Limits...
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 decreases during sporulation.(A) WT SK1 cells were synchronized in YPA medium and then transferred to sporulation medium. Cells were collected at indicated time points. Normalization was done with ACT1 levels. qRT-PCR analysis for SUB1 transcript (left panel). Semi qRT-PCR analysis for early (IME2), middle (SPS2) and late (DIT1) sporulation-specific genes (right panel). (B) Western blot analysis of endogenously TAP tagged Sub1 protein for indicated time points. Sub1 protein is highest at the time of shifting the cells to sporulation medium and subsequently decreases over the sporulation time-course. Actin was used as a loading control. (C) Quantitation of Sub1 protein levels shown in (B).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4492983&req=5

pone.0132350.g003: Sub1 decreases during sporulation.(A) WT SK1 cells were synchronized in YPA medium and then transferred to sporulation medium. Cells were collected at indicated time points. Normalization was done with ACT1 levels. qRT-PCR analysis for SUB1 transcript (left panel). Semi qRT-PCR analysis for early (IME2), middle (SPS2) and late (DIT1) sporulation-specific genes (right panel). (B) Western blot analysis of endogenously TAP tagged Sub1 protein for indicated time points. Sub1 protein is highest at the time of shifting the cells to sporulation medium and subsequently decreases over the sporulation time-course. Actin was used as a loading control. (C) Quantitation of Sub1 protein levels shown in (B).

Mentions: Sporulation requires a regulated temporal expression of genes that facilitate meiosis and spore formation. Based on temporal expression patterns, sporulation genes are classified as early, middle, mid-late and late genes [15,16]. To determine Sub1 expression pattern during the sporulation cascade, we determined its transcript and protein levels. These experiments were performed in the SK1 strain as a very efficient and synchronous passage through sporulation is necessary to examine the temporally ordered sporulation gene expression which are characteristics seen in this strain. SK1 cells were first synchronised by growth in YPA medium and then transfer to sporulation medium. Culture aliquots were collected at different time points during sporulation for RNA isolation and expression studies. The SUB1 transcript levels and transcript abundance for several other well-studied sporulation specific genes were measured by quantitative and semi-quantitative RT-PCR analysis respectively. SUB1 transcript levels decreased after 30 minutes of transfer to sporulation medium and remained low for the subsequent 5 hours. The transcript levels increased again at 7th hour and remained high in the later time course of sporulation (Fig 3A left panel). This is in agreement with the previously reported high-density tiling microarray data analysed in SK1 strain during sporulation [24]. The window wherein SUB1 transcripts were at the lowest, is correlated with the induction of expression of early and middle sporulation genes (Fig 3A right panel).


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

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

Sub1 decreases during sporulation.(A) WT SK1 cells were synchronized in YPA medium and then transferred to sporulation medium. Cells were collected at indicated time points. Normalization was done with ACT1 levels. qRT-PCR analysis for SUB1 transcript (left panel). Semi qRT-PCR analysis for early (IME2), middle (SPS2) and late (DIT1) sporulation-specific genes (right panel). (B) Western blot analysis of endogenously TAP tagged Sub1 protein for indicated time points. Sub1 protein is highest at the time of shifting the cells to sporulation medium and subsequently decreases over the sporulation time-course. Actin was used as a loading control. (C) Quantitation of Sub1 protein levels shown in (B).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0132350.g003: Sub1 decreases during sporulation.(A) WT SK1 cells were synchronized in YPA medium and then transferred to sporulation medium. Cells were collected at indicated time points. Normalization was done with ACT1 levels. qRT-PCR analysis for SUB1 transcript (left panel). Semi qRT-PCR analysis for early (IME2), middle (SPS2) and late (DIT1) sporulation-specific genes (right panel). (B) Western blot analysis of endogenously TAP tagged Sub1 protein for indicated time points. Sub1 protein is highest at the time of shifting the cells to sporulation medium and subsequently decreases over the sporulation time-course. Actin was used as a loading control. (C) Quantitation of Sub1 protein levels shown in (B).
Mentions: Sporulation requires a regulated temporal expression of genes that facilitate meiosis and spore formation. Based on temporal expression patterns, sporulation genes are classified as early, middle, mid-late and late genes [15,16]. To determine Sub1 expression pattern during the sporulation cascade, we determined its transcript and protein levels. These experiments were performed in the SK1 strain as a very efficient and synchronous passage through sporulation is necessary to examine the temporally ordered sporulation gene expression which are characteristics seen in this strain. SK1 cells were first synchronised by growth in YPA medium and then transfer to sporulation medium. Culture aliquots were collected at different time points during sporulation for RNA isolation and expression studies. The SUB1 transcript levels and transcript abundance for several other well-studied sporulation specific genes were measured by quantitative and semi-quantitative RT-PCR analysis respectively. SUB1 transcript levels decreased after 30 minutes of transfer to sporulation medium and remained low for the subsequent 5 hours. The transcript levels increased again at 7th hour and remained high in the later time course of sporulation (Fig 3A left panel). This is in agreement with the previously reported high-density tiling microarray data analysed in SK1 strain during sporulation [24]. The window wherein SUB1 transcripts were at the lowest, is correlated with the induction of expression of early and middle sporulation genes (Fig 3A right panel).

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