Evolution and Functional Trajectory of Sir1 in Gene Silencing

We used the budding yeastsSaccharomyces cerevisiaeandTorulaspora delbrueckiito examine the evolution of Sir-based silencing, focusing on Sir1, silencers, the molecular topography of silenced chromatin, and the roles ofSIRand RNA interference (RNAi) genes inT. delbrueckii. Chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) analysis of Sir proteins inT. delbrueckiirevealed a different topography of chromatin at theHMLandHMRloci than was observed inS. cerevisiae. S. cerevisiaeSir1, enriched at the silencers ofHMLα andHMRa, was absent from telomeres and did not repress subtelomeric genes. In contrast toS. cerevisiaeSIR1's partially dispensable role in silencing, theT. delbrueckiiSIR1paralogKOS3was essential for silencing.KOS3was also found at telomeres withT. delbrueckiiSir2 (Td-Sir2) and Td-Sir4 and repressed subtelomeric genes. Silencer mapping inT. delbrueckiirevealed single silencers atHMLandHMR, bound by Td-Kos3, Td-Sir2, and Td-Sir4. TheKOS3gene mapped nearHMR, and its expression was regulated by Sir-based silencing, providing feedback regulation of a silencing protein by silencing. In contrast to the prominent role of Sir proteins in silencing,T. delbrueckiiRNAi genesAGO1andDCR1did not function in heterochromatin formation. These results highlighted the shifting role of silencing genes and the diverse chromatin architectures underlying heterochromatin.

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A Silencer Promotes the Assembly of Silenced Chromatin Independently of Recruitment

Molecular and Cellular Biology ◽

10.1128/mcb.00983-08 ◽

2008 ◽

Vol 29 (1) ◽

pp. 43-56 ◽

Cited By ~ 17

Author(s):

Patrick J. Lynch ◽

Laura N. Rusche

Keyword(s):

Saccharomyces Cerevisiae ◽

Mating Type ◽

Chromatin Immunoprecipitation ◽

Protein Assembly ◽

The Other ◽

Sir Proteins ◽

Genomic Locations ◽

Nonlinear Fashion ◽

Silent Mating Type Loci

ABSTRACT In Saccharomyces cerevisiae, silenced chromatin occurs at telomeres and the silent mating-type loci HMR and HML. At these sites, the Sir proteins are recruited to a silencer and then associate with adjacent chromatin. We used chromatin immunoprecipitation to compare the rates of Sir protein assembly at different genomic locations and discovered that establishment of silenced chromatin was much more rapid at HMR than at the telomere VI-R. Silenced chromatin also assembled more quickly on one side of HMR-E than on the other. Despite differences in spreading, the Sir proteins were recruited to HMR-E and telomeric silencers at equivalent rates. Additionally, insertion of HMR-E adjacent to the telomere VI-R increased the rate of Sir2p association with the telomere. These data suggest that HMR-E functions to both recruit Sir proteins and promote their assembly across several kilobases. Observations that association of Sir2p occurs simultaneously throughout HMR and that silencing at HMR is insensitive to coexpression of catalytically inactive Sir2p suggest that HMR-E acts by enabling assembly to occur in a nonlinear fashion. The ability of silencers to promote assembly of silenced chromatin over several kilobases is likely an important mechanism for maintaining what would otherwise be unstable chromatin at the correct genomic locations.

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Molecular network of chromatin immunoprecipitation followed by deep sequencing-based vitamin D receptor target genes

Multiple Sclerosis Journal ◽

10.1177/1352458512471873 ◽

2013 ◽

Vol 19 (8) ◽

pp. 1035-1045 ◽

Cited By ~ 19

Author(s):

Jun-ichi Satoh ◽

Hiroko Tabunoki

Keyword(s):

Multiple Sclerosis ◽

Vitamin D ◽

Vitamin D Receptor ◽

Deep Sequencing ◽

Immune Cells ◽

Chromatin Immunoprecipitation ◽

Target Genes ◽

Molecular Network ◽

Genome Wide

Background: Vitamin D is a liposoluble vitamin essential for calcium metabolism. The ligand-bound vitamin D receptor (VDR), heterodimerized with retinoid X receptor, interacts with vitamin D response elements (VDREs) to regulate gene expression. Vitamin D deficiency due to insufficient sunlight exposure confers an increased risk for multiple sclerosis (MS). Objective: To study a protective role of vitamin D in multiple sclerosis (MS), it is important to characterize the global molecular network of VDR target genes (VDRTGs) in immune cells. Methods: We identified genome-wide VDRTGs collectively from two distinct chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq) datasets of VDR-binding sites derived from calcitriol-treated human cells of B cell and monocyte origins. We mapped short reads of next generation sequencing (NGS) data on hg19 with Bowtie, detected the peaks with Model-based Analysis of ChIP-Seq (MACS), and identified genomic locations by GenomeJack, a novel genome viewer for NGS platforms. Results: We found 2997 stringent peaks distributed on protein-coding genes, chiefly located in the promoter and the intron on VDRE DR3 sequences. However, the corresponding transcriptome data verified calcitriol-induced upregulation of only a small set of VDRTGs. The molecular network of 1541 calcitriol-responsive VDRTGs showed a significant relationship with leukocyte transendothelial migration, Fcγ receptor-mediated phagocytosis, and transcriptional regulation by VDR, suggesting a pivotal role of genome-wide VDRTGs in immune regulation. Conclusion: These results suggest the working hypothesis that persistent deficiency of vitamin D might perturb the complex network of VDRTGs in immune cells, being responsible for induction of an autoimmune response causative for MS.

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Interplay of a Ligand Sensor and an Enzyme in Controlling Expression of the Saccharomyces cerevisiae GAL Genes

Eukaryotic Cell ◽

10.1128/ec.05294-11 ◽

2011 ◽

Vol 11 (3) ◽

pp. 334-342 ◽

Cited By ~ 17

Author(s):

Dariusz Abramczyk ◽

Stacey Holden ◽

Christopher J. Page ◽

Richard J. Reece

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Transcriptional Control ◽

Transcriptional Response ◽

Content Type ◽

Considerable Debate ◽

Active Transcription ◽

Gal Genes ◽

The Subject

ABSTRACT The regulation of the Saccharomyces cerevisiae GAL genes in response to galactose as a source of carbon has served as a paradigm for eukaryotic transcriptional control over the last 50 years. Three proteins—a transcriptional activator (Gal4p), an inhibitor (Gal80p), and a ligand sensor (Gal3p)—control the switch between inert and active gene expression. The molecular mechanism by which the recognition of galactose within the cell is converted into a transcriptional response has been the subject of considerable debate. In this study, using a novel and powerful method of localizing active transcription factors within the nuclei of cells, we show that a short-lived complex between Gal4p, Gal80p, and Gal3p occurs soon after the addition of galactose to cells to activate GAL gene expression. Gal3p is subsequently replaced in this complex by Gal1p, and a Gal4p-Gal80p-Gal1p complex is responsible for the continued expression of the GAL genes. The transient role of the ligand sensor indicates that current models for the induction and continued expression of the yeast GAL genes need to be reevaluated.

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

mBio ◽

10.1128/mbio.01504-20 ◽

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.

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Effects of Pure and Mixed Autochthonous Torulaspora delbrueckii and Saccharomyces cerevisiae on Fermentation and Volatile Compounds of Narince Wines

Foods ◽

10.3390/foods7090147 ◽

2018 ◽

Vol 7 (9) ◽

pp. 147 ◽

Cited By ~ 7

Author(s):

Ebru Arslan ◽

Zeynep Çelik ◽

Turgut Cabaroğlu

Keyword(s):

Saccharomyces Cerevisiae ◽

Volatile Compounds ◽

Mixed Culture ◽

Volatile Acid ◽

Wine Aroma ◽

Mixed Culture Fermentation ◽

Black Sea Region ◽

Torulaspora Delbrueckii ◽

Pure Cultures

The cultivar of Narince is a native white grape variety of Vitis vinifera, grown in Tokat city, the Mid-Black Sea Region of Anatolia. In this study, the effects of pure and mixed autochthonous Torulaspora delbrueckii-214 and Saccharomyces cerevisiae-1088 cultures on the fermentation behavior and aroma compounds of Narince wines were investigated. Volatile compounds formed in wines were extracted using a liquid–liquid extraction method and determined by GC-MS-FID. Narince grape must was fermented in duplicate, under the following three conditions. Two pure cultures of T. delbrueckii-214 and S. cerevisiae-1088 and a mixture of T. delbrueckii-214 and S. cerevisiae-1088 (1:1). The presence of the non-Saccharomyces T. delbrueckii-214 yeast slowed down the fermentation and produced a lower level of ethanol and a higher levels of glycerol and volatile acid. Only the pure culture of T. delbrueckii-214 was unable to finish fermentation. On the other hand, mixed culture fermentation improved the aroma intensity and complexity of wine due to increased levels of higher alcohols and esters. According to sensory analysis, wine fermented with mixed culture was the most preferred wine followed by wine inoculated with pure S. cerevisiae-1088. This study confirms the role of T. delbrueckii in wine aroma and the potential of non-Saccharomyces use in winemaking.

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Ordered Nucleation and Spreading of Silenced Chromatin inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e02-03-0175 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2207-2222 ◽

Cited By ~ 183

Author(s):

Laura N. Rusché ◽

Ann L. Kirchmaier ◽

Jasper Rine

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromatin Immunoprecipitation ◽

Protein Assembly ◽

Structural Components ◽

Wild Type ◽

Boundary Formation ◽

Chromatin States ◽

Sir Proteins ◽

Data Support

In Saccharomyces cerevisiae, silencing at theHM loci depends on Sir proteins, which are structural components of silenced chromatin. To explore the structure and assembly of silenced chromatin, the associations of Sir proteins with sequences across the HMR locus were examined by chromatin immunoprecipitation. In wild-type cells, Sir2p, Sir3p, and Sir4p were spread throughout and coincident with the silenced region atHMR. Sir1p, in contrast, associated only with theHMR-E silencer, consistent with its role in establishment but not maintenance of silencing. Sir4p was required for the association of other Sir proteins with silencers. In contrast, in the absence of Sir2p or Sir3p, partial assemblies of Sir proteins could form at silencers, where Sir protein assembly began. Spreading acrossHMR required Sir2p and Sir3p, as well as the deacetylase activity of Sir2p. These data support a model for the spreading of silenced chromatin involving cycles of nucleosome deacetylation by Sir2p followed by recruitment of additional Sir2p, Sir3p, and Sir4p to the newly deacetylated nucleosome. This model suggests mechanisms for boundary formation, and for maintenance and inheritance of silenced chromatin. The principles are generalizable to other types of heritable chromatin states.

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A Role for theSaccharomyces cerevisiaeRENT Complex Protein Net1 inHMRSilencing

Genetics ◽

10.1093/genetics/161.4.1411 ◽

2002 ◽

Vol 161 (4) ◽

pp. 1411-1423

Author(s):

Daniela Kasulke ◽

Stefanie Seitz ◽

Ann E Ehrenhofer-Murray

Keyword(s):

Saccharomyces Cerevisiae ◽

Protein A ◽

Rdna Locus ◽

The Other ◽

Central Component ◽

Yeast Saccharomyces Cerevisiae ◽

Sir Proteins ◽

Complex Protein ◽

Subtelomeric Regions

AbstractSilencing in the yeast Saccharomyces cerevisiae is known in three classes of loci: in the silent mating-type loci HML and HMR, in subtelomeric regions, and in the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD+-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1, as well as NET1 expression from a 2μ-plasmid, restored repression at silencing-defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins and recruit other silencing factors to the silencer. In agreement with this, Net1 provided ORC-dependent, Sir1-independent silencing when artificially tethered to the silencer. In contrast, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 from the silenced loci toward HMR.

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RNA Interference in Fungi: Pathways, Functions, and Applications

Eukaryotic Cell ◽

10.1128/ec.05109-11 ◽

2011 ◽

Vol 10 (9) ◽

pp. 1148-1155 ◽

Cited By ~ 111

Author(s):

Yunkun Dang ◽

Qiuying Yang ◽

Zhihong Xue ◽

Yi Liu

Keyword(s):

Gene Regulation ◽

Rna Interference ◽

Small Rna ◽

Rnai Machinery ◽

Content Type ◽

Heterochromatin Formation ◽

Rna Molecules ◽

Cellular Processes ◽

Rna Biogenesis ◽

Core Components

ABSTRACT Small RNA molecules of about 20 to 30 nucleotides function in gene regulation and genomic defense via conserved eukaryotic RNA interference (RNAi)-related pathways. The RNAi machinery consists of three core components: Dicer, Argonaute, and RNA-dependent RNA polymerase. In fungi, the RNAi-related pathways have three major functions: genomic defense, heterochromatin formation, and gene regulation. Studies of Schizosaccharomyces pombe and Neurospora , and other fungi have uncovered surprisingly diverse small RNA biogenesis pathways, suggesting that fungi utilize RNAi-related pathways in various cellular processes to adapt to different environmental conditions. These studies also provided important insights into how RNAi functions in eukaryotic systems in general. In this review, we will discuss our current understanding of the fungal RNAi-related pathways and their functions, with a focus on filamentous fungi. We will also discuss how RNAi can be used as a tool in fungal research.

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Role of a 300-Kilodalton Nuclear Complex in the Maturation of Trypanosoma brucei Initiator Methionyl-tRNA

Eukaryotic Cell ◽

10.1128/ec.3.4.893-899.2004 ◽

2004 ◽

Vol 3 (4) ◽

pp. 893-899 ◽

Cited By ~ 5

Author(s):

George K. Arhin ◽

Shuiyuan Shen ◽

Henriette Irmer ◽

Elisabetta Ullu ◽

Christian Tschudi

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Interference ◽

Trypanosoma Brucei ◽

Unicellular Eukaryote ◽

Down Regulation ◽

Parasitic Protozoa ◽

Initiator Trna ◽

Nuclear Complex ◽

Posttranscriptional Processing

ABSTRACT tRNAs are transcribed as precursors containing 5′ leader and 3′ extensions that are removed by a series of posttranscriptional processing reactions to yield functional mature tRNAs. Here, we examined the maturation pathway of tRNAMet in Trypanosoma brucei, an early divergent unicellular eukaryote. We identified an approximately 300-kDa complex located in the nucleus of T. brucei that is required for trimming the 5′ leader of initiator tRNAMet precursors. One of the subunits of the complex (T. brucei MT40 [TbMT40]) is a putative methyltransferase and a homolog of Saccharomyces cerevisiae Gcd14, which is essential for 1-methyladenosine modification in tRNAs. Down-regulation of TbMT40 by RNA interference resulted in the accumulation of precursor initiator tRNAMet containing 5′ extensions but processed 3′ ends. In addition, immunoprecipitations with anti-La antibodies revealed initiator tRNAMet molecules with 5′ and 3′ extensions in TbMT40-silenced cells, albeit at a much lower level. Interestingly, silencing of TbMT40, as well as of TbMT53, a second subunit of the complex, led to an increase in the levels of mature elongator tRNAMet. Taken together, our data provide a glance at the maturation of tRNAs in parasitic protozoa and suggest that at least for initiator tRNAMet, 3′ trimming precedes 5′ processing.

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Pyrophosphate Stimulates the Phosphate-Sodium Symporter ofTrypanosoma bruceiAcidocalcisomes andSaccharomyces cerevisiaeVacuoles

mSphere ◽

10.1128/msphere.00045-19 ◽

2019 ◽

Vol 4 (2) ◽

Cited By ~ 2

Author(s):

Evgeniy Potapenko ◽

Ciro D. Cordeiro ◽

Guozhong Huang ◽

Roberto Docampo

Keyword(s):

Saccharomyces Cerevisiae ◽

Membrane Potential ◽

Trypanosoma Brucei ◽

Wild Type ◽

Inorganic Pyrophosphate ◽

Content Type ◽

Yeast Strains ◽

Outward Currents ◽

Sodium Dependent

ABSTRACTInorganic pyrophosphate (PPi) is a by-product of biosynthetic reactions and has bioenergetic and regulatory roles in a variety of cells. Here we show that PPiand other pyrophosphate-containing compounds, including polyphosphate (polyP), can stimulate sodium-dependent depolarization of the membrane potential and Piconductance inXenopusoocytes expressing aSaccharomyces cerevisiaeorTrypanosoma bruceiNa+/Pisymporter. PPiis not taken up byXenopusoocytes, and deletion of the TbPho91 SPX domain abolished its depolarizing effect. PPigenerated outward currents in Na+/Pi-loaded giant vacuoles prepared from wild-type orpho91Δ yeast strains expressingTbPHO91but not from thepho91Δstrains. Our results suggest that PPi, at physiological concentrations, can function as a signaling molecule releasing PifromS. cerevisiaevacuoles andT. bruceiacidocalcisomes.IMPORTANCEAcidocalcisomes, first described in trypanosomes and known to be present in a variety of cells, have similarities withS. cerevisiaevacuoles in their structure and composition. Both organelles share a Na+/Pisymporter involved in Pirelease to the cytosol, where it is needed for biosynthetic reactions. Here we show that PPi, at physiological cytosolic concentrations, stimulates the symporter expressed in eitherXenopusoocytes or yeast vacuoles via its SPX domain, revealing a signaling role of this molecule.

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