Biology subjects

Hebenstreit, D.

Publications and source records attributed to Hebenstreit, D..

11 recordsLinked to original sources

bioRxiv2026

Linking H3K36me3 to transcriptional dynamics

Genes are transcribed in bursts as they fluctuate between on and off states, in a process modulated by histone modifications. The mammalian histone methyltransferase SETD2 is solely responsible for depositing H3K36 trimethylation, which recruits histone deacetylases and regulates histone occupancy. Nucleosomes are known to act as a barrier to transcription elongation and have been observed to impact elongation rate. H3K36me3 has been associated with long pausing sites in gene bodies and is implicated in reducing speed of successive RNA polymerase II. However, the role of SETD2 and H3K36me3 in transcriptional dynamics are unknown. Here we show upon SETD2 knockout and H3K36me3 depletion in murine C2C12 myoblast cells that overall gene expression is reduced and splicing fidelity affected, as 5' mutually exclusive exons are favoured over 3' ones. Furthermore, transcriptional burst size is reduced, while bursting frequency increased. A simple mathematical model suggests that the observed changes in transcriptional dynamics can be explained by less stringently controlled polymerase pausing in the gene bodies of H3K36me3 depleted cells.

systems biology↗

bioRxiv2025

Immune cell de novo steroidogenesis regulates inflammation resolution and recovery in acute lung injury

Effective resolution of inflammation following acute lung infection or injury is critical for restoring immune and tissue homeostasis to ensure functional recovery. Prolonged or unresolved inflammation can impair lung repair, promote fibrosis, and contribute to pulmonary dysfunction. While systemic steroid signalling is known to modulate general immune responses, the specific role of immune cell-mediated steroidogenesis in regulating lung inflammation and repair remains unknown. Here, we show that immune cell de novo steroidogenesis is essential for resolving inflammation and promoting recovery in a murine model of acute lung injury. During the resolution phase, steroid-synthesizing immune cells, predominantly basophils, are enriched in the lung. Mice with immune cell-specific ablation of de novo steroidogenesis exhibit exacerbated lung injury, impaired resolution of inflammation, and defective tissue repair. These findings reveal a previously unrecognized immunoregulatory function of immune cell-derived steroids and identify immune cell steroidogenesis as a potential therapeutic target for promoting resolution and recovery in inflammatory lung diseases.

immunology↗

bioRxiv2022

Ceg1 depletion reveals mechanisms governing degradation of non-capped RNAs

Most functional eukaryotic mRNAs contain a 7-methylguanosine (m7G) cap which serves as a platform that recruits proteins to support essential biological functions such as mRNA processing, nuclear export and translation. Although capping is accomplished during the first steps of transcription the fate and turnover of uncapped transcripts have not been studied extensively. Here, we employed fast nuclear depletion of the capping enzymes in Saccharomyces cerevisiae to uncover the turnover of the transcripts that failed to be capped. We show that the levels of non-capped mRNAs are determined principally by the abundance of their synthesis. Nuclear depletion of the capping enzymes increases the levels of lowly expressed mRNAs and decreases mRNAs that are highly transcribed altogether mimicking the effects observed in cells lacking the predominantly cytoplasmic 5-3 exonuclease Xrn1. The nuclear 5-3 exonuclease Rat1 is not involved in the degradation of cap-defective transcripts and the lack of the capping does not affect the distribution of RNA Polymerase II on the chromatin. Our data indicate that the mRNAs that failed to be capped are not directed to a specific quality-control pathway and that the 5 cap role is associated with the Xrn1-dependent buffering of the cellular mRNA levels, along with protecting from 5-3 degradation.

molecular biology↗

bioRxiv2022

Synergising single-cell resolution and 4sU labelling boosts inference of transcriptional bursting

Despite the recent rise of RNA-seq datasets combining single-cell (sc) resolution with 4-thiouridine (4sU) labelling, analytical methods exploiting their power to dissect transcriptional bursting are lacking. Here, we present a mathematical model and Bayesian inference implementation to facilitate genome-wide joint parameter estimation and confidence quantification. We demonstrate that, unlike conventional scRNA-seq, 4sU scRNA-seq resolves temporal parameters and furthermore boosts inference of dimensionless parameters via a synergy between single-cell resolution and 4sU labelling. We applied our method to published 4sU scRNA-seq data and linked with ChIP-seq data, uncovering previously obscured associations between different parameters and histone modifications.

bioinformatics↗

bioRxiv2021

CMTr cap-adjacent 2`-O-ribose mRNA methyltransferases are required for reward learning and mRNA localization to synapses

Cap-adjacent nucleotides of animal, protist and viral mRNAs can be dynamically O-methylated at the 2 position of the ribose (cOMe). The functions of cOMe in animals, however, remain unknown. Here we show that the two cap methyltransferases (CMTr1 and CMTr2) of Drosophila can methylate the ribose of the first nucleotide in mRNA. Double-mutant flies lack cOMe but are viable. Consistent with prominent neuronal expression, they have a reward learning defect that can be rescued by conditional expression in mushroom body neurons before training. Among CMTr targets are cell adhesion and signaling molecules relevant for learning and cOMe is required for local translation of mRNAs at synapses. Hence, our study reveals a mechanism to co-transcriptionally prime mRNAs by cOMe for localized protein synthesis at synapses.

molecular biology↗

bioRxiv2021

RPRD Proteins Control Transcription in Human Cells

The regulation of transcription is an essential process that allows the cell to respond to various internal and external signals. RNA Polymerase II (Pol II) activity is controlled by a number of factors which bind to the C-terminal domain (CTD) of its largest subunit, RPB1, and stimulate or suppress RNA synthesis. Here, we demonstrate that CTD-interacting proteins, RPRD2, RPRD1B and RPRD1A act as negative regulators of transcription and their levels inversely correlate with the accumulation of nascent and newly transcribed RNA in human cells. We show that the RPRD proteins form mutually exclusive complexes with Pol II to coordinate their roles in transcriptional control. Our data indicate that RPRD2 exerts the most substantial impact on transcription and has the potential to alter key biological processes including the cellular stress response and cell growth.

molecular biology↗

bioRxiv2021

Non-coding RNAs Associated with Prader-Willi Syndrome Regulate Transcription of Neurodevelopmental Genes in Human Induced Pluripotent Stem Cells

Mutations and aberrant gene expression during cellular differentiation lead to neurodevelopmental disorders such as Prader-Willi syndrome (PWS) which results from the deletion of an imprinted locus on chromosome 15. We analysed chromatin-associated RNA in human induced pluripotent cells (iPSCs) upon depletion of hybrid small nucleolar long non-coding RNAs (sno-lncRNAs) and 5 snoRNA capped and polyadenylated long non-coding RNAs (SPA-lncRNAs) transcribed from the locus deleted in PWS. We found that rapid ablation of these lncRNAs affects transcription of specific gene classes. Downregulated genes contribute to neurodevelopment and neuronal maintenance while genes that are upregulated are predominantly involved in the negative regulation of cellular metabolism and apoptotic processes. Our data revealed the importance of SPA-lncRNAs and sno-lncRNAs in controlling gene expression in iPSCs and provided a platform for synthetic experimental approaches in PWS studies. We conclude that ncRNAs transcribed from the PWS locus are critical regulators of a transcriptional signature important for neuronal differentiation and development.

molecular biology↗

bioRxiv2021

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive and it correlates with Pol II transcription and mRNA expression levels. While changes in Pol II occupancy were detected at only some genes in a Upf1-deficient (upf1{Delta}) strain, there is an increased Ser2 Pol II signal at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1{Delta} cells are hypersensitive to the transcription elongation inhibitor 6-azauracil and display Pol II abnormalities suggestive of Pol II hyperphosphorylation. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1{Delta}. These data envisage that by operating on the nascent transcript Upf1 might influence Pol II phosphorylation and transcription.

molecular biology↗

bioRxiv2019

Polymerase recycling contributes to transcriptional noise

BackgroundTranscription in mammalian cells is a complex stochastic process involving shuttling of polymerase between genes and phase-separated liquid condensates. It occurs in bursts, which results in vastly different numbers of an mRNA species in isogenic cell populations. Several factors contributing to transcriptional bursting have been identified, usually classified as intrinsic, in other words local to single genes, or extrinsic, relating to the macroscopic state of the cell. However, some possible contributors have not been explored yet. Here, we focus on processes at the 3 and 5 ends of a gene that enable reinitiation of transcription upon termination. ResultsUsing Bayesian methodology, we measure the transcriptional bursting in inducible transgenes, showing that perturbation of polymerase shuttling typically reduces burst size, increases burst frequency, and thus limits transcriptional noise. Analysis based on paired-end tag sequencing (PolII ChIA-PET) suggests that this effect is genome wide. The observed noise patterns are also reproduced by a generative model that captures major characteristics of the polymerase flux between the ends of a gene and a phase-separated compartment. ConclusionsInteractions between the 3 and 5 ends of a gene, which facilitate polymerase recycling, are major contributors to transcriptional noise.

systems biology↗

bioRxiv2018

Timing Polymerase Pausing with TV-PRO-seq

Transcription of many genes in metazoans is subject to polymerase pausing, which is the transient stop of transcriptionally-engaged polymerases. This is known to mainly occur in promoter proximal regions but it is not well understood. In particular, a genome-wide measurement of pausing times at high resolution has been lacking. We present here the time-variant precision nuclear run-on and sequencing (TV-PRO-seq) assay, an extension of the standard PRO-seq that allows us to estimate genome-wide pausing times at single-base resolution. Its application to human cells demonstrates that, proximal to promoters, polymerases pause more frequently but for shorter times than in other genomic regions. Pausing release by the detergent sarkosyl, previously believed to be linked to the factor NELF at the promoter proximal region only, is independent of the latter. Comparison with single-cell gene expression data reveals that the polymerase pausing times are longer in highly expressed genes, while transcriptionally noisier genes have higher pausing frequencies and slightly longer pausing times. Analyses of histone modifications suggest that the marker H3K36me3 is related to the polymerase pausing.

systems biology↗

bioRxiv2018

The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required in nuclear processes of gene expression

UPF1 is an RNA helicase that is required for efficient nonsense-mediated mRNA decay (NMD) in eukaryotes, and the predominant view is that UPF1 mainly operates on the 3UTRs of mRNAs that are directed for NMD in the cytoplasm. Here we offer evidence, obtained from Drosophila, that UPF1 constantly moves between the nucleus and cytoplasm and that it has multiple functions in the nucleus. It is associated, genome-wide, with nascent RNAs at most of the active Pol II transcription sites and at some Pol III-transcribed genes, as demonstrated microscopically on the polytene chromosomes of salivary gland and by ChIP-seq analysis in S2 cells. Intron recognition seems to interfere with association and translocation of UPF1 on nascent pre-mRNA transcripts, and cells depleted of UPF1 show defects in several nuclear processes essential to correct gene expression - most strikingly, the release of mRNAs from transcription sites and mRNA export from the nucleus.

cell biology↗