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SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae.

Gupta R, Sadhale PP, Vijayraghavan U - PLoS ONE (2015)

Bottom Line: Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background.Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics.Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.

ABSTRACT
Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1Δ cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1Δ sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

No MeSH data available.


Related in: MedlinePlus

SUB1 deletion results in increased expression of middle sporulation genes.qRT-PCR analysis for expression of early (IME2), early-middle (NDT80), middle (SPS2, SMK1) and mid-late (DIT1, DIT2) sporulation specific genes. Fold change was calculated using ACT1 as control. (A) Cells were harvested at indicated time points from SK1 WT and sub1Δ cells. (B) Overexpression of SUB1 results in decrease in expression of middle sporulation genes. Cells were harvested at indicated time points from SK1 WT and WT overexpressing SUB1.
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pone.0132350.g004: SUB1 deletion results in increased expression of middle sporulation genes.qRT-PCR analysis for expression of early (IME2), early-middle (NDT80), middle (SPS2, SMK1) and mid-late (DIT1, DIT2) sporulation specific genes. Fold change was calculated using ACT1 as control. (A) Cells were harvested at indicated time points from SK1 WT and sub1Δ cells. (B) Overexpression of SUB1 results in decrease in expression of middle sporulation genes. Cells were harvested at indicated time points from SK1 WT and WT overexpressing SUB1.

Mentions: Because Sub1 levels show a significant reduction correlating with progression of meiosis, we investigated its likely downstream effects on sporulation gene expression. We generated homozygous diploid sub1Δ alleles in the SK1 genetic background, so as to achieve synchronous and high sporulation. The wild-type and sub1Δ/sub1Δ SK1 diploids were allowed to sporulate for 24 hours and the cells were collected at different time points during sporulation. Fluorescence microscopy of DAPI-stained cultures revealed that sub1Δ/sub1Δ diploid cells completed meiosis similar to wild-type, as observed by the presence of four distinct DAPI-stained foci (S3A Fig). Moreover, the sporulation efficiency and the germination ability of haploid spores from sub1Δ/sub1Δ diploids was comparable to characteristics of wild-type diploids (S3B and S3C Fig). By qRT-PCR analysis, we quantitated the expression levels of some genes chosen as representatives for each temporal class of sporulation genes. We compared the transcript abundance in wild-type and sub1Δ/sub1Δ deletion strains. The genes where transcript levels showed at least 2-fold change and statistical significance of P < 0.05 were considered affected. We observed that sub1Δ/sub1Δ cells have a significantly elevated transcript levels of middle sporulation genes (SPS2 and SMK1), that followed normal induction kinetics, i.e. 5 hours post transfer to sporulation medium (Fig 4A). The early gene IME2 and early-middle gene NDT80 displayed a marginal, but statistically insignificant increase in their transcript levels in sub1Δ/sub1Δ cells as compared to the wild-type. Moreover, the expression levels or timing for mid-late sporulation genes (DIT1, DIT2) did not differ in sub1Δ/sub1Δ strain as compared to the wild-type.


SUB1 Plays a Negative Role during Starvation Induced Sporulation Program in Saccharomyces cerevisiae.

Gupta R, Sadhale PP, Vijayraghavan U - PLoS ONE (2015)

SUB1 deletion results in increased expression of middle sporulation genes.qRT-PCR analysis for expression of early (IME2), early-middle (NDT80), middle (SPS2, SMK1) and mid-late (DIT1, DIT2) sporulation specific genes. Fold change was calculated using ACT1 as control. (A) Cells were harvested at indicated time points from SK1 WT and sub1Δ cells. (B) Overexpression of SUB1 results in decrease in expression of middle sporulation genes. Cells were harvested at indicated time points from SK1 WT and WT overexpressing SUB1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4492983&req=5

pone.0132350.g004: SUB1 deletion results in increased expression of middle sporulation genes.qRT-PCR analysis for expression of early (IME2), early-middle (NDT80), middle (SPS2, SMK1) and mid-late (DIT1, DIT2) sporulation specific genes. Fold change was calculated using ACT1 as control. (A) Cells were harvested at indicated time points from SK1 WT and sub1Δ cells. (B) Overexpression of SUB1 results in decrease in expression of middle sporulation genes. Cells were harvested at indicated time points from SK1 WT and WT overexpressing SUB1.
Mentions: Because Sub1 levels show a significant reduction correlating with progression of meiosis, we investigated its likely downstream effects on sporulation gene expression. We generated homozygous diploid sub1Δ alleles in the SK1 genetic background, so as to achieve synchronous and high sporulation. The wild-type and sub1Δ/sub1Δ SK1 diploids were allowed to sporulate for 24 hours and the cells were collected at different time points during sporulation. Fluorescence microscopy of DAPI-stained cultures revealed that sub1Δ/sub1Δ diploid cells completed meiosis similar to wild-type, as observed by the presence of four distinct DAPI-stained foci (S3A Fig). Moreover, the sporulation efficiency and the germination ability of haploid spores from sub1Δ/sub1Δ diploids was comparable to characteristics of wild-type diploids (S3B and S3C Fig). By qRT-PCR analysis, we quantitated the expression levels of some genes chosen as representatives for each temporal class of sporulation genes. We compared the transcript abundance in wild-type and sub1Δ/sub1Δ deletion strains. The genes where transcript levels showed at least 2-fold change and statistical significance of P < 0.05 were considered affected. We observed that sub1Δ/sub1Δ cells have a significantly elevated transcript levels of middle sporulation genes (SPS2 and SMK1), that followed normal induction kinetics, i.e. 5 hours post transfer to sporulation medium (Fig 4A). The early gene IME2 and early-middle gene NDT80 displayed a marginal, but statistically insignificant increase in their transcript levels in sub1Δ/sub1Δ cells as compared to the wild-type. Moreover, the expression levels or timing for mid-late sporulation genes (DIT1, DIT2) did not differ in sub1Δ/sub1Δ strain as compared to the wild-type.

Bottom Line: Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background.Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics.Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India.

ABSTRACT
Saccharomyces cerevisiae Sub1 is involved in several cellular processes such as, transcription initiation, elongation, mRNA processing and DNA repair. It has also been reported to provide cellular resistance during conditions of oxidative DNA damage and osmotic stress. Here, we report a novel role of SUB1 during starvation stress-induced sporulation, which leads to meiosis and spore formation in diploid yeast cells. Deletion of SUB1 gene significantly increased sporulation efficiency as compared to the wild-type cells in S288c genetic background. Whereas, the sporulation functions of the sub1(Y66A) missense mutant were similar to Sub1. SUB1 transcript and protein levels are downregulated during sporulation, in highly synchronized and sporulation proficient wild-type SK1 cells. The changes in Sub1 levels during sporulation cascade correlate with the induction of middle sporulation gene expression. Deletion of SUB1 increased middle sporulation gene transcript levels with no effect on their induction kinetics. In wild-type cells, Sub1 associates with chromatin at these loci in a temporal pattern that correlates with their enhanced gene expression seen in sub1Δ cells. We show that SUB1 genetically interacts with HOS2, which led us to speculate that Sub1 might function with Set3 repressor complex during sporulation. Positive Cofactor 4, human homolog of Sub1, complemented the sub1Δ sporulation phenotype, suggesting conservation of function. Taken together, our results suggest that SUB1 acts as a negative regulator of sporulation.

No MeSH data available.


Related in: MedlinePlus