strain 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 gta cct 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 KpnI and SmaI sites of the pJK210-Myc vector. Plasmid clones containing the desired C-terminal fragment were trans- formed 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. Snt1-GFP and Snt1-Myc as well as Hif2-GFP and Hif2-HA expressing strains were created using an analogous strategy as the epitope tagged Set3p strains. The forward and reverse primers used to create the Snt1-GFP integrant strain were 59-ggg ggg aat tct gag gtt ggg atg aaa aag aag aa-39 and 59-ggg ggc ccg ggt aca att tta tcg ttt ttg gac tg-39 respectively, and the forward and reverse primers used to create the Snt1-Myc integrant strain are 59-ggg ggg gta cct gag gtt ggg atg aaa aag aag aa-39 and 59-ggg ggc ccg ggt aca att tta tcg ttt ttg gac tg-39 respectively. The forward and reverse primers used to create the Hif2-GFP and Sif3-HA integrant strains are 5-ggg ggg aat tct gat cta gag gtg atg ctg gtg c-39 and 59-ggg ggc ccg ggc aga gaa tca tgt aaa aaa tca ca-39 respectively. Biochemical and Immunological Methods Cells of the indicated genotype were grown up to the mid-log phase at 30uC, collected by centrifugation, and resuspended in STOP buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 50 mM NaF, 10 mM EDTA, 1 mM NaN3). Cell pellets were stored at 280uC up to a maximum of 6 months. Cell pellets were subsequently thawed, and lysed using vortexing with glass beads in extraction buffer (1% IGEPAL CA630 (tetr-Octylphenoxy polyethanol), 150 mM NaCl, 50 mM Tris-HCl pH 8.0, 2 mM EDTA, 1 mM PMSF (phenylmethanesulphonylfluoride), 2 mM Benzamidine, 50 mM NaF, 0.1 mM Na3VO4, 50 mM B- glycerophosphate, 15 mM p-nitrophenyl phosphate, J Tablet Sigma Protease Inhibitors). Total cell extracts were subjected to SDS-PAGE, transferred to PVDF membranes and immunoblotted with anti-HA primary antibody (HA.11; Sigma) at a dilution of 1:2000. As a loading control, anti-tubulin (B14; Sigma) was used at a dilution of 1:2000, or primary antibody specific to the unphosphorylated C-terminal domain of RNA polymerase II (8WG16; Covance) was used at a dilution of 1:5000. Peroxidase conjugated anti-mouse IgG (Sigma) at 1:10000, was used as secondary antibody for HA, tubulin and RNA polymerase II blotted membranes. Blots were quantified using ImageJ 1.41 gel analyzer software (National Institutes of Health). In co-immunoprecipitation experiments cell extracts were obtained from the indicated strains as described above. Immuno- precipitations were performed using Protein G DynabeadsH (Invitrogen) according to the manufacturer’s protocol. Briefly, immunoprecipitations were performed by incubating anti-HA antibodies (HA.11; Sigma) with the Protein G Dynabeads using extraction buffer as the wash solution. Cell extracts were then added to the antibody-bound bead slurry. After incubation and repeated washing with extraction buffer, the bound proteins were eluted by incubation at 96uC for 5 minutes. The eluted proteins were then subjected to SDS-PAGE, transferred to PVDF membranes and probed with anti-Myc antibodies (9E10; Sigma). Global Gene Expression Analysis Strains of the indicated genotype were grown to mid-log phase and treated with 0.5 mM LatA or an equal volume of DMSO for SET Domain Protein Regulates S. pombe Cytokinesis 3 hours at 30uC. Total RNA extraction was performed using the Ambion RiboPureTM Yeast Kit (Ambion Inc). Three replicates were performed for each strain under each growth condition (plus or minus LatA). Bioanalysis of all samples showed that the isolated RNA was of sufficient quality to proceed to hybridizations (data not shown). GeneChipH Yeast Genome 2.0 Arrays purchased from Affymetrix were used for hybridization. Hybridizations were performed by the London Regional Genomic Centre using standard Affymetrix protocols. The quality of hybridization was analyzed using duplicate probes for the bioB, bioC, bioD, and cre genes. During hybridization, transcripts of these genes are ‘‘spiked’’ into the Affymetrix hybridization cocktail at concentra- tions of 1.5, 5, 25 and 100 pM, respectively. The level of hybridization as measured by the normalized signal values (data not shown) was consistent with the level of spiked transcript (i.e. bioB showed the lowest signal values and cre the highest). This demonstrated that the hybridizations had been performed successfully and that the data could be used for further analysis. Expression profiling data was obtained from London Regional Genomic Centre as .CEL files, which contained the raw hybridization signal-intensity values. Analysis of the data was done using Genespring GX 10.0.1 software provided by Agilent Technologies Inc. and Strand Life Sciences Pvt. Ltd. The .CEL files were first normalized with RMA (robust multi-array analysis) algorithm. RMA uses PM (perfect match) probes from the data and corrects the background by fitting a model that is the addition of an exponentially distributed signal and a normally dispersed background [48]. This generated normalized hybridization signal- intensity data for all twelve samples. Normalized expression data was subsequently used in the analysis. Data was grouped into two categories: genotype (wild type or set3D), and b) drug (LatA or DMSO treated). Thus, four experimental groups were used for comparison: 1) wild type, DMSO treated, 2) wild type, LatA treated, 3) set3D, DMSO treated, and 4) set3D, LatA treated. To identify differentially regulated genes, data was filtered using Volcano plot analysis employing the Benjamin-Hochberg multiple testing correction. Genes showing statistically significant differ- ences (p-value,0.05) and fold changes greater than 1.5 were identified as differentially regulated. Data is accessible from the GEO database under accession number GSE33228). Supporting Information Figure S1 ClustalW alignments of the Hif2p, Set3p, and Snt1p proteins with their human orthologues, TBL1X (A), MLL5 (B), and NCOR2 (C), respectively. Alignments in (B) and (C) were performed with conserved segments present in the N-terminal regions of the respective proteins. Asterisks (*) indicate identities. Colons (:) indicate conserved substitutions. Periods (.) indicate semi-conserved substitutions. (TIF) Figure S2 Deletion of the set3, snt1, or hif2 genes reduces the restrictive temperature of cdc15-140 mu- tants. Cells of the indicated genotype were cultured to logarithmic growth phase at 25uC. Ten-fold serial dilutions were subsequently plated onto solid YES media and incubated at 25uC, 31uC, or 36uC for 3 d. (TIF) Figure S3 The protein levels of Set3-HA, Snt1-HA, or Hif2-HA do not change as a function of cell cycle position. Strains of the indicated genotype were grown to early log phase in YES at 25uC and shifted to 36uC for 3 hours to arrest the cells at the G2/M transition. Cells were subsequently released PLoS ONE | www.plosone.org 14 February 2012 | Volume 7 | Issue 2 | e31224