Mutation in histone deacetylase clr6 promotes the survival of S. pombe cds1 null mutant in response to hydroxyurea.

Fission yeast Cds1 is responsible for the replication checkpoint activation and helps to protect replication fork collapse in response to hydroxyurea (HU). Here, we investigated the role of histone deacetylase in response to replication fork arrest and observed that in the presence of HU, the survival of cds1Δ cells was improved when the cells were simultaneously treated with histone deacetylase inhibitors. Furthermore, a mutation in the histone deacetylase gene, clr6, also suppresses the growth defect of cds1Δ cells in response to HU indicating a suppressive role of clr6-1 mutation in cds1 deletion background upon HU treatment. Interestingly, in response to HU, phosphorylation of Chk1 kinase and the number of Rad52YFP foci was reduced in cds1Δ clr6-1 double mutant as compared to cds1Δ single mutant indicating a decrease in the level of DNA damage in response to HU. Accordingly, the single-cell gel electrophoresis assay revealed a drastic reduction in the tail length of cds1Δ clr6-1 double mutant as compared to cds1Δ cells in the presence of HU suggesting the suppression of chromosomal defects in the double mutant. Taken together, we proposed that there could be transient suppression of fork collapse in cds1Δ clr6-1 double mutant upon HU treatment due to the delay in mitotic progression that leads to the facilitation of cell growth.

Kinetochore Components Required for Centromeric Chromatin Assembly Are Impacted by Msc1 in Schizosaccharomyces pombe.

Eukaryotic chromosome segregation requires a protein complex known as the kinetochore that mediates attachment between mitotic spindle microtubules and centromere-specific nucleosomes composed of the widely conserved histone variant CENP-A. Mutations in kinetochore proteins of the fission yeast Schizosaccharomyces pombe lead to chromosome missegregation such that daughter cells emerge from mitosis with unequal DNA content. We find that multiple copies of Msc1-a fission yeast homolog of the KDM5 family of proteins-suppresses the temperature-sensitive growth defect of several kinetochore mutants, including mis16 and mis18, as well as mis6, mis15, and mis17, components of the Constitutive Centromere Associated Network (CCAN). On the other hand, deletion of msc1 exacerbates both the growth defect and chromosome missegregation phenotype of each of these mutants. The C-terminal PHD domains of Msc1, previously shown to associate with a histone deacetylase activity, are necessary for Msc1 function when kinetochore mutants are compromised. We also demonstrate that, in the absence of Msc1, the frequency of localization to the kinetochore of Mis16 and Mis15 is altered from wild-type cells. As we show here for msc1, others have shown that elevating cnp1 levels acts similarly to promote survival of the CCAN mutants. The rescue of mis15 and mis17 by cnp1 is, however, independent of msc1 Thus, Msc1 appears to contribute to the chromatin environment at the centromere: the absence of Msc1 sensitizes cells to perturbations in kinetochore function, while elevating Msc1 overcomes loss of function of critical components of the kinetochore and centromere.

Microtubule dynamics decoded by the epigenetic state of centromeric chromatin.

Cell division with accurate chromosome segregation is fundamental to cell survival of all organisms. The precise molecular mechanisms that ensure accurate chromosome segregation are still being discovered using a variety of experimental systems and approaches. Microtubule attachment to the kinetochore is a prerequisite for mitotic progression, failure of which activates the spindle assembly checkpoint (SAC). The dynamic tension generated by interaction of the centromere, kinetochore and microtubules is a key regulator of the SAC. Here, in the context of current literature we discuss our recent observation in fission yeast that epigenetic alterations in centromeric and pericentromeric chromatin can compensate for altered dynamics of kinetochore-microtubule attachment to permit escape from mitotic arrest. A role for the spatial configuration of the centromere to influence the finely tuned regulators of mitotic progression opens up new avenues for research.

Escape from Mitotic Arrest: An Unexpected Connection Between Microtubule Dynamics and Epigenetic Regulation of Centromeric Chromatin in Schizosaccharomyces pombe.

Accurate chromosome segregation is necessary to ensure genomic integrity. Segregation depends on the proper functioning of the centromere, kinetochore, and mitotic spindle microtubules and is monitored by the spindle assembly checkpoint (SAC). In the fission yeast Schizosaccharomyces pombe, defects in Dis1, a microtubule-associated protein that influences microtubule dynamics, lead to mitotic arrest as a result of an active SAC and consequent failure to grow at low temperature. In a mutant dis1 background (dis1-288), loss of function of Msc1, a fission yeast homolog of the KDM5 family of proteins, suppresses the growth defect and promotes normal mitosis. Genetic analysis implicates a histone deacetylase (HDAC)-linked pathway in suppression because HDAC mutants clr6-1, clr3∆, and sir2∆, though not hos2∆, also promote normal mitosis in the dis1-288 mutant. Suppression of the dis phenotype through loss of msc1 function requires the spindle checkpoint protein Mad2 and is limited by the presence of the heterochromatin-associated HP1 protein homolog Swi6. We speculate that alterations in histone acetylation promote a centromeric chromatin environment that compensates for compromised dis1 function by allowing for successful kinetochore-microtubule interactions that can satisfy the SAC. In cells arrested in mitosis by mutation of dis1, loss of function of epigenetic determinants such as Msc1 or specific HDACs can promote cell survival. Because the KDM5 family of proteins has been implicated in human cancers, an appreciation of the potential role of this family of proteins in chromosome segregation is warranted.

The oxidative stress responsive transcription factor Pap1 confers DNA damage resistance on checkpoint-deficient fission yeast cells.

Eukaryotic cells invoke mechanisms to promote survival when confronted with cellular stress or damage to the genome. The protein kinase Chk1 is an integral and conserved component of the DNA damage response pathway. Mutation or inhibition of Chk1 results in mitotic death when cells are exposed to DNA damage. Oxidative stress activates a pathway that results in nuclear accumulation of the bZIP transcription factor Pap1. We report the novel finding that fission yeast Pap1 confers resistance to drug- and non-drug-induced DNA damage even when the DNA damage checkpoint is compromised. Multi-copy expression of Pap1 restores growth to chk1-deficient cells exposed to camptothecin or hydroxyurea. Unexpectedly, increased Pap1 expression also promotes survival of chk1-deficient cells with mutations in genes encoding DNA ligase (cdc17) or DNA polymerase δ (cdc6), but not DNA replication initiation mutants. The ability of Pap1 to confer resistance to DNA damage was not specific to chk1 mutants, as it also improved survival of rad1- and rad9-deficient cells in the presence of CPT. To confer resistance to DNA damage Pap1 must localize to the nucleus and be transcriptionally active.

A molecular evolution approach to study the roles of tropomyosin in fission yeast.

Tropomyosin, a coiled-coil protein that binds along the length of the actin filament, is a universal regulator of the actin cytoskeleton. We have taken a bioinformatics/proteomic approach to studying structure-function relationships in this protein. The presence of a single, essential tropomyosin gene, cdc8, in fission yeast, Schizosaccharomyces pombe, enables a systems-based approach to define the residues that are important for cellular functions. Using molecular evolution methodologies we identified the most conserved residues and related them to the coiled coil structure. Mutants in which one or more of 21 of the most conserved surface residues was mutated to Ala were tested for the ability to rescue growth of a temperature-sensitive cdc8 mutant when overexpressed at the restrictive temperature. Based on altered morphology of the septum and actin cytoskeleton, we selected three sets of mutations for construction of mutant cdc8 strains using marker reconstitution mutagenesis and analysis of recombinant protein in vitro: D16A.K30A, V114S.E117A.H118A and R121A.D131A.E138A. The mutations have sequence-specific effects on cellular morphology including cell length, organization of cytoskeletal structures (actin patches, actin cables and contractile rings), and in vitro actin affinity, lending credence to the proteomic approach introduced here. We propose that bioinformatics is a valid analysis tool for defining structure-function relationships in conserved proteins in this model organism.

Activity of a C-terminal plant homeodomain (PHD) of Msc1 is essential for function.

Msc1, a member of the Jarid1 family of putative histone demethylases, is required for chromosome stability in fission yeast. Msc1 associates with the Swr1 complex that facilitates deposition of histone H2A.Z into chromatin. To assess the function of Msc1 in the Swr1 complex, domains of Msc1 necessary for interaction with Swr1 were identified. The C-terminal plant homeodomain (PHD) 2 and PHD3 of Msc1 are sufficient to confer association with Swr1 and allow Msc1 to function in the context of kinetochore mutants. On the other hand, a mutant with a single amino acid substitution in PHD2 within the full-length Msc1 protein retains the ability to bind to Swr1 but eliminates the function of Msc1 in combination with kinetochore mutants. Thus, Swr1 association is critical but not sufficient for Msc1 function. An activity of Msc1 that depends on the cysteine residue within PHD2 of Msc1 is likewise critical for function. On the basis of our observation that the PHDs of Msc1 act as E3 ubiquitin ligases and that mutations of cysteine residues within those domains abolish ligase activity, we speculate that the ability of Msc1 to facilitate ubiquitin transfer is critical for the function it mediates through its association with Swr1.

Importance of a C-terminal conserved region of Chk1 for checkpoint function.

BACKGROUND: The protein kinase Chk1 is an essential component of the DNA damage checkpoint pathway. Chk1 is phosphorylated and activated in the fission yeast Schizosaccharomyces pombe when cells are exposed to agents that damage DNA. Phosphorylation, kinase activation, and nuclear accumulation are events critical to the ability of Chk1 to induce a transient delay in cell cycle progression. The catalytic domain of Chk1 is well-conserved amongst all species, while there are only a few regions of homology within the C-terminus. A potential pseudosubstrate domain exists in the C-terminus of S. pombe Chk1, raising the possibility that the C-terminus acts to inhibit the catalytic domain through interaction of this domain with the substrate binding site. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate this hypothesis, we characterized mutations in the pseudosubstrate region. Mutation of a conserved aspartic acid at position 469 to alanine or glycine compromises Chk1 function when the mutants are integrated as single copies, demonstrating that this domain of Chk1 is critical for function. Our data does not support, however, the hypothesis that the domain acts to inhibit Chk1 function as other mutations in the amino acids predicted to comprise the pseudosubstrate do not result in constitutive activation of the protein. When expressed in multi-copy, Chk1D469A remains non-functional. In contrast, multi-copy Chk1D469G confers cell survival and imposes a checkpoint delay in response to some, though not all forms of DNA damage. CONCLUSIONS/SIGNIFICANCE: Thus, we conclude that this C-terminal region of Chk1 is important for checkpoint function and predict that a limiting factor capable of associating with Chk1D469G, but not Chk1D469A, interacts with Chk1 to elicit checkpoint activation in response to a subset of DNA lesions.

Msc1 acts through histone H2A.Z to promote chromosome stability in Schizosaccharomyces pombe.

As a central component of the DNA damage checkpoint pathway, the conserved protein kinase Chk1 mediates cell cycle progression when DNA damage is generated. Msc1 was identified as a multicopy suppressor capable of facilitating survival in response to DNA damage of cells mutant for chk1. We demonstrate that loss of msc1 function results in an increased rate of chromosome loss and that an msc1 null allele exhibits genetic interactions with mutants in key kinetochore components. Multicopy expression of msc1 robustly suppresses a temperature-sensitive mutant (cnp1-1) in the centromere-specific histone H3 variant CENP-A, and localization of CENP-A to the centromere is compromised in msc1 null cells. We present several lines of evidence to suggest that Msc1 carries out its function through the histone H2A variant H2A.Z, encoded by pht1 in fission yeast. Like an msc1 mutant, a pht1 mutant also exhibits chromosome instability and genetic interactions with kinetochore mutants. Suppression of cnp1-1 by multicopy msc1 requires pht1. Likewise, suppression of the DNA damage sensitivity of a chk1 mutant by multicopy msc1 also requires pht1. We present the first genetic evidence that histone H2A.Z may participate in centromere function in fission yeast and propose that Msc1 acts through H2A.Z to promote chromosome stability and cell survival following DNA damage.

The plant homeodomain fingers of fission yeast Msc1 exhibit E3 ubiquitin ligase activity.

The DNA damage checkpoint pathway governs how cells regulate cell cycle progression in response to DNA damage. A screen for suppressors of a fission yeast chk1 mutant defective in the checkpoint pathway identified a novel Schizosaccharomyces pombe protein, Msc1. Msc1 contains 3 plant homeodomain (PHD) finger motifs, characteristically defined by a C4HC3 consensus similar to RING finger domains. PHD finger domains in viral proteins and in the cellular protein kinase MEKK1 (mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1) have been implicated as ubiquitin E3 protein ligases that affect protein stability. The close structural relationship of PHD fingers to RING fingers suggests that other PHD domain-containing proteins might share this activity. We show that each of the three PHD fingers of Msc1 can act as ubiquitin E3 ligases, reporting for the first time that PHD fingers from a nuclear protein exhibit E3 ubiquitin ligase activity. The function of the PHD fingers of Msc1 is needed to rescue the DNA damage sensitivity of a chk1Delta strain. Msc1 co-precipitates Rhp6, the S. pombe homologue of the human ubiquitin-conjugating enzyme Ubc2. Strikingly, deletion of msc1 confers complete suppression of the slow growth phenotype, UV and hydroxyurea sensitivities of an rhp6 deletion strain and restores deficient histone H3 methylation observed in the rhp6Delta mutant. We speculate that the target of the E3 ubiquitin ligase activity of Msc1 is likely to be a chromatin-associated protein.

Assaying cell cycle checkpoints: activity of the protein kinase Chk1.

Eukaryotic cells regulate progression through the cell cycle in response to DNA damage. Cell cycle checkpoints are the signal transduction pathways that couple the detection of DNA damage to the proteins that control transitions in the cell cycle. The protein kinase Chk1, originally discovered in fission yeast, but conserved in humans, is essential for preventing mitotic entry in the presence of DNA damage or blocks to DNA replication that cannot be reconciled. Chk1 is phosphorylated in response to DNA damage. Phosphorylation depends on the activity of conserved components of the checkpoint pathway including Rad3, a member of the ATM/ATR family of kinases. Phosphorylation leads to activation of Chk1 kinase activity. In this chapter, we describe an assay for monitoring the activity of Chk1 isolated.

Interaction of 14-3-3 protein with Chk1 affects localization and checkpoint function.

The protein kinase Chk1 is required for proper arrest of the cell cycle in response to DNA damage. We have previously shown in Schizosaccharomyces pombe, that upon DNA damage, phosphorylation of Chk1 correlates with checkpoint activation and that phosphorylated Chk1 is capable of interacting with the 14-3-3 proteins, Rad24 and Rad25. The interaction between Rad24 and Chk1 is stimulated tenfold after exposure to DNA damaging agents and we postulate that it is an important event in the DNA damage checkpoint response pathway in fission yeast. We identified a stretch of leucine residues as the domain in Chk1 that mediates the interaction with 14-3-3 proteins. Substitution of leucine residues with alanine disrupts the interaction with Rad24 and also prevents Chk1 from becoming phosphorylated in response to DNA damaging agents. Cells expressing the mutants are sensitive to UV radiation. In this study, we also show that Chk1 accumulates in the nucleus in response to DNA damage and this behavior is dependent on Rad24. Interestingly, the 14-3-3 binding domain mutants also fail to localize to the nucleus prompting a search for localization sequences within Chk1. Our investigations have identified the presence of both functional nuclear import and nuclear export sequences encoded in S. pombe Chk1 that, in conjunction with 14-3-3 proteins, may play a prominent role in regulating Chk1 localization and function.

Assaying the DNA damage checkpoint in fission yeast.

Cell cycle checkpoints exist to ensure the proper maintenance and stable inheritance of genomic information. The pathways that insure the faithful execution of these checkpoints are well conserved throughout evolution. In the fission yeast, Schizosaccharomyces pombe, a major cell cycle checkpoint exists that responds to the presence of damaged DNA and prevents this damage from being propagated to future generations. Fission yeast is an ideal system to investigate these pathways because there exist specific techniques that allow one to assay the fidelity of this DNA damage checkpoint pathway.

A novel protein with similarities to Rb binding protein 2 compensates for loss of Chk1 function and affects histone modification in fission yeast.

The conserved protein kinase Chk1 mediates cell cycle progression and consequently the ability of cells to survive when exposed to DNA damaging agents. Cells deficient in Chk1 are hypersensitive to such agents and enter mitosis in the presence of damaged DNA, whereas checkpoint-proficient cells delay mitotic entry to permit time for DNA repair. In a search for proteins that can improve the survival of Chk1-deficient cells exposed to DNA damage, we identified fission yeast Msc1, which is homologous to a mammalian protein that binds to the tumor suppressor Rb (RBP2). Msc1 and RBP2 each possess three PHD fingers, domains commonly found in proteins that influence the structure of chromatin. Msc1 is chromatin associated and coprecipitates a histone deacetylase activity, a property that requires the PHD fingers. Cells lacking Msc1 have a dramatically altered histone acetylation pattern, exhibit a 20-fold increase in global acetylation of histone H3 tails, and are readily killed by trichostatin A, an inhibitor of histone deacetylases. We postulate that Msc1 plays an important role in regulating chromatin structure and that this function modulates the cellular response to DNA damage.

Phosphorylation activates Chk1 and is required for checkpoint-mediated cell cycle arrest.

In the fission yeast Schizosaccharomyces pombe, the protein kinase Chk1 has an essential role in transducing a delay signal to the cell cycle machinery in the presence of DNA damage. Fission yeast cells lacking the chk1 gene do not delay progression of the cell cycle in response to damage and are thus sensitive to DNA damaging agents. We have previously shown that Chk1 is phosphorylated following DNA damage induced by a variety of agents and that this is dependent on the integrity of the DNA damage checkpoint pathway, including Rad3, the ATR homolog. Through a combination of mutagenesis and phospho-specific antibodies, we have shown that serine at position 345 (S345) is phosphorylated in vivo in response to DNA damage, and that S345 phosphorylation is required for an intact checkpoint response. We have developed a kinase assay for Chk1, and have shown that basal Chk1 kinase activity is increased in response to DNA damage and that this increase, but not the basal activity, is dependent on S345. Furthermore, we show that S345 phosphorylation is required for Chk1 to associate with Rad24, a 14-3-3 protein, upon DNA damage. These results are consistent with a model whereby Chk1 phosphorylation results in increased Chk1 kinase activity that is necessary for both checkpoint delay and cellular survival following damage to the genome. These data are similar to observations made in mammalian cells and Xenopus oocyte extracts, suggesting that mechanisms leading to Chk1 activation have been conserved in evolution.

The Ded1 DEAD box helicase interacts with Chk1 and Cdc2.

Ded1 is a fission yeast DEAD box protein involved in translation. We isolated Ded1 in a screen for multi-copy suppressors of a cold-sensitive, loss-of-function mutant of the cyclin-dependent kinase Cdc2. The checkpoint protein kinase Chk1, required for cell cycle arrest in response to DNA damage, was also isolated in this screen. Ded1 interacts with Chk1 in a two-hybrid screen, and this physical interaction can be recapitulated in Schizosaccharomyces pombe. The Ded1 polypeptide is modified in response to heat shock and depletion of carbon source. These two stressors appear to cause different modifications. Thus, the Ded1 protein appears to respond to particular types of cellular stress and may influence the activity of Cdc2 as a result.