SET Domain Protein Regulates S. pombe Cytokinesis Figure 5. Set3p, Snt1p and Hif2p levels increase two- to three-fold upon treatment with low doses of LatA. (A) Strains expressing the indicated fusion proteins were grown to early log phase in YES at 30uC and treated with 0.5 mM LatA (left panel) or 1 mM 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 and C) Strains expressing the indicated fusion proteins were grown to early log phase in YES at 30uC and treated with 0.5 mM 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. doi:10.1371/journal.pone.0031224.g005 constitute an environmental stress, the expression of the CESR genes was analyzed in wild-type cells to determine if the microarray analysis could correctly detect changes in CESR gene expression. The CESR is sub-divided into two groups: 136 genes that are up-regulated in response to stress (CESR-UP), and 104 that are down-regulated in response to stress (CESR-DN). When the expression of these genes was examined it was apparent that the majority of CESR-DN genes (96 out of 104) were down-regulated, whereas the majority of CESR-UP genes (120 out of 136) were up- regulated in LatA treated cultures relative to DMSO treated controls (Figure 6). Taken together these results indicated that the microarray analysis was providing accurate and biologically meaningful data. For subsequent analysis, the data was grouped into four categories for comparison: 1) wild type, DMSO treated, 2) wild type, LatA treated, 3) set3D, DMSO treated and 4) set3D, LatA treated. In order to identify genes differentially regulated by genotype, volcano plots (p-value,0.05; fold change .1.5) were used to compare the gene expression profiles of wild-type and set3D mutant strains in both DMSO and LatA treated conditions. We first examined the expression of 333 genes with known roles in cytokinesis and/or the cytoskeleton (File S1 for a complete list). None of the genes were identified as being differentially expressed in response to LatA treatment. Furthermore, scatterplots compar- ing wild-type and set3D strains treated with either DMSO or LatA (Figure 7) showed a strong correspondence of expression for the vast majority of cytokinesis genes. Thus, the gene expression data did not support a model in which the Set3p complex plays a role in Figure 6. The core environmental stress response genes (CESR) respond to LatA treatment in wild-type cells. Volcano plot analysis of the expression of the CESR genes in wild-type strains treated with 0.5 mM LatA. Blue diamonds represent CESR genes normally up- regulated in response to multiple stresses. Red squares represent CESR genes normally down-regulated in response to multiple stresses. doi:10.1371/journal.pone.0031224.g006 modulating the transcription of genes with roles in cytokinesis and/or the cytoskeleton. Since genes with roles in cytokinesis were not affected, we next examined the global data set to determine the genes that were differentially regulated by genotype. First, the expression profiles of set3D and wild-type strains were compared upon DMSO treatment. Surprisingly, only three genes were identified as being differentially regulated (p-value,0.05; fold change .1.5) (Figure 8A). As expected, this list included set3 itself, as well as mfm2, and the uncharacterized ORF, SPAC186.05c. Thus, under normal growth conditions – where the set3D mutant shows no obvious phenotype – gene expression patterns were not altered to a great extent. We next examined the difference in expression profiles in wild- type and set3D strains treated with LatA. Under these conditions 73 genes were identified as being differentially regulated (p- value,0.05; fold change .1.5) (Figure 8B). To determine if any of these genes shared any common functions, GO annotations were inspected. The gene ontology (GO) project is a bioinformatics initiative aimed at characterizing the attributes of genes and gene products across databases. This initiative describes all gene products with respect to their biological functions, molecular functions, and their cellular component (www.geneontology.org/). Interestingly, of the 73 differentially regulated genes, 40% (29 out of 73) were annotated by GO as having a role in the cellular response to stress (see File S1 for a complete list). To further explore the role of Set3p in affecting stress response genes, we analyzed the expression of the CESR genes in LatA or DMSO treated wild-type cells. As expected 144 out of 240 genes (,60%) were differentially expressed upon LatA treatment (Figure 9A). However, when the expression of CESR genes in LatA treated set3D cells was examined, only 1 out of 240 (,1%) were differentially regulated (Figure 9B). Next, we expanded the analysis to include any gene annotated by GO as having a cellular response to stress. In this sub-set, 113 out of 561 genes (,20%) were differentially regulated upon LatA treatment in wild-type cells, while only 7 out of 561 genes (,1%) were differentially regulated in set3D backgrounds (Figure 10A). Lastly, we performed scatter plot analysis on a combined set of genes that included both the CESR and genes annotated by GO as having a cellular response to stress. Interestingly, scatter plot analysis of the stress genes in set3D mutants revealed that the distribution of data points was closely clustered around median levels (Figure 10B). In wild-type, on the other hand, a similar analysis of the distribution revealed the existence of genes that were either up- (Figure 10B, blue oval) or down-regulated (Figure 10B, pink oval) in response to LatA. Taken together these data indicate that set3D cells are severely impaired in their ability to mount a proper transcriptional response to LatA treatment. Discussion Schizosaccharomyces pombe has proven to be an excellent model for understanding the regulatory modules that ensure the faithful and reliable execution of cytokinesis [1,2,3,4,6,7,8,9,10,12,13,22]. In this report we further expand our knowledge in this area by PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e31224