Biology subjects

Wilhelm, A.

Publications and source records attributed to Wilhelm, A..

6 recordsLinked to original sources

bioRxiv2026

Chromatin crosstalk between HDA19 and NuA4 sets thresholds for stress gene activation in Arabidopsis

Histone acetylation by the plant NuA4 complex promotes high expression of growth-related genes while preventing gene body H2A.Z depletion and spurious activation of stress-responsive loci. How this NuA4-dependent chromatin state is modulated by histone deacetylases (HDACs) in plants remains unclear. Here, we investigate the interplay between NuA4 and HDACs in Arabidopsis by generating a collection of HDAC loss-of-function mutants in NuA4-proficient and NuA4-deficient backgrounds. Loss of individual HDACs did not rescue the severe growth defects of the NuA4(-) mutant; instead, most combinations further aggravated the phenotype, indicating that HDACs predominantly support rather than counteract NuA4 function. Focusing on the hda19-5Atepl1ballele, we show that loss of HDA19 causes strong developmental defects and constitutive activation of biotic stress-responsive genes, but not canonical heat-response genes. 3' RNA-seq and ChIP-seq reveal that upregulated genes in hda19-5Atepl1bdisplay increased H3K9ac and H2A.Zac with largely unchanged gene-body H2A.Z, and substantially overlap with genes induced in the NuA4-null Atepl1-2 mutant and in wild-type plants exposed to elevated temperature. Integration of our datasets with published HDA19 ChIP-seq maps shows that H2A.Zac is selectively increased at HDA19-bound H2A.Z peaks in hda19-5Atepl1b, consistent with a direct role for HDA19 in H2A.Z deacetylation. However, elevated H2A.Zac is neither necessary nor sufficient for transcriptional activation, whereas changes in H3K9ac correlate strongly with gene induction. We propose that NuA4 and HDA19 cooperate to tune chromatin at stress-related loci, with H3K9 acetylation as the primary driver of transcription and H2A.Z acetylation acting as a modulatory mark that shapes stress gene responsiveness.

plant biology↗

bioRxiv2025

Modifying meiotic recombination by targeting chromatin modifiers to crossover hotspots in Arabidopsis

The impact of specific chromatin modifications on meiotic crossover frequency is usually inferred from correlative studies, leaving open the question of causality. To address this, we used a dCas9-based system to target histone H3 methylation modifiers to defined genomic loci. Targeting methyltransferases responsible for H3K9me3 and H3K27me3 had little effect on recombination at selected hotspots, whereas targeting H3K4me3- and H3K36me3-associated enzymes often silenced the endogenous genes. The strongest effect was observed with the demethylase JMJ14, which reduced local H3K4me3 levels and decreased crossover frequency within the targeted interval. This was accompanied by reduced transcription of a long non-coding RNA (lncRNA) located at the hotspot and altered crossover topology. Suppressed recombination was also seen at neighbouring, untargeted hotspots. Conversely, directing the transcriptional activator VP64 to the same region elevated lncRNA expression, increased crossover frequency, and raised H3K4me3 levels. Our results reveal a causal relationship between H3K4me3, transcription, and local crossover activity, demonstrating that H3K4me3 levels are tightly associated with both transcriptional output and recombination frequency at specific genomic sites.

genetics↗

bioRxiv2024

Intestinal interstitial fluid isolation provides novel insight into the human host-microbiome interface

AimsThe gastrointestinal (GI) tract is composed of distinct subregions which exhibit segment-specific differences in microbial colonization and (patho)physiological characteristics. Gut microbes can be collectively considered as an active endocrine organ. Microbes produce metabolites, which can be taken up by the host and can actively communicate with the immune cells in the gut lamina propria with consequences for cardiovascular health. Variation in bacterial load and composition along the GI tract may influence the mucosal microenvironment and thus be reflected its interstitial fluid (IF). Characterization of the segment-specific microenvironment is challenging and largely unexplored because of lack of available tools. Method and ResultsHere, we developed methods, namely tissue centrifugation and elution, to collect IF from the mucosa of different intestinal segments. These methods were first validated in rats and mice, and the tissue elution method was subsequently translated for use in humans. These new methods allowed us to quantify microbiota-derived metabolites, mucosa-derived cytokines, and proteins at their site-of-action. Quantification of short-chain fatty acids showed enrichment in the colonic IF. Metabolite and cytokine analyses revealed differential abundances within segments, often significantly increased compared to plasma, and proteomics revealed that proteins annotated to the extracellular phase were site-specifically identifiable in IF and were differentially expressed when compared to matched serum, all suggesting local synthesis. ConclusionCollection of IF from defined segments and the direct measurement of mediators at the site-of-action in rodents and humans bypasses the limitations of indirect analysis of fecal samples or serum, providing direct insight into this understudied compartment.

microbiology↗

bioRxiv2023

Cellular hnRNPA0 limits HIV-1 production by interference with LTR-activity and programmed ribosomal frame-shifting

The interplay between host factors and viral components has a profound impact on the viral replication efficiency and fitness. Heterogeneous nuclear ribonucleoproteins (hnRNPs), in particular members of the subfamily A/B, have been broadly studied as HIV-1 host dependency factors, however, the least related member hnRNPA0 has so far not been functionally studied in its potential role affecting viral replication. In this study, we revealed that hnRNPA0 overexpression in HEK293T cells significantly reduced HIV-1 long terminal repeat (LTR) activity up to 3.5-fold, leading to a significant decrease in total viral mRNA (5.5-fold) and protein levels (3-fold). Conversely, knockdown of hnRNPA0 enhanced LTR activity, suggesting its negative regulatory role in viral gene expression. Moreover, the splicing pattern of HIV-1 remained largely unaffected by altered hnRNPA0 levels indicating changes in viral mRNA expression predominantly occurred at the transcriptional level. Moreover, hnRNPA0 overexpression was found to significantly reduce the programmed ribosomal frameshift efficiency of HIV-1, resulting in a shift in the HIV-1 p55/p15 ratio, compromising viral fitness. Synergistic inhibition of LTR activity and thus reduced viral mRNA transcription and impaired ribosomal frameshifting efficiency, which is important for viral infectivity, were detrimental to HIV-1 replication. Additionally, our study revealed that hnRNPA0 levels were lower in therapy naive HIV-1-infected individuals compared to healthy controls and temporarily repressed after IFN-I treatment in HIV-1 target cells. Our findings highlight the significant role of hnRNPA0 in HIV-1 replication and suggests that its IFN-I regulated expression levels are decisive for viral fitness. ImportanceRNA binding proteins, in particular heterogeneous nuclear ribonucleoproteins (hnRNPs) have been extensively studied as host dependency factors for HIV-1 since they are involved in multiple cellular gene expression processes. However, the functional role of hnRNPA0, the least related member of the hnRNPA/B family, and its potential impact on viral replication remains unclear. For the first time, our findings demonstrate the significance of hnRNPA0 in restricting viral replication efficiency. We demonstrate that hnRNPA0 plays a pleiotropic role in limiting viral replication being a negative regulator of viral transcription and significantly impairing ribosomal frameshifting. Our study also revealed hnRNPA0 as an IFN-regulated host factor that is temporarily repressed after IFN-I treatment in HIV-1 target cells and lower expressed in therapy-naive HIV-1-infected individuals compared to healthy controls. Understanding the mode of action between hnRNPA0 and HIV-1 might help to identify novel therapeutically strategies against HIV-1 and other viruses.

microbiology↗

bioRxiv2021

Generation of a Sleeping Beauty transposon-based cellular system for rapid and sensitive identification of SARS-CoV-2 host dependency and restriction factors

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. The common methods to monitor and quantitate SARS-CoV-2 infectivity in cell culture are so far time-consuming and labor-intensive. Using the Sleeping Beauty transposase system, we generated a robust and versatile reporter cell system that allows SARS-CoV-2 infection experiments compatible for high-throughput and live cell imaging. The reporter cell is based on lung derived A549 cells, which show a profound interferon response and convenient cell culture characteristics. ACE2 and TMPRSS2 were introduced for constitutive expression in A549 cells. Subclones with varying levels of ACE2/TMPRSS2 were screened for optimal SARS-CoV2 susceptibility. Furthermore, extensive evaluation demonstrated that SARS-CoV-2 infected reporter cells were distinguishable from mock-infected cells and already showed approximately 12 h post infection a clear signal to noise ratio in terms of cell roughness, fluorescence and a profound visible cytopathic effect. Moreover, due to the high transfection efficiency and proliferation capacity, Sleeping Beauty transposase-based overexpression cell lines with a second inducible fluorescence reporter cassette (eGFP) can be generated in a very short time, enabling the investigation of host and restriction factors in a doxycycline-inducible manner. Thus, the novel reporter cell line allows rapid and sensitive detection of SARS-CoV-2 infection and the screening for host factors essential for viral replication. Highlights- Sleeping Beauty transposon-based cellular system was used to generate a highly susceptible cell line for monitoring SARS-CoV-2 infection - The versatile reporter cell line A549-AT is suitable for rapid and sensitive high-throughput assays - Additional gene specific expression cassettes allow the identification of SARS-CoV-2 host dependency and restriction factors

microbiology↗

bioRxiv2021

Fast, efficient and virus-free generation of TRAC-replaced CAR T cells

Chimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at early clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use.

immunology↗