Biology subjects

Vastenhouw, N.

Publications and source records attributed to Vastenhouw, N..

5 recordsLinked to original sources

bioRxiv2025

In vivo genome-wide CRISPRi-Seq in Streptococcus pneumoniae reveals host-adaptive pathways essential for meningitis and identifies new therapeutic targets

Streptococcus pneumoniae is an important human pathogen that causes invasive diseases and remains the leading cause of bacterial meningitis. However, the bacterial determinants required for survival within the central nervous system (CNS) are poorly understood. Here, we performed a genome-wide CRISPR interference screen (CRISPRi-Seq) in an in vivo zebrafish model to systematically map pneumococcal fitness determinants during meningitis. Using this approach, we investigated essential and non-essential genes contributing to the pathogenesis of pneumococcal meningitis. The screen identified 244 loci whose repression significantly reduced bacterial fitness in vivo, representing pathways involved in virulence, metabolism, cell-envelope biogenesis, translation, and stress adaptation. Comparative analysis with in vitro datasets showed that metabolic genes such as purA, proABC, thrC, glyA, and manLMN, as well as those involved in peptidoglycan and capsule biosynthesis (pbp3, cps2 operon) and oxidative stress response (nox, dpr), become selectively more essential in vivo. Functional validation confirmed that these pathways are critical for virulence, nutrient-dependent growth, and oxidative stress response during meningitis. Untargeted metabolomics of infected zebrafish corroborate several observed bacterial genetic sensitivities in the CNS with altered levels of key nutrients such as glucose and 4-aminobenzoic acid. Integration of the CRISPRi-Seq dataset with antibiotic-target annotations revealed both established and previously unrecognised vulnerabilities, including aminoacyl-tRNA synthetases such as leucyl-tRNA synthetase (LeuRS), whose inhibition by the benzoxaborole epetraborole improved host survival, highlighting its potential as a new therapeutic strategy for multidrug-resistant (MDR) S. pneumoniae. Together, these findings demonstrate the power of in vivo CRISPRi-Seq to define the pneumococcal essentialome under physiological conditions and reveal that metabolic adaptation, cell-envelope maintenance, and the oxidative stress response are central to bacterial survival in the CNS. This fitness map advances our understanding of pneumococcal adaptation in the CNS and identifies promising targets for developing therapies against S. pneumoniae.

microbiology↗

bioRxiv2022

Nanog organizes transcription bodies

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. How proteins of the transcriptional machinery come together to form such bodies, however, is unclear. Here, we take advantage of two large, isolated, and long-lived transcription bodies that reproducibly form during early zebrafish embryogenesis, to characterize the dynamics of transcription body formation. Once formed, these transcription bodies are enriched for initiating and elongating RNA polymerase II, as well as the transcription factors Nanog and Sox19b. Analyzing the events leading up to transcription, we find that Nanog and Sox19b cluster prior to transcription, and independently of RNA accumulation. The clustering of transcription factors is sequential; Nanog clusters first, and this is required for the clustering of Sox19b and the initiation of transcription. Mutant analysis revealed that both the DNA-binding domain, as well as one of the two intrinsically disordered regions of Nanog are required to organize the two bodies of transcriptional activity. Taken together, our data suggests that the clustering of transcription factors dictates the formation of transcription bodies. HIGHLIGHTSO_LITranscription factors cluster prior to, and independently of transcription C_LIO_LINanog organizes transcription bodies: it is required for the clustering of Sox19b as well as RNA polymerase II C_LIO_LIThis organizing activity requires its DNA binding domain as well as one of its intrinsically disordered regions C_LIO_LITranscription elongation results in the disassembly of transcription factor clusters C_LI

cell biology↗

bioRxiv2019

Quantitative measurements of chromatin modification dynamics during zygotic genome activation

Histone posttranslational modifications (PTMs) are key gene expression regulators, but their rapid dynamics during development remain difficult to capture. We describe an approach that images fluorescent antibody fragments to quantify PTM enrichment and active transcription at defined loci during zygotic genome activation in living zebrafish embryos. The technique reveals a drastic increase in histone H3 Lys27 acetylation (H3K27ac) before genome activation both at a specific locus and globally.

cell biology↗

bioRxiv2017

Transcription establishes microenvironments that organize euchromatin

Chromatin is organized into heterochromatin, which is transcriptionally inactive, and euchromatin, which can switch between transcriptionally active and inactive states. This switch in euchromatin activity is accompanied by changes in its spatial distribution. How euchromatin rearrangements are established is unknown. Here we use super-resolution and live-cell microscopy to show that transcriptionally inactive euchromatin moves away from transcriptionally active euchromatin. This movement is driven by the formation of RNA-enriched microenvironments that exclude inactive euchromatin. Using theory, we show that the segregation into RNA-enriched microenvironments and euchromatin domains can be considered an active microemulsion. The tethering of transcripts to chromatin via RNA polymerase II forms effective amphiphiles that intersperse the two segregated phases. Taken together with previous experiments, our data suggest that chromatin is organized in the following way: heterochromatin segregates from euchromatin by phase separation, while transcription organizes euchromatin similar to an active microemulsion.

cell biology↗

bioRxiv2017

Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

SUMMARYUpon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.

developmental biology↗