scholarly journals The Paf1 Complex Represses SER3 Transcription in Saccharomyces cerevisiae by Facilitating Intergenic Transcription-Dependent Nucleosome Occupancy of the SER3 Promoter

2011 ◽  
Vol 10 (10) ◽  
pp. 1283-1294 ◽  
Author(s):  
Justin A. Pruneski ◽  
Sarah J. Hainer ◽  
Kostadin O. Petrov ◽  
Joseph A. Martens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Remodeling ◽  
Promoter Region ◽  
Nucleosome Occupancy ◽  
Histone Chaperones ◽  
Elongation Complex ◽  
Noncoding Dna ◽  
Direct Role ◽  
Content Type ◽  
Paf1 Complex

ABSTRACT Previous studies have shown that repression of the Saccharomyces cerevisiae SER3 gene is dependent on transcription of SRG1 from noncoding DNA initiating within the intergenic region 5′ of SER3 and extending across the SER3 promoter region. By a mechanism dependent on the activities of the Swi/Snf chromatin remodeling factor, the HMG-like factor Spt2, and the Spt6 and Spt16 histone chaperones, SRG1 transcription deposits nucleosomes over the SER3 promoter to prevent transcription factors from binding and activating SER3 . In this study, we uncover a role for the Paf1 transcription elongation complex in SER3 repression. We find that SER3 repression is primarily dependent on the Paf1 and Ctr9 subunits of this complex, with minor contributions by the Rtf1, Cdc73, and Leo1 subunits. We show that the Paf1 complex localizes to the SRG1 transcribed region under conditions that repress SER3 , consistent with it having a direct role in mediating SRG1 transcription-dependent SER3 repression. Importantly, we show that the defect in SER3 repression in strains lacking Paf1 subunits is not a result of reduced SRG1 transcription or reduced levels of known Paf1 complex-dependent histone modifications. Rather, we find that strains lacking subunits of the Paf1 complex exhibit reduced nucleosome occupancy and reduced recruitment of Spt16 and, to a lesser extent, Spt6 at the SER3 promoter. Taken together, our results suggest that Paf1 and Ctr9 repress SER3 by maintaining SRG1 transcription-dependent nucleosome occupancy.

scholarly journals Cks1 Enhances Transcription Efficiency at the GAL1 Locus by Linking the Paf1 Complex to the 19S Proteasome

2013 ◽  
Vol 12 (9) ◽  
pp. 1192-1201 ◽  
Author(s):  
Yen-Ru Pan ◽  
Michael Sun ◽  
James Wohlschlegel ◽  
Steven I. Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Remodeling ◽  
Kinase Activity ◽  
Genetic Interactions ◽  
Physical Interaction ◽  
Subsequent Work ◽  
Content Type ◽  
Transcription Efficiency ◽  
Paf1 Complex ◽  
Inducible Genes

ABSTRACT Cks1 was originally identified based on genetic interactions with CDC28 , the gene that encodes Cdk1 in the budding yeast Saccharomyces cerevisiae . Subsequent work has shown that Cks1 binds Cdc28 and modulates its activity against certain substrates. However, the Cks1/Cdc28 complex also has a role in transcriptional chromatin remodeling not related to kinase activity. In order to elucidate protein networks associated with Cks1 transcriptional functions, proteomic analysis was performed on immunoaffinity-purified Cks1, identifying a physical interaction with the Paf1 complex. Specifically, we found that the Paf1 complex component Rtf1 interacts directly with Cks1 and that this interaction is essential for efficient recruitment of Cks1 to chromatin in the context of GAL1 gene induction. We further found that Cks1 in this capacity serves as an adaptor allowing Rtf1 to recruit 19S proteasome particles, shown to be required for efficient RNA production from some rapidly inducible genes such as GAL1 .

scholarly journals Nap1 Links Transcription Elongation, Chromatin Assembly, and Messenger RNP Complex Biogenesis

2008 ◽  
Vol 28 (7) ◽  
pp. 2113-2124 ◽  
Author(s):  
Brian C. Del Rosario ◽  
Lucy F. Pemberton
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Remodeling ◽  
Transcription Elongation ◽  
Mrna Export ◽  
Transcript Level ◽  
Chromatin Assembly ◽  
Regulation Of Transcription ◽  
Histone Chaperones ◽  
Coding Region ◽  
Rnp Complex

ABSTRACT Chromatin remodeling is central to the regulation of transcription elongation. We demonstrate that the conserved Saccharomyces cerevisiae histone chaperone Nap1 associates with chromatin. We show that Nap1 regulates transcription of PHO5, and the increase in transcript level and the higher phosphatase activity plateau observed for Δnap1 cells suggest that the net function of Nap1 is to facilitate nucleosome reassembly during transcription elongation. To further our understanding of histone chaperones in transcription elongation, we identified factors that regulate the function of Nap1 in this process. One factor investigated is an essential mRNA export and TREX complex component, Yra1. Nap1 interacts directly with Yra1 and genetically with other TREX complex components and the mRNA export factor Mex67. Additionally, we show that the recruitment of Nap1 to the coding region of actively transcribed genes is Yra1 dependent and that its recruitment to promoters is TREX complex independent. These observations suggest that Nap1 functions provide a new connection between transcription elongation, chromatin assembly, and messenger RNP complex biogenesis.

scholarly journals Gcn5p and Ubp8p Affect Protein Ubiquitylation and Cell Proliferation by Altering the Fermentative/Respiratory Flux Balance in Saccharomyces cerevisiae

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Antonella De Palma ◽  
Giulia Fanelli ◽  
Elisabetta Cretella ◽  
Veronica De Luca ◽  
Raffaele Antonio Palladino ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Consumption ◽  
Redox Balance ◽  
Glycolytic Enzymes ◽  
Glycolytic Flux ◽  
Direct Role ◽  
Content Type ◽  
Proteomic Approach ◽  
Altered Glucose

ABSTRACT Protein ubiquitylation regulates not only endocellular trafficking and proteasomal degradation but also the catalytic activity of enzymes. In Saccharomyces cerevisiae, we analyzed the composition of the ubiquitylated proteomes in strains lacking acetyltransferase Gcn5p, Ub-protease Ubp8p, or both to understand their involvement in the regulation of protein ubiquitylation. We analyzed His6Ub proteins with a proteomic approach coupling micro-liquid chromatography and tandem mass spectrometry (μLC-MS/MS) in gcn5Δ, ubp8Δ and ubp8Δ gcn5Δ strains. The Ub-proteome altered in the absence of Gcn5p, Ubp8p, or both was characterized, showing that 43% of the proteins was shared in all strains, suggesting their functional relationship. Remarkably, all major glycolytic enzymes showed increased ubiquitylation. Phosphofructokinase 1, the key enzyme of glycolytic flux, showed a higher and altered pattern of ubiquitylation in gcn5Δ and ubp8Δ strains. Severe defects of growth in poor sugar and altered glucose consumption confirmed a direct role of Gcn5p and Ubp8p in affecting the REDOX balance of the cell. IMPORTANCE We propose a study showing a novel role of Gcn5p and Ubp8p in the process of ubiquitylation of the yeast proteome which includes main glycolytic enzymes. Interestingly, in the absence of Gcn5p and Ubp8p glucose consumption and redox balance were altered in yeast. We believe that these results and the role of Gcn5p and Ubp8p in sugar metabolism might open new perspectives of research leading to novel protocols for counteracting the enhanced glycolysis in tumors.

scholarly journals Coordinated Action of Nap1 and RSC in Disassembly of Tandem Nucleosomes

2016 ◽  
Vol 36 (17) ◽  
pp. 2262-2271 ◽  
Author(s):  
Rashmi Prasad ◽  
Sheena D'Arcy ◽  
Arjan Hada ◽  
Karolin Luger ◽  
Blaine Bartholomew
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mutational Analysis ◽  
Histone Chaperone ◽  
Histone Chaperones ◽  
Content Type ◽  
Chromatin Remodelers ◽  
Coordinated Action

The SWI/SNF and RSC family of ATP-dependent chromatin remodelers disassembles nucleosomes by moving nucleosomes into the vicinity of adjoining nucleosomes. We found that the histone chaperone Nap1 efficiently promotes disassembly of adjacent nucleosomes with which RSC collides and not the disassembly of nucleosomes mobilized by RSC. Nap1 is specific to RSC, as it does not target SWI/SNF, its paralog inSaccharomyces cerevisiae. Extensive mutational analysis of Nap1 has revealed that Nap1 affinity for histones H2A-H2B and H3-H4 and its ability to displace histones from DNA are required for Nap1 to enhance RSC-mediated disassembly. Other histone chaperones, such as Vps75, that also bind histones are not able to enhance RSC-mediated disassembly. Our study suggests a mechanism by which Nap1 is recruited to actively transcribed regions and assists in the passage of the transcription complex through chromatin, and it provides a novel mechanism for the coordinated action of RSC and Nap1.

scholarly journals Siz2 Prevents Ribosomal DNA Recombination by Modulating Levels of Tof2 in Saccharomyces cerevisiae

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Neethu Maria Abraham ◽  
Kathirvel Ramalingam ◽  
Saketh Murthy ◽  
Krishnaveni Mishra
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Ribosomal Dna ◽  
Posttranslational Modifications ◽  
Dna Recombination ◽  
Dna Breaks ◽  
Direct Role ◽  
Content Type ◽  
Repetitive Nature ◽  
The Stability ◽  
Consequent Increase

ABSTRACT Ribosomal DNA (rDNA) recombination in budding yeast is regulated by multiple converging processes, including posttranslational modifications such as SUMOylation. In this study, we report that the absence of a SUMO E3 ligase, Siz2, results in increased unequal rDNA exchange. We show that Siz2 is enriched at the replication fork barrier (RFB) in the rDNA and also controls the homeostasis of Tof2 protein. siz2Δ resulted in increased accumulation of total Tof2 in the cell and a consequent increase in the enrichment of Tof2 at the rDNA. Overproducing Tof2 ectopically or conditional overexpression of Tof2 also resulted in higher levels of rDNA recombination, suggesting a direct role for Tof2. Additionally, our chromatin immunoprecipitation (ChIP) data indicate that the accumulation of Tof2 in a siz2Δ mutant resulted in an enhanced association of Fob1, an RFB binding protein at the rDNA at the RFB. This increased Fob1 association at the RFB may have resulted in the elevated rDNA recombination. Our study thus demonstrates that the Tof2 levels modulate recombination at the rDNA. IMPORTANCE The genes that encode rRNA in Saccharomyces cerevisiae are organized as multiple repeats. The repetitive nature and heavy transcription of this region make it prone to DNA breaks. DNA breaks could lead to recombination, which could result in either loss or gain of repeats with detrimental consequences to the cell. Multiple mechanisms operate to maintain the stability of rDNA. Earlier studies reported that the absence of Ulp2, a deSUMOylase, resulted in declining levels of Tof2 and thereby disrupted rDNA silencing. In contrast, our findings suggest that accumulation of Tof2 can also result in increased rDNA recombination, through a mechanism that involves Fob1, an RFB-bound protein. While our study has examined only Tof2, rDNA recombination could be regulated by other proteins through a mechanism similar to this.

scholarly journals A Genome-Wide Screen of Deletion Mutants in the Filamentous Saccharomyces cerevisiae Background Identifies Ergosterol as a Direct Trigger of Macrophage Pyroptosis

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Kristy Koselny ◽  
Nebibe Mutlu ◽  
Annabel Y. Minard ◽  
Anuj Kumar ◽  
Damian J. Krysan ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Wall ◽  
Fungal Pathogen ◽  
Innate Immune ◽  
Direct Role ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Genome Wide ◽  
A Genome

ABSTRACT Phagocytic cells such as macrophages play an important role in the host defense mechanisms mounted in response to the common human fungal pathogen Candida albicans. In vitro, C. albicans triggers macrophage NLRP3-Casp1/11-mediated pyroptosis, an inflammatory programmed cell death pathway. Here, we provide evidence that Casp1/11-dependent pyroptosis occurs in the kidney of infected mice during the early stages of infection. We have also used a genome-wide screen of nonessential Σ1278b Saccharomyces cerevisiae genes to identify genes required for yeast-triggered macrophage pyroptosis. The set of genes identified by this screen was enriched for those with functions in lipid and sterol homeostasis and trafficking. These observations led us to discover that cell surface localization and/or total levels of ergosterol correlate with the ability of S. cerevisiae, C. albicans, and Cryptococcus neoformans to trigger pyroptosis. Since the mammalian sterol cholesterol triggers NLRP3-mediated pyroptosis, we hypothesized that ergosterol may also do so. Consistent with that hypothesis, ergosterol-containing liposomes but not ergosterol-free liposomes induce pyroptosis. Cell wall mannoproteins directly bind ergosterol, and we found that Dan1, an ergosterol receptor mannoprotein, as well as specific mannosyltransferases, is required for pyroptosis, suggesting that cell wall-associated ergosterol may mediate the process. Taken together, these data indicate that ergosterol, like mammalian cholesterol, plays a direct role in yeast-mediated pyroptosis. IMPORTANCE Innate immune cells such as macrophages are key components of the host response to the human fungal pathogen Candida albicans. Macrophages undergo pyroptosis, an inflammatory, programmed cell death, in response to some species of pathogenic yeast. Prior to the work described in this report, yeast-triggered pyroptosis has been observed only in vitro; here, we show that pyroptosis occurs in the initial stages of murine kidney infection, suggesting that it plays an important role in the initial response of the innate immune system to invasive yeast infection. We also show that a key component of the fungal plasma membrane, ergosterol, directly triggers pyroptosis. Ergosterol is also present in the fungal cell wall, most likely associated with mannoproteins, and is increased in hyphal cells compared to yeast cells. Our data indicate that specific mannoproteins are required for pyroptosis. This is consistent with a potential mechanism whereby ergosterol present in the outer mannoprotein layer of the cell wall is accessible to the macrophage-mediated process. Taken together, our data provide the first evidence that ergosterol plays a direct role in the host-pathogen interactions of fungi.

scholarly journals Direct Role for the Rpd3 Complex in Transcriptional Induction of the Anaerobic DAN/TIR Genes in Yeast

2007 ◽  
Vol 27 (6) ◽  
pp. 2037-2047 ◽  
Author(s):  
Odeniel Sertil ◽  
Arvind Vemula ◽  
Sharon L. Salmon ◽  
Randall H. Morse ◽  
Charles V. Lowry
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Remodeling ◽  
Chromatin Immunoprecipitation ◽  
Histone H3 ◽  
Normal Function ◽  
Direct Role ◽  
Chromatin Remodeling Complex ◽  
Normal Expression ◽  
Repressor Complex ◽  
Transcriptional Induction

ABSTRACT Saccharomyces cerevisiae adapts to hypoxia by expressing a large group of “anaerobic” genes. Among these, the eight DAN/TIR genes are regulated by the repressors Rox1 and Mot3 and the activator Upc2/Mox4. In attempting to identify factors recruited by the DNA binding repressor Mot3 to enhance repression of the DAN/TIR genes, we found that the histone deacetylase and global repressor complex, Rpd3-Sin3-Sap30, was not required for repression. Strikingly, the complex was instead required for activation. In addition, the histone H3 and H4 amino termini, which are targets of Rpd3, were also required for DAN1 expression. Epistasis tests demonstrated that the Rpd3 complex is not required in the absence of the repressor Mot3. Furthermore, the Rpd3 complex was required for normal function and stable binding of the activator Upc2 at the DAN1 promoter. Moreover, the Swi/Snf chromatin remodeling complex was strongly required for activation of DAN1, and chromatin immunoprecipitation analysis showed an Rpd3-dependent reduction in DAN1 promoter-associated nucleosomes upon induction. Taken together, these data provide evidence that during anaerobiosis, the Rpd3 complex acts at the DAN1 promoter to antagonize the chromatin-mediated repression caused by Mot3 and Rox1 and that chromatin remodeling by Swi/Snf is necessary for normal expression.

scholarly journals The Paf1 Complex Represses ARG1 Transcription in Saccharomyces cerevisiae by Promoting Histone Modifications

2011 ◽  
Vol 10 (6) ◽  
pp. 712-723 ◽  
Author(s):  
Elia M. Crisucci ◽  
Karen M. Arndt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Modifications ◽  
Transcriptional Repression ◽  
Histone H3 ◽  
Coding Region ◽  
Histone H2b ◽  
Content Type ◽  
Active Transcription ◽  
Paf1 Complex ◽  
Mechanistic Basis

ABSTRACT The conserved multifunctional Paf1 complex is important for the proper transcription of numerous genes, and yet the exact mechanisms by which it controls gene expression remain unclear. While previous studies indicate that the Paf1 complex is a positive regulator of transcription, the repression of many genes also requires the Paf1 complex. In this study we used ARG1 as a model gene to study transcriptional repression by the Paf1 complex in Saccharomyces cerevisiae . We found that several members of the Paf1 complex contribute to ARG1 repression and that the complex localizes to the ARG1 promoter and coding region in repressing conditions, which is consistent with a direct repressive function. Furthermore, Paf1 complex-dependent histone modifications are enriched at the ARG1 locus in repressing conditions, and histone H3 lysine 4 methylation contributes to ARG1 repression. Consistent with previous reports, histone H2B monoubiquitylation, the mark upstream of histone H3 lysine 4 methylation, is also important for ARG1 repression. To begin to identify the mechanistic basis for Paf1 complex-mediated repression of ARG1 , we focused on the Rtf1 subunit of the complex. Through an analysis of RTF1 mutations that abrogate known Rtf1 activities, we found that Rtf1 mediates ARG1 repression primarily by facilitating histone modifications. Other members of the Paf1 complex, such as Paf1, appear to repress ARG1 through additional mechanisms. Together, our results suggest that Rtf1-dependent histone H2B ubiquitylation and H3 K4 methylation repress ARG1 expression and that histone modifications normally associated with active transcription can occur at repressed loci and contribute to transcriptional repression.

scholarly journals Influences of Histone Stoichiometry on the Target Site Preference of Retrotransposons Ty1 and Ty2 in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 761-776 ◽  
Author(s):  
Lori A Rinckel ◽  
David J Garfinkel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Promoter Region ◽  
Target Site ◽  
Site Preference ◽  
Wild Type ◽  
Histone Proteins ◽  
Protein Levels ◽  
In The Wild ◽  
Type Strains ◽  
Orientation Bias

Abstract In Saccharomyces cerevisiae, the target site specificity of the retrotransposon Ty1 appears to involve the Ty integration complex recognizing chromatin structures. To determine whether changes in chromatin structure affect Ty1 and Ty2 target site preference, we analyzed Ty transposition at the CAN1 locus in mutants containing altered levels of histone proteins. A Δhta1-htb1 mutant with decreased levels of H2A and H2B histone proteins showed a pattern of Ty1 and Ty2 insertions at CAN1 that was significantly different from that of both the wild-type and a Δhta2-htb2 mutant, which does not have altered histone protein levels. Altered levels of H2A and H2B proteins disrupted a dramatic orientation bias in the CAN1 promoter region. In the wild-type strains, few Ty1 and Ty2 insertions in the promoter region were oriented opposite to the direction of CAN1 transcription. In the Δhta1-htb1 background, however, numerous Ty1 and Ty2 insertions were in the opposite orientation clustered within the TATA region. This altered insertion pattern does not appear to be due to a bias caused by selecting canavanine resistant isolates in the different HTA1-HTB1 backgrounds. Our results suggest that reduced levels of histone proteins alter Ty target site preference and disrupt an asymmetric Ty insertion pattern.

scholarly journals Discovery of a Novel Class of Orally Active Antifungal β-1,3-d-Glucan Synthase Inhibitors

2011 ◽  
Vol 55 (11) ◽  
pp. 5099-5106 ◽  
Author(s):  
Scott S. Walker ◽  
Yiming Xu ◽  
Ilias Triantafyllou ◽  
Michelle F. Waldman ◽  
Cara Mendrick ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Glabrata ◽  
Pharmacokinetic Data ◽  
Morphological Response ◽  
Glucan Synthase ◽  
Content Type ◽  
Safety Issues ◽  
Spontaneous Mutants ◽  
Orally Active

ABSTRACTThe echinocandins are a class of semisynthetic natural products that target β-1,3-glucan synthase (GS). Their proven clinical efficacy combined with minimal safety issues has made the echinocandins an important asset in the management of fungal infection in a variety of patient populations. However, the echinocandins are delivered only parenterally. A screen for antifungal bioactivities combined with mechanism-of-action studies identified a class of piperazinyl-pyridazinones that target GS. The compounds exhibitedin vitroactivity comparable, and in some cases superior, to that of the echinocandins. The compounds inhibit GSin vitro, and there was a strong correlation between enzyme inhibition andin vitroantifungal activity. In addition, like the echinocandins, the compounds caused a leakage of cytoplasmic contents from yeast and produced a morphological response in molds characteristic of GS inhibitors. Spontaneous mutants ofSaccharomyces cerevisiaewith reduced susceptibility to the piperazinyl-pyridazinones had substitutions inFKS1. The sites of these substitutions were distinct from those conferring resistance to echinocandins; likewise, echinocandin-resistant isolates remained susceptible to the test compounds. Finally, we present efficacy and pharmacokinetic data on an example of the piperazinyl-pyridazinone compounds that demonstrated efficacy in a murine model ofCandida glabratainfection.

Sign in / Sign up

Export Citation Format

Share Document