Biology subjects

de Jonge, W. J.

Publications and source records attributed to de Jonge, W. J..

10 recordsLinked to original sources

bioRxiv2026

Spatial multi-omics reveals targetable immunosuppressive macrophage T-cell interactions in human AML bone marrow

The immunosuppressive bone marrow microenvironment is an important contributor to the limited success of immunotherapy in acute myeloid leukemia (AML), but the cellular interactions underlying AML immune evasion are incompletely understood. We therefore generated a single-cell spatial transcriptomic and proteomic atlas using 148 bone biopsies from 113 individuals comprising pediatric and adult AML at diagnosis and non-leukemic controls. We observed an expansion of regulatory T cells (Tregs) in AML, with stronger colocalization between Tregs and macrophages compared to non-leukemic bone marrow. Distinct cellular neighborhoods were enriched for myeloid progenitor-like cells together with macrophages and T cells, which correlated with higher macrophage and T cell immune checkpoint expression. Moreover, these neighborhoods were associated with specific AML subtypes, especially KMT2A-rearranged and RUNX1::RUNX1T1 AML. These spatial patterns were validated by identification of malignant cells via in situ fusion detection in RUNX1::RUNX1T1 cases. Functional experiments revealed that macrophages and AML cells not only actively recruit Tregs, but also promote naive T cell differentiation into Tregs. Spatially informed ligand-receptor analysis predicted the involvement of the Galectin-9 - CD44/TIM-3 axis in this immunosuppressive crosstalk, which was supported by in vitro inhibition of CD44 and/or TIM-3 preventing macrophage- and AML-induced Treg differentiation. Collectively, this comprehensive spatial map of the AML bone marrow identified tripartite crosstalk between AML, macrophages, and T cells mediated by the Galectin-9 - CD44/TIM-3 axis as a key component of the immunosuppressive microenvironment. Targeting Galectin-9 - CD44/TIM-3 interactions may be a promising strategy to overcome immune evasion and enhance immunotherapeutic success in AML. HighlightsO_LISpatial transcriptomic and proteomic atlas of pediatric and adult acute myeloid leukemia (AML) bone marrow C_LIO_LIIncreased colocalization of macrophages and regulatory T cells (Tregs) in AML C_LIO_LIMacrophages and AML cells induce differentiation of naive T cells to Tregs in vitro, which can be prevented by inhibition of CD44 or TIM-3 C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/743431v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@14f313aorg.highwire.dtl.DTLVardef@290bd3org.highwire.dtl.DTLVardef@c749baorg.highwire.dtl.DTLVardef@1b955d0_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

A Cooperative Mechanism of Eukaryotic Transcription Factor Target Search

Rapid gene activation requires transcription factors (TFs) to locate their target motifs within vast genomes. In bacteria, TF target search is accelerated by combining 3D diffusion with 1D sliding, called facilitated diffusion, yet whether eukaryotic TFs rely on similar strategies has remained unresolved due to the lack of direct measurements in living cells. Here, we directly visualize eukaryotic TF target search in living cells by labeling a single TF per nucleus and visualizing its binding to its endogenous locus. Using the budding yeast TF Gal4, we find that efficient target localization occurs near the diffusion limit and does not require facilitated diffusion. Instead, rapid association requires cooperative self-interactions mediated by an intrinsically-disordered central region (IDR), independent of the activation domain. Replacing the Gal4 IDR with human self-interacting IDRs (EWS or FUS) restores efficient search, demonstrating that self-interactions are a general and portable feature for search. A second structured dimerization domain further cooperatives with the IDR to stabilize binding at neighboring motifs, revealing two mechanistically separable forms of cooperativity that together govern TF function. These findings highlight that facilitated diffusion is not strictly required and establish cooperative IDR-driven self-interactions as a key mechanism to enable rapid target recognition in eukaryotic cells.

molecular biology↗

bioRxiv2024

Single-cell multiomics of pediatric BM reveals age-dependent differences in lineage differentiation linked to stromal cell heterogeneity

Childhood is critical for hematopoietic development and the onset of hematologic diseases. To explore hematopoietic changes from infancy through adolescence, we generated a multi-modal single-cell atlas capturing mRNA and surface protein expression of 90.710 bone marrow (BM) cells. This includes hematopoietic stem/progenitor cells and mesenchymal stromal cells, from seven pediatric individuals and two young adults. We demonstrate that young pediatric BM is distinct from adolescents/young adults (AYA), shifting from B-lineage dominance in early childhood to myeloid and T-lineage bias in adolescence. We uncover two distinct lymphoid progenitors (LyPs) subsets regulating this shift: CD127-positive LyPs with B-lineage output, most abundant in early childhood, and CD127-negative LyPs with lymphoid and myeloid features, more common in AYAs. Age-related changes in stromal composition and signaling, mediated by IL-7 and TGF-{beta}1, correspond with this lineage shift. This study provides an in-depth resource for understanding healthy hematologic development and potential early-life perturbations underlying pediatric hematologic diseases.

immunology↗

bioRxiv2024

Typing of feces-derived Candida albicans strains using a novel seven-locus microsatellite panel reveals associations with yeast phenotype in individuals with inflammatory bowel disease

Inflammatory diseases of the human gastrointestinal tract are affected by the microbes that reside in the mucosal surfaces along this tract. Patients with inflammatory bowel diseases (IBD) have altered bacterial and fungal intestinal compositions, including higher levels of fecal Candida yeasts. Ongoing research indicates that genetic and phenotypic diversity of Candida albicans may be linked with disease severity. In this study, we set out to investigate feces-derived C. albicans strains from individuals with Crohns disease, ulcerative colitis, and healthy volunteers through microsatellite-based genotyping and phenotypic assays. A seven-locus microsatellite panel was applied, of which six loci were newly developed. Combined interpretation of minimum spanning networks and principal component analysis (PCA) led to indications that there is no specific lineage of C. albicans that is associated with IBD, but rather that the three study populations do have distinguishable distributions of genotypes. In addition, phenotypic characterization by means of enzyme release assays revealed trends between genotypes and virulence-related enzyme activity. Furthermore, correlations were observed between the clinical inflammation biomarker fecal calprotectin and serum anti- Saccharomyces cerevisiae antibodies, as well as between lipase activity of feces-derived C. albicans strains and serum 1,3-{beta}-glucan levels. We thus show that microsatellite typing can describe genetic diversity of feces-derived C. albicans strains, and that phenotypic diversity of these strains may indeed correlate with fungal genotype or disease. This study opens further possibilities to investigate fecal fungi in relation to severity of inflammation in IBD or in other (intestinal) diseases.

microbiology↗

bioRxiv2022

Transcription factor clusters enable target search but do not contribute to target gene activation

Many transcription factors (TFs) localize in nuclear clusters of locally increased concentrations, but how TF clustering is regulated and how it influences gene expression is not well understood. Here, we use quantitative microscopy in living cells to study the regulation and function of clustering of the budding yeast TF Gal4 in its endogenous context. Our results show that Gal4 cluster formation is facilitated by, but does not completely depend on DNA binding and intrinsically disordered regions. Gal4 cluster properties are regulated by the Gal4-inhibitor Gal80 and Gal4 concentration. Moreover, we discover that clustering acts as a double-edged sword: self-interactions aid TF recruitment to target genes, but recruited Gal4 molecules that are not DNA-bound do not contribute to, and may even inhibit, transcription activation. We propose that cells need to balance the different effects of TF clustering on target search and transcription activation to facilitate proper gene expression.

molecular biology↗

bioRxiv2020

Modulation of macrophage inflammatory function through selective inhibition of the epigenetic reader protein SP140

Speckled 140 KDa (SP140) is a nuclear body protein, mainly expressed in immune cells, which contains multiple domains suggestive of an epigenetic reader function; namely a bromodomain, a PHD domain and a SAND domain. Single nucleotide polymorphisms and epigenetic modifications in the SP140 locus have been linked to autoimmune and inflammatory diseases including Crohns disease (CD). However, little is known about the cellular function of SP140; this is due in part to the fact that, unlike for other many other epigenetic proteins, no small molecule inhibitors have been available to investigate the biological role of SP140. We report the discovery of the first small molecule SP140 inhibitor (GSK761) and utilize this to elucidate SP140 function in innate immune cells. We show that SP140 is highly expressed in CD68+ CD mucosal macrophages and in in vitro-generated inflammatory macrophages. SP140 inhibition through GSK761 reduced monocyte differentiation into inflammatory macrophages and lipopolysaccharide (LPS)-induced inflammatory activation, whilst inducing the generation of CD206+ regulatory macrophages that mark anti-TNF remission induction in CD patients. ChIP-seq analyses revealed that SP140 preferentially occupies transcriptional start sites (TSS) in inflammatory macrophages, with enrichment at gene loci encoding pro-inflammatory cytokines/chemokines and inflammatory pathways. GSK761 specifically reduced SP140 binding and thereby expression of SP140-dependent downstream inflammatory genes. Notably, in CD14+ macrophages isolated from CD intestinal-mucosa, GSK761 inhibited the spontaneous expression of cytokines, including TNF. Together, this study identifies SP140 as a druggable epigenetic reader and therapeutic target for CD.

immunology↗

bioRxiv2020

Intestinal transkingdom analysis on the impact of antibiotic perturbation in health and critical illness

Bacterial microbiota play a critical role in mediating local and systemic immunity, and shifts in these microbial communities have been linked to impaired outcomes in critical illness. Emerging data indicate that other intestinal organisms, including bacteriophages, viruses of eukaryotes, fungi, and protozoa, are closely interlinked with the bacterial microbiota and their host, yet their collective role during antibiotic perturbation and critical illness remains to be elucidated. Here, multi-omics factor analysis (MOFA), a novel computational strategy to systematically integrate viral, fungal and bacterial sequence data, we describe the functional impact of exposure to broad-spectrum antibiotics in healthy volunteers and critically ill patients. We observe that a loss of the anaerobic intestinal environment is directly correlated with an overgrowth of aerobic pathobionts and their corresponding bacteriophages, as well as an absolute enrichment of opportunistic yeasts capable of causing invasive disease. These findings further illustrate the complexity of transkingdom interactions within the intestinal environment, and show that modulation of the bacterial component of the microbiome has implications extending beyond this kingdom alone.

microbiology↗

bioRxiv2020

Genome-wide off-rates reveal how DNA binding dynamics shape transcription factor function

Protein-DNA interactions are dynamic and these dynamics are an important aspect of chromatin-associated processes such as transcription or replication. Due to a lack of methods to study on- and off-rates across entire genomes, protein-DNA interaction dynamics have not been studied extensively. Here we determine in vivo off-rates for the Saccharomyces cerevisiae chromatin organising factor Abf1, at 191 sites simultaneously across the yeast genome. Average Abf1 residence times span a wide-range, varying between 4.5 and 37 minutes. Sites with different off-rates are associated with different functional characteristics. This includes their transcriptional dependency on Abf1, nucleosome positioning and the size of the nucleosome-free region, as well as the ability to roadblock RNA polymerase II for termination. The results show how off-rates contribute to transcription factor function and that DIVORSEQ (Determining In Vivo Off-Rates by SEQuencing) is a meaningful way of investigating protein-DNA binding dynamics genome-wide.

molecular biology↗

bioRxiv2019

An extensively optimized chromatin immunoprecipitation protocol for quantitatively comparable and robust results

Chromatin immunoprecipitation (ChIP) is a commonly used technique to investigate which parts of a genome are bound by a particular protein. The result of ChIP is often interpreted in a binary manner: bound or not bound. Due to this focus, ChIP protocols frequently lack the ability to quantitatively compare samples with each other, for example in a time series or under different growth conditions. Here, using the yeast S. cerevisiae transcription factors Cbf1, Abf1, Reb1, Mcm1 and Sum1, we optimized the five major steps of a commonly used ChIP protocol: cross-linking, quenching, cell lysis, fragmentation and immunoprecipitation. Quenching with glycine is inefficient and can lead to large degrees of variability, an issue that is resolved by using tris(hydroxymethyl)aminomethane (Tris). Another source of variability is degradation of the protein of interest during the procedure. Enzymatic cell lysis with zymolyase can lead to extensive protein degradation, which is greatly reduced by mechanical lysis through bead beating. Degradation also occurs during sonication of chromatin, affecting large proteins in particular. An optimal mix of protease inhibitors and cross-linking with a higher percentage of formaldehyde reduces the extent of this degradation. Finally we also show that the immunoprecipitation step itself can be greatly improved with magnetic beads and optimized incubation/washing steps. The study results in a highly optimized protocol, which is shorter, easier to perform and has a stronger, more reproducible signal with less background. This protocol is presented in detail. In addition, the results highlight the greatest sources of variability in many other protocols, showing which steps are important to focus on for reproducible and quantitatively comparable ChIP experiments.

molecular biology↗

bioRxiv2019

Defining tissue- and disease-associated macrophages using a transcriptome-based classification model

Macrophages are heterogeneous multifunctional leukocytes which are regulated in a tissue-and disease-specific context. Many different studies have been published using in vitro macrophage models to study disease. Here, we aggregated public expression data to define consensus expression profiles for eight commonly-used in vitro macrophage models. Altogether, we observed well-known but also novel markers for different macrophage subtypes. Using these data we subsequently built the classifier macIDR, capable of distinguishing macrophage subsets with high accuracy (>0.95). This classifier was subsequently applied to transcriptional profiles of tissue-isolated and disease-associated macrophages to specifically define macrophage characteristics in vivo. Classification of these in vivo macrophages showed that alveolar macrophages displayed high resemblance to interleukin-10 activated macrophages, whereas macrophages from patients with chronic obstructive pulmonary disease patients displayed a drop in interferon-{gamma} signature. Adipose tissue-derived macrophages were classified as unstimulated macrophages, but resembled LPS-activated macrophages more in diabetic-obese patients. Finally, rheumatoid arthritic synovial macrophages showed characteristics of both interleukin-10 or interferon-{gamma} signatures. Altogether, our results suggest that macIDR is capable of identifying macrophage-specific changes as a result of tissue-and disease-specific stimuli and thereby can be used to better define and model populations of macrophages that contribute to disease.

immunology↗