scholarly journals Altered Mediator dynamics during heat shock in budding yeast

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.

Mediator dynamics during heat shock in budding yeast

2021 ◽  
pp. gr.275750.121
Author(s):  
Debasish Sarkar ◽  
Z. Iris Zhu ◽  
Elisabeth R. Knoll ◽  
Emily Paul ◽  
David Landsman ◽  
...  
Keyword(s):  
Heat Shock ◽  
Rna Polymerase Ii ◽  
Budding Yeast ◽  
Core Promoter ◽  
Growth Conditions ◽  
Promoter Regions ◽  
Pol Ii ◽  
A Genome ◽  
Wide Scale ◽  
Stable Association

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.

scholarly journals High similarity among ChEC-seq datasets

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.

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

eLife ◽  
2020 ◽  
Vol 9 ◽  
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.

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

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.

scholarly journals Genome-Wide Mapping of Boundary Element-Associated Factor (BEAF) Binding Sites in Drosophila melanogaster Links BEAF to Transcription

2009 ◽  
Vol 29 (13) ◽  
pp. 3556-3568 ◽  
Author(s):  
Nan Jiang ◽  
Eldon Emberly ◽  
Olivier Cuvier ◽  
Craig M. Hart
Keyword(s):  
Boundary Element ◽  
Rna Polymerase Ii ◽  
Cultured Cells ◽  
Polytene Chromosomes ◽  
Elongation Factor ◽  
Promoter Regions ◽  
Tiling Microarrays ◽  
Reverse Transcription Pcr ◽  
Genome Wide ◽  
A Genome

ABSTRACT Insulator elements play a role in gene regulation that is potentially linked to nuclear organization. Boundary element-associated factors (BEAFs) 32A and 32B associate with hundreds of sites on Drosophila polytene chromosomes. We hybridized DNA isolated by chromatin immunoprecipitation to genome tiling microarrays to construct a genome-wide map of BEAF binding locations. A distinct difference in the association of 32A and 32B with chromatin was noted. We identified 1,820 BEAF peaks and found that more than 85% were less than 300 bp from transcription start sites. Half are between head-to-head gene pairs. BEAF-associated genes are transcriptionally active as judged by the presence of RNA polymerase II, dimethylated histone H3 K4, and the alternative histone H3.3. Forty percent of these genes are also associated with the polymerase negative elongation factor NELF. Like NELF-associated genes, most BEAF-associated genes are highly expressed. Using quantitative reverse transcription-PCR, we found that the expression levels of most BEAF-associated genes decrease in embryos and cultured cells lacking BEAF. These results provide an unexpected link between BEAF and transcription, suggesting that BEAF plays a role in maintaining most associated promoter regions in an environment that facilitates high transcription levels.

scholarly journals CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation

2020 ◽  
Vol 48 (14) ◽  
pp. 7712-7727
Author(s):  
Michael Tellier ◽  
Justyna Zaborowska ◽  
Livia Caizzi ◽  
Eusra Mohammad ◽  
Taras Velychko ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Pol Ii ◽  
Terminal Domain ◽  
Carboxyl Terminal Domain ◽  
Genome Wide ◽  
A Genome ◽  
Carboxyl Terminal ◽  
Rna Polymerase Ii Transcription

Abstract Cyclin-dependent kinase 12 (CDK12) phosphorylates the carboxyl-terminal domain (CTD) of RNA polymerase II (pol II) but its roles in transcription beyond the expression of DNA damage response genes remain unclear. Here, we have used TT-seq and mNET-seq to monitor the direct effects of rapid CDK12 inhibition on transcription activity and CTD phosphorylation in human cells. CDK12 inhibition causes a genome-wide defect in transcription elongation and a global reduction of CTD Ser2 and Ser5 phosphorylation. The elongation defect is explained by the loss of the elongation factors LEO1 and CDC73, part of PAF1 complex, and SPT6 from the newly-elongating pol II. Our results indicate that CDK12 is a general activator of pol II transcription elongation and indicate that it targets both Ser2 and Ser5 residues of the pol II CTD.

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

2020 ◽  
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

AbstractThe 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 of noncoding RNAs prior to premature termination, in addition to transcription of mRNAs, are 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.

scholarly journals F-Box Genes in the Wheat Genome and Expression Profiling in Wheat at Different Developmental Stages

Genes ◽  
2020 ◽  
Vol 11 (10) ◽  
pp. 1154
Author(s):  
Min Jeong Hong ◽  
Jin-Baek Kim ◽  
Yong Weon Seo ◽  
Dae Yeon Kim
Keyword(s):  
Developmental Stages ◽  
Brachypodium Distachyon ◽  
Triticum Aestivum L ◽  
Wheat Genome ◽  
Post Translational Modification ◽  
Genome Wide ◽  
A Genome ◽  
High Sequence Homology ◽  
Wide Scale

Genes of the F-box family play specific roles in protein degradation by post-translational modification in several biological processes, including flowering, the regulation of circadian rhythms, photomorphogenesis, seed development, leaf senescence, and hormone signaling. F-box genes have not been previously investigated on a genome-wide scale; however, the establishment of the wheat (Triticum aestivum L.) reference genome sequence enabled a genome-based examination of the F-box genes to be conducted in the present study. In total, 1796 F-box genes were detected in the wheat genome and classified into various subgroups based on their functional C-terminal domain. The F-box genes were distributed among 21 chromosomes and most showed high sequence homology with F-box genes located on the homoeologous chromosomes because of allohexaploidy in the wheat genome. Additionally, a synteny analysis of wheat F-box genes was conducted in rice and Brachypodium distachyon. Transcriptome analysis during various wheat developmental stages and expression analysis by quantitative real-time PCR revealed that some F-box genes were specifically expressed in the vegetative and/or seed developmental stages. A genome-based examination and classification of F-box genes provide an opportunity to elucidate the biological functions of F-box genes in wheat.

scholarly journals Genome-wide mapping of unexplored essential regions in the Saccharomyces cerevisiae genome: evidence for hidden synthetic lethal combinations in a genetic interaction network

2014 ◽  
Vol 42 (15) ◽  
pp. 9838-9853 ◽  
Author(s):  
Saeed Kaboli ◽  
Takuya Yamakawa ◽  
Keisuke Sunada ◽  
Tao Takagaki ◽  
Yu Sasano ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Genetic Interaction ◽  
Interaction Network ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Lethal Gene ◽  
Synthetic Lethal ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale

Abstract Despite systematic approaches to mapping networks of genetic interactions in Saccharomyces cerevisiae, exploration of genetic interactions on a genome-wide scale has been limited. The S. cerevisiae haploid genome has 110 regions that are longer than 10 kb but harbor only non-essential genes. Here, we attempted to delete these regions by PCR-mediated chromosomal deletion technology (PCD), which enables chromosomal segments to be deleted by a one-step transformation. Thirty-three of the 110 regions could be deleted, but the remaining 77 regions could not. To determine whether the 77 undeletable regions are essential, we successfully converted 67 of them to mini-chromosomes marked with URA3 using PCR-mediated chromosome splitting technology and conducted a mitotic loss assay of the mini-chromosomes. Fifty-six of the 67 regions were found to be essential for cell growth, and 49 of these carried co-lethal gene pair(s) that were not previously been detected by synthetic genetic array analysis. This result implies that regions harboring only non-essential genes contain unidentified synthetic lethal combinations at an unexpectedly high frequency, revealing a novel landscape of genetic interactions in the S. cerevisiae genome. Furthermore, this study indicates that segmental deletion might be exploited for not only revealing genome function but also breeding stress-tolerant strains.

Genome-wide regulations of the pre-initiation complex formation and elongating RNA polymerase II by an E3 ubiquitin ligase, San1.

2021 ◽  
Author(s):  
Priyanka Barman ◽  
Rwik Sen ◽  
Amala Kaja ◽  
Jannatul Ferdoush ◽  
Shalini Guha ◽  
...  
Keyword(s):  
Quality Control ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Ubiquitin Ligase ◽  
Nuclear Protein ◽  
Protein Quality ◽  
Initiation Complex ◽  
Pol Ii ◽  
Intrinsically Disordered ◽  
Genome Wide

San1 ubiquitin ligase is involved in nuclear protein quality control via its interaction with intrinsically disordered proteins for ubiquitylation and proteasomal degradation. Since several transcription/chromatin regulatory factors contain intrinsically disordered domains and can be inhibitory to transcription when in excess, San1 might be involved in transcription regulation. To address this, we analyzed the role of San1 in genome-wide association of TBP [that nucleates pre-initiation complex (PIC) formation for transcription initiation] and RNA polymerase II (Pol II). Our results reveal the roles of San1 in regulating TBP recruitment to the promoters and Pol II association with the coding sequences, and hence PIC formation and coordination of elongating Pol II, respectively. Consistently, transcription is altered in the absence of San1. Such transcriptional alteration is associated with impaired ubiquitylation and proteasomal degradation of Spt16 and gene association of Paf1, but not the incorporation of centromeric histone, Cse4, into the active genes in Δsan1 . Collectively, our results demonstrate distinct functions of a nuclear protein quality control factor in regulating the genome-wide PIC formation and elongating Pol II (and hence transcription), thus unraveling new gene regulatory mechanisms.

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