Differential gene expression signatures for cell wall integrity found in chitin synthase II (chs2 Δ) and myosin II (myo1 Δ) deficient cytokinesis mutants of Saccharomyces cerevisiae
© Rodríguez-Medina et al; licensee BioMed Central Ltd. 2009
Received: 16 March 2009
Accepted: 09 May 2009
Published: 09 May 2009
Myosin II-dependent contraction of the cytokinetic ring and primary septum formation by chitin synthase II are interdependent processes during cytokinesis in Saccharomyces cerevisiae. Hence, null mutants of myosin II (myo1 Δ) and chitin synthase II (chs2 Δ) share multiple morphological and molecular phenotypes. To understand the nature of their interdependent functions, we will seek to identify genes undergoing transcriptional regulation in chs2 Δ strains and to establish a transcription signature profile for comparison with myo1 Δ strains.
A total of 467 genes were commonly regulated between myo1Δ and chs2Δ mutant strains (p ≤ 0.01). Common regulated biological process categories identified by Gene Set Enrichment Analysis (GSEA) in both gene expression profiles were: protein biosynthesis, RNA processing, and stress response. Expression of 17/20 genes in the main transcriptional fingerprint for cell wall stress was confirmed in the chs2Δ strain versus 5/20 for the myo1Δ strain. One of these genes, SLT2/MPK1, was up-regulated in both strains and both strains accumulated the hyperphosphorylated form of Slt2p thereby confirming that the PKC1 cell wall integrity pathway (CWIP) was activated by both mutations. The SLT2/MPK1 gene, essential for myo1Δ strains, was not required in the chs2Δ strain.
Comparison of the chs2Δ and myo1 Δ gene expression profiles revealed similarities in the biological process categories that respond to the chs2Δ and myo1 Δ gene mutations. This supports the view that these mutations affect a common function in cytokinesis. Despite their similarities, these mutants exhibited significant differences in expression of the main transcriptional fingerprint for cell wall stress and their requirement of the CWIP for survival.
In the budding yeast Saccharomyces cerevisiae, myosin type II (Myo1p) and chitin synthase II (Chs2p) are essential proteins for the formation of the normal cytokinetic apparatus. These proteins participate in assembly of the cytokinetic ring and synthesis of the primary septum, respectively, during cytokinesis [1–3]. Previous studies revealed that contraction of the cytokinetic ring and closure of the primary septum by Chs2p are interdependent processes that occur at adjacent sites on the plasma membrane and at similar times of the cell cycle . Previous studies also showed that Chs2p at the bud neck is required to maintain the stability of the actomyosin ring and for normal completion of the cytokinetic process [2, 4]. Thus, MYO1 and CHS2 deficient cells, (myo1 Δ and chs2 Δ respectively), have been described to be cytokinesis mutant strains  that share multiple phenotypes associated with their respective cytokinesis defect . Both mutants require expression of chitin synthase III (Chs3p), [3, 6, 7] an enzyme that synthesizes ~90% of the cell wall chitin, which implies the existence of cell wall stress conditions. Cell wall stress can be overcome by activation of the PKC1- dependent cell wall integrity pathway [8, 9]. Readouts of the pathway in yeast cells include up-regulation of cell wall biosynthetic enzymes, heat shock proteins, and increased production of other cell wall components .
In this study we will establish a signature transcription profile for chs2 Δ strains and compare it with the signature profile in myo1 Δ strains previously published . To accomplish this goal, we have performed a comparative oligonucleotide microarray analysis of mRNAs extracted from a chs2 Δ strain and wild-type controls. When compared to the profiles previously reported in myo1 Δ strains, we observed that both mutants alter the expression of similar groups of genes associated with specific biological process categories. However, these mutants exhibit significant differences in expression of the main transcriptional fingerprint for cell wall stress and differ in their requirement of the cell wall integrity pathway for survival.
Strains and culture conditions
Strains used in this study.
MAT α trp1–289 ura3–52 leu2–3, 112 his3delta1 ADE+ ARG cyh R
MAT α trp1–289 ura3–52 leu2–3, 112 his3delta1 ADE+ ARG cyh R myo1delta::HIS5+ parental MGD353-46D
MAT α trp1–289 ura3–52 leu2–3, 112 his3delta1 ADE+ ARG cyh R chs2delta::KAN R parental MGD353-46D
(chs2 Δslt2 Δ)
MAT α trp1–289 ura3–52 leu2–3, 112 his3delta1 ADE+ ARG cyh R chs2delta::KAN R slt2delta::URA3+ parental MGD353-46D
(myo1 Δslt2 Δ pRS316-MYO1)
MAT α trp1–289 ura3–52 leu2–3, 112 his3delta1 ADE+ ARG cyh R myo1delta::HIS5+ parental MGD353-46D, pRS316-MYO1, slt2delta::KAN R
MAT α trp1–289 ura3–52 leu2–3, 112 his3delta1 ADE+ ARG cyh R , slt2delta::KAN R parental MGD353-46D
SLT2 gene disruption
A SLT2 gene disruption was created in the myo1 Δ pRS316-MYO1 strain (Table 1) by replacing the SLT2 gene with a KanMX4 module by homologous recombination. The myo1 Δ slt2 Δ pRS316-MYO1 strain was grown in CSM 5-FOA to uncover the myo1 Δslt2 Δ mutant . Mutant chs2 Δ strains were transformed with URA3 cassette, to perform the SLT2 gene disruption. All colonies were confirmed by PCR.
RNA extraction procedure
Total RNA was extracted from cells derived from five biological replicate cultures of wild type and chs2 Δ strains as described previously . RNA concentrations, purity and integrity were determined using a Nanodrop spectrophotometer (Nanodrop Technologies, Wilmington, DE) and an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) respectively.
Oligonucleotide microarray experiments
Oligonucleotide microarray experiments and data analysis were performed as described previously . Briefly, 1.0 μg of total RNA from each sample was amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies, Palo Alto, CA), and then it was labeled with 10 mM Cy3 or Cy5. Labeled cRNA's were hybridized, and then microarray slides were washed and scanned with a VersArray Chip Reader (BioRad, Hercules, CA). The microarrays raw data was generated with Imagene 3.0 and then analyzed using Limma software  as previously described . 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 and FDR test  were performed. The p-value ≤ 0.01 cutoff was established for differential expression. Gene Set Enrichment Analysis (GSEA)  was performed using the Limma package of Bioconductor as described previously . A corrected p-value was obtained from the analysis using the Bonferroni correction p-value ≤ 0.0004. Microarray raw and processed data are available at the Gene Expression Omnibus (GEO) site of NCBI (GSE5931 and GSE12994 for myo1 Δ and chs2 Δ, respectively) .
Real time RT-PCR experiments
Primers used in this study for real time RT-PCR and genetic deletions
Western blot analysis of hyperphosphorylated Slt2p levels
Yeast strains were grown in selective medium between 0.5–0.8 OD600 at 26°C. Cells treatment, protein extraction and quantification methods were performed as described [6, 9, 10, 17]. Total protein extracts (75 μg) were separated in a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane at 70 V for 2 h at 4°C. The membrane was incubated with anti-phospho-p42/44 MAP kinase monoclonal antibody (1:1000) (Cell Signaling Technologies, Danvers, MA). The membrane was stripped and reprobed with a rabbit polyclonal antibody against Slt2p (1:1000) and mouse monoclonal antibody against Pgk1p (1:500) (Molecular probes, Invitrogen, Danvers, MA).
Results and Discussion
Comparison between transcriptional profiles of chs2 Δ and myo1 Δ strains
Confirmation of microarray data by real time RT-PCR assay on a selected set of genes for chs2 Δ and myo1 Δ (p ≤ 0.01)
Analysis of the PKC1-dependent cell wall integrity pathway requirement
Twenty genes representing the main transcriptional fingerprint for cell wall stress .
In this study the global mRNA expression profile of a chs2 Δ mutant strain was determined and analyzed by GSEA for further comparison with the previously reported myo1 Δ strain profile . The GSEA identified four biological categories affected by each mutant condition, where protein biosynthesis and RNA processing categories were down- regulated while stress response and autophagy were up-regulated. This analysis revealed that genes involved in the autophagy process were significantly up-regulated exclusively in the chs2 Δ strain. We did not detect any growth impairment in an autophagy-deficient strain chs2Δ atg9Δ (data not shown) suggesting that although this represented a differentially regulated biological process in the chs2 Δ strain, autophagy was not essential for growth of this mutant under our culture conditions.
We have demonstrated that the PKC1- dependent cell integrity pathway was activated in both myo1Δ and chs2 Δ strains. Slt2p/Mpk1p, a gene product of the signal transduction biological process category, was not essential for cell viability in the chs2Δ mutant. Nonetheless, the chs2 Δ strain exhibited higher resistance to cell lysis by exogenously added β-1, 3 glucanase than a wild-type strain suggesting that it most likely has modified its cell wall composition (data not shown). A positive contribution of the cell wall integrity pathway to these putative modifications may be inferred from the transcriptional fingerprint for this mutant.
In summary, a comparison of the chs2Δ and myo1 Δ gene expression profiles revealed similarities in the biological process categories that respond to the chs2Δ and myo1 Δ gene mutations. This supports the view that these mutations may affect common functions in cytokinesis. Despite their similarities, these mutants exhibited significant differences in expression of the main transcriptional fingerprint for cell wall stress and in their requirement of these genes for survival. These differences provide insight to how S. cerevisiae circumvents cell death and may help in the development of novel antifungal treatment.
The authors thank Dr. Rafael Irizarry for continuous advice in microarray data analysis during the course of JFRQ's graduate training. We also thank Sahily González-Crespo and Lilliam Villanueva-Alicea for outstanding technical support. This work was supported by a USPHS grant to JRRM from NIGMS/NIAID (1SC1AI081658-01) with partial support from NCRR-RCMI (G12RR03051). JFRQ received support from MBRS-RISE (R25GM61838).
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