Biology subjects

Vermeulen, M.

Publications and source records attributed to Vermeulen, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Enhancer plasticity in endometrial tumorigenesis demarcates non-coding driver mutations and alterations in 3D genome organization to stimulate oncogene expression

The incidence and mortality of Endometrial Cancer (EC) is on the rise. 85% of ECs depend on Estrogen Receptor alpha (ER) for proliferation, but little is known about its transcriptional regulation in these tumors. We generated epigenomics, transcriptomics and Hi-C datastreams in healthy and tumor endometrial tissues, identifying robust ER reprogramming and profound alterations in 3D genome organization that lead to a gain of tumor-specific enhancer activity during EC development. Integration with endometrial cancer risk single-nucleotide polymorphisms, as well as WGS data from primary tumors and metastatic samples revealed a striking enrichment of risk variants and non-coding somatic mutations at tumor-enriched ER sites. Through machine learning-based predictions and interaction proteomics analyses, we identified an enhancer mutation which alters 3D genome conformation, impairing recruitment of the transcriptional repressor EHMT2/G9a/KMT1C, thereby alleviating transcriptional repression of ESR1 in EC. In summary, we identified a complex genomic-epigenomic interplay in EC development and progression, altering 3D genome organization to enhance expression of the critical driver ER.

cancer biology↗

bioRxiv2023

Single-cell transcriptomic analysis of NK cell dynamics in myeloma patients reveal persistent reduction of cytotoxic NK cells from diagnosis to relapse

Natural killer (NK) cells mediate the cytotoxic immune response against multiple myeloma and are important effector cells in immune therapies through antibody-dependent cellular cytotoxicity. Here, we used single-cell transcriptomics, flow cytometry and functional assays to investigate the bone marrow NK cell compartment of myeloma patients at diagnosis, during treatment and after relapse. The bone marrow of myeloma patients is characterized by a reduction in conventional cytotoxic NK cells that persists throughout treatment. We show in 20% of newly diagnosed myeloma patients that an altered balance between cytotoxic and cytokine-producing NK cells translates into a reduced cytotoxic ability in response to therapeutic antibodies. The relative loss of cytotoxic NK cells persists at relapse and is accompanied by an expansion of IFN-responsive NK cells. These findings reveal previously unappreciated alterations in bone marrow NK cell composition and highlight the importance of understanding the bone marrow immune system in patients receiving immunotherapies. Statement of significanceThe bone marrow of multiple myeloma patients is characterized by a persistent reduction in cytotoxic CD56dim NK cells, accompanied by inferior in vitro responses to therapeutic antibodies at diagnosis and an increase in IFN-responsive NK cells at relapse. These findings highlight the importance of understanding the BM microenvironment in multiple myeloma patients receiving immunotherapies.

immunology↗

bioRxiv2023

Glycolytic imprinting of monocytes impairs HIF1α-driven migration of dendritic cells in tuberculosis

During tuberculosis, migration of dendritic cells (DCs) from the site of infection to the draining lymph nodes is known to be impaired, hindering the rapid development of protective T-cell mediated immunity. However, the mechanisms involved in the delayed migration of DCs during tuberculosis (TB) are still poorly defined. Here, we found that infection of DCs with Mycobacterium tuberculosis (Mtb) triggers HIF-1-mediated aerobic glycolysis in a TLR2-dependent manner, and that this metabolic profile is essential for DC migration. In particular, the lactate dehydrogenase (LDH) inhibitor oxamate and the HIF-1 inhibitor PX-478 abrogated Mtb-induced DC migration in vitro to the lymphoid tissue-specific chemokine CCL21, and in vivo to lymph nodes in mice. Strikingly, we found that although monocytes from TB patients are inherently biased toward glycolysis metabolism, they differentiate into poorly glycolytic and poorly migratory DCs, compared with healthy subjects. Taken together, these data suggest that because of their preexisting glycolytic state, circulating monocytes from TB patients are refractory to differentiation into migratory DCs, which may explain the delayed migration of these cells during the disease and opens avenues for host-directed therapies for TB. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/535400v6_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@9c6849org.highwire.dtl.DTLVardef@82981dorg.highwire.dtl.DTLVardef@1d0b7faorg.highwire.dtl.DTLVardef@1deb76f_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

An IL-1β driven neutrophil-stromal cell axis fosters a BAFF-rich microenvironment in multiple myeloma

The bone marrow permanently harbors high numbers of neutrophils, and a tumor-supportive bias of these cells could significantly impact bone marrow-confined malignancies. In multiple myeloma, the bone marrow is characterized by inflammatory stromal cells with the potential to influence neutrophils. We investigated myeloma-associated alterations in marrow neutrophils and the impact of stromal inflammation on neutrophil function. Mature neutrophils in myeloma marrow are activated and tumor-supportive, transcribing increased levels of IL-1{beta}, and myeloma cell survival factor BAFF. Interactions with inflammatory stromal cells can induce neutrophil activation, including BAFF secretion, in a STAT3-dependent manner and once activated, neutrophils gain the ability to reciprocally induce stromal activation. After first-line myeloid-depleting treatment, patient bone marrow retains residual stromal inflammation and newly-formed neutrophils are reactivated. Combined, we identify a neutrophil-stromal cell feed-forward loop driving tumor-supportive inflammation that persists after treatment and warrants novel strategies to target both stromal and immune microenvironments in multiple myeloma.

immunology↗

bioRxiv2023

A novel antifolate suppresses growth of FPGS-deficient cells and overcomes methotrexate resistance

Cancer cells make extensive use of the folate cycle to sustain increased anabolic metabolism. Multiple chemotherapeutic drugs interfere with the folate cycle, including methotrexate and 5-fluorouracil that are commonly applied for the treatment of leukemia and colorectal cancer (CRC), respectively. Despite high success rates, therapy-induced resistance causes relapse at later disease stages. Depletion of folylpolyglutamate synthase (FPGS), which normally promotes intracellular accumulation and activity of both natural folates and methotrexate, is linked to methotrexate and 5-fluorouracil resistance and its association with relapse illustrates the need for improved intervention strategies. In this study, we characterize a novel antifolate (C1) that, like methotrexate, potently inhibits dihydrofolate reductase (DHFR) and downstream one-carbon metabolism. Contrary to methotrexate, however, C1 displays optimal efficacy in FPGS-deficient contexts, due to decreased competition with intracellular folate concentrations for interaction with DHFR. Indeed, we show that FPGS-deficient patient-derived CRC organoids display enhanced sensitivity to C1-induced growth inhibition, while FPGS-high CRC organoids are more sensitive to methotrexate. Our results thus argue that polyglutamylation-independent antifolates can be applied to exert selective pressure on FPGS-deficient cells during chemotherapy, employing a vulnerability created by polyglutamylation deficiency.

biochemistry↗

bioRxiv2022

Dynamics and composition of small heat shock protein condensates and aggregates

Small heat shock proteins (sHSPs) are essential ATP-independent chaperones that protect the cellular proteome during stress. These proteins assemble into polydisperse oligomeric structures, the composition of which dramatically affects their chaperone activity. The biomolecular consequences of variations in sHSP ratios, especially inside living cells, remain elusive. Here, we study the consequences of altering the relative expression levels of HspB2 and HspB3. These chaperones are partners in a hetero-oligomeric complex, and genetic mutations that abolish their mutual interaction are associated with myopathic disorders. HspB2 displays three distinct phenotypes when co-expressed with HspB3 at varying ratios. Expression of HspB2 alone lead to formation of liquid nuclear condensates, while shifting the stoichiometry towards HspB3 resulted in the formation of large solid-like aggregates. Only cells co-expressing HspB2 with a limited amount of HspB3 showed a homogeneous nuclear distribution of HspB2. Strikingly, both condensates and aggregates were reversible, as shifting the HspB2:HspB3 balance in situ resulted in dissolution of these structures. To uncover the molecular composition of HspB2 condensates and aggregates, we used APEX-mediated proximity labelling. Most proteins interact transiently with the condensates and were neither enriched nor depleted. In contrast, we found that HspB2:HspB3 aggregates sequestered several disordered proteins among which autophagy factors, suggesting that the cell is actively attempting to clear these aggregates. This study presents a striking example of how changes in the relative expression levels of interacting proteins affects their phase behavior. Our approach can be a useful tool to study the role of protein stoichiometry in other biomolecular condensates. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/519563v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@bf5787org.highwire.dtl.DTLVardef@b7a18org.highwire.dtl.DTLVardef@17cd64corg.highwire.dtl.DTLVardef@d9392a_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Small heat shock protein hetero-oligomerization affects their chaperone function - The HspB2:HspB3 expression ratio determines phase separation and aggregation - HspB2 condensates and HspB2:HspB3 aggregates are fully reversible - Proximity labelling unveils autophagy factor recruitment to HspB2:HspB3 aggregates - Stoichiometry-dependant regulation of phase behaviour may be widespread in biology

molecular biology↗

bioRxiv2022

ONECUT2 restricts Microfold cell numbers in the small intestine; a multi-omics study

Microfold (M) cells reside in the intestinal epithelium of Peyers patches. Their unique ability to take up and transport antigens from the intestinal lumen to the underlying lymphoid tissue is key in the regulation of the gut-associated immune response. Here, we applied a (single-cell) multi-omics approach to investigate the molecular mechanisms that drive M cell differentiation in mouse small intestinal organoids. We generated a comprehensive profile of chromatin accessibility changes and transcription factor dynamics during in vitro M cell differentiation, allowing us to uncover numerous cell type-specific regulatory elements and associated transcription factors. Single-cell RNA sequencing resulted in the identification of an M cell precursor population. Our new computational tool SCEPIA determined that these precursor cells were characterized by high expression of and motif activity for the transcription factor ONECUT2. Subsequent perturbation experiments revealed that ONECUT2 acts downstream of the RANK/RANKL signalling to support Enterocyte differentiation and restrict M cell lineage specification in vitro and in vivo, thereby regulating mucosal immunity. This study provides a useful blueprint for future investigations of cell fate switches in the intestinal epithelium.

molecular biology↗

bioRxiv2022

Galectin-9 interacts with Vamp-3 to regulate cytokine secretion in dendritic cells

Intracellular vesicle transport is essential for cellular homeostasis and is partially mediated by SNARE proteins. Endosomal trafficking to the plasma membrane ensures cytokine secretion in dendritic cells (DCs) and the initiation of immune responses. Despite its critical importance, the specific molecular agents that regulate DC cytokine secretion are poorly characterised. Galectin-9, a {beta}-galactoside-binding protein, has emerged as a novel cellular modulator although its exact intracellular roles in regulating (immune) cell homeostasis and vesicle transport are virtually unknown. We investigated galectin-9 function in primary human DCs and report that galectin-9 is essential for intracellular cytokine trafficking to the cell surface. Galectin-9-depleted DCs accumulate cytokine-containing vesicles in the Golgi complex that eventually undergo lysosomal degradation. We observed galectin-9 to molecularly interact with Vamp-3 using immunoprecipitation-mass-spectrometry and identified galectin-9 was required for rerouting Vamp-3-containing endosomes upon DC activation as the underlying mechanism. Overall, this study identifies galectin-9 as a necessary mechanistic component for intracellular trafficking. This may impact our general understanding of vesicle transport and shed new light into the multiple roles galectins play in governing cell function.

cell biology↗

bioRxiv2022

Chromatin regulates genome-wide transcription factor binding affinities

Transcription factor binding across the genome is regulated by DNA sequence and chromatin features. However, it is not yet possible to quantify the impact of chromatin context on genome-wide transcription factor binding affinities. Here we report the establishment of a method to determine genome-wide absolute apparent binding affinities of transcription factors to native, chromatinized DNA. Our experiments revealed that DNA accessibility is the main determinant of transcription factor binding in the genome, which largely restricts nanomolar affinity binding of YY1, SP1 and MYC/MAX to promoters, while FOXA1 also interacts with non-promoter elements with high affinity. Furthermore, whereas consensus DNA binding motifs for transcription factors are important to establish very high-affinity binding sites, these motifs are not always strictly required to generate nanomolar affinity interactions in the genome. Finally, we uncovered transcription factor concentration dependent binding to specific gene classes, suggesting transcription factor concentration dependent effects on gene expression and cell fate. Importantly, our method adds a quantitative dimension to transcription factor biology which enables stratification of genomic targets based on transcription factor concentration and prediction of transcription factor binding sites under non-physiological conditions, such as disease associated overexpression of (onco)genes.

genomics↗

bioRxiv2022

THRONCAT: Efficient metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog

Profiling the nascent cellular proteome and capturing early proteomic changes in response to external stimuli provides valuable insight into cellular physiology. Existing metabolic protein labeling approaches based on bioorthogonal methionine-or puromycin analogs allow for the selective visualization and enrichment of the newly synthesized proteins. However, their applications are limited as they require methionine-free conditions, auxotrophic cells and/or are toxic to cells. Here, we introduce THRONCAT, a novel threonine-derived non-canonical amino acid tagging method based on bioorthogonal threonine analog {beta}-ethynylserine ({beta}ES) that enables efficient and non-toxic labeling of the nascent proteome in complete growth media within minutes. We used THRONCAT for the visualization and enrichment of nascent proteins in bacteria, mammalian cells and Drosophila melanogaster. We profiled immediate proteome dynamics of Ramos B-cells in response to receptor activation, demonstrating the ease-of-use of the method and its potential to address diverse biological questions. In addition, using a Drosophila model of Charcot-Marie-Tooth peripheral neuropathy, we show that THRONCAT enables visualization and quantification of relative protein synthesis rates in vivo.

cell biology↗

bioRxiv2022

Growth inhibition of Akkermansia muciniphila by a secreted pathobiont sialidase

Akkermansia muciniphila is considered a key constituent of a healthy gut microbiota. In inflammatory bowel disease (IBD), A. muciniphila has a reduced abundance while other, putative pathogenic, mucus colonizers bloom. We hypothesized that interbacterial competition may contribute to this observation. By screening the supernatants of a panel of enteric bacteria, we discovered that a previously uncharacterized Allobaculum species potently inhibits the growth of A. muciniphila. Mass spectrometry analysis identified a secreted Allobaculum sialidase as inhibitor of A. muciniphila growth. The sialidase targets sialic acids on casein O-glycans, thereby altering the accessibility of nutrients critical for A. muciniphila. The altered glycometabolic niche results in distorted A. muciniphila cell division and efficiently arrests its growth. The identification of a novel mechanism of A. muciniphila growth inhibition by a competing bacterial pathobiont may provide a rationale for interventions aimed at restoring and maintaining a healthy microbiota symbiosis in patients with intestinal disease.

microbiology↗

bioRxiv2022

Arbovirus-vector protein interactomics identifies Loquacious as a co-factor for dengue virus replication in Aedes mosquitoes

Efficient virus replication in Aedes vector mosquitoes is essential for the transmission of arboviral diseases such as dengue virus (DENV) in human populations. Like in vertebrates, virus-host protein-protein interactions are essential for viral replication and immune evasion in the mosquito vector. Here, 79 mosquito host proteins interacting with DENV non-structural proteins NS1 and NS5 were identified by label-free mass spectrometry, followed by a functional screening. We confirmed interactions with host factors previously observed in mammals, such as the oligosaccharyltransferase complex, and we identified protein-protein interactions that seem to be specific for mosquitoes. Among the interactors, the double-stranded RNA (dsRNA) binding protein Loquacious (Loqs), an RNA interference (RNAi) cofactor, was found to be essential for efficient replication of DENV and Zika virus (ZIKV) in mosquito cells. Loqs did not affect viral RNA stability or translation of a DENV replicon and its proviral activity was independent of its RNAi regulatory activity. Interestingly, Loqs colocalized with DENV dsRNA in viral replication organelles in infected cells and directly interacted with high affinity with DENV RNA in the 3 untranslated region in vitro (KD = 100-200 nM). Our study provides an interactome for DENV NS1 and NS5 and identifies Loqs as a key proviral host factor in mosquitoes. We propose that DENV hijacks a factor of the RNAi mechanism for replication of its own RNA. AUTHOR SUMMARYDengue virus is a mosquito-transmitted virus endemic to the tropics and subtropics, affecting an estimated 390 million people yearly. While the mechanisms of infection, pathogenesis and immune evasion have been extensively studied in humans, replication in Aedes mosquitoes has received much less attention, despite being a critical step in the arbovirus transmission cycle. Here, we used a proteomic approach to identify Aedes mosquito proteins recruited by dengue virus non- structural proteins NS1 and NS5. In addition to previously established host proteins that interact with DENV in mammals, we identified Loquacious, a double-stranded RNA binding protein involved in the antiviral RNAi immune response of mosquitoes. Unexpectedly, our data showed Loquacious functions as a proviral factor that is recruited to replication organelles to facilitate viral RNA replication. We propose that DENV exploits host immune components, such as Loquacious, for its own benefit.

microbiology↗

bioRxiv2022

m6A reader Pho92 is recruited co-transcriptionally and couples translation efficacy to mRNA decay to promote meiotic fitness in yeast

N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.

biochemistry↗

bioRxiv2021

Single-embryo phosphoproteomics reveals the importance of intrinsic disorder in cell cycle dynamics

Switch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

cell biology↗

bioRxiv2021

SARS-CoV-2 501Y.V2 escapes neutralization by South African COVID-19 donor plasma

SARS-CoV-2 501Y.V2 (B.1.351), a novel lineage of coronavirus causing COVID-19, contains substitutions in two immunodominant domains of the spike protein. Here, we show that pseudovirus expressing 501Y.V2 spike protein completely escapes three classes of therapeutically relevant antibodies. This pseudovirus also exhibits substantial to complete escape from neutralization, but not binding, by convalescent plasma. These data highlight the prospect of reinfection with antigenically distinct variants and foreshadows reduced efficacy of spike-based vaccines.

immunology↗

bioRxiv2020

Chromosomal instability by mutations in a novel specificity factor of the minor spliceosome

Aneuploidy is the leading cause of miscarriage and congenital birth defects, and a hallmark of cancer. Despite this strong association with human disease, the genetic causes of aneuploidy remain largely unknown. Through exome sequencing of patients with constitutional mosaic aneuploidy, we identified biallelic truncating mutations in CENATAC (CCDC84). We show that CENATAC is a novel component of the minor (U12-dependent) spliceosome that promotes splicing of a specific, rare minor intron subtype. This subtype is characterized by AT-AN splice sites and relatively high basal levels of intron retention. CENATAC depletion or expression of disease mutants resulted in excessive retention of AT-AN minor introns in ~100 genes enriched for nucleocytoplasmic transport and cell cycle regulators, and caused chromosome segregation errors. Our findings reveal selectivity in minor intron splicing with a specific impact on the chromosome segregation process, and show how defects herein can cause constitutional aneuploidy.

molecular biology↗

bioRxiv2020

SALL4 controls cell fate in response to DNA base composition

SO_SCPLOWUMMARYC_SCPLOWMammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4 which contains multiple zinc-fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely up-regulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4-null cells, including precocious differentiation of embryonic stem cells and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by down-regulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive by-product of genome evolution, but constitutes a signal that aids control of cell fate. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/179481v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1ec4eb9org.highwire.dtl.DTLVardef@1246a36org.highwire.dtl.DTLVardef@1b9134dorg.highwire.dtl.DTLVardef@1a24904_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2020

The m6A reader Ythdf restricts axonal growth in Drosophila through target selection modulation of the Fragile X mental retardation protein

N6-methyladenosine (m6A) regulates a variety of physiological processes through modulation of RNA metabolism. The modification is particularly enriched in the nervous system of several species, and its dysregulation has been associated with neurodevelopmental defects and neural dysfunctions. In Drosophila, loss of m6A alters fly behavior albeit the underlying mechanism and the role of m6A during nervous system development have remained elusive. Here we find that impairment of the m6A pathway leads to axonal overgrowth and misguidance at larval neuromuscular junctions as well as in the adult mushroom bodies. We identify Ythdf as the main m6A reader in the nervous system being required for limiting axonal growth. Mechanistically, we show that Ythdf directly interacts with Fragile X mental retardation protein to inhibit the translation of key transcripts involved in axonal growth regulation. Altogether, this study demonstrates that the m6A pathway controls development of the nervous system by modulating Fmr1 target selection.

molecular biology↗