Biology subjects

Guenther, T.

Publications and source records attributed to Guenther, T..

9 recordsLinked to original sources

bioRxiv2026

Temporal Profiling of Upper-Layer Neurons Reveals Changes in the Molecular Landscape Upon Maternal Immune Activation

Neuronal differentiation is a dynamic, multi-layered process that transforms progenitor cells into functionally integrated neurons, yet the coordinated molecular programs underlying this transition remain incompletely understood. Here, we define the developmental trajectory of murine upper-layer cortical neurons using an integrated multi-omics approach combining transcriptomics and proteomics across key stages of neurogenesis. We find that neuronal identity emerges gradually through coordinated transitions from RNA processing and splicing programs toward synaptic and metabolic maturation. Integration of matched transcriptomic and proteomic datasets reveals a compact set of concordant molecular modules that define this trajectory, highlighting post-transcriptional regulation as a central driver of neuronal maturation. We further show that maternal immune activation (MIA), a model of prenatal inflammation, deranges this developmental program. MIA induces sustained upregulation of Wnt signalling pathways alongside a downregulation of synaptic regulators, without detectable global alterations in DNA methylation. These molecular changes are accompanied by defects in neuronal positioning during cortical development, linking altered molecular trajectories to functional outcomes. Together, our findings establish a temporal molecular framework of neuronal differentiation and demonstrate that prenatal environmental perturbations reshape cortical development primarily through post-transcriptional and signalling-based mechanisms.

neuroscience↗

bioRxiv2025

Merkel cell polyomavirus infection and persistence modelled in skin organoids

Merkel cell polyomavirus (MCPyV) causes most Merkel cell carcinomas (MCC). The virus is one of the few known human tumor viruses, and due to its direct role in this skin cancer development, it is a bona fide model for viral tumorigenesis and tumorigenesis in general. Chronic viruses in humans such as MCPyV are highly adapted to their host and current models to study infection, persistence and pathogenesis are highly limited. We here use an induced pluripotent stem cell (iPSC)-derived hair-bearing skin organoid (SkO) system to demonstrate efficient viral infection, progression and spread of MCPyV. Using bulk-, single cell - and spatial-transcriptomics, combined with immunostaining and nucleic acid hybridization technologies, we show that MCPyV ensures persistence due to a quasi-latency state within the majority of dermal fibroblasts carry the viral genome. Further, we identify the cell type of productive infection with papillary fibroblasts and dermal sheath fibroblasts supporting viral replication and progeny production. Our high-resolution methods demonstrate that the virus in these cells evades the innate immune response, as evidenced by the efficacy of interferon-beta treatment or ruxolitinib, a JAK/STAT inhibitor, in suppressing or stimulating viral replication. We show that iPSC-derived SkOs are able to support infection and long-term persistence of the virus under conditions very similar to those found in humans. Thus, this infection model provides a robust platform for understanding and characterizing the interaction of this virus with the immune system in infection, testing treatment strategies to control reactivation and map processes involved in tumor development.

molecular biology↗

bioRxiv2025

Polycomb group proteins protect latent Kaposi sarcoma-associated herpesvirus from episome clearance and HUSH-dependent chromatin silencing

Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of a number of human neoplasms. KSHV causes lifelong chronic infections by establishing persistent reservoirs of latently infected cells in which the viral double-stranded DNA genome is present in the form of an extrachromosomal episome. Polycomb group protein (PcG) mediated gene repression is detected early after entry of the CpG-rich and epigenetically naive viral DNA into the host nucleus. Here we show that the establishment and maintenance of KSHV latency is strongly influenced by Polycomb Repressive Complexes 1 and 2 (PRC1, and PRC2, respectively). Using a series of genetic deletion models, we dissect the functional interdependence of PRC1 and PRC2 on KSHV and identify KSHV episomes as a prime target for independent as well as collaborative de novo PRC recruitment. Loss of PRC function leads to a severely altered chromatin state on KSHV and we show that PRC mediated gene repression is not only preventing transcription of viral genomes but also protects KSHV episomes from detection by the human silencing hub (HUSH). Furthermore, we demonstrate that PRC complexes play an important role in ensuring episomal maintenance in dividing cells. Taken together, our results show that absence of PRC complexes creates a metastable state of KSHV latency and suggest early chromatinization as an integral step for KSHV latency establishment.

microbiology↗

bioRxiv2025

Spatiotemporal analysis of de novo KSHV infection using Crispr/Cas9-based 3D live cell imaging at single episome resolution

Kaposi Sarcoma-associated herpesvirus (KSHV) persists as a latent episome in infected cells. While the virus efficiently infects established cell lines and primary cells in vitro, the early events guiding establishment of latent infection and the dynamic interplay between viral episomes and host factors remain incompletely understood. Here, we describe the development and application of a CRISPR/Cas9-based 3D live cell imaging system capable of tracking single KSHV episomes in real-time. Our approach exploits the SunTag technology, wherein deactivated Cas9 (dCas9) molecules are fused to repetitive epitope arrays recognized by superfolder GFP-fused single-chain antibodies. By targeting these complexes to terminal repeat units of KSHV, we achieve high level signal amplification, allowing us not only to detect newly incoming viral genomes within the first hours of de novo infection, but also to follow their spatiotemporal trajectories through different stages of the viral lifecycle. Furthermore, to facilitate efficient generation of stable reporter cell lines, we adapted the transposon-based piggyBac system to combine all SunTag components into a single-vector targeting system (SunSeT). Using these systems, we demonstrate the ability to observe both transient and stable interactions between KSHV episomes and key cellular regulators, including the variant polycomb-repressive complex 1 (vPRC) component KDM2B and the innate immune sensor IFI16. Furthermore, our platform allows detailed visualization of episodic changes in episome localization, abundance and distribution during de novo and long-term infection, providing critical insights into how viral genome positioning and dynamics correlate with host subnuclear environments. Overall, our study introduces a robust and adaptable imaging platform to dissect the earliest events of KSHV infection. The ability to track viral episomes in living cells offers a powerful tool to advance our understanding of the spatial and temporal regulation of individual KSHV genomes, shedding light on fundamental mechanisms of herpesvirus latency and persistence.

microbiology↗

bioRxiv2025

Intrinsic immunity against HAdV is achieved by a novel epigenetic silencing complex

The DNA double-stranded genome of human adenoviruses (HAdV) is preferentially targeted by host factors involved in chromatin remodeling, such as SPOC1 and KAP1 to inhibit efficient viral gene expression. HAdV genomes undergo alterations through association with host histones and epigenetic modifications; however, the precise underlying mechanism remains elusive. A recently discovered silencing mechanism for retrotransposons and retroviruses involves the Human Silencing Hub (HUSH) complex. This complex of MPP8, TASOR, and PPHLN1 safeguards the human genome by utilizing histone H3 Lys9 trimethylation (H3K9me3) to block transcription. Through the recruitment of SETDB1 and MORC2, the HUSH complex silences host genes and condenses target genomes to combat infections such as HIV, MLV, and AAV. Here, we present evidence that the HUSH complex effectively restricts HAdV infection. To counteract the repressive function of this epigenetic silencing complex, HUSH factors are inhibited through binding of HAdV proteins and subsequent relocalization. We observe that MPP8 is targeted by the adenoviral E3 ubiquitin ligase, thus recruited by the viral early proteins E1B-55K and E4orf6 for proteasomal degradation. In summary, we provide evidence that the HUSH complex is a previously unrecognized host factor that restricts HAdV gene expression and replication. Based on these novel findings, we propose that HUSH represents a promising therapeutic target to combat HAdV infection.

cell biology↗

bioRxiv2025

E2A SUMOylation promotes HAdV mediated inhibition of the tumor suppressor p53 during infection

HAdV express early viral genes to modulate the activity of the cellular tumor suppressor p53 and ensure efficient viral replication, through various processes, whilst some of them are not completely understood. HAdV oncoprotein E1B-55K interactions with SUMO, PML-IV/V and Sp100A are tightly connected to the transforming potential of the viral factor in non-lytic infections. SUMO modified HAdV DNA binding protein (DBP) E2A interacts with PML and Sp100A acting as a molecular bridge between viral replication centers (RCs) and PML tracks. Here, we provide a novel concept showing that HAdV E2A is involved in the inhibition of p53 activity and apoptosis; thus we identified a novel route of exhibiting virus induced oncogenic potential. We revealed an E1B-55K independent localization of p53 within HAdV replication centers marked by E2A. In the absence of any functional E1B-55K protein expression, p53 interacts with E2A. E2A SUMOylation promotes HAdV- mediated inhibition of p53/DNA binding and p53-dependent transactivation by supporting p53 degradation, E1B-55K interaction and colocalization with PML-V that leads to E1B-55K induced upregulation of p53 SUMOylation. In sum, we provide evidence for a novel mechanism by which the HAdV DBP E2A protein inhibits p53- mediated transcriptional activation and apoptotic host cell response. Intriguingly, these findings change the longstanding dogma of host DNA damage response (DDR) inactivation by HAdV infection and offer crucial insights to improve future p53 selective oncolytic virus therapies and adenoviral vectors.

cell biology↗

bioRxiv2023

The Cytomegalovirus M35 Protein Modulates Transcription of Ifnb1 and Other IRF3-Driven Genes by Direct Promoter Binding

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defence a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system. M35 localizes to the nucleus and interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35s function. Using electrophoretic mobility shift assays (EMSA), we demonstrate that purified M35 protein specifically binds to the regulatory DNA element that governs transcription of the first type I IFN gene induced in non-immune cells, Ifnb1. Determination of M35s crystal structure combined with reverse genetics revealed that homodimerisation is a key feature for M35s immunomodulatory activity. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signalling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signalling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labelled transcripts (SLAM-seq), and assessed M35s global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically down-regulated basal expression of IRF3-dependent genes, and during MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonises gene induction by IRF3 and impairs the antiviral response more broadly than formerly recognised. ImportanceReplication of the ubiquitous human cytomegalovirus (CMV) in healthy individuals mostly goes unnoticed, but can impair foetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes. Unrelated proteins from other herpesviruses employ the same mechanism, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.

molecular biology↗

bioRxiv2021

Epigenetic reprogramming of human macrophages by an intestinal pathogen

Various pathogens systematically reprogram gene expression in macrophages, but the underlying mechanisms are largely unknown. We investigated whether the enteropathogen Yersinia enterocolitica alters chromatin states to reprogram gene expression in primary human macrophages. Genome-wide chromatin immunoprecipitation (ChIP) seq analyses showed that pathogen-associated molecular patterns (PAMPs) induced up- or down-regulation of histone modifications (HMod) at approximately 14500 loci in promoters and enhancers. Effectors of Y. enterocolitica reorganized about half of these dynamic HMod, with the effector YopP being responsible for about half of these modulatory activities. The reorganized HMod were associated with genes involved in immune response and metabolism. Remarkably, the altered HMod also associated with 61 % of all 534 known Rho GTPase pathway genes, revealing a new level in Rho GTPase regulation and a new aspect of bacterial pathogenicity. Changes in HMod were associated to varying degrees with corresponding gene expression, e. g. depending on chromatin localization and cooperation of the HMod. In summary, infection with Y. enterocolitica remodels HMod in human macrophages to modulate key gene expression programs of the innate immune response. Author SummaryHuman pathogenic bacteria can affect epigenetic histone modifications to modulate gene expression in host cells. However, a systems biology analysis of this bacterial virulence mechanism in immune cells has not been performed. Here we analyzed genome-wide epigenetic histone modifications and associated gene expression changes in primary human macrophages infected with enteropathogenic Yersinia enterocolitica. We demonstrate that Yersinia virulence factors extensively modulate histone modifications and associated gene expression triggered by the pathogen-associated molecular patterns (PAMPs) of the bacteria. The epigenetically modulated genes are involved in several key pathways of the macrophage immune response, including the Rho GTPase pathway, revealing a novel level of Rho GTPase regulation by a bacterial pathogen. Overall, our findings provide an in-depth view of epigenetic and gene expression changes during host-pathogen interaction and might have further implications for understanding of the innate immune memory in macrophages.

microbiology↗

bioRxiv2020

SARS-CoV-2 infects carotid arteries: implications for vascular disease and organ injury in COVID-19

Stroke and central nervous system dysfunction are cardinal symptoms in critically ill corona virus disease 19 (COVID-19) patients. In an autopsy series of 32 COVID-19 patients, we investigated whether carotid arteries were infected with SARS-CoV-2 by employing genomic, virologic, histochemical and transcriptomic analyses. We show that SARS-CoV-2 productively infects and modulates vascular responses in carotid arteries. This finding has far reaching implications for the understanding and clinical treatment of COVID-19.

microbiology↗