Abo1, a conserved bromodomain AAA-ATPase, maintains global nucleosome occupancy and organisation.

Maintenance of the correct level and organisation of nucleosomes is crucial for genome function. Here, we uncover a role for a conserved bromodomain AAA-ATPase, Abo1, in the maintenance of nucleosome architecture in fission yeast. Cells lacking abo1(+) experience both a reduction and mis-positioning of nucleosomes at transcribed sequences in addition to increased intragenic transcription, phenotypes that are hallmarks of defective chromatin re-establishment behind RNA polymerase II. Abo1 is recruited to gene sequences and associates with histone H3 and the histone chaperone FACT. Furthermore, the distribution of Abo1 on chromatin is disturbed by impaired FACT function. The role of Abo1 extends to some promoters and also to silent heterochromatin. Abo1 is recruited to pericentromeric heterochromatin independently of the HP1 ortholog, Swi6, where it enforces proper nucleosome occupancy. Consequently, loss of Abo1 alleviates silencing and causes elevated chromosome mis-segregation. We suggest that Abo1 provides a histone chaperone function that maintains nucleosome architecture genome-wide.

The longevity and reversibility of quiescence in Schizosaccharomyces pombe are dependent upon the HIRA histone chaperone.

Quiescence (G0) is a reversible non-dividing state that facilitates cellular survival in adverse conditions. Here, we demonstrate that the HIRA histone chaperone complex is required for the reversibility and longevity of nitrogen starvation-induced quiescence in Schizosaccharomyces pombe. The HIRA protein, Hip1 is not required for entry into G0 or the induction of autophagy. Although hip1Δ cells retain metabolic activity in G0, they rapidly lose the ability to resume proliferation. After a short period in G0 (1 day), hip1Δ mutants can resume cell growth in response to the restoration of a nitrogen source but do not efficiently reenter the vegetative cell cycle. This correlates with a failure to induce the expression of MBF transcription factor-dependent genes that are critical for S phase. In addition, hip1Δ G0 cells rapidly progress to a senescent state in which they can no longer re-initiate growth following nitrogen source restoration. Analysis of a conditional hip1 allele is consistent with these findings and indicates that HIRA is required for efficient exit from quiescence and prevents an irreversible cell cycle arrest.

Heterochromatin in the fungal plant pathogen, Zymoseptoria tritici: Control of transposable elements, genome plasticity and virulence.

Heterochromatin is a repressive chromatin state that plays key roles in the functional organisation of eukaryotic genomes. In fungal plant pathogens, effector genes that are required for host colonization tend to be associated with heterochromatic regions of the genome that are enriched with transposable elements. It has been proposed that the heterochromatin environment silences effector genes in the absence of host and dynamic chromatin remodelling facilitates their expression during infection. Here we discuss this model in the context of the key wheat pathogen, Zymoseptoria tritici. We cover progress in understanding the deposition and recognition of heterochromatic histone post translational modifications in Z. tritici and the role that heterochromatin plays in control of genome plasticity and virulence.

Response of Schizosaccharomyces pombe to zinc deficiency.

A component of the cellular response to zinc deficiency operates via control of transcript abundance. Therefore, microarray analysis was employed to identify Schizosaccharomyces pombe genes whose mRNA levels are regulated by intracellular zinc status. A set of 57 genes whose mRNA levels were substantially reduced in response to zinc deficiency was identified, while the mRNA levels of 63 genes were increased by this condition. In order to investigate the mechanisms that control these responses, a genetic screen was employed to identify mutants with defective zinc-responsive gene expression. Two strains (II-1 and V7) that were identified by this screen harbor mutations that are linked to zrt1+, which encodes a putative Zrt/IRT-like protein (ZIP) zinc uptake transporter. Importantly, zrt1+ mRNA levels are increased in response to zinc deprivation, and cells lacking functional Zrt1 are highly impaired in their ability to proliferate at limiting zinc concentrations. Furthermore, zrt1 null cells were found to have severely reduced zinc contents, indicating that Zrt1 functions as a key regulator of intracellular zinc levels in fission yeast. The deletion of fet4+, another zinc-responsive gene encoding a putative metal ion transporter, exacerbated the phenotypes associated with the loss of Zrt1, suggesting that Fet4 also plays a role in zinc uptake under limiting conditions.

The Schizosaccharomyces pombe HIRA-like protein Hip1 is required for the periodic expression of histone genes and contributes to the function of complex centromeres.

HIRA-like (Hir) proteins are evolutionarily conserved and are implicated in the assembly of repressive chromatin. In Saccharomyces cerevisiae, Hir proteins contribute to the function of centromeres. However, S. cerevisiae has point centromeres that are structurally different from the complex centromeres of metazoans. In contrast, Schizosaccharomyces pombe has complex centromeres whose domain structure is conserved with that of human centromeres. Therefore, we examined the functions of the fission yeast Hir proteins Slm9 and the previously uncharacterised protein Hip1. Deletion of hip1(+) resulted in phenotypes that were similar to those described previously for slm9 Delta cells: a cell cycle delay, synthetic lethality with cdc25-22, and poor recovery from nitrogen starvation. However, while it has previously been shown that Slm9 is not required for the periodic expression of histone H2A, we found that loss of Hip1 led to derepression of core histone genes expression outside of S phase. Importantly, we found that deletion of either hip1(+) or slm9(+) resulted in increased rates of chromosome loss, increased sensitivity to spindle damage, and reduced transcriptional silencing in the outer centromeric repeats. Thus, S. pombe Hir proteins contribute to pericentromeric heterochromatin, and our data thus suggest that Hir proteins may be required for the function of metazoan centromeres.

Role of fission yeast Tup1-like repressors and Prr1 transcription factor in response to salt stress.

In Schizosaccharomyces pombe, the Sty1 mitogen-activated protein kinase and the Atf1 transcription factor control transcriptional induction in response to elevated salt concentrations. Herein, we demonstrate that two repressors, Tup11 and Tup12, and the Prr1 transcription factor also function in the response to salt shock. We find that deletion of both tup genes together results in hypersensitivity to elevated cation concentrations (K(+) and Ca(2+)) and we identify cta3(+), which encodes an intracellular cation transporter, as a novel stress gene whose expression is positively controlled by the Sty1 pathway and negatively regulated by Tup repressors. The expression of cta3(+) is maintained at low levels by the Tup repressors, and relief from repression requires the Sty1, Atf1, and Prr1. Prr1 is also required for KCl-mediated induction of several other Sty1-dependent genes such as gpx1(+) and ctt1(+). Surprisingly, the KCl-mediated induction of cta3(+) expression occurs independently of Sty1 in a tup11Delta tup12Delta mutant and so the Tup repressors link induction to the Sty1 pathway. We also report that in contrast to a number of other Sty1- and Atf1-dependent genes, the expression of cta3(+) is induced only by high salt concentrations. However, in the absence of the Tup repressors this specificity is lost and a range of stresses induces cta3(+) expression.

Restriction of Retrotransposon Mobilization in Schizosaccharomyces pombe by Transcriptional Silencing and Higher-Order Chromatin Organization.

Uncontrolled propagation of retrotransposons is potentially detrimental to host genome integrity. Therefore, cells have evolved surveillance mechanisms to restrict the mobility of these elements. In Schizosaccharomyces pombe the Tf2 LTR retrotransposons are transcriptionally silenced and are also clustered in the nucleus into structures termed Tf bodies. Here we describe the impact of silencing and clustering on the mobility of an endogenous Tf2 element. Deletion of genes such as set1(+) (histone H3 lysine 4 methyltransferase) or abp1(+) (CENP-B homolog) that both alleviate silencing and clustering, result in a corresponding increase in mobilization. Furthermore, expression of constitutively active Sre1, a transcriptional activator of Tf2 elements, also alleviates clustering and induces mobilization. In contrast, clustering is not disrupted by loss of the HIRA histone chaperone, despite high levels of expression, and in this background, mobilization frequency is only marginally increased. Thus, mutations that compromise transcriptional silencing but not Tf bodies are insufficient to drive mobilization. Furthermore, analyses of mutant alleles that separate the transcriptional repression and clustering functions of Set1 are consistent with control of Tf2 propagation via a combination of silencing and spatial organization. Our results indicate that host surveillance mechanisms operate at multiple levels to restrict Tf2 retrotransposon mobilization.

The impact of the HIRA histone chaperone upon global nucleosome architecture.

HIRA is an evolutionarily conserved histone chaperone that mediates replication-independent nucleosome assembly and is important for a variety of processes such as cell cycle progression, development, and senescence. Here we have used a chromatin sequencing approach to determine the genome-wide contribution of HIRA to nucleosome organization in Schizosaccharomyces pombe. Cells lacking HIRA experience a global reduction in nucleosome occupancy at gene sequences, consistent with the proposed role for HIRA in chromatin reassembly behind elongating RNA polymerase II. In addition, we find that at its target promoters, HIRA commonly maintains the full occupancy of the -1 nucleosome. HIRA does not affect global chromatin structure at replication origins or in rDNA repeats but is required for nucleosome occupancy in silent regions of the genome. Nucleosome organization associated with the heterochromatic (dg-dh) repeats located at the centromere is perturbed by loss of HIRA function and furthermore HIRA is required for normal nucleosome occupancy at Tf2 LTR retrotransposons. Overall, our data indicate that HIRA plays an important role in maintaining nucleosome architecture at both euchromatic and heterochromatic loci.

Quantitative Fitness Analysis Identifies exo1∆ and Other Suppressors or Enhancers of Telomere Defects in Schizosaccharomyces pombe.

Synthetic genetic array (SGA) has been successfully used to identify genetic interactions in S. cerevisiae and S. pombe. In S. pombe, SGA methods use either cycloheximide (C) or heat shock (HS) to select double mutants before measuring colony size as a surrogate for fitness. Quantitative Fitness Analysis (QFA) is a different method for determining fitness of microbial strains. In QFA, liquid cultures are spotted onto solid agar and growth curves determined for each spot by photography and model fitting. Here, we compared the two S. pombe SGA methods and found that the HS method was more reproducible for us. We also developed a QFA procedure for S. pombe. We used QFA to identify genetic interactions affecting two temperature sensitive, telomere associated query mutations (taz1Δ and pot1-1). We identify exo1∆ and other gene deletions as suppressors or enhancers of S. pombe telomere defects. Our study identifies known and novel gene deletions affecting the fitness of strains with telomere defects. The interactions we identify may be relevant in human cells.

An Atg10-like E2 enzyme is essential for cell cycle progression but not autophagy in Schizosaccharomyces pombe.

Many proteins involved in autophagy have been identified in the yeast Saccharomyces cerevisiae. For example, Atg3 and Atg10 are two E2 enzymes that facilitate the conjugation of the ubiquitin-like proteins (Ubls) Atg8 and Atg12, respectively. Here, we describe the identification and characterization of the predicted Atg10 homolog (SpAtg10) of the evolutionarily distant Schizosaccharomyces pombe. Unexpectedly, SpAtg10 is not essential for autophagy. Instead, we find that SpAtg10 is essential for normal cell cycle progression, and for responses to various stress conditions that perturb the cell cycle, independently of Atg12 conjugation. Taken together, our data indicate that autophagic Ubl conjugation pathways differ between eukaryotes and, furthermore, that enzymes such as Atg10 may have additional functions in controlling key cellular processes such as cell cycle progression. Atg10-related proteins are found from yeast to humans, and, thus, this study has implications for understanding the functions of this protein family in Ubl conjugation in eukaryotes.

A homeodomain transcription factor regulates the DNA replication checkpoint in yeast.

Checkpoints monitor the successful completion of cell cycle processes, such as DNA replication, and also regulate the expression of cell cycle-dependent genes that are required for responses. In the model yeast Schizosaccharomyces pombe G 1/S phase-specific gene expression is regulated by the MBF (also known as DSC1) transcription factor complex and is also activated by the mammalian ATM/ATR-related Rad3 DNA replication checkpoint. Here, we show that the Yox1 homeodomain transcription factor acts to co-ordinate the expression of MBF-regulated genes during the cell division cycle. Moreover, our data suggests that Yox1 is inactivated by the Rad3 DNA replication checkpoint via phosphorylation by the conserved Cds1 checkpoint kinase. Collectively, our data has implications for understanding the mechanisms underlying the coordination of cell cycle processes in eukaryotes.

Silencing mediated by the Schizosaccharomyces pombe HIRA complex is dependent upon the Hpc2-like protein, Hip4.

BACKGROUND: HIRA (or Hir) proteins are conserved histone chaperones that function in multi-subunit complexes to mediate replication-independent nucleosome assembly. We have previously demonstrated that the Schizosaccharomyces pombe HIRA proteins, Hip1 and Slm9, form a complex with a TPR repeat protein called Hip3. Here we have identified a new subunit of this complex. METHODOLOGY/PRINCIPAL FINDINGS: To identify proteins that interact with the HIRA complex, rapid affinity purifications of Slm9 were performed. Multiple components of the chaperonin containing TCP-1 complex (CCT) and the 19S subunit of the proteasome reproducibly co-purified with Slm9, suggesting that HIRA interacts with these complexes. Slm9 was also found to interact with a previously uncharacterised protein (SPBC947.08c), that we called Hip4. Hip4 contains a HRD domain which is a characteristic of the budding yeast and human HIRA/Hir-binding proteins, Hpc2 and UBN1. Co-precipitation experiments revealed that Hip4 is stably associated with all of the other components of the HIRA complex and deletion of hip4(+) resulted in the characteristic phenotypes of cells lacking HIRA function, such as temperature sensitivity, an elongated cell morphology and hypersensitivity to the spindle poison, thiabendazole. Moreover, loss of Hip4 function alleviated the heterochromatic silencing of reporter genes located in the mating type locus and centromeres and was associated with increased levels of non-coding transcripts derived from centromeric repeat sequences. Hip4 was also found to be required for the distinct form of silencing that controls the expression of Tf2 LTR retrotransposons. CONCLUSIONS/SIGNIFICANCE: Overall, these results indicate that Hip4 is an integral component of the HIRA complex that is required for transcriptional silencing at multiple loci.

tRNA genes in eukaryotic genome organization and reorganization.

The primary function of tRNA genes is to provide the templates for the transcription of essential tRNA molecules. However, there is now evidence that these dispersed repetitive elements have the potential to mediate the spatial and functional organization of the genome and to drive genome change and evolution. Indeed, tRNA genes and related Pol III promoter elements can occupy distinct subnuclear positions and also provide barriers which functionally separate domains of chromatin. Furthermore, tRNA genes can also represent barriers to DNA replication fork progression and accordingly, tRNA genes can contribute to the formation of genomic fragile sites and have been implicated in genome evolution. Here we give insight into our current understanding of these "extra transcriptional" functions of tRNA genes and discuss how these functions may impact upon genome regulation and evolution.

The fission yeast HIRA histone chaperone is required for promoter silencing and the suppression of cryptic antisense transcripts.

The assembly of nucleosomes by histone chaperones is an important component of transcriptional regulation. Here, we have assessed the global roles of the HIRA histone chaperone in Schizosaccharomyces pombe. Microarray analysis indicates that inactivation of the HIRA complex results in increased expression of at least 4% of fission yeast genes. HIRA-regulated genes overlap with those which are normally repressed in vegetatively growing cells, such as targets of the Clr6 histone deacetylase and silenced genes located in subtelomeric regions. HIRA is also required for silencing of all 13 intact copies of the Tf2 long terminal repeat (LTR) retrotransposon. However, the role of HIRA is not restricted to bona fide promoters, because HIRA also suppresses noncoding transcripts from solo LTR elements and spurious antisense transcripts from cryptic promoters associated with transcribed regions. Furthermore, the HIRA complex is essential in the absence of the quality control provided by nuclear exosome-mediated degradation of illegitimate transcripts. This suggests that HIRA restricts genomic accessibility, and consistent with this, the chromosomes of cells lacking HIRA are more susceptible to genotoxic agents that cause double-strand breaks. Thus, the HIRA histone chaperone is required to maintain the protective functions of chromatin.

Hip3 interacts with the HIRA proteins Hip1 and Slm9 and is required for transcriptional silencing and accurate chromosome segregation.

The fission yeast HIRA proteins Hip1 and Slm9 are members of an evolutionarily conserved family of histone chaperones that are implicated in nucleosome assembly. Here we have used single-step affinity purification and mass spectrometry to identify factors that interact with both Hip1 and Slm9. This analysis identified Hip3, a previously uncharacterized 187-kDa protein, with similarity to S. cerevisiae Hir3. Consistent with this, cells disrupted for hip3+ exhibit a range of growth defects that are similar to those associated with loss of Hip1 and Slm9. These include temperature sensitivity, a cell cycle delay, and synthetic lethality with cdc25-22. Furthermore, genetic analysis also indicates that disruption of hip3+ is epistatic with mutation of hip1+ and slm9+. Mutation of hip3+ alleviates transcriptional silencing at several heterochromatic loci, including in the outer (otr) centromeric repeats, indicating that Hip3 is required for the integrity of pericentric heterochromatin. As a result, loss of Hip3 function leads to high levels of minichromosome loss and an increased frequency of lagging chromosomes during mitosis. Importantly, the function of Hip1, Slm9, and Hip3 is not restricted to constitutive heterochromatic loci, since these proteins also repress the expression of a number of genes, including the Tf2 retrotransposons.

Surplus zinc is handled by Zym1 metallothionein and Zhf endoplasmic reticulum transporter in Schizosaccharomyces pombe.

Homeostatic mechanisms prevent the accumulation of free zinc in the cytoplasm, raising questions regarding where surplus zinc is stored and how it is delivered to and from these stores. A genetic screen for zinc hypersensitivity in Schizosaccharomyces pombe identified a missense mutation truncating Zhf, an endoplasmic reticulum transporter. These cells were approximately 5-fold more zinc-sensitive than other independent mutants. The targeted disruption of zhf prevented growth on low zinc medium and caused hypersensitivity to elevated zinc/cobalt but resistance to cadmium. The exposure to elevated zinc but not copper also promotes the accumulation of transcripts encoding a metallothionein designated Zym1. The Sty1 pathway is required for maximal zym1 expression but is not obligatory for zinc perception. The targeted disruption of zym1 impaired cadmium tolerance but only slightly impaired zinc tolerance, whereas zym1 overexpression substantially rescued zinc hypersensitivity of zhf(-) cells. Four equivalents of zinc were displaced from Zym1 by up to 12 equivalents of p-(hydroxymercuri)phenylsulphonate. Zym1 thiols react rapidly with 5,5'-dithiobis-(2-nitrobenzoic acid) compared with bacterial zinc metallothionein (6.8 and 0.2 x 10(-4) s(-1), respectively). Zym1 is unlike known fungal metallothioneins that are induced by and sequester copper but not zinc. Less zinc but normal cadmium was accumulated by zym1Delta, consistent with zinc sequestration by Zym1 in vivo.

Suppressors of an adenylate cyclase deletion in the fission yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe utilizes two opposing signaling pathways to sense and respond to its nutritional environment. Glucose detection triggers a cyclic AMP signal to activate protein kinase A (PKA), while glucose or nitrogen starvation activates the Spc1/Sty1 stress-activated protein kinase (SAPK). One process controlled by these pathways is fbp1+ transcription, which is glucose repressed. In this study, we isolated strains carrying mutations that reduce high-level fbp1+ transcription conferred by the loss of adenylate cyclase (git2delta), including both wis1- (SAPK kinase) and spc1- (SAPK) mutants. While characterizing the git2delta suppressor strains, we found that the git2delta parental strains are KCl sensitive, though not osmotically sensitive. Of 102 git2delta suppressor strains, 17 strains display KCl-resistant growth and comprise a single linkage group, carrying mutations in the cgs1+ PKA regulatory subunit gene. Surprisingly, some of these mutants are mostly wild type for mating and stationary-phase viability, unlike the previously characterized cgs1-1 mutant, while showing a significant defect in fbp1-lacZ expression. Thus, certain cgs1- mutant alleles dramatically affect some PKA-regulated processes while having little effect on others. We demonstrate that the PKA and SAPK pathways regulate both cgs1+ and pka1+ transcription, providing a mechanism for cross talk between these two antagonistically acting pathways and feedback regulation of the PKA pathway. Finally, strains defective in both the PKA and SAPK pathways display transcriptional regulation of cgs1+ and pka1+, suggesting the presence of a third glucose-responsive signaling pathway.

The forkhead transcription factor Fkh2 regulates the cell division cycle of Schizosaccharomyces pombe.

In eukaryotes the regulation of gene expression plays a key role in controlling cell cycle progression. Here, we demonstrate that a forkhead transcription factor, Fkh2, regulates the periodic expression of cdc15(+) and spo12(+) in the M and G(1) phases of the cell division cycle in the fission yeast Schizosaccharomyces pombe. We also show that Fkh2 is important for several cell cycle processes, including cell morphology and cell separation, nuclear structure and migration, and mitotic spindle function. We find that the expression of fkh2(+) is itself regulated in a cell cycle-dependent manner in G(1) coincident with the expression of cdc18(+), a Cdc10-regulated gene. However, fkh2(+) expression is independent of Cdc10 function. Fkh2 was found to be phosphorylated during the cell division cycle, with a timing that suggests that this posttranslational modification is important for cdc15(+) and spo12(+) expression. Related forkhead proteins regulate G(2) and M phase-specific gene expression in the evolutionarily distant Saccharomyces cerevisiae, suggesting that these proteins play conserved roles in regulating cell cycle processes in eukaryotes.