Slm9, a novel nuclear protein involved in mitotic control in fission yeast

In the fission yeast Schizosaccharomyces pombe, as in other eukaryotic cells, Cdc2/cyclin B complex is the key regulator of mitosis. Perhaps the most important regulation of Cdc2 is the inhibitory phosphorylation of tyrosine-15 that is catalyzed by Wee1 and Mik1. Cdc25 and Pyp3 phosphatases dephosphorylate tyrosine-15 and activate Cdc2. To isolate novel activators of Cdc2 kinase, we screened synthetic lethal mutants in a cdc25-22 background at the permissive temperature (25 degrees ). One of the genes, slm9, encodes a novel protein of 807 amino acids. Slm9 is most similar to Hir2, the histone gene regulator in budding yeast. Slm9 protein level is constant and Slm9 is localized to the nucleus throughout the cell cycle. The slm9 disruptant is delayed at the G(2)-M transition as indicated by cell elongation and analysis of DNA content. Inactivation of Wee1 fully suppressed the cell elongation phenotype caused by the slm9 mutation. The slm9 mutant is defective in recovery from G(1) arrest after nitrogen starvation. The slm9 mutant is also UV sensitive, showing a defect in recovery from the cell cycle arrest after UV irradiation.     

Separation-of-function mutation in HPC2, a member of the HIR complex in S. cerevisiae, results in derepression of the histone genes but does not confer cryptic TATA phenotypes

The HIR complex, which is comprised of the four proteins Hir1, Hir2, Hir3 and Hpc2, was first characterized as a repressor of three of the four histone gene loci in Saccharomyces cerevisiae. Using a bioinformatical approach, previous studies have identified a region of Hpc2 that is conserved in Schizosaccharomyces pombe and humans. Using a similar approach, we identified two additional domains, CDI and CDII, of the Hpc2 protein that are conserved among yeast species related to S. cerevisiae. We showed that the N terminal CDI domain (spanning amino acids 63-79) is dispensable for HIR complex assembly, but plays an essential role in the repression of the histone genes by recruiting the HIR complex to the HIR-dependent histone gene loci. The second conserved domain, CDII (spanning amino acids 452-480), is required for the stability of the Hpc2 protein itself as well as for the assembly of the HIR complex. In addition, we report a novel separation-of-function mutation within CDI of Hpc2, which causes derepression of the histone genes but does not confer other reported hir/hpc- phenotypes (such as Spt phenotypes, heterochromatin silencing defects and repression of cryptic promoters). This is the first direct demonstration that a separation-of-function mutation exists within the HIR complex.     

The fission yeast TOR homolog, tor1+, is required for the response to starvation and other stresses via a conserved serine

Targets of rapamycin (TORs) are conserved phosphatidylinositol kinase-related kinases that are involved in the coordination between nutritional or mitogenic signals and cell growth. Here we report the initial characterization of two Schizosaccharomyces pombe TOR homologs, tor1(+) and tor2(+). tor2(+) is an essential gene, whereas tor1(+) is required only under starvation and other stress conditions. Specifically, Deltator1 cells fail to enter stationary phase or undergo sexual development and are sensitive to cold, osmotic stress, and oxidative stress. In complex with the prolyl isomerase FKBP12, the drug rapamycin binds a conserved domain in TORs, FRB, thus inhibiting some of the functions of TORs. Mutations at a conserved serine within the FRB domain of Saccharomyces cerevisiae TOR proteins led to rapamycin resistance but did not otherwise affect the functions of the proteins. The S. pombe tor1(+) exhibits different features; substitution of the conserved serine residue, Ser(1834), with arginine compromises its functions and has no effect on the inhibition that rapamycin exerts on sexual development in S. pombe.     

Sir2 regulates histone H3 lysine 9 methylation and heterochromatin assembly in fission yeast

Hypoacetylated histones are a hallmark of heterochromatin in organisms ranging from yeast to humans. Histone deacetylation is carried out by both NAD(+)-dependent and NAD(+)-independent enzymes. In the budding yeast Saccharomyces cerevisiae, deacetylation of histones in heterochromatic chromosomal domains requires Sir2, a phylogenetically conserved NAD(+)-dependent deacetylase. In the fission yeast Schizosaccharomyces pombe, NAD(+)-independent histone deacetylases are required for the formation of heterochromatin, but the role of Sir2-like deacetylases in this process has not been evaluated. Here, we show that spSir2, the S. pombe Sir2-like protein that is the most closely related to the S. cerevisiae Sir2, is an NAD(+)-dependent deacetylase that efficiently deacetylates histone H3 lysine 9 (K9) and histone H4 lysine 16 (K16) in vitro. In sir2 Delta cells, silencing at the donor mating-type loci, telomeres, and the inner centromeric repeats (imr) is abolished, while silencing at the outer centromeric repeats (otr) and rDNA is weakly reduced. Furthermore, Sir2 is required for hypoacetylation and methylation of H3-K9 and for the association of Swi6 with the above loci in vivo. Our findings suggest that the NAD(+)-dependent deacetylase Sir2 plays an important and conserved role in heterochromatin assembly in eukaryotes.     

Replication-independent histone deposition by the HIR complex and Asf1

The orderly deposition of histones onto DNA is mediated by conserved assembly complexes, including chromatin assembly factor-1 (CAF-1) and the Hir proteins . CAF-1 and the Hir proteins operate in distinct but functionally overlapping histone deposition pathways in vivo . The Hir proteins and CAF-1 share a common partner, the highly conserved histone H3/H4 binding protein Asf1, which binds the middle subunit of CAF-1 as well as to Hir proteins . Asf1 binds to newly synthesized histones H3/H4 , and this complex stimulates histone deposition by CAF-1 . In yeast, Asf1 is required for the contribution of the Hir proteins to gene silencing . Here, we demonstrate that Hir1, Hir2, Hir3, and Hpc2 comprise the HIR complex, which copurifies with the histone deposition protein Asf1. Together, the HIR complex and Asf1 deposit histones onto DNA in a replication-independent manner. Histone deposition by the HIR complex and Asf1 is impaired by a mutation in Asf1 that inhibits HIR binding. These data indicate that the HIR complex and Asf1 proteins function together as a conserved eukaryotic pathway for histone replacement throughout the cell cycle.     

Schizosaccharomyces pombe histone acetyltransferase Mst1 (KAT5) is an essential protein required for damage response and chromosome segregation

Schizosaccharomyces pombe Mst1 is a member of the MYST family of histone acetyltransferases and is the likely ortholog of Saccharomyces cerevisiae Esa1 and human Tip60 (KAT5). We have isolated a temperature-sensitive allele of this essential gene. mst1 cells show a pleiotropic phenotype at the restrictive temperature. They are sensitive to a variety of DNA-damaging agents and to the spindle poison thiabendazole. mst1 has an increased frequency of Rad22 repair foci, suggesting endogenous damage. Two-hybrid results show that Mst1 interacts with a number of proteins involved in chromosome integrity and centromere function, including the methyltransferase Skb1, the recombination mediator Rad22 (Sc Rad52), the chromatin assembly factor Hip1 (Sc Hir1), and the Msc1 protein related to a family of histone demethylases. mst1 mutant sensitivity to hydroxyurea suggests a defect in recovery following HU arrest. We conclude that Mst1 plays essential roles in maintenance of genome stability and recovery from DNA damage.     

Multiple interactions among the components of the recombinational DNA repair system in Schizosaccharomyces pombe

Schizosaccharomyces pombe Rhp55 and Rhp57 are RecA-like proteins involved in double-strand break (DSB) repair. Here we demonstrate that Rhp55 and Rhp57 proteins strongly interact in vivo, similar to Saccharomyces cerevisiae Rad55p and Rad57p. Mutations in the conserved ATP-binding/hydrolysis folds of both the Rhp55 and Rhp57 proteins impaired their function in DNA repair but not in cell proliferation. However, when combined, ATPase fold mutations in Rhp55p and Rhp57p resulted in severe defects of both functions, characteristic of the deletion mutants. Yeast two-hybrid analysis also revealed other multiple in vivo interactions among S. pombe proteins involved in recombinational DNA repair. Similar to S. cerevisiae Rad51p-Rad54p, S. pombe Rhp51p and Rhp54p were found to interact. Both putative Rad52 homologs in S. pombe, Rad22p and Rti1p, were found to interact with the C-terminal region of Rhp51 protein. Moreover, Rad22p and Rti1p exhibited mutual, as well as self-, interactions. In contrast to the S. cerevisiae interacting pair Rad51p-Rad55p, S. pombe Rhp51 protein strongly interacted with Rhp57 but not with Rhp55 protein. In addition, the Rti1 and Rad22 proteins were found to form a complex with the large subunit of S. pombe RPA. Our data provide compelling evidence that most, but not all, of the protein-protein interactions found in S. cerevisiae DSB repair are evolutionarily conserved.     

Human CABIN1 is a functional member of the human HIRA/UBN1/ASF1a histone H3.3 chaperone complex

The mammalian HIRA/UBN1/ASF1a complex is a histone chaperone complex that is conserved from yeast (Saccharomyces cerevisiae) to humans. This complex preferentially deposits the histone variant H3.3 into chromatin in a DNA replication-independent manner and is implicated in diverse chromatin regulatory events from gene activation to heterochromatinization. In yeast, the orthologous complex consists of three Hir proteins (Hir1p, Hir2p, and Hir3p), Hpc2p, and Asf1p. Yeast Hir3p has weak homology to CABIN1, a fourth member of the human complex, suggesting that Hir3p and CABIN1 may be orthologs. Here we show that HIRA and CABIN1 interact at ectopic and endogenous levels of expression in cells, and we isolate the quaternary HIRA/UBN1/CABIN1/ASF1a (HUCA) complex, assembled from recombinant proteins. Mutational analyses support the view that HIRA acts as a scaffold to bring together UBN1, ASF1a, and CABIN1 into a quaternary complex. We show that, like HIRA, UBN1, and ASF1a, CABIN1 is involved in heterochromatinization of the genome of senescent human cells. Moreover, in proliferating cells, HIRA and CABIN1 regulate overlapping sets of genes, and these genes are enriched in the histone variant H3.3. In sum, these data demonstrate that CABIN1 is a functional member of the human HUCA complex and so is the likely ortholog of yeast Hir3p.     

An alpha-amylase homologue, aah3, encodes a GPI-anchored membrane protein required for cell wall integrity and morphogenesis in Schizosaccharomyces pombe

Glycosylphosphatidylinositol (GPI)-anchored proteins are essential for normal cellular morphogenesis and have an additional role in mediating cross-linking of glycoproteins to cell wall glucan in yeast cells. Although many GPI-anchored proteins have been characterized in Saccharomyces cerevisiae, none have been reported for well-characterized GPI-anchored proteins in Schizosaccharomyces pombe to date. Among the putative GPI-anchored proteins in S. pombe, four alpha-amylase homologs (Aah1p-Aah4p) have putative signal sequences and C-terminal GPI anchor addition signals. Disruption of aah3(+) resulted in a morphological defect and hypersensitivity to cell wall-degrading enzymes. Biochemical analysis showed that Aah3p is an N-glycosylated, GPI-anchored membrane protein localized in the membrane and cell wall fractions. Conjugation and sporulation were not affected by the aah3(+) deletion, but the ascal wall of aah3Delta cells was easily lysed by hydrolases. Expression of aah3 alleles in which the conserved aspartic acid and glutamic acid residues required for hydrolase activity were replaced with alanine residues failed to rescue the morphological and ascal wall defects of aah3Delta cells. Taken together, these results indicate that Aah3p is a GPI-anchored protein and is required for cell and ascal wall integrity in S. pombe.     

A new role for the transcriptional corepressor SIN3; regulation of centromeres

Centromeres play a vital role in maintaining the genomic stability of eukaryotes by coordinating the equal distribution of chromosomes to daughter cells during mitosis and meiosis. Fission yeast (S. pombe) centromeres consist of a 4-9 kb central core region and 30-100 kb of flanking inner (imr/B) and outer (otr/K) repeats. These sequences direct a laminar kinetochore structure similar to that of human centromeres. Centromeric heterochromatin is generally underacetylated. We have previously shown that inhibition of histone deacetylases (HDACs) caused hyperacetylation of centromeres and defective chromosome segregation. SIN3 is a HDAC corepressor that has the ability to mediate HDAC targeting in the repression of promoters. In this study, we have characterized S. pombe sin three corepressors (Pst1p and Pst2p) to investigate whether SIN3-HDAC is required in the regulation of centromeres. We show that only pst1-1 and not pst2Delta cells displayed anaphase defects and thiabendazole sensitivity. pst1-1 cells showed reduced centromeric silencing, increased histone acetylation in centromeric chromatin, and defective centromeric sister chromatid cohesion. The HDAC Clr6p and Pst1p coimmunoprecipitated, and Pst1p colocalized with centromeres, particularly in binucleate cells. These data are consistent with a model in which Pst1p-Clr6p temporally associate with centromeres to carry out the initial deacetylation necessary for subsequent steps in heterochromatin formation.     

Vesicle-mediated protein transport pathways to the vacuole in Schizosaccharomyces pombe

The vacuole of Saccharomyces cerevisiae plays essential roles not only for osmoregulation and ion homeostasis but also down-regulation (degradation) of cell surface proteins and protein and organellar turnover. Genetic selections and genome-wide screens in S. cerevisiae have resulted in the identification of a large number of genes required for delivery of proteins to the vacuole. Although the complete genome sequence of the fission yeast Schizosaccharomyces pombe has been reported, there have been few reports on the proteins required for vacuolar protein transport and vacuolar biogenesis in S. pombe. Recent progress in the S. pombe genome project of has revealed that most of the genes required for vacuolar biogenesis and protein transport are conserved between S. pombe and S. cerevisiae. This suggests that the basic machinery of vesicle-mediated protein delivery to the vacuole is conserved between the two yeasts. Identification and characterization of the fission yeast counterparts of the budding yeast Vps and Vps-related proteins have facilitated our understanding of protein transport pathways to the vacuole in S. pombe. This review focuses on the recent advances in vesicle-mediated protein transport to the vacuole in S. pombe.     

Fission yeast homologs of human CENP-B have redundant functions affecting cell growth and chromosome segregation

Two functionally important DNA sequence elements in centromeres of the fission yeast Schizosaccharomyces pombe are the centromeric central core and the K-type repeat. Both of these DNA elements show internal functional redundancy that is not correlated with a conserved DNA sequence. Specific, but degenerate, sequences in these elements are bound in vitro by the S. pombe DNA-binding proteins Abp1p (also called Cbp1p) and Cbhp, which are related to the mammalian centromere DNA-binding protein CENP-B. In this study, we determined that Abp1p binds to at least one of its target sequences within S. pombe centromere II central core (cc2) DNA with an affinity (K(s) = 7 x 10(9) M(-1)) higher than those of other known centromere DNA-binding proteins for their cognate targets. In vivo, epitope-tagged Cbhp associated with centromeric K repeat chromatin, as well as with noncentromeric regions. Like abp1(+)/cbp1(+), we found that cbh(+) is not essential in fission yeast, but a strain carrying deletions of both genes (Deltaabp1 Deltacbh) is extremely compromised in growth rate and morphology and missegregates chromosomes at very high frequency. The synergism between the two null mutations suggests that these proteins perform redundant functions in S. pombe chromosome segregation. In vitro assays with cell extracts with these proteins depleted allowed the specific assignments of several binding sites for them within cc2 and the K-type repeat. Redundancy observed at the centromere DNA level appears to be reflected at the protein level, as no single member of the CENP-B-related protein family is essential for proper chromosome segregation in fission yeast. The relevance of these findings to mammalian centromeres is discussed.     

Schizosaccharomyces pombe Hat1 (Kat1) Is Associated with Mis16 and Is Required for Telomeric Silencing

The Hat1 histone acetyltransferase has been implicated in the acetylation of histone H4 during chromatin assembly. In this study, we have characterized the Hat1 complex from the fission yeast Schizosaccharomyces pombe and have examined its role in telomeric silencing. Hat1 is found associated with the RbAp46 homologue Mis16, an essential protein. The Hat1 complex acetylates lysines 5 and 12 of histone H4, the sites that are acetylated in newly synthesized H4 in a wide range of eukaryotes. Deletion of hat1 in S. pombe is itself sufficient to cause the loss of silencing at telomeres. This is in contrast to results obtained with an S. cerevisiae hat1Δ strain, which must also carry mutations of specific acetylatable lysines in the H3 tail domain for loss of telomeric silencing to occur. Notably, deletion of hat1 from S. pombe resulted in an increase of acetylation of histone H4 in subtelomeric chromatin, concomitant with derepression of this region. A similar loss of telomeric silencing was also observed after growing cells in the presence of the deacetylase inhibitor trichostatin A. However, deleting hat1 did not cause loss of silencing at centromeres or the silent mating type locus. These results point to a direct link between Hat1, H4 acetylation, and the establishment of repressed telomeric chromatin in fission yeast.     

A novel member of the Swi6p family of fission yeast chromo domain-containing proteins associates with the centromere in vivo and affects chromosome segregation

We previously used a genetic approach to identify a new class of Schizosaccharomyces pombe genes (chromosome loss when overexpressed; clo genes) that, when present in elevated dosage, cause the loss of an otherwise stable cen1 linear minichromosome at high rates. Here we report the identities of two clo genes; one encodes histone H3.3 and the other, designated clo2, encodes a novel protein with significant homology to fission yeast Swi6p, human and Drosophila HP1 heterochromatin proteins, and other chromo domain-containing proteins. Members of this group have been shown to localize to heterochromatic DNA, including centromeres, and to play roles in chromatin formation and organization. The S. pombe Clo2 protein localizes to centromere DNA in vivo, and overexpression of clo2 leads to a dramatic increase in the rate of mitotic loss of an artificial chromosome. Clo2p is not essential for mitotic growth, however, even in cells that also lack Swi6p. Thus, fission yeast appears to utilize multiple, functionally redundant, HP1-related proteins for heterochromatin-associated activities at centromeres and perhaps elsewhere in the genome.