The Hos2p Histone De-acetylase Promotes the Successful Completion of Cytokinesis in Schizosaccharomyces pombe.

A cell cycle checkpoint at the G2/M boundary of fission yeast cells ensures that the G2/M transition of daughter cells only occurs after successful cytokinesis in the mother cell. The Lst complex is necessary for proper functioning of this checkpoint and is orthologous to a human histone de-aceylase complex (HDAC3/NCOR2-SMRT), with a known role in cytokinesis. However, the fission yeast orthologue of the human HDAC3 histone de-acetylase, Hos2p, was never previously characterized with respect to the Lst complex. Through a combination of phenotypic analyses on hos2 mutants, live-cell imaging of Hos2p localization, live-cell imaging of cytokinesis dynamics as they relate to Hos2p’s cytokinetic regulatory function, and co-immunoprecipitation experiments, I showed that Hos2p is indeed a member of the Lst complex, and ensures faithful cytokinesis through its de-acetylase activity. Additionally, I used Western Blotting to show that the Lst complex is a stress-responsive complex that up-regulates expression of its Lstlp sub-unit in response to a sub-set of environmental stressors known to turn on the fission yeast core environmental stress response (CESR). Finally, I show that Hos2p inhibits growth and produces various abnormal phenotypes in a dose-dependent manner, although the biological significance of these observations is unclear

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

Hif2p, Set3p, and Snt1p form a nuclear-localized complex.

<p>(<b>A</b>) Cells expressing the indicated fusion proteins were grown to mid-log phase at 30°C in YES media, fixed, and then stained with DAPI and observed using the DAPI and GFP filter sets. Bar, 5 µm. (<b>B</b>) Cells expressing the indicated fusion proteins were grown to mid-log phase in YES, lysed under native conditions, and subjected to anti-Myc immunoprecipitations. Both total lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies specific for the HA epitope.</p

<i>hif2</i>, <i>set3</i>, and <i>snt1</i> deletion mutants are hyper-sensitive to LatA treatment.

<p>(<b>A</b>) Ten-fold serial dilutions of logarithmically growing cells of the indicated genotype were plated onto YES plates containing 0.5 µM LatA or DMSO (solvent control) at 30°C for 3 d. (<b>B</b>) Cells of the indicated genotype were grown to mid-log phase at 30°C and then treated with 0.5 µM LatA for 5 h before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Bar, 10 µm. (<b>C</b>) Quantitation of phenotypes of cells treated as in B. Between 200 and 500 cells were counted for each genotypic class.</p

Set3p, Snt1p and Hif2p levels increase two- to three-fold upon treatment with low doses of LatA.

<p>(<b>A</b>) Strains expressing the indicated fusion proteins were grown to early log phase in YES at 30°C and treated with 0.5 µM LatA (left panel) or 1 µM LatA (right panel). Extracts were subjected to SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-HA antibody. Tubulin was used as a loading control. (<b>B and C</b>) Strains expressing the indicated fusion proteins were grown to early log phase in YES at 30°C and treated with 0.5 µM LatA. Extracts were subjected to SDS-PAGE, transferred to PVDF membranes, and immunoblotted with anti-HA antibody. Tubulin or the RNA pol II carboxy-terminal domain (CTD) were used as loading controls. Signal intensities relative to the loading controls of three independent trials were quantified using ImageJ 1.41 Gel Analyzer software and are plotted below representative blots. Error bars indicate sd.</p

Identification of <i>S. pombe</i> genes differentially expressed with respect to genotype.

<p>Volcano plot (left panels) and scatter plot (right panels) analysis of the expression of all <i>S. pombe</i> genes in DMSO (<b>A</b>) or LatA (<b>B</b>) treated cells of the indicated genotype. Horizontal green lines in volcano plots represent a p-value of 0.05. Vertical green lines in volcano plots represent threshold for a 1.5 fold change in expression. Red squares in volcano plots indicate differentially expressed genes. Diagonal green lines in scatter plots represent the threshold for a 1.5 fold change in expression. The color of the squares in scatter plots indicates the level of expression of that gene in DMSO (<b>A</b>) or LatA (<b>B</b>) treated wild-type cells.</p

Domain structure of Set3p, Snt1p, and Hif2p.

<p>Structures are based upon Uniprot database predictions. Arabic numerals to the right of each schematic indicate the length of the protein in amino acids. Arabic numerals below each schematic indicate the amino acid position of that domain within the protein. Schematics are not drawn to scale.</p

The expression levels of cytokinesis related genes are not differentially regulated.

<p>(<b>A</b>) Cells of the indicated genotypes were grown to mid-log phase at 30°C in YES, and then treated with DMSO (<b>A</b>) or 0.5 µM LatA (<b>B</b>) for 3 hours. Total RNA was extracted and used in expression profiling using Affymetrix Yeast 2.0. Genechips. Graphs shows scatter plot analysis comparing the expression of cytokinesis related genes in wild-type and <i>set3Δ</i> strains. Green lines represent the threshold for a 1.5 fold change in transcript levels. The color of the squares indicates the level of expression of that gene in DMSO (<b>A</b>) or LatA (<b>B</b>) treated wild-type cells.</p

<i>set3</i> functions in a pathway parallel to those defined by <i>clp1</i> and <i>lsk1</i>.

<p>Cells of the indicated genotype were grown to mid-log phase at 30°C and then treated with 0.1 µM LatA for 5 h before being fixed and stained with DAPI (nuclei) and aniline blue (cell wall/septa). Two hundred cells were counted for each genotypic class.</p