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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 recruitment at SMK1 and SPS2 middle gene loci.Chromatin immunoprecipitation analysis for recruitment of Sub1 at the promoters of early-middle (NDT80) and middle (SMK1 and SPS2) sporulation genes at different time points (T—0, 2 and 5 hours post induction) during sporulation. Fold enrichment was calculated by normalization to ACT1 promoter region.
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pone.0132350.g005: Sub1 recruitment at SMK1 and SPS2 middle gene loci.Chromatin immunoprecipitation analysis for recruitment of Sub1 at the promoters of early-middle (NDT80) and middle (SMK1 and SPS2) sporulation genes at different time points (T—0, 2 and 5 hours post induction) during sporulation. Fold enrichment was calculated by normalization to ACT1 promoter region.

Mentions: In mitotically growing vegetative cells, Sub1 has been shown to associate with the promoter and coding regions of several constitutively—transcribed RNA Pol II and Pol III genes [3,4,6]. Moreover, Sub1 gets recruited to IMD2 gene and thereby represses its expression [6,25]. These prior reports and our data highlighting the relationship between Sub1 and gene expression cascade during sporulation led us to hypothesize that Sub1 may be directly binding to the middle sporulation genes. To experimentally test this, we performed chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) in wild-type SK1 cells wherein the endogenous SUB1 ORF was TAP tagged. These diploids were sporulated and chromatin was prepared from cells at 0, 2 and 5 hours after transfer to sporulation medium and subjected to immunoprecipitation. We detect significant enrichment of Sub1 at the promoter DNA sequence of SMK1 and SPS2 genes, notably at 5th hour post transfer to sporulation medium (Fig 5). At the same time point the association of Sub1 was lowered at the NDT80 promoter sequences as compared to the levels at SMK1 and SPS2 loci. This difference in Sub1 occupancy at NDT80, SMK1 and SPS2 loci temporally correlates with the increased transcript levels of SMK1 and SPS2 genes and the unaltered levels of NDT80 transcripts in sub1Δ/sub1Δ SK1 cells. These data suggests that Sub1 shows a stronger association with middle sporulation genes and thereby regulates their expression levels.


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

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

Sub1 recruitment at SMK1 and SPS2 middle gene loci.Chromatin immunoprecipitation analysis for recruitment of Sub1 at the promoters of early-middle (NDT80) and middle (SMK1 and SPS2) sporulation genes at different time points (T—0, 2 and 5 hours post induction) during sporulation. Fold enrichment was calculated by normalization to ACT1 promoter region.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492983&req=5

pone.0132350.g005: Sub1 recruitment at SMK1 and SPS2 middle gene loci.Chromatin immunoprecipitation analysis for recruitment of Sub1 at the promoters of early-middle (NDT80) and middle (SMK1 and SPS2) sporulation genes at different time points (T—0, 2 and 5 hours post induction) during sporulation. Fold enrichment was calculated by normalization to ACT1 promoter region.
Mentions: In mitotically growing vegetative cells, Sub1 has been shown to associate with the promoter and coding regions of several constitutively—transcribed RNA Pol II and Pol III genes [3,4,6]. Moreover, Sub1 gets recruited to IMD2 gene and thereby represses its expression [6,25]. These prior reports and our data highlighting the relationship between Sub1 and gene expression cascade during sporulation led us to hypothesize that Sub1 may be directly binding to the middle sporulation genes. To experimentally test this, we performed chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) in wild-type SK1 cells wherein the endogenous SUB1 ORF was TAP tagged. These diploids were sporulated and chromatin was prepared from cells at 0, 2 and 5 hours after transfer to sporulation medium and subjected to immunoprecipitation. We detect significant enrichment of Sub1 at the promoter DNA sequence of SMK1 and SPS2 genes, notably at 5th hour post transfer to sporulation medium (Fig 5). At the same time point the association of Sub1 was lowered at the NDT80 promoter sequences as compared to the levels at SMK1 and SPS2 loci. This difference in Sub1 occupancy at NDT80, SMK1 and SPS2 loci temporally correlates with the increased transcript levels of SMK1 and SPS2 genes and the unaltered levels of NDT80 transcripts in sub1Δ/sub1Δ SK1 cells. These data suggests that Sub1 shows a stronger association with middle sporulation genes and thereby regulates their expression levels.

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