Signature gene expression profiles for cytokinesis mutants in the budding yeast Saccharomyces cerevisiae

During cytokinesis in the budding yeast Saccharomyces cerevisiae, contraction of the cytokinetic ring and primary septum synthesis by chitin synthase II (Chs2p) are coupled processes. Myosin II (Myo1p), is involved in the actomyosin ring formation, required for proper cytokinesis, while Chs2p is responsible for the chitin primary septum formation which is necessary to stabilize the cytokinetic ring during its contraction. Morphological phenotypes of myo1∆ and chs2∆ mutants are therefore similar particularly in that both are unable to complete normal cytokinesis. A comparison between the global mRNA transcription profiles of myo1∆ and chs2∆ strains was conducted using oligonucleotide microarrays to establish if these cytokinesis mutants exhibited similar mRNA expression patterns and signature profiles were later generated from gene set enrichment analysis (GSEA). Genetic experiments were conducted to further test predictions based on the GSEA results. As reported previously in myo1∆ strains, a significant number of protein biosynthesis and RNA processing genes that may be attributed to regulation by cell integrity pathway(s) were also down-regulated in the chs2∆ strain. Genes coding for proteins involved in autophagy were coordinately upregulated only in the chs2∆ strain yet an ATG9 genetic knockout that completely blocks autophagy was viable in chs2∆ and myo1∆ genetic backgrounds.. However, the chs2∆ strain was significantly more susceptible to lysis by B-1,3-glucanase than myo1∆ and fks1 control strains. We interpret this result to indicate that these two cytokinesis mutant strains possess different cell wall properties. This interpretation was further supported by the observation that Slt2p hyperphosphorylation was detected in the chs2∆ strain yet expression of SLT2 was not required for cell viability. We conclude that in contrast to myo1∆ strains, chs2∆ strains do not activate the cell integrity pathway. All the experiments were performed using Saccharomyces cerevisiae wild type and chs2 strains. Five biological replicates were performed for this experiment. YFR23 is a chs2::KANMX4 strain generated by homologous recombination in the parental haploid wild type strain, MGD-353-46D using a PCR based method. Cultures were grown overnight at 26ºC to an optical density between 0.5-0.8 (OD600) in complete synthetic media (CSM, 2% glucose, 1X Nitrogen base) or histidine dropout media (CSM-HIS-) with continuous shaking at 200 rpm. Total RNA was extracted from 4 X 10^7 cells derived from triplicate biological replicate cultures of strains MGD343-56D and YFR23 using the RNeasy Mini Kit for isolation of total RNA following manufacturer’s instructions. RNA concentrations were determined by measuring absorbance at 260nm using a Nanodrop spectrophotometer (Nanodrop Technologies). The purity and integrity of the RNA was monitored using an Agilent Bioanalyzer (Agilent Technologies) following manufacturer’s instructions. 1.0 ug of total RNA from each sample was amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies). The amplified cRNA was labeled with 10mM Cyanine 5-CTP (Cy5) or Cyanine 3-CTP (Cy3) (Perkin Elmer Life Sciences). Labeled cRNA’s were purified with Qiagen RNeasy mini spin columns and dye incorporation was monitored on an Agilent Bioanalyzer. Hybridization of Cy5 and Cy3 labeled cRNA’s were performed using Yeast Oligo Microarray slides and hybridization kit from Agilent Technologies in 1012AG hybridization chambers (Sheldon Manufacturing) at 60ºC for 17 hours. Slides were washed at high stringency and scanned with a VersArray Chip Reader system (BioRad, Hercules, CA) at a resolution of 5um with detector sensitivity values between 704-800 and laser power at 85%. Scanned images were transferred to the Imagene 3.0 software (Biodiscovery) for further analysis to locate spots, adjust the appropriate grid, and obtain the Cy3 and Cy5 TIFF files. The microarrays raw data generated with Imagene 3.0 were analyzed using Limma software (Bioconductor Package 1.7). The data was prepared for analysis by correcting for background intensity. The individual data sets were normalized using the locally weighted linear regression (Lowess) within each array. After normalization, the difference between the experimental and control signal was calculated, replicates were combined, and their averages were calculated. The fold change in gene expression was calculated by 2^(M), where M is the log2-fold change after background correction and normalization. An Empirical Bayes Statistics for differential expression analysis (eBayes statistics) was performed by Limma. Genes with a p-value ≤ 0.01 were established as a cutoff for differential expression. In addition, a false discovery rate (FDR) test was performed by Limma program.