Biology subjects

van den Berg, J.

Publications and source records attributed to van den Berg, J..

13 recordsLinked to original sources

bioRxiv2026

Unified function of FACT in mammalian chromatin replication and transcription, dissolving and restoring nucleosomes to counteract genome aggregation

Nucleosomes with their associated modifications underlie genome organisation and regulation. Replication and transcription require nucleosome disassembly to access the DNA template. How this is orchestrated without jeopardizing chromatin function remains unknown. Here, we reveal a general, global requirement of the histone chaperone FACT in mammalian replication, transcription and chromatin maintenance. Upon acute FACT depletion, replisome and RNA polymerase progression is halted genome-wide and chromatin structure in their wake collapses with reduced nucleosome occupancy, irregular spacing and intermediate assemblies. Chromatin states deteriorate as modified histones are lost due to a lack of histone recycling. Chromatin fiber disorder further manifests in the 3D genome, triggering active genes to coalesce in aberrant microcompartments. This establishes a unifying mechanistic basis for mammalian chromatin replication and transcription, with FACT mediating nucleosome disruption and re-assembly and thereby guarding against spurious chromatin aggregation. Nucleosome organization can therefore dynamically regulate genome architecture.

molecular biology↗

bioRxiv2025

THE NSL COMPLEX PROMOTES NEURAL DEVELOPMENT BY PREVENTING R-LOOP INDUCED REPLICATION STRESS

Early neuronal development relies on the proliferation of neural progenitor cells, making this developmental stage particularly vulnerable to DNA replication impediments. Here, we identify a non-canonical role for the non-specific lethal (NSL) complex in safeguarding DNA replication during neurodevelopment. The NSL complex, which acetylates histone 4 at gene promoters and is mutated in Koolen-de Vries syndrome (KdVS), prevents unscheduled R-loop accumulation at weakly transcribed promoters that are typically devoid of R-loops. Single-cell sequencing reveals that loss of NSL causes replisome stalling and delayed S-phase progression. Neural organoids derived from KdVS patients exhibit impaired DNA replication, developmental abnormalities, and reduced synapse formation, driven entirely by unscheduled R-loop accumulation. These findings reveal that faithful DNA replication is critical for early neurodevelopment.

genomics↗

bioRxiv2025

WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

The WEE1 kinase negatively regulates CDK1/2 to control DNA replication and mitotic entry. Genetic factors that determine sensitivity to WEE1 inhibitors (WEE1i) are largely unknown. A genome-wide insertional mutagenesis screen revealed that mutation of EIF2A, a translation regulator, sensitized to WEE1i. Mechanistically, WEE1i treatment triggers a translational shut-down, which is lethal in combination with the reduced translation of EIF2AKO cells. A genome-wide CRISPR-Cas9 screen revealed that inactivation of integrated stress response (ISR) kinases GCN1/2 rescued WEE1i-mediated cytotoxicity. WEE1i induced GCN2 activation, ATF4 upregulation, and altered ribosome dynamics. Loss of the collided ribosome sensor ZNF598 conversely increased sensitivity to WEE1i. Notably, the ISR was not required for WEE1i to induce DNA damage, premature mitotic entry or sensitization to DNA-damaging chemotherapeutics. ISR activation was independent of CDK1/2 activation. Importantly, WEE1i-mediated ISR activation was independent of WEE1 presence, pointing at off-target effects, which are shared by multiple chemically distinct WEE1i. This response was also observed in peripheral blood mononuclear cells. Importantly, low-dose WEE1 inhibition did not induce ISR activation, while it still synergized with PKMYT1 inhibition. Taken together, WEE1i triggers toxic ISR activation and translational shutdown, which can be prevented by low-dose or combination treatments, while retaining the cell cycle checkpoint-perturbing effects.

cancer biology↗

bioRxiv2024

Acute multi-level response to defective de novo chromatin assembly in S-phase

Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly, with single-cell genomics, quantitative proteomics, and live-microscopy to uncover these initiating mechanisms in human cells. CAF-1 loss immediately slows down DNA replication speed and renders nascent DNA hyper-accessible. A rapid cellular response, distinct from canonical DNA damage signaling, is triggered and lowers histone mRNAs. As a result, histone variants usage and their modifications are altered, limiting transcriptional fidelity and delaying chromatin maturation within a single S-phase. This multi-level response induces a cell-cycle arrest after mitosis. Our work reveals the immediate consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered. HighlightsO_LIThe histone chaperone CAF-1 sustains DNA replication speed in single cells. C_LIO_LICAF-1 loss alters histone repertoire and delays chromatin maturation. C_LIO_LIH3K9me3 and H3K27me3 regions respond differently to acute CAF-1 depletion. C_LIO_LIImpaired S-phase chromatin assembly triggers an immediate response and a G0 arrest. C_LI

cell biology↗

bioRxiv2024

Assessing the effects of acute temperature changes of sea water temperature due to climate change on Scottish farmed Atlantic salmon and investigating genetic mitigation

Changes in growth, survival, maturation and health in Atlantic salmon post-smolts were observed under moderate and more extreme heat-wave conditions for the west coast of Scotland. A Scottish Atlantic salmon breeding programme population of 518 salmon of age 13-14 months, was observed for 4 weeks in summer, in tanks with ambient water temperature or [~] 4 degrees and [~] 8 degrees above ambient temperature. Data were recorded for the fish before and after the challenge. All fish were genotyped using a custom SNP (single nucleotide polymorphism) array (8,978 SNP genotypes after quality control). Temperature-dependant genotype-by-environment interactions and the potential for selective breeding to improve resilience were investigated. Statistical analyses revealed significant differences between thermal environments for body weight, average daily weight gain, and survival, while higher temperature induced earlier maturation, and an increase of gill health scores. QPCR (quantitative polymerase chain reaction) analyses revealed the presence of Aeromonas hydrophila, a pathogen typically more prevalent in warmer climates. Using genomic relationships and animal mixed models, body weight and average daily weight gain provided moderate heritabilities, while between-tank genetic correlations were close to 1, indicating no significant re-ranking of genotypes between the different thermal environments. These findings suggest that even short-term exposure to heat stress may be sufficient to negatively impact survival and gill health, and induce earlier maturation. However, observations took place within a commercial farm where having replicates for each thermal environment was not possible, hence further experiments with larger populations, exposed to more prolonged heat stress are needed.

genetics↗

bioRxiv2022

Acceleration of genome replication uncovered by single-cell nascent DNA sequencing

In a human cell thousands of replication forks simultaneously coordinate the duplication of the entire genome. The rate at which this process occurs, might depend on the epigenetic state of the genome and vary between, or even within, cell types. To accurately measure DNA replication speeds, we developed a technology to detect recently replicated DNA using single-cell sequencing. Replication speed is not constant but increases during S-phase of the cell cycle. Using genetic and pharmacological perturbations we are able to alter this acceleration of replication and conclude that DNA damage inflicted by the process of transcription limits the speed of replication during early S-phase. In late S-phase, during which less transcription occurs, replication accelerates and approaches its maximum speed.

molecular biology↗

bioRxiv2022

Druggable Growth Dependencies and Tumor Evolution Analysis in Patient-Derived Organoids of Neuroendocrine Cancer

Neuroendocrine neoplasms (NENs) comprise well-differentiated neuroendocrine tumors and poorly-differentiated carcinomas. Treatment options for patients with NENs are limited, in part due to lack of accurate models. To address this need we established the first patient-derived tumor organoids (PDTOs) from pulmonary neuroendocrine tumors and derived PDTOs from an understudied NEN subtype, large cell neuroendocrine carcinoma (LCNEC). PDTOs maintain the gene expression patterns, intra-tumoral heterogeneity, and evolutionary processes of parental tumors. Through drug sensitivity analyses, we uncover therapeutic sensitivities to an inhibitor of NAD salvage biosynthesis and to an inhibitor of BCL-2. Finally, we identify a dependency on EGF in pulmonary neuroendocrine tumor PDTOs. Consistent with these findings, analysis of an independent cohort showed that approximately 50% of pulmonary neuroendocrine tumors expressed EGFR. This study identifies a potentially actionable vulnerability for a subset of NENs, and further highlights the utility of these novel PDTO models for the study of NENs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=188 HEIGHT=200 SRC="FIGDIR/small/514549v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@14bed0borg.highwire.dtl.DTLVardef@1b7791forg.highwire.dtl.DTLVardef@7120a6org.highwire.dtl.DTLVardef@116b21d_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPDTOs of pulmonary NETs and LCNEC were established C_LIO_LIPDTOs recapitulate intra-tumoral heterogeneity and evolution of parental tumors C_LIO_LIDrug assays reveal therapeutic vulnerabilities and biomarkers C_LIO_LIPulmonary NET PDTOs are dependent on EGF C_LI

cancer biology↗

bioRxiv2020

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

DNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

molecular biology↗

bioRxiv2020

Secreted autotaxin through LPA suppresses chemotaxis and tumor infiltration of CD8+ T Cells

Autotaxin (ATX) is secreted by diverse cell types to produce lysophosphatidic acid (LPA) that regulates multiple biological functions via G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis mainly via LPAR1; however, its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T-cell repulsion predominantly involves G12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T-cell responses but suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from patient samples are consistent with intra-tumor ATX acting as a T-cell repellent. These studies highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T-cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

cancer biology↗

bioRxiv2020

A FOXO-dependent replication checkpoint restricts proliferation of damaged cells

DNA replication is challenged by numerous exogenous and endogenous factors that can interfere with the progression of replication forks. Stalling or slowing of the replication fork as a result of replication stress leads to formation of aberrant single-stranded DNA (ssDNA) stretches and potentially DNA double-stranded-breaks (DSBs). Accumulation of ssDNA activates the ATR-dependent DNA replication stress checkpoint response that slows progression from S/G2- to M-phase to protect genomic integrity (1). However, whether mild replication stress restricts proliferation remains controversial (2-6). Here we identify a novel cell cycle exit mechanism, that prevents S/G2 phase arrested cells from undergoing mitosis after exposure to mild replication stress through premature activation of the CDH1 bound Anaphase Promoting Complex / Cyclosome (APC/CCDH1). We find that replication stress causes a gradual decrease of the levels of the APC/CCDH1 inhibitor EMI1/FBXO5 through Forkhead Box O (FOXOs) mediated repression of its transcriptional regulator E2F1. By doing so, FOXOs limit the time during which the replication stress checkpoint is reversible, and thereby play an important role in maintaining genomic stability.

molecular biology↗

bioRxiv2019

DNA end-resection in highly accessible chromatin produces a toxic break

The authors have withdrawn their manuscript whilst they perform additional experiments to test some of their conclusions further. Despite repetitive attempts to reproduce the data shown in figure 2I, we have failed to obtain convincing evidence that chemical inhibition of Mre11 can improve the fitness of cells exposed to the so-called "halt" breaks. Upon re-analysis of the raw data used to compose figure 2I, we noted inconsistencies in the inclusion of datapoints, for which a satisfactory motivation was not apparent. In order to reproduce the experiments with the DNA-PK inhibitor, we ordered new batches of this compound, but noticed that the IC50 was significantly lower than that of the earlier batch (as determined in suppl. fig.1). While this might be due to a difference in batches, the IC50 of the newer batches was more comparable to the IC50 of this compound described for other cell lines. This sheds doubts on the quality of the batch used for the experiments shown in our manuscript, and we no longer wish to draw any conclusions based on these experiments. The authors hope that future experiments can resolve if open chromatin does indeed produce a potentially more toxic DNA break. The authors do not wish this work to be cited as reference for the project. If you have any questions, please contact the corresponding author.

cell biology↗

bioRxiv2019

Diversity of fall armyworm, Spodoptera fugiperda and their gut bacterial community in Kenya

The invasive fall armyworm (FAW), Spodoptera frugiperda (J.E. Smith) is a polyphagous pest that causes widespread damage particularly to maize and sorghum in Africa. The microbiome associated with S. frugiperda could play a role in their success and adaptability. However, these bacterial communities remain poorly studied, especially for S. frugiperda in Africa. We investigated the composition, abundance and diversity of microbiomes associated with larval and adult specimens of S. frugiperda collected from four maize growing regions in Kenya through high throughput sequencing of bacterial 16S rRNA gene. We identified Proteobacteria and Firmicutes as the most dominant phyla and lesser proportions of Bacteroidetes and Actinobacteria. We also observed differences in bacterial microbiome diversity between larvae and adults that are a likely indication that some prominent larval bacterial groups are lost during metamorphosis. Several bacterial groups were found in both adults and larvae suggesting that they are transmitted across developmental stages. Reads corresponding to several known entomopathogenic bacterial clades as well as the non-bacterial entomopathogen, Metarhizium rileyi (Farl.) Kepler, Rehner & Humber (2014), were observed. Mitochondrial DNA haplotyping of the S. frugiperda population in Kenya indicated the presence of both Rice and Corn strains, with a higher prevalence of the Rice strain. Insights into the microbiota may ultimately provide alternative avenues for controlling of this pest.

molecular biology↗

bioRxiv2018

A limited number of double-strand DNA breaks are sufficient to delay cell cycle progression.

DNA damaging agents cause a variety of lesions, of which DNA double-strand breaks (DSBs) are the most genotoxic. Unbiased approaches aimed at investigating the relationship between the number of DSBs and outcome of the DNA damage response have been challenging due to the random nature in which damage is induced by classical DNA damaging agents. Here we describe a CRISPR/Cas9-based system that permits us to efficiently introduce DSBs at defined sites in the genome. Using this system, we show that a guide RNA targeting only a single site in the human genome can trigger a checkpoint response that is potent enough to delay cell cycle progression. Abrogation of this checkpoint leads to DNA breaks in mitosis which give rise to micronucleated daughter cells.

cell biology↗