HIGHLIGHTSO_LICombined scRNA-seq and FCA on KO mESCs in vitro differentiation enabled unbiased identification of the respective TF functions during specific stages of hematoendothelial lineage specification. C_LIO_LIAtf3-KO: Increased abundance of mesodermal lineages and decreased endothelial lineages and EMPs. - Downregulation of key TF-encoding genes (e.g., Runx1, Mafb, Egr1, Jun, Jund, Fos, Batf3, Zf608) explain the effects on EMPs. - Generation of a distinct mesodermal subpopulation with high expression of sets of interferon and antiviral-related genes. - Upregulation of interferon and anti-viral-related genes, indicating Atf3 acts as a negative regulator of these genes. C_LIO_LIZfp711-KO: Increased numbers of blood progenitor and erythroid cells. Decreased numbers of endothelial cells. - Shift in mesodermal populations: Hoxa expressing mesoderm decreased, and Hoxb expressing mesoderm increased. - Hoxa9, Hoxa10, Hoxa13, Jun, Jund and Atf3 amongst the downregulated genes. The Atf3 promoter has a potential Zfp711-binding site. - Increased expression in a given Endothelium subtype in endocardium in vivo and in vitro. C_LIO_LIBcl6b-KO: No observable impact on early hematopoiesis. C_LI

developmental biology↗

bioRxiv2025

In vivo deletion of a GWAS-identified Myb distal enhancer acts on Myb expression, globin switching, and clinical erythroid parameters in beta-thalassemia

Genome-wide association studies (GWAS) have identified numerous genetic variants linked to human diseases, mostly located in non-coding regions of the genome, particularly in putative enhancers. However, functional assessment of the non-coding GWAS variants has progressed at slow pace, since the functions of the vast majority of genomic enhancers have not been defined, impeding interpretation of disease-susceptibility variants. The HBS1L-MYB intergenic region harbors multiple SNPs associated with clinical erythroid parameters, including fetal hemoglobin levels, a feature impacting disease severity of beta-hemoglobinopathies such as sickle cell anemia and beta-thalassemia. HBS1L-MYB variants cluster in the vicinity of several MYB enhancers, altering MYB expression and globin switching. We and others have highlighted the conserved human MYB -84kb enhancer, known as the -81kb enhancer in the mouse, as likely candidate linked to these traits. We report here the generation of a Myb -81kb enhancer knock-out mouse model, and shed light for the first time on its impact on steady state erythropoiesis and in beta-thalassemia in vivo.

genetics↗

bioRxiv2025

A Novel XACT lncRNA Transcript with Functions Transcending X-Chromosome Inactivation.

X-chromosome inactivation (XCI) is initiated during early mammalian embryogenesis by a long non-coding RNA (lncRNA) XIST, which coats one of the two X-chromosomes and facilitates epigenetic transcriptional silencing. A second, evolutionarily recent primate-specific lncRNA XACT was proposed to antagonize XISTs ability to induce XCI. XACT expression is restricted to pluripotent states and early embryonic stages and coats the active X-chromosome in both females and males. Here, we report a novel XACT transcript expressed in normal and cancerous somatic cells from both the inactive (Xi) and active (Xa) X chromosomes. It coexists with XIST on the Xi without affecting XIST expression or (re)activating X-linked genes inactivated by XCI. During hematopoietic stem cell (HSC) differentiation, XACT is primarily expressed in myeloid progenitors in both sexes. XACT expression is activated in HSCs and peaks in megakaryocyte-erythrocyte progenitor cells (MEPs) before rapidly declining as the MEPs differentiate into megakaryocytes or erythrocytes. By combining CRISPR-based XACT perturbation with epigenomic and transcriptional studies, we revealed the key role of XACT in the self-renewal and differentiation of erythroid progenitors into erythrocytes, by recruiting cis-regulatory proteins and regulating transcription through ETS and AP-1 transcription factors. Furthermore, XACT is expressed in a subset of acute myeloid leukemia (AML) patients, with high levels found in erythroid-megakaryocytic blast cells; suggesting XACTs potential as a marker for AML subtypes. Thus, we identified a novel XACT transcript with a unique expression profile and important roles in normal and cancer cells, revealing XACT lncRNA functions beyond its previously proposed role in XCI during embryogenesis.

genetics↗

bioRxiv2024

Fc-dependent protective efficacy of non-inhibitory antibodies targeting influenza A virus neuraminidase is limited by epitope availability

Antibodies targeting hemagglutinin and neuraminidase (NA) are key components of the adaptive immune response against influenza A virus (IAV). However, antigenic drift allows the virus to escape inhibition by such antibodies. In this study, we aimed to isolate antibodies with cross-subtype reactivity against human H1N1 and H3N2 IAVs from transgenic mice bearing genes encoding the human immunoglobulin variable regions. We immunized these mice with recombinant N1 and N2 NA proteins, presenting them either as unconjugated soluble proteins or conjugated to self-assembling protein nanoparticles. This approach yielded a panel of NA-specific monoclonal antibodies (mAbs) with various levels of intra-and inter-subtype reactivity for N1 and N2 NA. Three of these mAbs, which collectively recognize two distinct epitopes, were cross-reactive against N1 and N2 NAs in ELISA, but did not inhibit NA enzymatic activity. Two of these mAbs, 21H8 and 45D9, were selected for further characterization. These recognized different epitopes and induced Fc-mediated effector functions to varying extents. Prophylactic administration of 21H8, but not 45D9, protected mice against challenge with H1N1 IAV, while neither mAb protected against a H3N2 challenge. The observed protective efficacy correlated with the mAbs capacity, or lack thereof, to bind membrane-associated full-length NA. The introduction of Fc silencing mutations in mAb 21H8 resulted in an inability to activate NK cells or mediate phagocytosis in vitro and significantly reduced protection in vivo, indicating that the protective efficacy of mAb 21H8 is Fc-dependent. However, mAb 21H8 expressed with reduced core fucosylation of its Fc N-glycan, which specifically enhanced NK cell activation in vitro, failed to improve protection against H1N1 challenge in vivo. Future work is needed to decipher in more detail the mechanism of Fc-mediated protection against influenza via NA-specific antibodies and to identify the optimal strategies for their enhancement.

microbiology↗

bioRxiv2022

An ACE2-blocking antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

The ongoing evolution of SARS-CoV-2 has resulted in the emergence of Omicron, which displays striking immune escape potential. Many of its mutations localize to the spike protein ACE2 receptor-binding domain, annulling the neutralizing activity of most therapeutic monoclonal antibodies. Here we describe a receptor-blocking human monoclonal antibody, 87G7, that retains ultrapotent neutralization against SARS-CoV-2 variants including the Alpha, Beta, Gamma, Delta and Omicron (BA.1/BA.2) Variants-of-Concern (VOCs). Structural analysis reveals that 87G7 targets a patch of hydrophobic residues in the ACE2-binding site that are highly conserved in SARS-CoV-2 variants, explaining its broad neutralization capacity. 87G7 protects mice and/or hamsters against challenge with all current SARS-CoV-2 VOCs. Our findings may aid the development of sustainable antibody-based strategies against COVID-19 that are more resilient to SARS-CoV-2 antigenic diversity. One sentence summaryA human monoclonal antibody confers broad neutralization and protection against Omicron and other SARS-CoV-2 variants

microbiology↗

bioRxiv2020

Isolation of cross-reactive monoclonal antibodies against divergent human coronaviruses that delineate a conserved and vulnerable site on the spike protein

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry. As such, it is an attractive target for the development of protective antibodies and vaccines. Here we describe two human monoclonal antibodies, 1.6C7 and 28D9, that display a remarkable cross-reactivity against distinct species from three Betacoronavirus subgenera, capable of binding the spike proteins of SARS-CoV and SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both antibodies, derived from immunized transgenic mice carrying a human immunoglobulin repertoire, blocked MERS-CoV infection in cells, whereas 28D9 also showed weak cross-neutralizing potential against HCoV-OC43, SARS-CoV and SARS-CoV-2 in a neutralization-sensitive virus pseudotyping system, but not against authentic virus. Both cross-reactive monoclonal antibodies were found to target the stem helix in the spike protein S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle, that is antibody-accessible. We demonstrate that administration of these antibodies in mice protects from a lethal MERS-CoV challenge in both prophylactic and/or therapeutic models. Collectively, these antibodies delineate a conserved, immunogenic and vulnerabe site on the spike protein which spurs the development of broad-range diagnostic, preventive and therapeutic measures against coronaviruses.

microbiology↗

bioRxiv2020

SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

Effective clinical intervention strategies for COVID-19 are urgently needed. Although several clinical trials have evaluated the use of convalescent plasma containing virus-neutralizing antibodies, the effectiveness has not been proven. We show that hamsters treated with a high dose of human convalescent plasma or a monoclonal antibody were protected against weight loss showing reduced pneumonia and pulmonary virus replication compared to control animals. However, a ten-fold lower dose of convalescent plasma showed no protective effect. Thus, variable and relatively low levels of virus neutralizing antibodies in convalescent plasma may limit their use for effective antiviral therapy, favouring concentrated, purified (monoclonal) antibodies.

microbiology↗

bioRxiv2020

A human monoclonal 1 antibody blocking SARS-CoV-2 infection

The emergence of the novel human coronavirus SARS-CoV-2 in Wuhan, China has caused a worldwide epidemic of respiratory disease (COVID-19). Vaccines and targeted therapeutics for treatment of this disease are currently lacking. Here we report a human monoclonal antibody that neutralizes SARS-CoV-2 (and SARS-CoV). This cross-neutralizing antibody targets a communal epitope on these viruses and offers potential for prevention and treatment of COVID-19.

microbiology↗

bioRxiv2018

The mouse Klf1 Nan variant impairs nuclear condensation and erythroid maturation

Kruppel-like factor 1 (KLF1) is an essential transcription factor for erythroid development, as demonstrated by Klf1 knockout mice which die around E14 due to severe anemia. In humans, >65 KLF1 variants, causing different erythroid phenotypes, have been described. The Klf1 Nan variant, a single amino acid substitution (p.E339D) in the DNA binding domain, causes hemolytic anemia and is dominant over wildtype KLF1. Here we describe the effects of the Nan variant during fetal development. We show that Nan embryos have defects in erythroid maturation. RNA-sequencing of the Nan fetal liver cells revealed that Exportin 7 (Xpo7) was among the ~780 deregulated genes. This nuclear exportin is implicated in terminal erythroid differentiation; in particular it is involved in nuclear condensation. Indeed, KLF1 Nan fetal liver cells had larger nuclei and reduced chromatin condensation. Knockdown of XPO7 in wildtype erythroid cells caused a similar phenotype. We conclude that reduced expression of XPO7 is partially responsible for the erythroid defects observed in Nan erythroid cells.

developmental biology↗