Mediator dynamics during heat shock in budding yeast

The Mediator complex is central to transcription by RNA polymerase II (Pol II) in eukaryotes. In budding yeast (Saccharomyces cerevisiae), Mediator is recruited by activators and associates with core promoter regions, where it facilitates pre-initiation complex (PIC) assembly, only transiently prior to Pol II escape. Interruption of the transcription cycle by inactivation or depletion of Kin28 inhibits Pol II escape and stabilizes this association. However, Mediator occupancy and dynamics have not been examined on a genome-wide scale in yeast grown in nonstandard conditions. Here we investigate Mediator occupancy following heat shock or CdCl2 exposure, with and without depletion of Kin28. We find that Pol II occupancy exhibits similar dependence on Mediator under normal and heat shock conditions. However, while Mediator association increases at many genes upon Kin28 depletion under standard growth conditions, little or no increase is observed at most genes upon heat shock, indicating a more stable association of Mediator after heat shock. Mediator remains associated upstream of the core promoter at genes repressed by heat shock or CdCl2 exposure whether or not Kin28 is depleted, suggesting that Mediator is recruited by activators but is unable to engage PIC components at these repressed targets. This persistent association is strongest at promoters that bind the HMGB family member Hmo1, and is reduced but not eliminated in hmo1∆ yeast. Finally, we show a reduced dependence on PIC components for Mediator occupancy at promoters after heat shock, further supporting altered dynamics or stronger engagement with activators under these conditions.

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Altered Mediator dynamics during heat shock in budding yeast

10.1101/2020.08.25.267088 ◽

2020 ◽

Author(s):

Debasish Sarkar ◽

Z. Iris Zhu ◽

Emily Paul ◽

David Landsman ◽

Randall H. Morse

Keyword(s):

Heat Shock ◽

Rna Polymerase Ii ◽

Growth Conditions ◽

Proximal Promoter ◽

Promoter Regions ◽

Pol Ii ◽

Genome Wide ◽

A Genome ◽

Similar Dependence ◽

Wide Scale

AbstractThe Mediator complex is central to transcription by RNA polymerase II (Pol II) in eukaryotes. In yeast, Mediator is recruited by activators via its tail module and then facilitates assembly of the pre-initiation complex (PIC), including Pol II, setting the stage for productive transcription. Mediator occupies proximal promoter regions only transiently prior to Pol II escape; interruption of the transcription cycle by inactivation or depletion of Kin28 inhibits Pol II escape and stabilizes Mediator occupancy at promoters. However, whether Mediator occupancy and dynamics differ for gene cohorts induced by stress or alternative growth conditions has not been examined on a genome-wide scale. Here we investigate Mediator occupancy following heat shock or CdCl2 induction, with or without depletion of Kin28. We find that Pol II occupancy exhibits similar dependence on Mediator under normal and heat shock conditions; however, Mediator occupancy does not increase upon Kin28 depletion at most genes active during heat shock, indicating altered dynamics. Furthermore, Mediator occupancy persists at genes repressed by heat shock or CdCl2 induction and exhibits peaks upstream of the proximal promoter whether or not Kin28 is depleted, suggesting that Mediator is recruited by activators but is unable to engage PIC components at these repressed targets. Finally, we show a reduced dependence on PIC components for Mediator occupancy at promoters after heat shock, further supporting an altered dynamics or stronger engagement with activators under these conditions.

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CRISPR-based transcriptional activation tool for silent genes in filamentous fungi

Scientific Reports ◽

10.1038/s41598-020-80864-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

László Mózsik ◽

Mirthe Hoekzema ◽

Niels A. W. de Kok ◽

Roel A. L. Bovenberg ◽

Yvonne Nygård ◽

...

Keyword(s):

Filamentous Fungi ◽

Transcriptional Activation ◽

Core Promoter ◽

Crop Protection ◽

Gene Clusters ◽

Growth Conditions ◽

Biosynthetic Gene ◽

Biosynthetic Gene Clusters ◽

Fluorescent Reporter ◽

A Genome

AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.

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Computational identification and experimental characterization of preferred downstream positions in human core promoters

10.1101/2021.02.02.429413 ◽

2021 ◽

Author(s):

René Dreos ◽

Nati Malachi ◽

Anna Sloutskin ◽

Philipp Bucher ◽

Tamar Juven-Gershon

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Motif Discovery ◽

Core Promoter ◽

Sequence Motifs ◽

Promoter Element ◽

Pol Ii ◽

Core Promoters ◽

The Core

AbstractMetazoan core promoters, which direct the initiation of transcription by RNA polymerase II (Pol II), may contain short sequence motifs termed core promoter elements/motifs (e.g. the TATA box, initiator (Inr) and downstream core promoter element (DPE)), which recruit Pol II via the general transcription machinery. The DPE was discovered and extensively characterized in Drosophila, where it is strictly dependent on both the presence of an Inr and the precise spacing from it. Since the Drosophila DPE is recognized by the human transcription machinery, it is most likely that some human promoters contain a downstream element that is similar, though not necessarily identical, to the Drosophila DPE. However, only a couple of human promoters were shown to contain a functional DPE, and attempts to computationally detect human DPE-containing promoters have mostly been unsuccessful. Using a newly-designed motif discovery strategy based on Expectation-Maximization probabilistic partitioning algorithms, we discovered preferred downstream positions (PDP) in human promoters that resemble the Drosophila DPE. Available chromatin accessibility footprints revealed that Drosophila and human Inr+DPE promoter classes are not only highly structured, but also similar to each other, particularly in the proximal downstream region. Clustering of the corresponding sequence motifs using a neighbor-joining algorithm strongly suggests that canonical Inr+DPE promoters could be common to metazoan species. Using reporter assays we demonstrate the contribution of the identified downstream positions to the function of multiple human promoters. Furthermore, we show that alteration of the spacing between the Inr and PDP by two nucleotides results in reduced promoter activity, suggesting a strict spacing dependency of the newly discovered human PDP on the Inr. Taken together, our strategy identified novel functional downstream positions within human core promoters, supporting the existence of DPE-like motifs in human promoters.Author summaryTranscription of genes by the RNA polymerase II enzyme initiates at a genomic region termed the core promoter. The core promoter is a regulatory region that may contain diverse short DNA sequence motifs/elements that confer specific properties to it. Interestingly, core promoter motifs can be located both upstream and downstream of the transcription start site. Variable compositions of core promoter elements have been identified. The initiator (Inr) motif and the downstream core promoter element (DPE) is a combination of elements that has been identified and extensively characterized in fruit flies. Although a few Inr+DPE - containing human promoters have been identified, the presence of transcriptionally important downstream core promoter positions within human promoters has been a matter of controversy in the literature. Here, using a newly-designed motif discovery strategy, we discovered preferred downstream positions in human promoters that resemble fruit fly DPE. Clustering of the corresponding sequence motifs in eight additional species indicated that such promoters could be common to multicellular non-plant organisms. Importantly, functional characterization of the newly discovered preferred downstream positions supports the existence of Inr+DPE-containing promoters in human genes.

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High similarity among ChEC-seq datasets

10.1101/2021.02.04.429774 ◽

2021 ◽

Author(s):

Chitvan Mittal ◽

Matthew J. Rossi ◽

B. Franklin Pugh

Keyword(s):

Rna Polymerase Ii ◽

Transcriptional Regulators ◽

Micrococcal Nuclease ◽

High Similarity ◽

Dna Interactions ◽

Promoter Regions ◽

Genome Wide ◽

A Genome ◽

Protein Dna Interactions ◽

Negative Controls

AbstractChEC-seq is a method used to identify protein-DNA interactions across a genome. It involves fusing micrococcal nuclease (MNase) to a protein of interest. In principle, specific genome-wide interactions of the fusion protein with chromatin result in local DNA cleavages that can be mapped by DNA sequencing. ChEC-seq has been used to draw conclusions about broad gene-specificities of certain protein-DNA interactions. In particular, the transcriptional regulators SAGA, TFIID, and Mediator are reported to generally occupy the promoter/UAS of genes transcribed by RNA polymerase II in yeast. Here we compare published yeast ChEC-seq data performed with a variety of protein fusions across essentially all genes, and find high similarities with negative controls. We conclude that ChEC-seq patterning for SAGA, TFIID, and Mediator differ little from background at most promoter regions, and thus cannot be used to draw conclusions about broad gene specificity of these factors.

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HSFs drive stress type-specific transcription of genes and enhancers

10.1101/2021.11.23.469701 ◽

2021 ◽

Author(s):

Samu V Himanen ◽

Mikael C Puustinen ◽

Alejandro J Da Silva ◽

Anniina Vihervaara ◽

Lea Sistonen

Keyword(s):

Oxidative Stress ◽

Heat Shock ◽

Rna Polymerase Ii ◽

Molecular Mechanisms ◽

Gene Promoters ◽

Heat Shock Factors ◽

Pol Ii ◽

Bona Fide ◽

Stressed Cells ◽

First Time

Reprogramming of transcription is critical for the survival under cellular stress. Heat shock has provided an excellent model to investigate nascent transcription in stressed cells, but the molecular mechanisms orchestrating RNA synthesis during other types of stress are unknown. We utilized PRO-seq and ChIP-seq to study how Heat Shock Factors, HSF1 and HSF2, coordinate transcription at genes and enhancers upon oxidative stress and heat shock. We show that pause-release of RNA polymerase II (Pol II) is a universal mechanism regulating gene transcription in stressed cells, while enhancers are activated at the level of Pol II recruitment. Moreover, besides functioning as conventional promoter-binding transcription factors, HSF1 and HSF2 bind to stress-induced enhancers to trigger Pol II pause-release from poised gene promoters. Importantly, HSFs act at distinct genes and enhancers in a stress type-specific manner. HSF1 binds to many chaperone genes upon oxidative and heat stress but activates them only in heat-shocked cells. Under oxidative stress, HSF1 and HSF2 trans-activate genes independently of each other, demonstrating, for the first time, that HSF2 is a bona fide transcription factor. Taken together, we show that HSFs function as multi-stress-responsive factors that activate specific genes and enhancers when encountering changes in temperature and redox state.

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The miR-430 locus with extreme promoter density is a transcription body organizer, which facilitates long range regulation in zygotic genome activation

10.1101/2021.08.09.455629 ◽

2021 ◽

Author(s):

Yavor Hadzhiev ◽

Lucy Wheatley ◽

Ledean Cooper ◽

Federico Ansaloni ◽

Celina Whalley ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Gene Cluster ◽

Core Promoter ◽

Single Copy ◽

Early Embryo ◽

Zygotic Genome Activation ◽

Pol Ii ◽

A Minor ◽

Genome Activation ◽

Zygotic Genome

In anamniote embryos the major wave of zygotic genome activation (ZGA) starts during the mid-blastula transition. This major wave of ZGA is facilitated by several mechanisms, including dilution of repressive maternal factors and accumulation of activating transcription factors during the fast cell division cycles preceding the mid-blastula transition. However, a set of genes escape global genome repression and are activated substantially earlier, during what is called, the minor wave of genome activation. While the mechanisms underlying the major wave of genome activation have been studied extensively, the minor wave of genome activation is little understood. In zebrafish the earliest expressed RNA polymerase II (Pol II) transcribed genes are activated in a pair of large transcription bodies depleted of chromatin, abundant in elongating Pol II and nascent RNAs (Hadzhiev et al., 2019; Hilbert et al., 2021). This transcription body includes the miR-430 gene cluster required for maternal mRNA clearance. Here we explored the genomic, chromatin organisation and cis-regulatory mechanisms of the minor wave of genome activation occurring in the transcription body. By long read genome sequencing we identified a remarkable cluster of miR-430 genes with over 300 promoters and spanning 0.6 Mb, which represent the highest promoter density of the genome. We demonstrate that the miR-430 gene cluster is required for the formation of the transcription body and acts as a transcription organiser for minor wave activation of a set of zinc finger genes scattered on the same chromosome arm, which share promoter features with the miR-430 cluster. These promoter features are shared among minor wave genes overall and include the TATA-box and sharp transcription start site profile. Single copy miR-430 promoter transgene reporter experiments indicate the importance of promoter-autonomous mechanisms regulating escape from global repression of the early embryo. These results together suggest that formation of the transcription body in the early embryo is the result of high promoter density coupled to a minor wave-specific core promoter code for transcribing key minor wave ZGA genes, which are required for the overhaul of the transcriptome during early embryonic development.

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Perturbations of transcription and gene expression-associated processes alter distribution of cell size values in Saccharomyces cerevisiae

10.1101/461210 ◽

2018 ◽

Author(s):

Nairita Maitra ◽

Jayamani Anandhakumar ◽

Heidi M. Blank ◽

Craig D. Kaplan ◽

Michael Polymenis

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Rna Polymerase Ii ◽

Cell Size ◽

Gamma Distribution ◽

Single Gene ◽

Growth Conditions ◽

Wild Type ◽

Pol Ii ◽

Diploid Cells

ABSTRACTThe question of what determines whether cells are big or small has been the focus of many studies because it is thought that such determinants underpin the coupling of cell growth with cell division. In contrast, what determines the overall pattern of how cell size is distributed within a population of wild type or mutant cells has received little attention. Knowing how cell size varies around a characteristic pattern could shed light on the processes that generate such a pattern and provide a criterion to identify its genetic basis. Here, we show that cell size values of wild type Saccharomyces cerevisiae cells fit a gamma distribution, in haploid and diploid cells, and under different growth conditions. To identify genes that influence this pattern, we analyzed the cell size distributions of all single-gene deletion strains in Saccharomyces cerevisiae. We found that yeast strains which deviate the most from the gamma distribution are enriched for those lacking gene products functioning in gene expression, especially those in transcription or transcription-linked processes. We also show that cell size is increased in mutants carrying altered activity substitutions in Rpo21p/Rpb1, the largest subunit of RNA polymerase II (Pol II). Lastly, the size distribution of cells carrying extreme altered activity Pol II substitutions deviated from the expected gamma distribution. Our results are consistent with the idea that genetic defects in widely acting transcription factors or Pol II itself compromise both cell size homeostasis and how the size of individual cells is distributed in a population.

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Live-cell single particle imaging reveals the role of RNA polymerase II in histone H2A.Z eviction

eLife ◽

10.7554/elife.55667 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 5

Author(s):

Anand Ranjan ◽

Vu Q Nguyen ◽

Sheng Liu ◽

Jan Wisniewski ◽

Jee Min Kim ◽

...

Keyword(s):

Rna Polymerase ◽

Rna Polymerase Ii ◽

Single Molecule ◽

Transcription Initiation ◽

General Mechanism ◽

Pol Ii ◽

Promoter Escape ◽

Genome Wide ◽

A Genome ◽

Particle Imaging

The H2A.Z histone variant, a genome-wide hallmark of permissive chromatin, is enriched near transcription start sites in all eukaryotes. H2A.Z is deposited by the SWR1 chromatin remodeler and evicted by unclear mechanisms. We tracked H2A.Z in living yeast at single-molecule resolution, and found that H2A.Z eviction is dependent on RNA Polymerase II (Pol II) and the Kin28/Cdk7 kinase, which phosphorylates Serine 5 of heptapeptide repeats on the carboxy-terminal domain of the largest Pol II subunit Rpb1. These findings link H2A.Z eviction to transcription initiation, promoter escape and early elongation activities of Pol II. Because passage of Pol II through +1 nucleosomes genome-wide would obligate H2A.Z turnover, we propose that global transcription at yeast promoters is responsible for eviction of H2A.Z. Such usage of yeast Pol II suggests a general mechanism coupling eukaryotic transcription to erasure of the H2A.Z epigenetic signal.

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Stress-Induced Transcriptional Memory Accelerates Promoter-Proximal Pause-Release and Decelerates Termination over Mitotic Divisions

10.1101/576959 ◽

2019 ◽

Cited By ~ 1

Author(s):

Anniina Vihervaara ◽

Dig Bijay Mahat ◽

Samu V. Himanen ◽

Malin A.H. Blom ◽

John T. Lis ◽

...

Keyword(s):

Heat Shock ◽

Rna Polymerase Ii ◽

Transcriptional Response ◽

Regulatory Elements ◽

List Type ◽

Pol Ii ◽

Daughter Cells ◽

Heat Shocks ◽

Transcriptional Memory ◽

Pol Ii Pausing

SummaryHeat shock triggers an instant reprogramming of gene and enhancer transcription, but whether cells encode a memory to stress, at the level of nascent transcription, has remained unknown. Here, we measured transcriptional response to acute heat stress in unconditioned cells and in daughters of cells that had been exposed to a single or multiple heat shocks. Tracking RNA Polymerase II (Pol II) genome-wide at nucleotide-resolution revealed that cells precisely remember their transcriptional identity throughout stress, restoring Pol II distribution at gene bodies and enhancers upon recovery. However, single heat shock primed faster gene-induction in the daughter cells by increasing promoter-proximal Pol II pausing, and accelerating the pause-release. In repeatedly stressed cells, both basal and inducible transcription was refined, and pre-mRNA processing decelerated, which retained transcripts on chromatin and reduced recycling of the transcription machinery. These results mechanistically uncovered how the steps of pause-release and termination maintain transcriptional memory over mitosis.Highlights-Cell type-specific transcription precisely recovers after heat-induced reprogramming-Single heat shock primes genes for accelerated induction over mitotic divisionsviaincreased promoter-proximal Pol II pausing and faster pause-release-Multiple heat shocks refine basal and inducible transcription over mitotic divisions to support survival of the daughter cells-Decelerated termination at active genes reduces recycling of Pol II to heat-activated promoters and enhancers-HSF1 increases the rate of promoter-proximal pause-releaseviadistal and proximal regulatory elements

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Characterization of the radiation desiccation response regulon of the radioresistant bacterium Deinococcus radiodurans by integrative genomic analyses.

10.1101/2021.07.07.451423 ◽

2021 ◽

Author(s):

Nicolas Eugenie ◽

Yvan Zivanovic ◽

Gaelle Lelandais ◽

Genevieve Coste ◽

Claire Bouthier de la Tour ◽

...

Keyword(s):

Deinococcus Radiodurans ◽

Normal Growth ◽

Growth Conditions ◽

Rna Seq ◽

Genome Wide ◽

A Genome ◽

Genomic Analyses ◽

Number Of Genes ◽

Wide Scale

Numerous genes are overexpressed in the radioresistant bacterium Deinococcus radiodurans after exposure to radiation or prolonged desiccation. The DdrO and IrrE proteins play a major role in regulating the expression of approximately predicted twenty of these genes. The transcriptional repressor DdrO blocks the expression of these genes under normal growth conditions. After exposure to genotoxic agents, the IrrE metalloprotease cleaves DdrO and relieves gene repression. Bioinformatic analyzes showed that this mechanism seems to be conserved in several species of Deinococcus, but many questions remain as such the number of genes regulated by DdrO. Here, by RNA-seq and CHiP-seq assays performed at a genome-wide scale coupled with bioinformatic analyses, we show that, the DdrO regulon in D. radiodurans includes many other genes than those previously described. These results thus pave the way to better understand the radioresistance mechanisms encoded by this bacterium.

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