cytokinesis phenotypes were the result of defects in the transcription of genes involved in cytokinesis and/or the cytoskeleton (Figure 7). Importantly, while expression profiling clearly showed that this was not the case, a careful examination of the microarray data did reveal several interesting findings. First, wild-type cells respond to LatA with a general cell stress response (Figures 6, 8,9,10). This was evident by the strong induction/repression of the fission yeast CESR genes. The CESR genes are predicted to modulate cellular metabolic pathways and to limit growth related processes [25]. It is hypothesized that activation of the CESR may promote survival against potentially lethal doses of a given stress and in doing so provide a means for the cell to adapt to its new environment [25,39]. In stark contrast to wild-type cells, the set3D mutant exhibited a significantly reduced capacity to modulate the expression of stress response genes upon LatA treatment (Figures 8,9,10). Thus, cytokinetic failure in set3D mutants may be a manifestation of the mutant cells inability to properly adapt to the presence of LatA leading to direct and/or indirect effects on the function of the cytokinetic machinery. It is also of interest to note that, in addition to LatA, set3D strains show sensitivity to the calcineurin inhibitor, FK506 [40]. Intriguingly, calcineurin mutants in S. pombe have been shown to affect cytokinesis, cell polarity, and spindle pole body positioning [41]. A role in the stress response, may be an evolutionarily conserved feature since the Set3p complex in budding yeast is required to respond to secretory stress [42]. Furthermore, the budding yeast class I histone deacetylase Rpd3p, and its associated Rpd3-L complex, is required for activation and repression of environmen- tal stress response genes [43]. Taking this into consideration, we favour a model in which the observed defects in cytokinesis are related to the impaired ability of the mutants to modulate gene expression so as to properly counter the effects of LatA induced stress. This is supported by the observation that the protein levels of all three complex members increase in response to LatA (Figure 5), as well as the observation that wild-type cells modulate the expression of a large sub-set of genes with a role in the stress response (Figures 6, 9 and 10). In any event, we suspect that future analysis of this system might translate into a theoretical framework for understanding how the orthologous MLL5 complex functions to regulate cytokinesis in human cells, as well as how its dysfunction might lead to genomic instability. Materials and Methods Yeast Methods All Schizosaccharomyces pombe strains used in this work (Table S1) originated from previous studies [12,44,45], were created during the course of this work, or were purchased from Bioneer Corporation (Alameda, CA). Schizosaccharomyces pombe cells were cultured in either YES or Edinburgh Minimal Media (EMM) (Forsburg and Rhind 2006) with the appropriate supplements (Adenine, Histidine, Leucine, or Uracil). Liquid cultures were grown with shaking (200 rpm) at 30uC. Genetic crosses were performed using standard methods [46]. In experiments involving Latrunculin treatment, S. pombe cells were grown to mid log phase (O.D. 0.2) and treated with 0.2–0.5 mM of Latrunculin A (Enzo Life Sciences International, Plymouth Meeting, Pennsylvania) dissolved in DMSO. Cells were grown at 30uC with shaking at 200 rpm for 3–6 hrs, before being fixed. All experiments were repeated a minimum of three times. Plasmid vectors were transformed into S. pombe using the lithium acetate protocol according to Forsburg and Rhind [46]. In block and release experiments, cdc25-22 cells were grown to logarithmic phase in SET Domain Protein Regulates S. pombe Cytokinesis YES at 25uC, shifted to 36uC for 3 hours to block at the G2/M transition, and then shifted back to 25uC to effect release of the block. The cell cycle progress of the strains was subsequently monitored by determining the level of bi-nucleate cells every 30 minutes after the shift. Fluorescence Microscopy S. pombe cells expressing Lsg1-GFP fusions, were fixed using ethanol fixation [46] and stored in PBS pH 7.4. To observe nuclei and cell wall/septa material, cells were mixed with 0.02 mg/mL 496,-diamidino-2-phenylindole (DAPI) and 1 mg/mL aniline blue. Fluorescent images were obtained using Zeiss Axioskop 2 microscope driven by ImageJ 1.41 software (National Institutes of Health) and Scion CFW Monochrome CCD Firewire Camera (Scion Corporation, Frederick Maryland) using DAPI and GFP filter sets. Cloning Methods The snt1 gene deletion mutant was created using a PCR based cloning strategy. A 286 bp region upstream of the snt1 start codon was PCR amplified using High-Fidelity PCR Enzyme Mix (Fermentas Life Sciences) with the forward primer 59-ggg ggg gta cca aat gaa ggg gat tcc ttg g-39 and reverse primer 59-ggg ggc tcg agt gtc aga gga ggc act aca gc-39 and cloned into the pSKURA4 vector upstream of the ura4 selectable marker using the restriction enzymes KpnI and XhoI (Fermentas Life Sciences). Next, a 259 bp region downstream of the snt1 stop codon was PCR amplified using the forward primer 59-ggg ggt cta gat gtg tcg ggt tat gat ggt g-39 and reverse primer 59-ggg ggg agc tca ttt ttg gtg tcg gtt ttg c-39 and cloned downstream of the ura4 selectable marker in pSKURA4 using the restriction enzymes XbaI and SacI. Molecular cloning of the desired fragments was confirmed by restriction digestion and DNA sequencing. The ura4 selectable marker flanked by upstream and downstream regions of snt1 was excised with restriction enzymes KpnI and SacI to isolate a linear dsDNA deletion cassette. The deletion cassette was transformed into S. pombe strain MBY1343 (ura4-D18). Ura4+ integrants were selected for by growth on EMM lacking uracil and subjected to colony PCR to identify clones in which the construct had integrated into the genome via homologous recombination. Strains bearing gene deletions of set3 and hif2 were purchased from Bioneer Corporation (Alameda, CA). Genotypes were verified by colony PCR. The hif2::natMX gene deletion was created using the high throughput knockout strategy devised by the Kim Nasmyth lab [47]. Primers, plasmids and a detailed protocol are available at the S. pombe deletion web server (http://mendel.imp.ac.at/Pombe_ deletion/). S. pombe strains expressing carboxy-terminal epitope tagged fusion protein were constructed using a PCR based cloning strategy. To create the Set3-GFP and Set3-HA expressing strains a C-terminal fragment of the set3 gene was PCR amplified using High-Fidelity PCR Enzyme Mix (Fermentas Life Sciences) from S. pombe genomic DNA with the forward primer 59-ggg ggg aat tct gaa ata ctt caa gaa gcg aaa aca ag-39 and reverse primer 59-ggg ggc ccg ggt cgc gta aat gaa ggg tta g-39 and cloned in frame into the EcoRI and SmaI sites of the pJK210-GFP and pJK210-HA vectors respectively. Molecular cloning of the desired C-terminal fragments was confirmed by restriction digestion and DNA sequencing. Plasmid clones containing the desired C-terminal fragment were transformed into S. pombe strain MBY1343 (ura4- D18). Ura4+ integrants were selected for by growth on EMM lacking uracil and subjected to colony PCR to identify clones in which the construct had integrated into the genome via homologous recombination. To create the Set3-myc expressing PLoS ONE | www.plosone.org 13 February 2012 | Volume 7 | Issue 2 | e31224