Systematic profiling of subtelomeric silencing factors in budding yeast.

Subtelomeric gene silencing is the negative transcriptional regulation of genes located close to telomeres. This phenomenon occurs in a variety of eukaryotes with salient physiological implications, such as cell adherence, virulence, immune-system escape, and ageing. The process has been widely studied in the budding yeast Saccharomyces cerevisiae, where genes involved in this process have been identified mostly on a gene-by-gene basis. Here, we introduce a quantitative approach to study gene silencing, that couples the classical URA3 reporter with GFP monitoring, amenable to high-throughput flow cytometry analysis. This dual silencing reporter was integrated into several subtelomeric loci in the genome, where it showed a gradual range of silencing effects. By crossing strains with this dual reporter at the COS12 and YFR057W subtelomeric query loci with gene-deletion mutants, we carried out a large-scale forward screen for potential silencing factors. The approach was replicable and allowed accurate detection of expression changes. Results of our comprehensive screen suggest that the main players influencing subtelomeric silencing were previously known, but additional potential factors underlying chromatin conformation are involved. We validate and report the novel silencing factor LGE1, a protein with unknown molecular function required for histone H2B ubiquitination. Our strategy can be readily combined with other reporters and gene perturbation collections, making it a versatile tool to study gene silencing at a genome-wide scale.

Spt3 and Spt8 Are Involved in the Formation of a Silencing Boundary by Interacting with TATA-Binding Protein.

In Saccharomyces cerevisiae, a heterochromatin-like chromatin structure called the silencing region is present at the telomere as a complex of Sir2, Sir3, and Sir4. Although spreading of the silencing region is blocked by histone acetylase-mediated boundary formation, the details of the factors and mechanisms involved in the spread and formation of the boundary at each telomere are unknown. Here, we show that Spt3 and Spt8 block the spread of the silencing regions. Spt3 and Spt8 are members of the Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, which has histone acetyltransferase activity. We performed microarray analysis of the transcriptome of spt3Δ and spt8Δ strains and RT-qPCR analysis of the transcript levels of genes from the subtelomeric region in mutants in which the interaction of Spt3 with TATA-binding protein (TBP) is altered. The results not only indicated that both Spt3 and Spt8 are involved in TBP-mediated boundary formation on the right arm of chromosome III, but also that boundary formation in this region is DNA sequence independent. Although both Spt3 and Spt8 interact with TBP, Spt3 had a greater effect on genome-wide transcription. Mutant analysis showed that the interaction between Spt3 and TBP plays an important role in the boundary formation.

SIR telomere silencing depends on nuclear envelope lipids and modulates sensitivity to a lysolipid.

The nuclear envelope (NE) is important in maintaining genome organization. The role of lipids in communication between the NE and telomere regulation was investigated, including how changes in lipid composition impact gene expression and overall nuclear architecture. Yeast was treated with the non-metabolizable lysophosphatidylcholine analog edelfosine, known to accumulate at the perinuclear ER. Edelfosine induced NE deformation and disrupted telomere clustering but not anchoring. Additionally, the association of Sir4 at telomeres decreased. RNA-seq analysis showed altered expression of Sir-dependent genes located at sub-telomeric (0-10 kb) regions, consistent with Sir4 dispersion. Transcriptomic analysis revealed that two lipid metabolic circuits were activated in response to edelfosine, one mediated by the membrane sensing transcription factors, Spt23/Mga2, and the other by a transcriptional repressor, Opi1. Activation of these transcriptional programs resulted in higher levels of unsaturated fatty acids and the formation of nuclear lipid droplets. Interestingly, cells lacking Sir proteins displayed resistance to unsaturated-fatty acids and edelfosine, and this phenotype was connected to Rap1.

Regulation of telomere silencing by the core histones-autophagy-Sir2 axis.

Telomeres contain compacted heterochromatin, and genes adjacent to telomeres are subjected to transcription silencing. Maintaining telomere structure integrity and transcription silencing is important to prevent the occurrence of premature aging and aging-related diseases. How telomere silencing is regulated during aging is not well understood. Here, we find that the four core histones are reduced during yeast chronological aging, leading to compromised telomere silencing. Mechanistically, histone loss promotes the nuclear export of Sir2 and its degradation by autophagy. Meanwhile, reducing core histones enhances the autophagy pathway, which further accelerates autophagy-mediated Sir2 degradation. By screening the histone mutant library, we identify eight histone mutants and one histone modification (histone methyltransferase Set1-catalyzed H3K4 trimethylation) that regulate telomere silencing by modulating the core histones-autophagy-Sir2 axis. Overall, our findings reveal core histones and autophagy as causes of aging-coupled loss of telomere silencing and shed light on dynamic regulation of telomere structure during aging.

Limits to transcriptional silencing in Saccharomyces cerevisiae.

Mating-type switching in the budding yeast Saccharomyces cerevisiae relies on the Sir protein complex to silence HML and HMR, the two loci containing copies of the alleles of the mating type locus, MAT. Sir-based transcriptional silencing has been considered locus-specific, but the recent discovery of rare and transient escapes from silencing at HMLα2 with a sensitive assay called to question if these events extend to the whole locus. Adapting the same assay, we measured that transient silencing failures at HML were more frequent for the α2 gene than α1, similarly to their expression level in unsilenced cells. By coupling a mating assay, at HML we found that one of the two genes at that locus can be transiently expressed while the other gene is maintained silent. Thus, transient silencing loss can be a property of the gene rather than the locus. Cells lacking the SIR1 gene experience epigenetic bistability at HML and HMR. Our previous result led us to ask if HML could allow for two independent epigenetic states within the locus in a sir1Δ mutant. A simple construct using a double fluorescent reporter at HMLα1 and HMLα2 ruled out this possibility. Each HML locus displayed a single epigenetic state. We revisited the question of the correlation between the states of two HML loci in diploid cells, and showed they were independent. Finally, we determined the relative strength of gene repression achieved by Sir-based silencing with that achieved by the a1-α2 repressor.

Multiple phage resistance systems inhibit infection via SIR2-dependent NAD+ depletion.

Defence-associated sirtuins (DSRs) comprise a family of proteins that defend bacteria from phage infection via an unknown mechanism. These proteins are common in bacteria and harbour an N-terminal sirtuin (SIR2) domain. In this study we report that DSR proteins degrade nicotinamide adenine dinucleotide (NAD+) during infection, depleting the cell of this essential molecule and aborting phage propagation. Our data show that one of these proteins, DSR2, directly identifies phage tail tube proteins and then becomes an active NADase in Bacillus subtilis. Using a phage mating methodology that promotes genetic exchange between pairs of DSR2-sensitive and DSR2-resistant phages, we further show that some phages express anti-DSR2 proteins that bind and repress DSR2. Finally, we demonstrate that the SIR2 domain serves as an effector NADase in a diverse set of phage defence systems outside the DSR family. Our results establish the general role of SIR2 domains in bacterial immunity against phages.

Sir2 and Reb1 antagonistically regulate nucleosome occupancy in subtelomeric X-elements and repress TERRAs by distinct mechanisms.

Telomere chromatin structure is pivotal for maintaining genome stability by regulating the binding of telomere-associated proteins and inhibiting the DNA damage response. In Saccharomyces cerevisiae, silent information regulator (Sir) proteins bind to terminal repeats and to subtelomeric X-elements, resulting in transcriptional silencing. Herein, we show that sir2 mutant strains display a specific loss of a nucleosome residing in the X-elements and that this deficiency is remarkably consistent between different telomeres. The X-elements contain several binding sites for the transcription factor Reb1 and we found that Sir2 and Reb1 compete for stabilizing/destabilizing this nucleosome, i.e. inactivation of Reb1 in a sir2 background reinstated the lost nucleosome. The telomeric-repeat-containing RNAs (TERRAs) originate from subtelomeric regions and extend into the terminal repeats. Both Sir2 and Reb1 repress TERRAs and in a sir2 reb1 double mutant, TERRA levels increased synergistically, showing that Sir2 and Reb1 act in different pathways for repressing TERRAs. We present evidence that Reb1 restricts TERRAs by terminating transcription. Mapping the 5'-ends of TERRAs from several telomeres revealed that the Sir2-stabilized nucleosome is the first nucleosome downstream from the transcriptional start site for TERRAs. Finally, moving an X-element to a euchromatic locus changed nucleosome occupancy and positioning, demonstrating that X-element nucleosome structure is dependent on the local telomere environment.

Distinct silencer states generate epigenetic states of heterochromatin.

Heterochromatic loci can exhibit different transcriptional states in genetically identical cells. A popular model posits that the inheritance of modified histones is sufficient for inheritance of the silenced state. However, silencing inheritance requires silencers and therefore cannot be driven by the inheritance of modified histones alone. To address these observations, we determined the chromatin architectures produced by strong and weak silencers in Saccharomyces. Strong silencers recruited Sir proteins and silenced the locus in all cells. Strikingly, weakening these silencers reduced Sir protein recruitment and stably silenced the locus in some cells; however, this silenced state could probabilistically convert to an expressed state that lacked Sir protein recruitment. Additionally, changes in the constellation of silencer-bound proteins or the concentration of a structural Sir protein modulated the probability that a locus exhibited the silenced or expressed state. These findings argued that distinct silencer states generate epigenetic states and regulate their dynamics.

Distinct structural groups of histone H3 and H4 residues have divergent effects on chronological lifespan in Saccharomyces cerevisiae.

We have performed a comprehensive analysis of the involvement of histone H3 and H4 residues in the regulation of chronological lifespan in yeast and identify four structural groups in the nucleosome that influence lifespan. We also identify residues where substitution with an epigenetic mimic extends lifespan, providing evidence that a simple epigenetic switch, without possible additional background modifications, causes longevity. Residues where substitution result in the most pronounced lifespan extension are all on the exposed face of the nucleosome, with the exception of H3E50, which is present on the lateral surface, between two DNA gyres. Other residues that have a more modest effect on lifespan extension are concentrated at the extremities of the H3-H4 dimer, suggesting a role in stabilizing the dimer in its nucleosome frame. Residues that reduce lifespan are buried in the histone handshake motif, suggesting that these mutations destabilize the octamer structure. All residues exposed on the nucleosome disk face and that cause lifespan extension are known to interact with Sir3. We find that substitution of H4K16 and H4H18 cause Sir3 to redistribute from telomeres and silent mating loci to secondary positions, often enriched for Rap1, Abf1 or Reb1 binding sites, whereas H3E50 does not. The redistribution of Sir3 in the genome can be reproduced by an equilibrium model based on primary and secondary binding sites with different affinities for Sir3. The redistributed Sir3 cause transcriptional repression at most of the new loci, including of genes where null mutants were previously shown to extend chronological lifespan. The transcriptomic profiles of H4K16 and H4H18 mutant strains are very similar, and compatible with a DNA replication stress response. This is distinct from the transcriptomic profile of H3E50, which matches strong induction of oxidative phosphorylation. We propose that the different groups of residues are involved in binding to heterochromatin proteins, in destabilizing the association of the nucleosome DNA, disrupting binding of the H3-H4 dimer in the nucleosome, or disrupting the structural stability of the octamer, each category impacting on chronological lifespan by a different mechanism.

Discovery and identification of genes involved in DNA damage repair in yeast.

DNA repair defects are common in tumour cells and can lead to misrepair of double-strand breaks (DSBs), posing a significant challenge to cellular integrity. The overall mechanisms of DSB have been known for decades. However, the list of the genes that affect the efficiency of DSB repair continues to grow. Additional factors that play a role in DSB repair pathways have yet to be identified. In this study, we present a computational approach to identify novel gene functions that are involved in DNA damage repair in Saccharomyces cerevisiae. Among the primary candidates, GAL7, YMR130W, and YHI9 were selected for further analysis since they had not previously been identified as being active in DNA repair pathways. Originally, GAL7 was linked to galactose metabolism. YHI9 and YMR130W encode proteins of unknown functions. Laboratory testing of deletion strains gal7Δ, ymr130wΔ, and yhi9Δ implicated all 3 genes in Homologous Recombination (HR) and/or Non-Homologous End Joining (NHEJ) repair pathways, and enhanced sensitivity to DNA damage-inducing drugs suggested involvement in the broader DNA damage repair machinery. A subsequent genetic interaction analysis revealed interconnections of these three genes, most strikingly through SIR2, SIR3 and SIR4 that are involved in chromatin regulation and DNA damage repair network.

Glycerol 3-phosphate dehydrogenase regulates heat shock response in Saccharomyces cerevisiae.

The aim of this work was to identify elements of adaptive regulatory mechanism for basal level of yeast histone deacetylase Sir2. Heat shock response (HSR) was altered in the absence of the NAD-dependent glycerol 3-phosphate dehydrogenase (Gpd1). Increase in HSR was lower in ΔGpd1 cells than wild-type cells. An inverse correlation existed between Gpd1 and Sir2; Sir2-deleted cells showed higher expression of Gpd1 while deletion of Gpd1 led to higher expression of Sir2. In the absence of Gpd1, basal activity of Sir2 promoter was higher and was increased further upon heat shock, suggesting higher Sir2 levels. No interaction between Gpd1 and Sir2 was detected without or with heat shock using immunoprecipitation. The results show that Gpd1 regulates HSR in yeast cells and likely blocks its uncontrolled activation. As uncontrolled stress adversely affects the cellular adaptive response, Gpd1 may be a component of the cell's catalogue to ensure a balanced response to unmitigated thermal stress.

Distinguishing between recruitment and spread of silent chromatin structures in Saccharomyces cerevisiae.

The formation of heterochromatin at HML, HMR, and telomeres in Saccharomyces cerevisiae involves two main steps: the recruitment of Sir proteins to silencers and their spread throughout the silenced domain. We developed a method to study these two processes at single basepair resolution. Using a fusion protein between the heterochromatin protein Sir3 and the nonsite-specific bacterial adenine methyltransferase M.EcoGII, we mapped sites of Sir3-chromatin interactions genome-wide using long-read Nanopore sequencing to detect adenines methylated by the fusion protein and by ChIP-seq to map the distribution of Sir3-M.EcoGII. A silencing-deficient mutant of Sir3 lacking its Bromo-Adjacent Homology (BAH) domain, sir3-bah∆, was still recruited to HML, HMR, and telomeres. However, in the absence of the BAH domain, it was unable to spread away from those recruitment sites. Overexpression of Sir3 did not lead to further spreading at HML, HMR, and most telomeres. A few exceptional telomeres, like 6R, exhibited a small amount of Sir3 spreading, suggesting that boundaries at telomeres responded variably to Sir3-M.EcoGII overexpression. Finally, by using a temperature-sensitive allele of SIR3 fused to M.ECOGII, we tracked the positions first methylated after induction and found that repression of genes at HML and HMR began before Sir3 occupied the entire locus.

Spreading-dependent or independent Sir2-mediated gene silencing in budding yeast.

BACKGROUND: In the budding yeast Saccharomyces cerevisiae, a silent chromatin structure is formed at three distinct loci, including telomeres, rDNA, and mating-type loci, which silence the expression of genes within their structures. Sir2 is the only common factor, regulating the three silent chromatin regions. S. cerevisiae has 32 telomeres, but studies on gene silencing in budding yeast have been performed using some reporter genes, artificially inserted in the telomeric regions. Therefore, insights into the global landscape of Sir-dependent silencing of genes within the silent chromatin regions are required. OBJECTIVE: This study aimed to obtain global insights into Sir2-dependent gene silencing on all silent chromatin regions in budding yeast. METHODS: RNA-sequencing was performed to identify genes that are silenced by Sir2. By comparing with the chromatin immunoprecipitation-sequencing (ChIP-seq) of Sir2 in the wild-type strain, we confirmed Sir2-regulated genes. RESULTS: Using Sir2 ChIP-seq data, we identified that the Sir2 binding domain length caused by Sir2 spreading from the chromosomal end is different in each telomere in budding yeast. Expression of most subtelomeric genes increased in the ∆sir2 strain. Some Sir2-regulated subtelomeric genes were positioned within the telomeric Sir2-binding domain, while the others were outside the Sir2-binding domain. In addition, Sir2 was bound to the mating-type loci and rDNA region, and gene expression increased in the ∆sir2 strain. CONCLUSION: We concluded that S. cerevisiae has two modes of Sir2-mediated gene silencing: one is dependent on chromatin binding and spreading of Sir2, and the other is independent of spreading of Sir2.

Sir3 heterochromatin protein promotes non-homologous end joining by direct inhibition of Sae2.

Heterochromatin is a conserved feature of eukaryotic chromosomes, with central roles in gene expression regulation and maintenance of genome stability. How heterochromatin proteins regulate DNA repair remains poorly described. In the yeast Saccharomyces cerevisiae, the silent information regulator (SIR) complex assembles heterochromatin-like chromatin at sub-telomeric chromosomal regions. SIR-mediated repressive chromatin limits DNA double-strand break (DSB) resection, thus protecting damaged chromosome ends during homologous recombination (HR). As resection initiation represents the crossroads between repair by non-homologous end joining (NHEJ) or HR, we asked whether SIR-mediated heterochromatin regulates NHEJ. We show that SIRs promote NHEJ through two pathways, one depending on repressive chromatin assembly, and the other relying on Sir3 in a manner that is independent of its heterochromatin-promoting function. Via physical interaction with the Sae2 protein, Sir3 impairs Sae2-dependent functions of the MRX (Mre11-Rad50-Xrs2) complex, thereby limiting Mre11-mediated resection, delaying MRX removal from DSB ends, and promoting NHEJ.

Measuring the buffering capacity of gene silencing in Saccharomyces cerevisiae.

Gene silencing in budding yeast is mediated by Sir protein binding to unacetylated nucleosomes to form a chromatin structure that inhibits transcription. Transcriptional silencing is characterized by the high-fidelity transmission of the silent state. Despite its relative stability, the constituent parts of the silent state are in constant flux, giving rise to a model that silent loci can tolerate such fluctuations without functional consequences. However, the level of tolerance is unknown, and we developed methods to measure the threshold of histone acetylation that causes the silent chromatin state to switch to the active state as well as to measure the levels of the enzymes and structural proteins necessary for silencing. We show that loss of silencing required 50 to 75% acetyl-mimic histones, though the precise levels were influenced by silencer strength and upstream activating sequence (UAS) enhancer/promoter strength. Measurements of repressor protein levels necessary for silencing showed that reducing SIR4 gene dosage two- to threefold significantly weakened silencing, though reducing the gene copy numbers for Sir2 or Sir3 to the same extent did not significantly affect silencing suggesting that Sir4 was a limiting component in gene silencing. Calculations suggest that a mere twofold reduction in the ability of acetyltransferases to acetylate nucleosomes across a large array of nucleosomes may be sufficient to generate a transcriptionally silent domain.

Genetic screen for suppressors of increased silencing in rpd3 mutants in Saccharomyces cerevisiae identifies a potential role for H3K4 methylation.

Several studies have identified the paradoxical phenotype of increased heterochromatic gene silencing at specific loci that results from deletion or mutation of the histone deacetylase (HDAC) gene RPD3. To further understand this phenomenon, we conducted a genetic screen for suppressors of this extended silencing phenotype at the HMR locus in Saccharomyces cerevisiae. Most of the mutations that suppressed extended HMR silencing in rpd3 mutants without completely abolishing silencing were identified in the histone H3 lysine 4 methylation (H3K4me) pathway, specifically in SET1, BRE1, and BRE2. These second-site mutations retained normal HMR silencing, therefore, appear to be specific for the rpd3Δ extended silencing phenotype. As an initial assessment of the role of H3K4 methylation in extended silencing, we rule out some of the known mechanisms of Set1p/H3K4me mediated gene repression by HST1, HOS2, and HST3 encoded HDACs. Interestingly, we demonstrate that the RNA Polymerase III complex remains bound and active at the HMR-tDNA in rpd3 mutants despite silencing extending beyond the normal barrier. We discuss these results as they relate to the interplay among different chromatin-modifying enzyme functions and the importance of further study of this enigmatic phenomenon.

Breakers and amplifiers in chromatin circuitry: acetylation and ubiquitination control the heterochromatin machinery.

Eukaryotic genomes are segregated into active euchromatic and repressed heterochromatic compartments. Gene regulatory networks, chromosomal structures, and genome integrity rely on the timely and locus-specific establishment of active and silent states to protect the genome and provide the basis for cell division and specification of cellular identity. Here, we focus on the mechanisms and molecular machinery that establish heterochromatin in Schizosaccharomyces pombe and compare it with Saccharomyces cerevisiae and the mammalian polycomb system. We present recent structural and mechanistic evidence, which suggests that histone acetylation protects active transcription by disrupting the positive feedback loops used by the heterochromatin machinery and that H2A and H3 monoubiquitination actively drives heterochromatin, whereas H2B monoubiquitination mobilizes the defenses to quench heterochromatin.

Cell volume homeostatically controls the rDNA repeat copy number and rRNA synthesis rate in yeast.

The adjustment of transcription and translation rates to the changing needs of cells is of utmost importance for their fitness and survival. We have previously shown that the global transcription rate for RNA polymerase II in budding yeast Saccharomyces cerevisiae is regulated in relation to cell volume. Total mRNA concentration is constant with cell volume since global RNApol II-dependent nascent transcription rate (nTR) also keeps constant but mRNA stability increases with cell size. In this paper, we focus on the case of rRNA and RNA polymerase I. Contrarily to that found for RNA pol II, we detected that RNA polymerase I nTR increases proportionally to genome copies and cell size in polyploid cells. In haploid mutant cells with larger cell sizes, the rDNA repeat copy number rises. By combining mathematical modeling and experimental work with the large-size cln3 strain, we observed that the increasing repeat copy number is based on a feedback mechanism in which Sir2 histone deacetylase homeostatically controls the amplification of rDNA repeats in a volume-dependent manner. This amplification is paralleled with an increase in rRNA nTR, which indicates a control of the RNA pol I synthesis rate by cell volume.

A novel allele of SIR2 reveals a heritable intermediate state of gene silencing.

Genetic information acquires additional meaning through epigenetic regulation, the process by which genetically identical cells can exhibit heritable differences in gene expression and phenotype. Inheritance of epigenetic information is a critical step in maintaining cellular identity and organismal health. In Saccharomyces cerevisiae, one form of epigenetic regulation is the transcriptional silencing of two mating-type loci, HML and HMR, by the SIR-protein complex. To focus on the epigenetic dimension of this gene regulation, we conducted a forward mutagenesis screen to identify mutants exhibiting an epigenetic or metastable silencing defect. We utilized fluorescent reporters at HML and HMR, and screened yeast colonies for epigenetic silencing defects. We uncovered numerous independent sir1 alleles, a gene known to be required for stable epigenetic inheritance. More interestingly, we recovered a missense mutation within SIR2, which encodes a highly conserved histone deacetylase. In contrast to sir1Δ, which exhibits states that are either fully silenced or fully expressed, this sir2 allele exhibited heritable states that were either fully silenced or expressed at an intermediate level. The heritable nature of this unique silencing defect was influenced by, but not completely dependent on, changes in rDNA copy number. Therefore, this study revealed a heritable state of intermediate silencing and linked this state to a central silencing factor, Sir2.

Simulated microgravity accelerates aging in Saccharomyces cerevisiae.

The human body experiences physiological changes under microgravity environment that phenocopy aging on Earth. These changes include early onset osteoporosis, skeletal muscle atrophy, cardiac dysfunction, and immunosenescence, and such adaptations to the space environment may pose some risk to crewed missions to Mars. To investigate the effect of microgravity on aging, many model organisms have been used such as the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and mice. Herein we report that the budding yeast Saccharomyces cerevisiae show decreased replicative lifespan (RLS) under simulated microgravity in a clinostat. The reduction of yeast lifespan is not a result of decreased tolerance to heat shock or oxidative stress and could be overcome either by deletion of FOB1 or calorie restriction, two known interventions that extend yeast RLS. Deletion of the sirtuin gene SIR2 worsens the simulated microgravity effect on RLS, and together with the fob1Δ mutant phenotype, it suggests that simulated microgravity augments the formation of extrachromosomal rDNA circles, which accumulate in yeast during aging. We also show that the chronological lifespan in minimal medium was not changed when cells were grown in the clinostat. Our data suggest that the reduction in longevity due to simulated microgravity is conserved in yeast, worms, and flies, and these findings may have potential implications for future crewed missions in space, as well as the use of microgravity as a model for human aging.