Biology subjects

Prevorovsky, M.

Publications and source records attributed to Prevorovsky, M..

10 recordsLinked to original sources

bioRxiv2024

Expanding the CarD interaction network: CrsL is a novel transcription factor in Mycobacterium smegmatis

Bacterial transcription regulation is critical for adaptation and survival. CarD is an essential transcription factor in mycobacteria involved in regulation of gene expression. We searched for CarD interaction partners in the model organism Mycobacterium smegmatis and identified two proteins: ApeB (MSMEG_5828) and an uncharacterized protein, which we named CrsL (MSMEG_5890). While ApeB interacted with CarD only when CarD was overexpressed, CrsL associated with CarD at its physiological levels. CrsL is a 5.7 kDa protein shown by NMR to be intrinsically disordered. CrsL homologs are present in actinobacteria including pathogenic species such as Mycobacterium tuberculosis. CrsL directly interacts with CarD and binds RNAP. ChIP-seq showed that CrsL associates with promoters of actively transcribed genes and [~]75 % of these regions are also associated with CarD. RNA-seq showed [~]50% and [~]66% overlap in differentially expressed genes between CrsL and CarD knockdowns during exponential and stationary phases, respectively. CrsL represses expression of DesA desaturase (MSMEG_5773) and DEAD/DEAH-box RNA helicase MSMEG_1930, which are important for adaptation to cold stress. Furthermore, CrsL promotes the growth of M. smegmatis at elevated temperature. In summary, this study identifies CrsL as a novel actinobacterial transcription factor and provides a basis for its further investigation.

microbiology↗

bioRxiv2024

HelD is a Global Transcription Factor Enhancing Gene Expression in Rapidly Growing Mycobacteria

HelD protein, also named HelR (encoded by MSMEG_2174 in Mycobacterium smegmatis), interacts with mycobacterial RNA polymerase (RNAP) and affects rifampicin resistance in Mycobacterium abscessus. Here, we provide data on rifampicin resistance and helD presence in the genomes of other clinically relevant nontuberculous mycobacteria. We show that helD is primarily found in rapidly growing mycobacteria, such as M. smegmatis, where we detected HelD at a subset of promoters that can also associate with CarD and RbpA. Transcriptome analysis of a helD deletion strain using RNA-seq revealed that HelD enhances gene expression during exponential growth and decreases it in stationary phase, during which we observed reduced levels of CarD, RbpA, and GTP, the initiation nucleotide for the majority of M. smegmatis transcripts. We propose a model in which HelD releases abortive RNAP complexes and confirm that HelD dissociates RNAP from the promoter in vitro. HelD not only helps mycobacteria overcome rifampicin treatment but also supports efficient transcription during rapid growth, which indicates a dual role of this transcription regulator.

microbiology↗

bioRxiv2024

Cbf11 and Mga2 function as a single regulatory entity to activate transcription of lipid metabolism genes and promote mitotic fidelity in fission yeast

Within a eukaryotic cell, both lipid homeostasis and faithful cell cycle progression are meticulously orchestrated. The fission yeast Schizosaccharomyces pombe provides a powerful platform to study the intricate regulatory mechanisms governing these fundamental processes. In S. pombe, the Cbf11 and Mga2 proteins are transcriptional activators of non-sterol lipid metabolism genes, with Cbf11 also known as a cell cycle regulator. Despite sharing a common set of target genes, little was known about their functional relationship. This study reveals that Cbf11 and Mga2 function together as a single regulatory entity critical for both lipid metabolism and mitotic fidelity. Deletion of either gene results in a similar array of defects, including slow growth, dysregulated lipid homeostasis, impaired cell cycle progression (cut phenotype), abnormal cell morphology, perturbed transcriptomic and proteomic profiles, and compromised response to the stressors camptothecin and thiabendazole. Remarkably, the double deletion mutant does not exhibit a more severe phenotype compared to the single mutants, suggesting that Cbf11 and Mga2 work together in the same pathway. In addition, ChIP-nexus analysis reveals that both Cbf11 and Mga2 bind to nearly identical positions within the promoter regions of target genes. Interestingly, Mga2 binding appears to be dependent on the presence of Cbf11 and Cbf11 likely acts as a tether to DNA, while Mga2 is needed to activate the target genes. In addition, the study explores the distribution of Cbf11 and Mga2 homologs across fungi. The presence of both Cbf11 and Mga2 homologs in Basidiomycota contrasts with Ascomycota, which mostly lack Cbf11 but retain Mga2. This suggests an evolutionary rewiring of the regulatory circuitry governing lipid metabolism and mitotic fidelity. In conclusion, this study offers compelling support for Cbf11 and Mga2 functioning jointly as a single regulator of lipid metabolism and mitotic fidelity in fission yeast. SUMMARY STATEMENTCbf11 and Mga2, transcriptional activators of non-sterol lipid metabolism genes, function as a single regulatory unit and are both required for proper cell cycle progression in the fission yeast Schizosaccharomyces pombe.

molecular biology↗

bioRxiv2023

Nitrogen availability and TOR signalling are important for preventing catastrophic mitosis in fission yeast

Mitosis is a critical stage in the cell cycle, controlled by a vast network of regulators responding to multiple internal and external factors. The fission yeast Schizosaccharomyces pombe may demonstrate catastrophic mitotic phenotypes due to mutations or drug treatments. One of the factors provoking catastrophic mitosis is a disturbed lipid metabolism, resulting from e.g. mutations in acetyl-CoA/biotin carboxylase (cut6), in fatty acid synthase (fas2/lsd1), or in the transcriptional regulator of lipid metabolism (cbf11) genes, as well as treatment with inhibitors of fatty acid synthesis. It was previously shown that mitotic fidelity in lipid metabolism mutants can be partially rescued by ammonium chloride. In this study we demonstrate that mitotic fidelity can be improved by multiple good nitrogen sources. Moreover, this rescue is not limited to lipid metabolism disturbances but also applies to a number of unrelated mitotic mutants. Interestingly, the rescue is not achieved by restoring the lipid metabolism state, but rather indirectly. We found that the TOR regulatory network plays a major role in mediating such rescue, highlighting a novel role for TOR in mitotic fidelity.

cell biology↗

bioRxiv2023

Critical importance of DNA binding for CSL protein functions in fission yeast

CSL (CBF1/RBP-J{kappa}/Suppressor of Hairless/LAG-1) proteins are conserved transcription factors found in animals and fungi. In fission yeast, they regulate various cellular processes, including cell cycle progression, lipid metabolism, and cell adhesion. CSL proteins bind to DNA through their N-terminal Rel-like domain and central beta-trefoil domain. Here, we investigated the importance of DNA binding for CSL functions in the fission yeast Schizosaccharomyces pombe. We created CSL mutants with disrupted DNA binding and found that the vast majority of CSL functions depend on intact DNA binding. Specifically, DNA binding is crucial for the regulation of cell adhesion, lipid metabolism, cell cycle progression, long non-coding RNA expression, and genome integrity maintenance. Interestingly, perturbed lipid metabolism leads to chromatin structure changes, potentially linking lipid metabolism to the diverse CSL-associated phenotypes. Our study highlights the critical role of DNA binding for CSL protein functions in fission yeast. SUMMARY STATEMENTCSL transcription factors regulate a diverse set of processes, but the mechanisms are not always clear. We show that S. pombe CSL proteins need the ability to bind DNA for most of their roles.

molecular biology↗

bioRxiv2023

POMBOX: a fission yeast toolkit for molecular and synthetic biology

Schizosaccharomyces pombe is a popular model organism in molecular biology and cell physiology. With its ease of genetic manipulation and growth, supported by in-depth functional annotation in the PomBase database and genome-wide metabolic models, S. pombe is an attractive option for synthetic biology applications. However, S. pombe currently lacks modular tools for generating genetic circuits with more than one transcriptional unit. We have developed a toolkit to address this issue. Adapted from the MoClo- YTK plasmid kit for Saccharomyces cerevisiae and using the same Golden Gate grammar, our POMBOX toolkit is designed to facilitate the fast, efficient and modular construction of genetic circuits in S. pombe. It allows for interoperability when working with DNA sequences that are functional in both S. cerevisiae and S. pombe (e.g. protein tag, antibiotic resistance cassette, coding sequences). Moreover, POMBOX enables the modular assembly of multi-gene pathways and increases possible pathway length from 6 to 12 transcriptional units. We also adapted the stable integration vector homology arms to Golden Gate assembly and tested the genomic integration success rate depending on different sequence sizes, from four to twenty-four kilobases. We included fourteen S. pombe promoters that we characterized for two fluorescent proteins, in both minimal defined media (EMM2) and complex media (YES). Then we tested six S. cerevisiae and six synthetic terminators in S. pombe. Finally, we used the POMBOX kit for a synthetic biology application in metabolic engineering and expressed plant enzymes in S. pombe to produce specialized metabolite precursors, namely methylxanthine, amorpha-4,11-diene and cinnamic acid from the purine, mevalonate and amino acid pathways.

synthetic biology↗

bioRxiv2022

Altered cohesin dynamics and histone H3K9 modifications contribute to mitotic defects in the cbf11Δ lipid metabolism mutant

Mitotic fidelity is crucial for the faithful distribution of genetic information into the daughter cells. Many fungal species, including the fission yeast Schizosaccharomyces pombe, undergo a closed form of mitosis, during which the nuclear envelope does not break down. In S. pombe numerous processes have been identified that contribute to successful completion of mitosis. Notably, perturbations of lipid metabolism can lead to catastrophic mitosis and the "cut" phenotype. It was suggested that these mitotic defects are caused by insufficient membrane phospholipid supply during the anaphase nuclear expansion. However, it is not clear whether additional factors are involved. In this study we characterized in detail the mitosis in an S. pombe mutant lacking the Cbf11 transcription factor, which regulates lipid metabolism genes. We show that in cbf11{Delta} cells mitotic defects appear already prior to anaphase, before the nuclear expansion begins. Moreover, we identify altered cohesin dynamics and centromeric chromatin structure as additional factors affecting mitotic fidelity in cells with disrupted lipid homeostasis, providing new insights into this fundamental biological process.

cell biology↗

bioRxiv2022

SAGA histone acetyltransferase module facilitates chromatin accessibility to SMC5/6

Structural Maintenance of Chromosomes (SMC) complexes are molecular machines driving chromatin organization at higher levels. In eukaryotes, three SMC complexes (cohesin, condensin, and SMC5/6) play key roles in cohesion, condensation, replication, transcription and DNA repair. Here, we performed a genetic screen in fission yeast to identify novel factors required for SMC5/6 binding to DNA. We identified 79 genes of which histone acetyltransferases (HATs) were the most represented. Genetic and phenotypic analyses suggested a particularly strong functional relationship between the SMC5/6 and SAGA complexes. Furthermore, several SMC5/6 subunits physically interacted with SAGA HAT module components Gcn5 and Ada2. As Gcn5-dependent acetylation facilitates the accessibility of chromatin to DNA repair proteins, we first analysed the formation of DNA damage-induced SMC5/6 foci in the {Delta}gcn5 mutant. The SMC5/6 foci formed normally in {Delta}gcn5, suggesting SAGA-independent SMC5/6 localization to DNA-damaged sites. Next, we used Nse4-FLAG chromatin-immunoprecipitation (ChIP-seq) analysis in unchallenged cells to assess SMC5/6 distribution. A significant portion of SMC5/6 accumulated within gene regions in wild-type cells, which was reduced in {Delta}gcn5 and {Delta}ada2 mutants. The drop in SMC5/6 levels was also observed in gcn5-E191Q acetyltransferase-dead mutant. Altogether, our data suggest that the SAGA HAT module may facilitate chromatin accessibility to SMC5/6 at gene regions. Author SummaryGenomes of all eukaryotes must be folded and packed into their relatively small nuclear spaces. Histones first pack free genomic DNA into nucleosomes and their arrays. Other complexes like histone modifiers and remodelers can regulate nucleosome positions and their packing within chromatin fibres. They assist in the relative opening or condensation of chromatin fibres and facilitate their accessibility to DNA-binding proteins. The highly conserved Structural Maintenance of Chromosomes (SMC) complexes (cohesin, condensin, and SMC5/6) compact further chromatin fibres at higher levels. These molecular machines can loop chromatin fibres, which need access to segments of free DNA for their physical binding to DNA. Here, we studied genetic and physical interactions between histone-modifying SAGA complex and SMC5/6. We show that the SAGA histone acetyltransferase module may facilitate chromatin access to SMC5/6.

genetics↗

bioRxiv2022

Perturbed fatty-acid metabolism is linked to localized chromatin hyperacetylation, increased stress-response gene expression and resistance to oxidative stress

Oxidative stress is associated with cardiovascular and neurodegenerative diseases, diabetes, cancer, psychiatric disorders and aging. In order to counteract, eliminate and/or adapt to the sources of stress, cells possess elaborate stress-response mechanisms, which also operate at the level of regulating transcription. Interestingly, it is becoming apparent that the metabolic state of the cell and certain metabolites can directly control the epigenetic information and gene expression. In the fission yeast Schizosaccharomyces pombe, the conserved Sty1 stress-activated protein kinase cascade is the main pathway responding to most types of stresses, and regulates the transcription of hundreds of genes via the Atf1 transcription factor. Here we report that fission yeast cells defective in fatty acid synthesis (cbf11, mga2 and ACC/cut6 mutants) show increased expression of a subset of stress-response genes. This altered gene expression depends on Sty1, and the Gcn5 and Mst1 histone acetyltransferases, is associated with increased acetylation of histone H3 at lysine 9 in the corresponding gene promoters, and results in increased cellular resistance to oxidative stress. Since both fatty-acid synthesis and histone acetylation compete for the same substrate, acetyl-CoA, we propose that changes in lipid metabolism can regulate the chromatin and transcription of specific stress-response genes, which in turn might help cells to maintain redox homeostasis.

molecular biology↗

bioRxiv2021

NAD+ capping of RNA in Archaea and Mycobacteria

Chemical modifications of RNA affect essential properties of transcripts, such as their translation, localization and stability. 5-end RNA capping with the ubiquitous redox cofactor nicotinamide adenine dinucleotide (NAD+) has been discovered in organisms ranging from bacteria to mammals. However, the hypothesis that NAD+ capping might be universal in all domains of life has not been proven yet, as information on this RNA modification is missing for Archaea. Likewise, this RNA modification has not been studied in the clinically important Mycobacterium genus. Here, we demonstrate that NAD+ capping occurs in the archaeal and mycobacterial model organisms Methanosarcina barkeri and Mycobacterium smegmatis. Moreover, we identify the NAD+-capped transcripts in M. smegmatis, showing that this modification is more prevalent in stationary phase, and revealing that mycobacterial NAD+-capped transcripts include non-coding small RNAs, such as Ms1. Furthermore, we show that mycobacterial RNA polymerase incorporates NAD+ into RNA, and that the genes of NAD+-capped transcripts are preceded by promoter elements compatible with {sigma}A/{sigma}F dependent expression. Taken together, our findings demonstrate that NAD+ capping exists in the archaeal domain of life, suggesting that it is universal to all living organisms, and define the NAD+-capped RNA landscape in mycobacteria, providing a basis for its future exploration.

molecular biology↗