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.     

Crystal structures of fission yeast histone chaperone Asf1 complexed with the Hip1 B-domain or the Cac2 C terminus

The assembly of core histones onto eukaryotic DNA is modulated by several histone chaperone complexes, including Asf1, CAF-1, and HIRA. Asf1 is a unique histone chaperone that participates in both the replication-dependent and replication-independent pathways. Here we report the crystal structures of the apo-form of fission yeast Asf1/Cia1 (SpAsf1N; residues 1-161) as well as its complexes with the B-domain of the fission yeast HIRA orthologue Hip1 (Hip1B) and the C-terminal region of the Cac2 subunit of CAF-1 (Cac2C). The mode of the fission yeast Asf1N-Hip1B recognition is similar to that of the human Asf1-HIRA recognition, suggesting that Asf1N recognition of Hip1B/HIRA is conserved from yeast to mammals. Interestingly, Hip1B and Cac2C show remarkably similar interaction modes with Asf1. The binding between Asf1N and Hip1B was almost completely abolished by the D37A and L60A/V62A mutations in Asf1N, indicating the critical role of salt bridge and van der Waals contacts in the complex formation. Consistently, both of the aforementioned Asf1 mutations also drastically reduced the binding to Cac2C. These results provide a structural basis for a mutually exclusive Asf1-binding model of CAF-1 and HIRA/Hip1, in which Asf1 and CAF-1 assemble histones H3/H4 (H3.1/H4 in vertebrates) in a replication-dependent pathway, whereas Asf1 and HIRA/Hip1 assemble histones H3/H4 (H3.3/H4 in vertebrates) in a replication-independent pathway.     

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.     

Hus1p, a conserved fission yeast checkpoint protein, interacts with Rad1p and is phosphorylated in response to DNA damage.

The hus1+ gene is one of six fission yeast genes, termed the checkpoint rad genes, which are essential for both the S-M and DNA damage checkpoints. Classical genetics suggests that these genes are required for activation of the PI-3 kinase-related (PIK-R) protein, Rad3p. Using a dominant negative allele of hus1+, we have demonstrated a genetic interaction between hus1+ and another checkpoint rad gene, rad1+. Hus1p and Rad1p form a stable complex in wild-type fission yeast, and the formation of this complex is dependent on a third checkpoint rad gene, rad9+, suggesting that these three proteins may exist in a discrete complex in the absence of checkpoint activation. Hus1p is phosphorylated in response to DNA damage, and this requires rad3+ and each of the other checkpoint rad genes. Although there is no gene related to hus1+ in the Saccharomyces cerevisiae genome, we have identified closely related mouse and human genes, suggesting that aspects of the checkpoint control mechanism are conserved between fission yeast and higher eukaryotes.     

The SET domain protein, Set3p, promotes the reliable execution of cytokinesis in Schizosaccharomyces pombe

In response to perturbation of the cell division machinery fission yeast cells activate regulatory networks that ensure the faithful completion of cytokinesis. For instance, when cells are treated with drugs that impede constriction of the actomyosin ring (low doses of Latrunculin A, for example) these networks ensure that cytokinesis is complete before progression into the subsequent mitosis. Here, we identify three previously uncharacterized genes, hif2, set3, and snt1, whose deletion results in hyper-sensitivity to LatA treatment and in increased rates of cytokinesis failure. Interestingly, these genes are orthologous to TBL1X, MLL5, and NCOR2, human genes that encode components of a histone deacetylase complex with a known role in cytokinesis. Through co-immunoprecipitation experiments, localization studies, and phenotypic analysis of gene deletion mutants, we provide evidence for an orthologous complex in fission yeast. Furthermore, in light of the putative role of the complex in chromatin modification, together with our results demonstrating an increase in Set3p levels upon Latrunculin A treatment, global gene expression profiles were generated. While this analysis demonstrated that the expression of cytokinesis genes was not significantly affected in set3Δ backgrounds, it did reveal defects in the ability of the mutant to regulate genes with roles in the cellular response to stress. Taken together, these findings support the existence of a conserved, multi-protein complex with a role in promoting the successful completion of cytokinesis.     

Thematic review series: sphingolipids. New insights into sphingolipid metabolism and function in budding yeast

Our understanding of sphingolipid metabolism and functions in the baker's yeast Saccharomyces cerevisiae has progressed substantially in the past 2 years. Yeast sphingolipids contain a C26-acyl moiety, all of the genes necessary to make these long-chain fatty acids have been identified, and a mechanism for how chain length is determined has been proposed. Advances in understanding how the de novo synthesis of ceramide and complex sphingolipids is regulated have been made, and they demonstrate that the Target Of Rapamycin Complex 2 (TORC2) controls ceramide synthase activity. Other work shows that TORC2 regulates the level of complex sphingolipids in a pathway using the Slm1 and Slm2 proteins to control the protein phosphatase calcineurin, which regulates the breakdown of complex sphingolipids. The activity of Slm1 and Slm2 has also been shown to be regulated during heat stress by phosphoinositides and TORC2, along with sphingoid long-chain bases and the Pkh1 and Pkh2 protein kinases, to control the actin cytoskeleton, the trafficking of nutrient transporters, and cell viability. Together, these results provide the first molecular insights into understanding previous genetic interaction data that indicated a connection between sphingolipids and the TORC2 and phosphoinositide signaling networks. This new knowledge provides a foundation for greatly advancing our understanding of sphingolipid biology in yeast.     

Human UBN1 Is an Ortholog of Yeast Hpc2p and Has an Essential Role in the HIRA/ASF1a Chromatin-Remodeling Pathway in Senescent Cells

Cellular senescence is an irreversible proliferation arrest, tumor suppression process and likely contributor to tissue aging. Senescence is often characterized by domains of facultative heterochromatin, called senescence-associated heterochromatin foci (SAHF), which repress expression of proliferation-promoting genes. Given its likely contribution to tumor suppression and tissue aging, it is essential to identify all components of the SAHF assembly pathway. Formation of SAHF in human cells is driven by a complex of histone chaperones, namely, HIRA and ASF1a. In yeast, the complex orthologous to HIRA/ASF1a contains two additional proteins, Hpc2p and Hir3p. Using a sophisticated approach to search for remote orthologs conserved in multiple species through evolution, we identified the HIRA-associated proteins, UBN1 and UBN2, as candidate human orthologs of Hpc2p. We show that the Hpc2-related domain of UBN1, UBN2, and Hpc2p is an evolutionarily conserved HIRA/Hir-binding domain, which directly interacts with the N-terminal WD repeats of HIRA/Hir. UBN1 binds to proliferation-promoting genes that are repressed by SAHF and associates with histone methyltransferase activity that methylates lysine 9 of histone H3, a site that is methylated in SAHF. UBN1 is indispensable for formation of SAHF. We conclude that UBN1 is an ortholog of yeast Hpc2p and a novel regulator of senescence.