Search

In Saccharomyces cerevisiae, the Rpd3L complex includes the histone deacetylase Rpd3 and the DNA binding proteins Ume6 and Ash1 and serves as a transcriptional silencer or enhancer. In S. cerevisiae, the transcription of PDR5, which encodes a major drug efflux pump, and pleiotropic drug resistance (PDR) are hyperactivated by the transcription factor Pdr3 in ρ0/− cells, which lack mitochondrial DNA. We previously showed that RPD3 and UME6 are required for the activation of PDR5 transcription and PDR in S. cerevisiae ρ0 cells. Here, using real-time PCR analysis, we revealed that RPD3 and UME6 are responsible for the activated basal expression of the ABC transporter-encoding genes SNQ2, PDR15, and PDR5 in S. cerevisiae ρ0 cells. Furthermore, using real-time PCR analysis and a spot dilution assay, we found that Ume6 increases the basal expression of PDR5 and PDR15 and induces PDR in a manner dependent on RPD3 and PDR3 in ρ0 cells. This finding may contribute to the elucidation of the relationships between the molecules required for the activation of ABC transporter genes in S. cerevisiae ρ0/− cells and in pathogenic Candida species.

In Saccharomyces cerevisiae, the Rpd3L complex includes the histone deacetylase Rpd3 and the DNA binding proteins Ume6 and Ash1 and serves as a transcriptional silencer or enhancer. In S. cerevisiae, the transcription of PDR5, which encodes a major drug efflux pump, and pleiotropic drug resistance (PDR) are hyperactivated by the transcription factor Pdr3 in ρ0/− cells, which lack mitochondrial DNA. We previously showed that RPD3 and UME6 are required for the activation of PDR5 transcription and PDR in S. cerevisiae ρ0 cells. Here, using real-time PCR analysis, we revealed that RPD3 and UME6 are responsible for the activated basal expression of the ABC transporter-encoding genes SNQ2, PDR15, and PDR5 in S. cerevisiae ρ0 cells. Furthermore, using real-time PCR analysis and a spot dilution assay, we found that Ume6 increases the basal expression of PDR5 and PDR15 and induces PDR in a manner dependent on RPD3 and PDR3 in ρ0 cells. This finding may contribute to the elucidation of the relationships between the molecules required for the activation of ABC transporter genes in S. cerevisiae ρ0/− cells and in pathogenic Candida species. [ABSTRACT FROM AUTHOR]

Abstract Background In Saccharomyces cerevisiae, the retrograde signalling pathway is activated in ρ0/− cells, which lack mitochondrial DNA. Within this pathway, the activation of the transcription factor Pdr3 induces transcription of the ATP-binding cassette (ABC) transporter gene, PDR5, and causes pleiotropic drug resistance (PDR). Although a histone deacetylase, Rpd3, is also required for cycloheximide resistance in ρ0/− cells, it is currently unknown whether Rpd3 and its DNA binding partners, Ume6 and Ash1, are involved in the activation of PDR5 transcription and PDR in ρ0/− cells. This study investigated the roles of RPD3, UME6, and ASH1 in the activation of PDR5 transcription and PDR by retrograde signalling in ρ0 cells. Results ρ0 cells in the rpd3∆ and ume6∆ strains, with the exception of the ash1∆ strain, were sensitive to fluconazole and cycloheximide. The PDR5 mRNA levels in ρ0 cells of the rpd3∆ and ume6∆ strains were significantly reduced compared to the wild-type and ash1∆ strain. Transcriptional expression of PDR5 was reduced in cycloheximide-exposed and unexposed ρ0 cells of the ume6∆ strain; the transcriptional positive response of PDR5 to cycloheximide exposure was also impaired in this strain. Conclusions RPD3 and UME6 are responsible for enhanced PDR5 mRNA levels and PDR by retrograde signalling in ρ0 cells of S. cerevisiae.

In Saccharomycescerevisiae, the Rpd3L complex contains a histone deacetylase, Rpd3, and the DNA binding proteins, Ume6 and Ash1, and acts as a transcriptional repressor or activator. We previously showed that RPD3 and UME6 are required for the activation of PDR5, which encodes a major efflux pump, and pleiotropic drug resistance (PDR) in ρ0/− cells, which lack mitochondrial DNA. However, there are inconsistent reports regarding whether RPD3 and UME6 are required for Pdr5-mediated PDR in ρ+ cells with mitochondrial DNA. Since PDR5 expression or PDR in the ρ+ cells of the rpd3Δ and ume6Δ mutants have primarily been examined using fermentable media, mixed cultures of ρ+ and ρ0/− cells could be used. Therefore, we examined whether RPD3 and UME6 are required for basal and drug-induced PDR5 transcription and PDR in ρ+ cells using fermentable and nonfermentable media. UME6 suppresses the basal transcription levels of the ABC transporters, including PDR5, and drug resistance in ρ+ cells independent of the carbon source used in the growth medium. In contrast, RPD3 is required for drug resistance but did not interfere with the basal PDR5 mRNA levels. UME6 is also required for the cycloheximide-induced transcription of PDR5 in nonfermentable media but not in fermentable media. [ABSTRACT FROM AUTHOR]

Background: In Saccharomyces cerevisiae, the retrograde signalling pathway is activated in ρ0/− cells, which lack mitochondrial DNA. Within this pathway, the activation of the transcription factor Pdr3 induces transcription of the ATP-binding cassette (ABC) transporter gene, PDR5, and causes pleiotropic drug resistance (PDR). Although a histone deacetylase, Rpd3, is also required for cycloheximide resistance in ρ0/− cells, it is currently unknown whether Rpd3 and its DNA binding partners, Ume6 and Ash1, are involved in the activation of PDR5 transcription and PDR in ρ0/− cells. This study investigated the roles of RPD3, UME6, and ASH1 in the activation of PDR5 transcription and PDR by retrograde signalling in ρ0 cells. Results: ρ0 cells in the rpd3∆ and ume6∆ strains, with the exception of the ash1∆ strain, were sensitive to fluconazole and cycloheximide. The PDR5 mRNA levels in ρ0 cells of the rpd3∆ and ume6∆ strains were significantly reduced compared to the wild-type and ash1∆ strain. Transcriptional expression of PDR5 was reduced in cycloheximide-exposed and unexposed ρ0 cells of the ume6∆ strain; the transcriptional positive response of PDR5 to cycloheximide exposure was also impaired in this strain. Conclusions: RPD3 and UME6 are responsible for enhanced PDR5 mRNA levels and PDR by retrograde signalling in ρ0 cells of S. cerevisiae. [ABSTRACT FROM AUTHOR]

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 rpd3D 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 chromatinmodifying enzyme functions and the importance of further study of this enigmatic phenomenon. [ABSTRACT FROM AUTHOR]

ABSTRACT Acetylation and deacetylation of histones are key epigenetic mechanisms for gene regulation in response to environmental stimuli. RPD3 is a well-conserved class I histone deacetylase (HDAC) that is involved in diverse biological processes. Here, we investigated the roles of the Magnaporthe oryzae RPD3 (MoRPD3) gene, an ortholog of Saccharomyces cerevisiae Rpd3, during development and pathogenesis in the model plant-pathogenic fungus Magnaporthe oryzae. We demonstrated that the MoRPD3 gene is able to functionally complement the yeast Rpd3 deletion mutant despite the C-terminal extension of the MoRPD3 protein. MoRPD3 localizes primarily to the nuclei of vegetative hyphae, asexual spores, and invasive hyphae. Deletion of MoRPD3 appears to be lethal. Depletion of MoRPD3 transcripts via gene silencing (MoRPD3kd, where “kd” stands for “knockdown”) has opposing effects on asexual and sexual reproduction. Although conidial germination and appressorium formation rates of the mutants were almost comparable to those of the wild type, in-depth analysis revealed that the appressoria of mutants are smaller than those of the wild type. Furthermore, the MoRPD3kd strain shows a significant reduction in pathogenicity, which can be attributed to the delay in appressorium-mediated penetration and impaired invasive growth. Interestingly, MoRPD3 does not regulate potassium transporters, as shown for Rpd3 of S. cerevisiae. However, it functioned in association with the target of rapamycin (TOR) kinase pathway, resulting in the dependency of appressorium formation on hydrophilic surfaces and on TOR’s inhibition by MoRPD3. Taken together, our results uncovered distinct and evolutionarily conserved roles of MoRPD3 in regulating fungal reproduction, infection-specific development, and virulence. IMPORTANCE RPD3 is an evolutionarily conserved class I histone deacetylase (HDAC) that plays a pivotal role in diverse cellular processes. In filamentous fungal pathogens, abrogation of the gene encoding RPD3 results in either lethality or severe growth impairment, making subsequent genetic analyses challenging. Magnaporthe oryzae is a causal agent of rice blast disease, which is responsible for significant annual yield losses in rice production. Here, we characterized the RPD3 gene of M. oryzae (MoRPD3) in unprecedented detail using a gene-silencing approach. We provide evidence that MoRPD3 is a bona fide HDAC regulating fungal reproduction and pathogenic development by potentially being involved in the TOR-mediated signaling pathway. To the best of our knowledge, this work is the most comprehensive genetic dissection of RPD3 in filamentous fungal pathogens. Our work extends and deepens our understanding of how an epigenetic factor is implicated in the development and virulence of fungal pathogens of plants.

In response to nutrient starvation, the budding yeast Saccharomyces cerevisiae abandons mitotic proliferation and embarks on a differentiation process that leads through meiosis to the formation of haploid spores. This process is driven by cascading waves of meiosis-specific-gene expression. The early meiosis-specific genes are repressed during mitotic proliferation by the DNA-binding protein Ume6 in combination with repressors Rpd3 and Sin3. The expression of meiosis-specific transcription factor Ime1 leads to activation of the early meiosis-specific genes. We investigated the stability and promoter occupancy of Ume6 in sporulating cells and determined that it remains bound to early meiosis-specific gene promoters when those genes are activated. Furthermore, we find that the repressor Rpd3 remains associated with Ume6 after the transactivator Ime1 has joined the complex and that the Gcn5 and Tra1 components of the SAGA complex bind to the promoter of IME2 in an Ime1-dependent fashion to induce transcription of the early meiosis-specific genes. Our investigation supports a model whereby Ume6 provides a platform allowing recruitment of both activating and repressing factors to coordinate the expression of the early meiosis-specific genes in Saccharomyces cerevisiae. [ABSTRACT FROM AUTHOR]

PIF1 family helicases are evolutionarily conserved among prokaryotes and eukaryotes. These enzymes function to support genome integrity by participating in multiple DNA transactions that can be broadly grouped into DNA replication, DNA repair, and telomere maintenance roles. However, the levels of PIF1 activity in cells must be carefully controlled, as Pif1 over-expression in Saccharomyces cerevisiae is toxic, and knockdown or over-expression of human PIF1 (hPIF1) supports cancer cell growth. This suggests that PIF1 family helicases must be subject to tight regulation in vivo to direct their activities to where and when they are needed, as well as to maintain those activities at proper homeostatic levels. Previous work shows that C-terminal phosphorylation of S. cerevisiae Pif1 regulates its telomere maintenance activity, and we recently identified that Pif1 is also regulated by lysine acetylation. The over-expression toxicity of Pif1 was exacerbated in cells lacking the Rpd3 lysine deacetylase, but mutation of the NuA4 lysine acetyltransferase subunit Esa1 ameliorated this toxicity. Using recombinant proteins, we found that acetylation stimulated the DNA binding affinity, ATPase activity, and DNA unwinding activities of Pif1. All three domains of the helicase were targets of acetylation in vitro, and multiple lines of evidence suggest that acetylation drives a conformational change in the N-terminal domain of Pif1 that impacts this stimulation. It is currently unclear what triggers lysine acetylation of Pif1 and how this modification impacts the many in vivo functions of the helicase, but future work promises to shed light on how this protein is tightly regulated within the cell. [ABSTRACT FROM AUTHOR]

In Saccharomycescerevisiae, the Rpd3L complex contains a histone deacetylase, Rpd3, and the DNA binding proteins, Ume6 and Ash1, and acts as a transcriptional repressor or activator. We previously showed that RPD3 and UME6 are required for the activation of PDR5, which encodes a major efflux pump, and pleiotropic drug resistance (PDR) in ρ0/− cells, which lack mitochondrial DNA. However, there are inconsistent reports regarding whether RPD3 and UME6 are required for Pdr5-mediated PDR in ρ+ cells with mitochondrial DNA. Since PDR5 expression or PDR in the ρ+ cells of the rpd3Δ and ume6Δ mutants have primarily been examined using fermentable media, mixed cultures of ρ+ and ρ0/− cells could be used. Therefore, we examined whether RPD3 and UME6 are required for basal and drug-induced PDR5 transcription and PDR in ρ+ cells using fermentable and nonfermentable media. UME6 suppresses the basal transcription levels of the ABC transporters, including PDR5, and drug resistance in ρ+ cells independent of the carbon source used in the growth medium. In contrast, RPD3 is required for drug resistance but did not interfere with the basal PDR5 mRNA levels. UME6 is also required for the cycloheximide-induced transcription of PDR5 in nonfermentable media but not in fermentable media.

A subset of Saccharomyces cerevisiae cells in a stationary phase culture achieve a unique quiescent state characterized by increased cell density, stress tolerance, and longevity. Trehalose accumulation is necessary but not sufficient for conferring this state, and it is not recapitulated by abrupt starvation. The fraction of cells that achieve this state varies widely in haploids and diploids and can approach 100%, indicating that both mother and daughter cells can enter quiescence. The transition begins when about half the glucose has been taken up from the medium. The high affinity glucose transporters are turned on, glycogen storage begins, the Rim15 kinase enters the nucleus and the accumulation of cells in G1 is initiated. After the diauxic shift (DS), when glucose is exhausted from the medium, growth promoting genes are repressed by the recruitment of the histone deacetylase Rpd3 by quiescence‐specific repressors. The final division that takes place post‐DS is highly asymmetrical and G1 arrest is complete after 48 h. The timing of these events can vary considerably, but they are tightly correlated with total biomass of the culture, suggesting that the transition to quiescence is tightly linked to changes in external glucose levels. After 7 days in culture, there are massive morphological changes at the protein and organelle level. There are global changes in histone modification. An extensive array of condensin‐dependent, long‐range chromatin interactions lead to genome‐wide chromatin compaction that is conserved in yeast and human cells. These interactions are required for the global transcriptional repression that occurs in quiescent yeast. Take Away: The transition to quiescence begins before glucose limitation.Abrupt glucose starvation does not trigger quiescence.Quiescence‐specific repressors recruit Rpd3L histone deacetylase.Histones undergo massive ubiquitylation, methylation and acetylation changes.Quiescent chromatin compaction is conserved in yeast and human cells. [ABSTRACT FROM AUTHOR]

Yeast Hsl7 is recognized as a homolog of human arginine methyltransferase 5 (PRMT5) and shows type II PRMT activity by forming symmetric dimethylarginine residues on histones. Previously, we reported that Hsl7 is responsible for in vivo symmetric dimethylation on histone H4 arginine 3 (H4R3me2s) in a transcriptionally repressed state, possibly in association with histone deacetylation by Rpd3. Here, we investigated the function of Hsl7 during cell cycle progression. We found that the accumulation of Hsl7-mediated H4R3me2s is maintained by the histone deacetylase Rpd3 during transcriptional repression and that the low level of H4R3me2s is required for proper asymmetric cell growth during cell division. Our results suggest that the hypoacetylated state of histones is connected to the function of Hsl7 in regulating properly polarized cell growth during cell division and provide new insight into the epigenetic modifications that are important for cell cycle morphogenesis checkpoint control based on the repressive histone crosstalk between symmetric arginine methylation of H4 and histone deacetylation. [ABSTRACT FROM AUTHOR]

• H3 acetylations on the RNA polymerase III-transcribed genes increase with repression. • Mutants with lower SNR6 gene expression show higher H3K9ac levels in active state. • H3K9 and H3K14 acetylations on SNR6 gene are reversible but show different dynamics. • H3K9ac marks the transcriptional status; H3K14ac denotes chromatin structure changes. Several covalent modifications are found associated with the transcriptionally active chromatin regions constituted by the genes transcribed by RNA polymerase (pol) II. Pol III-transcribed genes code for the small, stable RNA species, which participate in many cellular processes, essential for survival. Pol III transcription is repressed under most of the stress conditions by its negative regulator Maf1. We found that most of the histone acetylations increase with starvation-induced repression on several genes transcribed by the yeast pol III. On one of these genes, SNR6 (coding for the U6snRNA), a strongly positioned nucleosome in the gene upstream region plays regulatory role under repression. On this nucleosome, the changes in H3K9 and H3K14 acetylations show different dynamics. During repression, acetylation levels on H3K9 show steady increase whereas H3K14 acetylation increases with a peak at 40 min after which levels reduce. Both the levels settle by 2 hr to a level higher than the active state, which revert to normal levels with nutrient repletion. The increase in H3 acetylations is seen in the mutants reported to show reduced SNR6 transcription but not in the maf1Δ cells. This increase on a regulatory nucleosome may be part of the signaling mechanisms, which prepare cells for the stress-related quick repression as well as reactivation. The contrasting association of the histone acetylations with pol II and pol III transcription may be an important consideration to make in research studies focused on drug developments targeting histone modifications. [ABSTRACT FROM AUTHOR]

Protein arginine methylation, an evolutionarily conserved post-translational modification, serves critical cellular functions by transferring a methyl group to a variety of substrates, including histones and some transcription factors. In budding yeast, Hsl7 (histone synthetic lethal 7) displays type II PRMT (protein arginine methyltransferase) activity by generating symmetric dimethylarginine residues on histone H2A in vitro. However, identification of the in vivo substrate of Hsl7 and how it contributes to important cellular processes remain largely unexplored. In the present study, we show that Hsl7 has a repressive role in transcription. We found that Hsl7 is responsible for in vivo symmetric dimethylation of histone H4 arginine 3 (H4R3me2s) in a transcriptionally repressed state. Tandem affinity purification further demonstrated that Hsl7 physically interacts with histone deacetylase Rpd3, and both similarly repress transcription. Our results suggest that H4R3me2s generation by the type II PRMT Hsl7 is required for transcriptional repression, possibly in cooperation with histone deacetylation by Rpd3. [ABSTRACT FROM AUTHOR]

Cellular quiescence is a reversible differentiation state when cells are changing the gene expression program to reduce metabolic functions and adapt to a new cellular environment. When fission yeast cells are deprived of nitrogen in the absence of any mating partner, cells can reversibly arrest in a differentiated G0-like cellular state, called quiescence. This change is accompanied by a marked alteration of nuclear organization and a global reduction of transcription. Using high-throughput flow cytometry combined with genetic analysis, we describe the results of a comprehensive screen for genes encoding chromatin components and regulators that are required for the entry and the maintenance of cellular quiescence. We show that the histone acetylase and deacetylase complexes, SAGA and Rpd3, have key roles both for G0 entry and survival during quiescence. We reveal a novel function for the Ino80 nucleosome remodeling complex in cellular quiescence. Finally, we demonstrate that components of the MRN complex, Rad3, the nonhomologous end-joining, and nucleotide excision DNA repair pathways are essential for viability in G0.

Autophagy is a catabolic process to maintain homeostasis, and involved in cell differentiation and development. Autophagy is tightly regulated in response to nutrient availability but the underlying mechanism is not completely understood. Recently, we identified the chromatin remodeling complex INO80 (inositol-requiring mutant 80) and histone variant H2A.Z as new autophagy regulators and uncover how histone deacetylase Rpd3L (reduced potassium dependency 3 large) complex represses autophagy by deacetylating Ino80 and H2A.Z. In particular, Rpd3L complex deacetylates Ino80 at lysine 929, which protects Ino80 from being degraded by autophagy. The stabilized Ino80 then evicts H2A.Z from autophagy-related (ATG) genes, leading to their transcriptional repression. In parallel, Rpd3L complex also deacetylates H2A.Z, which further reduces its association with ATG gene promoters and repress ATG gene transcription. Under nutrient-rich conditions, Rpd3L-mediated deacetylation of Ino80 K929 and H2A.Z is enhanced by the TORC1 complex (target of rapamycin complex 1). Under nitrogen-starvation condition, TORC1 is inactivated, leading to reduced activity of Rpd3L complex and increased acetylation of Ino80 and H2A.Z, which in turn induce the transcription of ATG genes. These results reveal a critical role of chromatin remodelers and histone variants in regulating autophagy in response to nutrient availability. Abbreviations: INO80: inositol-requiring mutant 80; Rpd3: reduced potassium dependency 3; H2A.Z: histone H2A variant; Rpd3L complex: Rpd3 large complex; H4K16: H4 lysine 16; H3R17: H3 arginine 17; H3T11: H3 threonine 11; TORC1 complex: target of rapamycin complex 1; ATG: autophagy-related; SWI/SNF: switch/sucrose non-fermentable; SWR1: Swi2/Snf2-related ATPase complex; RSC: remodel the structure of chromatin; ISWI: imitation switch; CHD1: chromodomain helicase DNA binding protein 1; Arp8: actin-related protein 8; Sds3: suppressor of defective silencing 3; Ume6: unscheduled meiotic gene expression 6.

Development, tissue renewal and long term survival of multi-cellular organisms is dependent upon the persistence of stem cells that are quiescent, but retain the capacity to re-enter the cell cycle to self-renew, or to produce progeny that can differentiate and re-populate the tissue. Deregulated release of these cells from the quiescent state, or preventing them from entering quiescence, results in uncontrolled proliferation and cancer. Conversely, loss of quiescent cells, or their failure to re-enter cell division, disrupts organ development and prevents tissue regeneration and repair. Understanding the quiescent state and how cells control the transitions in and out of this state is of fundamental importance. Investigations into the mechanics of G1 arrest during the transition to quiescence continue to identify striking parallels between the strategies used by yeast and mammals to regulate this transition. When cells commit to a stable but reversible arrest, the G1/S genes responsible for promoting S phase must be inhibited. This process, from yeast to humans, involves the formation of quiescence-specific complexes on their promoters. In higher cells, these so-called DREAM complexes of E2F4/DP/RBL/MuvB recruit the highly conserved histone deacetylase HDAC1, which leads to local histone deacetylation and repression of S phase-promoting transcripts. Quiescent yeast cells also show pervasive histone deacetylation by the HDAC1 counterpart Rpd3. In addition, these cells contain quiescence-specific regulators of G1/S genes: Msa1 and Msa2, which can be considered components of the yeast equivalent of the DREAM complex. Despite a lack of physical similarities, the goals and the strategies used to achieve a reversible transition to quiescence are highly conserved. This motivates a detailed study of this process in the simple model organism: budding yeast. [ABSTRACT FROM AUTHOR]

BACKGROUND: Rpd3 is a conserved histone deacetylase that removes acetyl groups from lysine residues within histones and other proteins. Reduction or inhibition of Rpd3 extends longevity in yeast, worms, and flies. Previous studies in flies suggest an overlap with the mechanism of lifespan extension by dietary restriction. However, the mechanism of rpd3's effects on longevity remains unclear. OBJECTIVES: In this study we investigated how rpd3 reduction affects fly spontaneous physical activity, fecundity, and stress resistance. METHODS: We examined the effects of rpd3 reduction on fly spontaneous physical activity by using population monitors, we determined female fecundity by counting daily egg laying, and we determined fly survivorship in response to starvation and paraquat. RESULTS: In flies, rpd3 reduction increases peak spontaneous physical activity of rpd3def male flies at a young age but does not affect total 24 hour activity. Male and female rpd3def mutants are more resistant to starvation on low and high calorie diets. In addition, increased resistance to paraquat was observed in females of one allele. A decrease in rpd3 levels does not affect female fecundity. CONCLUSIONS: A decrease in rpd3 levels mirrors some but not all changes associated with calorie restriction, illustrated by an increased peak of spontaneous activity in rpd3def /+ heterozygous male flies but no effect on total spontaneous activity and fecundity. [ABSTRACT FROM AUTHOR]

The epigenetic regulation of DNA structure and function is essential for changes in gene expression involved in development, growth, and maintenance of cellular function. Epigenetic changes include histone modifications such as methylation, acetylation, ubiquitination, and phosphorylation. Histone deacetylase (HDAC) proteins have a major role in epigenetic regulation of chromatin structure. HDACs are enzymes that catalyze the removal of acetyl groups from lysine residues within histones, as well as a range of other proteins including transcriptional factors. HDACs are highly conserved proteins divided into two families and based on sequence similarity in four classes. Here we will discuss the roles of Rpd3 in physiology and longevity with emphasis on its role in flies. Rpd3, the Drosophila HDAC1 homolog, is a class I lysine deacetylase and a member of a large family of HDAC proteins. Rpd3 has multiple functions including control of proliferation, development, metabolism, and aging. Pharmacological and dietary HDAC inhibitors have been used as therapeutics in psychiatry, cancer, and neurology. [ABSTRACT FROM AUTHOR]

Summary Chromatin immunoprecipitation followed by sequencing (ChIP-seq) has been instrumental to our current view of chromatin structure and function. It allows genome-wide mapping of histone marks, which demarcate biologically relevant domains. However, ChIP-seq is an ensemble measurement reporting the average occupancy of individual marks in a cell population. Consequently, our understanding of the combinatorial nature of chromatin states relies almost exclusively on correlation between the genomic distributions of individual marks. Here, we report the development of combinatorial-iChIP to determine the genome-wide co-occurrence of histone marks at single-nucleosome resolution. By comparing to a null model, we show that certain combinations of overlapping marks (H3K36me3 and H3K79me3) co-occur more frequently than would be expected by chance, while others (H3K4me3 and H3K36me3) do not, reflecting differences in the underlying chromatin pathways. We further use combinatorial-iChIP to illuminate aspects of the Set2-RPD3S pathway. This approach promises to improve our understanding of the combinatorial complexity of chromatin. [ABSTRACT FROM AUTHOR]

As the only catalytic member of the Sir-protein gene-silencing complex, Sir2's catalytic activity is necessary for silencing. The only known role for Sir2's catalytic activity in Saccharomyces cerevisiae silencing is to deacetylate N-terminal tails of histones H3 and H4, creating high-affinity binding sites for the Sir-protein complex, resulting in association of Sir proteins across the silenced domain. This histone deacetylation model makes the simple prediction that preemptively removing Sir2's H3 and H4 acetyl substrates, by mutating these lysines to unacetylatable arginines, or removing the acetyl transferase responsible for their acetylation, should restore silencing in the Sir2 catalytic mutant. However, this was not the case. We conducted a genetic screen to explore what aspect of Sir2's catalytic activity has not been accounted for in silencing. Mutation of a nonsirtuin histone deacetylase, Rpd3, restored Sir-proteinbased silencing in the absence of Sir2's catalytic activity. Moreover, this antagonism could be mediated by either the large or the small Rpd3-containing complex. Interestingly, this restoration of silencing appeared independent of any known histone H3 or H4 substrates of Rpd3. Investigation of Sir-protein association in the Rpd3 mutant revealed that the restoration of silencing was correlated with an increased association of Sir proteins at the silencers, suggesting that Rpd3 was an antagonist of Sir2's function in nucleation of Sir proteins to the silencer. Additionally, restoration of silencing by Rpd3 was dependent on another sirtuin family member, Hst3, indicating multiple antagonistic roles for deacetylases in S. cerevisiae silencing. [ABSTRACT FROM AUTHOR]

Inactivation of HDAC1 and its homolog HDAC2 or addition of HDAC inhibitors in mammalian systems induces apoptosis, cell cycle arrest, and developmental defects. Although these phenotypes have been extensively characterized, the precise underlying mechanisms remain unclear, particularly in in vivo settings. In this study, we show that inactivation of Rpd3, the only HDAC1 and HDAC2 ortholog in Drosophila , induced apoptosis and clone elimination in the developing eye and wing imaginal discs. Depletion of Rpd3 by RNAi cell-autonomously increased JNK activities and decreased activities of Yki, the nuclear effecter of Hippo signaling pathway. In addition, inhibition of JNK activities largely rescued Rpd3 RNAi -induced apoptosis, but did not affect its inhibition of Yki activities. Conversely, increasing the Yki activities largely rescued Rpd3 RNAi -induced apoptosis, but did not affect its induction of JNK activities. Furthermore, inactivation of Mi-2, a core component of the Rpd3-containing NuRD complex strongly induced JNK activities; while inactivation of Sin3A, a key component of the Rpd3-containing Sin3 complex, significantly inhibited Yki activities. Taken together, these results reveal that inactivation of Rpd3 independently regulates JNK and Yki activities and that both Hippo and JNK signaling pathways contribute to Rpd3 RNAi -induced apoptosis. [ABSTRACT FROM AUTHOR]

Chromatin modification enzymes are important regulators of gene expression and some are evolutionarily conserved from yeast to human. Saccharomyces cerevisiae is a major model organism for genome-wide studies that aim at the identification of target genes under the control of conserved epigenetic regulators. Ume6 interacts with the upstream repressor site 1 (URS1) and represses transcription by recruiting both the conserved histone deacetylase Rpd3 (through the co-repressor Sin3) and the chromatin-remodeling factor Isw2. Cells lacking Ume6 are defective in growth, stress response, and meiotic development. RNA profiling studies and in vivo protein-DNA binding assays identified mRNAs or transcript isoforms that are directly repressed by Ume6 in mitosis. However, a comprehensive understanding of the transcriptional alterations, which underlie the complex ume6Δ mutant phenotype during fermentation, respiration, or sporulation, is lacking. We report the protein-coding transcriptome of a diploid MAT a/α wild-type and ume6/ ume6 mutant strains cultured in rich media with glucose or acetate as a carbon source, or sporulation-inducing medium. We distinguished direct from indirect effects on mRNA levels by combining GeneChip data with URS1 motif predictions and published high-throughput in vivo Ume6-DNA binding data. To gain insight into the molecular interactions between successive waves of Ume6-dependent meiotic genes, we integrated expression data with information on protein networks. Our work identifies novel Ume6 repressed genes during growth and development and reveals a strong effect of the carbon source on the derepression pattern of transcripts in growing and developmentally arrested ume6/ ume6 mutant cells. Since yeast is a useful model organism for chromatin-mediated effects on gene expression, our results provide a rich source for further genetic and molecular biological work on the regulation of cell growth and cell differentiation in eukaryotes. [ABSTRACT FROM AUTHOR]

The tripartite Rpd3/Sin3/Ume6 complex represses meiotic isoforms during mitosis. We asked if it also controls starvation-induced isoforms. We report that VTH1/VTH2 encode acetate-inducible isoforms with extended 5′-regions overlapping antisense long non-coding RNAs. Rpd3 and Ume6 repress the long isoform of VTH2 during fermentation. Cells metabolising glucose contain Vth2, while the protein is undetectable in acetate and during sporulation. VTH2 is a useful model locus to study mechanisms implicating promoter directionality, lncRNA transcription and post-transcriptional control of gene expression via 5′-UTRs. Since mammalian genes encode transcript isoforms and Rpd3 is conserved, our findings are relevant for gene expression in higher eukaryotes. [ABSTRACT FROM AUTHOR]

Lysine deacetylases (KDACs) inhibitors may have therapeutic value in anti-malarial combination therapies with artemisinin. To evaluate connections between KDACs and artemisinin, Saccharomyces cerevisiae deletion mutants in KDAC genes were assayed. Deletion of RPD3 , but not other KDAC genes, resulted in strong sensitivity to artemisinin, which was also observed in sit4 Δ mutants with impaired endoplasmic reticulum (ER) to Golgi protein trafficking. Decreased accumulation of the transporters Pdr5p, Fur4p, and Tat2p was observed in rpd3 Δ and sit4 Δ cells. The unfolded protein response is induced in rpd3 Δ cells consistent with retention of proteins in the ER. Disruption of protein trafficking appears to sensitize cells to artemisinin and targeting these pathways may be useful as part of artemisinin based anti-malarial therapy. [ABSTRACT FROM AUTHOR]

When mitophagy is induced in Saccharomyces cerevisiae, the mitochondrial outer membrane protein ScAtg32 interacts with the cytosolic adaptor protein ScAtg11. ScAtg11 then delivers the mitochondria to the pre-autophagosomal structure for autophagic degradation. Despite the importance of ScAtg32 for mitophagy, the expression and functional regulation of ScAtg32 are poorly understood. In this study, we identified and characterized the ScAtg32 homolog in Pichia pastoris (PpAtg32). Interestingly, we found that PpAtg32 was barely expressed before induction of mitophagy and was rapidly expressed after induction of mitophagy by starvation. Additionally, PpAtg32 was phosphorylated when mitophagy was induced. We found that PpAtg32 expression was suppressed by Tor and the downstream PpSin3-PpRpd3 complex. Inhibition of Tor by rapamycin induced PpAtg32 expression, but could neither phosphorylate PpAtg32 nor induce mitophagy. Based on these findings, we conclude that the Tor and PpSin3- PpRpd3 pathway regulates PpAtg32 expression, but not PpAtg32 phosphorylation. [ABSTRACT FROM AUTHOR]

Most eukaryotes contain many tandem repeats of ribosomal RNA genes of which only a subset is transcribed at any given time. Current biochemical methods allow for the determination of the fraction of transcribing repeats (ON) versus non‐transcribing repeats (OFF) but do not provide any dynamical information and obscure any transcription activity at the single‐cell level. Here, we use a fluorescence in situ hybridization (FISH) technique that allows the detection of single‐RNA molecules in individual yeast cells. We use this method complemented with theoretical modeling to determine the rate of switching from OFF to ON (activation rate) and the average number of RNA molecules produced during each transcriptional burst (burst size). We explore how these two variables change in mutants and different growth conditions, and show that this method resolves changes in these two variables even when the average rDNA expression is unaltered. These phenotypic changes could not have been detected by traditional biochemical assays.

Drosophila nasuta nasuta (2 n = 8) and D. n. albomicans (2 n = 6) are morphologically identical, cross fertile and karyotypically dissimilar pair of chromosomal races belonging to nasuta subgroup of immigrans group of Drosophila. Interracial hybridization between these two races yielded karyotypically stabilized newly evolved Cytoraces with new combinations of chromosomes and DNA content, and are called nasuta- albomicans complex of Drosophila. Along with many other features, striking plasticity in the lifespan has been observed in the karyotypically stabilized members of nasuta- albomicans complex of Drosophila. These findings provide a strong background to understand any changes at the molecular levels. In view of this, we cloned and characterized Sod1 and Rpd3 in the members of nasuta- albomicans complex of Drosophila. The evolution of Sod1 and Rpd3 in D. n. nasuta and D. n. albomicans is contrasting with the other species of Drosophila, at the level of synonymous mutations, intron variation, InDels and secondary structure changes in protein. In the members of NAC of Drosophila there were synonymous changes, variations in intron sequences of Sod1, whereas, in Rpd3, synonymous, nonsynonymous, intron variation, and secondary structure changes in protein were observed. The contrasting differences in the levels of Rpd3 (and Sir2) proteins were also noticed among short-lived and long-lived Cytoraces. The Cytoraces have exhibited not only specific changes in Sod1 and Rpd3, but also show pronounced changes in the levels of synthesis of these proteins, which indicates rapid evolution of these Cytoraces in laboratory. Further these Cytoraces have become a model system to understand the process of anagenesis. [ABSTRACT FROM AUTHOR]

Telomeres are specialized structures at the end of eukaryotic chromosomes that are required to preserve genome integrity, chromosome stability and nuclear architecture. Telomere maintenance and function are established epigenetically in several eukaryotes. However, the exact chromatin enzymatic modifications regulating telomere homeostasis are poorly understood. In Drosophila melanogaster, telomere length and stability are maintained through the retrotransposition of specialized telomeric sequences and by the specific loading of protecting capping proteins, respectively. Here, we show that the loss of the essential and evolutionarily conserved histone deacetylase Rpd3, the homolog of mammalian HDAC1, causes aberrant telomeric fusions on polytene chromosome ends. Remarkably, these telomere fusion defects are associated with a marked decrease of histone H4 acetylation, as well as an accumulation of heterochromatic epigenetic marks at telomeres, including histone H3K9 trimethylation and the heterochromatic protein HP2. Our work suggests that Drosophila telomere structure is epigenetically regulated by the histone deacetylase Rpd3. [ABSTRACT FROM AUTHOR]

Most eukaryotes contain many tandem repeats of ribosomal RNA genes of which only a subset is transcribed at any given time. Current biochemical methods allow for the determination of the fraction of transcribing repeats (ON) versus non-transcribing repeats (OFF) but do not provide any dynamical information and obscure any transcription activity at the single-cell level. Here, we use a fluorescence in situ hybridization (FISH) technique that allows the detection of single-RNA molecules in individual yeast cells.We use this method complemented with theoretical modeling to determine the rate of switching from OFF to ON (activation rate) and the average number of RNA molecules produced during each transcriptional burst (burst size). We explore how these two variables change in mutants and different growth conditions, and show that this method resolves changes in these two variables even when the average rDNA expression is unaltered. These phenotypic changes could not have been detected by traditional biochemical assays. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae cells control their cell size at a point in late G1 called Start. Here, we describe a negative role for the Sin3/Rpd3 histone deacetylase complex in the regulation of cell size at Start. Initiation of G1/S-specific transcription of CLN1, CLN2 and PCL1 in a sin3Δ strain occurs at a reduced cell size compared with a wild-type strain. In addition, inactivation of the transcriptional regulator SIN3 partially suppressed a cln3Δ mutant, causing sin3Δcln3Δ double mutants to start the cell cycle at wild-type size. Chromatin immunoprecipitation results demonstrate that Sin3 and Rpd3 are recruited to promoters of SBF (Swi4/Swi6)-regulated genes, and reveal that binding of Sin3 to SBF-specific promoters is cell-cycle regulated. We observe that transcriptional repression of SBF-dependent genes in early G1 coincides with the recruitment of Sin3 to specific promoters, whereas binding of Sin3 is abolished from Swi4/Swi6-regulated promoters when transcription is activated at the G1 to S phase transition. We conclude that the Sin3/Rpd3 histone deacetylase complex helps to prevent premature activation of the S phase in daughter cells. [ABSTRACT FROM AUTHOR]

The article presents a study which examines how the modification of the histone tail regulates gene expression. It monitors the H3 acetylation level and the presence of the histone deacetylases (HDAC) at the site of seven non-meiotic and one meiotic gene from microarray analyses. Moreover, it also notes the protein-interaction analysis which indicates that more than 50 gene products physically interacts with Sin3.

To shed light on the specific contribution of HDA101 in modulating metabolic pathways in the maize seed, changes in the metabolic profiles of kernels obtained from hda101 mutant plants have been investigated by a metabonomic approach. Dynamic properties of chromatin folding can be mediated by enzymes that modify DNA and histones. The enzymes responsible for the steady-state of histone acetylation are histone acetyltransferase and histone deacetylase (HDA). Therefore, it is interesting to evaluate the effects of up- and down-regulation of a Rpd-3 type HDA on the development of maize seeds in terms of metabolic changes. This has been reached by analysing nuclear magnetic resonance spectra by different chemometrician approaches, such as Orthogonal Projection to Latent Structure-Discriminant Analysis, Parallel Factors Analysis, and Multi-way Partial Least Squares-Discriminant Analysis (N-PLS-DA). In particular, the latter approaches were chosen because they explicitly take time into account, organizing data into a set of slices that refer to different steps of the developing process. The results show the good discriminating capabilities of the N-PLS-DA approach, even if the number of samples ought be increased to obtain better predictive capabilities. However, using this approach, it was possible to show differences in the accumulation of metabolites during development and to highlight the changes occuring in the modified seeds. In particular, the results confirm the role of this gene in cell cycle control. [ABSTRACT FROM PUBLISHER]

The PDR5 gene encodes the major multidrug resistance efflux pump in Saccharomyces cerevisiae. In drug-resistant cells, the hyperactive Pdr1p or Pdr3p transcriptional activators are responsible for the PDR5 upregulation. In this work, it is shown that the RPD3 gene encoding the histone deacetylase that functions as a transcriptional corepressor at many promoters and the ROM2 gene coding for the GDP/GTP exchange protein for Rho1p and Rho2p participating in signal transduction pathways are required for PDR5 transcription under cycloheximide-induced and noninduced conditions. Transposon insertion mutations in ROM2, RPD3 and some other genes encoding specific subunits of the large Rpd3L protein complex resulted in enhanced susceptibility of mutant cells to antifungals. In the rpd3Δ and rom2Δ mutants, the level of PDR5 mRNA and the rate of rhodamine 6G efflux were reduced. Unlike rpd3Δ, in rom2Δ mutant cells the drug hypersensitivity and the defect in PDR5 expression were suppressed by PDR1 or PDR3 overexpressed from heterologous promoters and by the hyperactive pdr3-9 mutant allele. The results indicate that Rpd3p histone deacetylase participating in chromatin remodeling and Rom2p participating in the cell integrity pathway are involved in the control of PDR5 expression and modulation of multidrug resistance in yeast. [ABSTRACT FROM AUTHOR]

Arabidopsis thaliana histone deacetylase 1 (AtHD1 or AtHDA19), a homolog of yeast RPD3, is a global regulator of many physiological and developmental processes in plants. In spite of the genetic evidence for a role of AtHD1 in plant gene regulation and development, the biochemical and cellular properties of AtHD1 are poorly understood. Here we report cellular localization patterns of AtHD1 in vivo and histone deacetylase activity in vitro. The transient and stable expression of a green fluorescent protein (GFP)-tagged AtHD1 in onion cells and in roots, seeds and leaves of the transgenic Arabidopsis, respectively, revealed that AtHD1 is localized in the nucleus presumably in the euchromatic regions and excluded from the nucleolus. The localization patterns of AtHD1 are different from those of AtHD2 and AtHDA6 that are involved in nucleolus formation and silencing of transgenes and repeated DNA elements, respectively. In addition, a histone deacetylase activity assay showed that the recombinant AtHD1 produced in bacteria demonstrated a specific histone deacetylase activity in vitro. The data suggest that AtHD1 is a nuclear protein and possesses histone deacetylase activities responsible for global transcriptional regulation important to plant growth and development.Cell Research (2006) 16: 479–488. doi:10.1038/sj.cr.7310059; published online 15 May 2006 [ABSTRACT FROM AUTHOR]

Presents a study that investigated whether the modulation of heterochromatin-mediated gene silencing via alterations in the expression level of for heterochromatin protein 1 is sufficient to affect survival of adult Drosophila melanogaster. Inhibition of heterochromatin-mediated gene silencing; Survivorship and mortality rate in adults with one allele of Su(var)2-5; Mediation of lifespan by Sir2.

rRNA transcription in Saccharomyces cerevisiae is performed by RNA polymerase I and regulated by changes in growth conditions. During log phase, ∼50% of the ribosomal DNA (rDNA) genes in each cell are transcribed and maintained in an open, psoralen-accessible conformation. During stationary phase, the percentage of open rDNA genes is greatly reduced. In this study we found that the Rpd3 histone deacetylase was required to inactivate (close) individual rDNA genes as cells entered stationary phase. Even though ∼50% of the rDNA genes remained open during stationary phase in rpd3Δ mutants, overall rRNA synthesis was stilt reduced. Using electron microscopy of Miller chromatin spreads, we found that the number of RNA polymerases transcribing each open gene in the rpd3Δ mutant was significantly reduced when cells grew past tog phase. Bulk levels of histone H3 and H4 acetylation were reduced during stationary phase in an RPD3-dependent manner. However, histone H3 and H4 acetylation was not significantly altered at the rDNA locus in an rpd3Δ mutant. Rpd3 therefore regulates the number of open rDNA repeats. [ABSTRACT FROM AUTHOR]

Caloric restriction has been demonstrated to extend life span and postpone aging in a variety of species. The recent extension of the caloric restriction paradigm to yeast places the emphasis of the search for the longevity effectors at the cellular level. To narrow the range of potential effectors of the caloric restriction response, we have examined the effects of the histone deacetylases Rpd3p, Hda1p, and Sir2p, which have distinguishable but partially overlapping influences on global patterns of gene expression, on the life extension afforded by caloric restriction. Deletion of the RPD3 gene extended life span, and there was no additive effect of caloric restriction. Deletion of HDA1 had no effect of its own on longevity but acted synergistically with caloric restriction to increase life span. SIR2 deletion shortened life span but did not prevent extension of life span by caloric restriction. The results suggest that Rpd3p affects both processes that play an obligate and those that play a synergistic role in life extension by caloric restriction, while Hda1p and Sir2p affect processes that are not the obligate longevity effectors of caloric restriction but instead synergize with them, although in opposite directions. From the known patterns of gene expression elicited by rpd3Δ, hda1Δ, and sir2Δ, we propose that the major longevity effectors of caloric restriction in yeast involve carbohydrate/energy metabolism and mitochondrial function. [Copyright &y& Elsevier]

We examined the consequences of the deacetylase inhibitor trichostatin A (TSA) on the development of Drosophila melanogaster. When fed to flies, TSA caused lethality and delayed development at concentrations as low as 5 μM, had stronger effects on males than females, and acted synergistically with mutations in the gene encoding the RPD3 deacetylase to cause notched wings, but did not appear to affect a SINA signaling pathway that is normally repressed by the SIN3 corepressor. These findings suggest that deacetylated histones play an important role in normal developmental progression and establish parameters for genetic screens to dissect the role of deacetylases in this process. [ABSTRACT FROM AUTHOR]

Acetylation of core histone N-terminal tails influences chromatin condensation and transcription. To examine how the SIN3-RPD3 deacetylase complex contributes to these events in vivo, we examined binding of SIN3 and RPD3 to Drosophila salivary gland polytene chromosomes. The binding patterns of SIN3 and RPD3 were highly coincident, suggesting that the SIN3-RPD3 complex is the most abundant chromatin-bound RPD3 complex in salivary gland cells. SIN3-RPD3 binding was restricted to less condensed, hypo-acetylated euchromatic interbands and was absent from moderately condensed, hyperacetylated euchromatic bands and highly condensed, differentially acetylatcd centric heterochromatin. Consistent with its demonstrated role in transcriptional repression, SIN3-RPD3 did not co-localize with RNA polymerase II. Chromatin binding of the complex, mediated by SMRTER, decreased upon ecdysone-induced transcriptional activation but was restored when transcription was reduced. These results implicate SIN3-RPD3 in maintaining histone acetylation levels or patterns within less condensed chromatin domains and suggest that SIN3-RPD3 activity is required, in the absence of an activation signal, to repress transcription of particular genes within transcriptionally active chromatin domains. [ABSTRACT FROM AUTHOR]

Histone acetylation is modulated through the action of histone acetyltransferase and deacetylase, which play key roles in the regulation of eukaryotic gene expression. We have screened the expressed sequence tag database with the yeast histone deacetylase RPD3 sequence and identified two Arabidopsis homologues, AtRPD3A and AtRPD3B. The deduced amino acid sequences of AtRPD3A and AtRPD3B show high overall homology (55% identity) to each other. AtRPD3A encodes a putative protein of 502 amino acids with 49% identity to the yeast RPD3. AtRPD3B encodes a putative protein of 471 amino acids and shares 55% amino acid identity with the yeast RPD3. Northern analysis indicated that AtRPD3A was highly expressed in the leaves, stems, flowers and young siliques of Arabidopsis plants, whereas the AtRPD3B transcript was not detected in these organs. An AtRPD3A fusion protein repressed transcription when directed to a promoter driving a reporter gene, indicating a role for AtRPD3A protein in gene repression. Arabidopsis plants were transformed with a gene construct comprising a truncated AtRPD3A cDNA in the antisense orientation driven by a strong constitutive promoter, −394tCUP. Antisense expression of AtRPD3A resulted in decreased endogenous AtRPD3A transcript and delayed flowering in transgenic Arabidopsis plants, suggesting that the transition from the vegetative to reproductive phase of development could be affected by histone acetylation. Our study demonstrates the important role of histone deacetylases in plant growth and development. [ABSTRACT FROM AUTHOR]