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Functional overlap among distinct G1/S inhibitory pathways allows robust G1 arrest by yeast mating pheromones.

Pope PA, Pryciak PM - Mol. Biol. Cell (2013)

Bottom Line: In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1.Deleting SIC1 alone strongly disrupts Far1-independent G1 arrest, revealing that inhibition of B-type cyclin-Cdk activity can empower weak arrest pathways.Overall our findings illustrate how multiple distinct G1/S-braking mechanisms help to prevent premature cell cycle commitment and ensure a robust signal-induced G1 arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605.

ABSTRACT
In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1. Alternate pathways must also exist, however, because deleting the cyclin CLN2 restores pheromone arrest to far1 cells. Here we probe whether these alternate pathways require the G1/S transcriptional repressors Whi5 and Stb1 or the CKI protein Sic1, whose metazoan analogues (Rb or p27) antagonize cell cycle entry. Removing Whi5 and Stb1 allows partial escape from G1 arrest in far1 cln2 cells, along with partial derepression of G1/S genes, which implies a repressor-independent route for inhibiting G1/S transcription. This route likely involves pheromone-induced degradation of Tec1, a transcriptional activator of the cyclin CLN1, because Tec1 stabilization also causes partial G1 escape in far1 cln2 cells, and this is additive with Whi5/Stb1 removal. Deleting SIC1 alone strongly disrupts Far1-independent G1 arrest, revealing that inhibition of B-type cyclin-Cdk activity can empower weak arrest pathways. Of interest, although far1 cln2 sic1 cells escaped G1 arrest, they lost viability during pheromone exposure, indicating that G1 exit is deleterious if the arrest signal remains active. Overall our findings illustrate how multiple distinct G1/S-braking mechanisms help to prevent premature cell cycle commitment and ensure a robust signal-induced G1 arrest.

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Effects of Far1, Whi5, and Stb1 on G1/S mRNA levels. The effects of Whi5 and Stb1 on G1/S transcript levels were measured in FAR1 cln2∆ (left) and far1∆ cln2∆ (right) backgrounds. PGAL1-CDC20 strains were arrested in M phase and released with or without α factor. At 30-min intervals, mRNA levels were measured by RT-qPCR (see Materials and Methods). Five G1/S transcripts (CLN1, YOX1, RNR1, POL1, and CDC21) and one M/G1 transcript (SIC1) were monitored. mRNA levels at each time point were plotted relative to the levels present in the M-phase–arrested cultures (t = 0). The drop in G1/S transcript levels from M phase (t = 0) to G1 (t = 30 min) was unexpected because these genes are not believed to be active during mitosis; this behavior might reflect imperfect synchronization in M phase, or it might indicate that maximal repression of these genes requires nuclear localization of Swi6 and DNA binding by SBF/MBF, which are inhibited by high Cdk activity in M phase (Sidorova et al., 1995; Koch et al., 1996; Queralt and Igual, 2003; Geymonat et al., 2004). See Figure 4 for further analyses.
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Figure 3: Effects of Far1, Whi5, and Stb1 on G1/S mRNA levels. The effects of Whi5 and Stb1 on G1/S transcript levels were measured in FAR1 cln2∆ (left) and far1∆ cln2∆ (right) backgrounds. PGAL1-CDC20 strains were arrested in M phase and released with or without α factor. At 30-min intervals, mRNA levels were measured by RT-qPCR (see Materials and Methods). Five G1/S transcripts (CLN1, YOX1, RNR1, POL1, and CDC21) and one M/G1 transcript (SIC1) were monitored. mRNA levels at each time point were plotted relative to the levels present in the M-phase–arrested cultures (t = 0). The drop in G1/S transcript levels from M phase (t = 0) to G1 (t = 30 min) was unexpected because these genes are not believed to be active during mitosis; this behavior might reflect imperfect synchronization in M phase, or it might indicate that maximal repression of these genes requires nuclear localization of Swi6 and DNA binding by SBF/MBF, which are inhibited by high Cdk activity in M phase (Sidorova et al., 1995; Koch et al., 1996; Queralt and Igual, 2003; Geymonat et al., 2004). See Figure 4 for further analyses.

Mentions: We considered two possible explanations for why removing the transcriptional repressors did not fully eliminate G1 arrest in far1∆ cln2∆ cells: 1) pheromone signaling might still be able to inhibit G1/S transcription even without the repressors; and 2) the G1/S transcripts could be fully derepressed, but pheromone signaling might exert nontranscriptional effects that inhibit exit from G1. To test these possibilities, we analyzed G1/S transcript levels via real-time quantitative PCR (RT-qPCR). We chose five representative genes (CLN1, YOX1, POL1, RNR1, and CDC21) that are induced by SBF and/or MBF at the G1/S transition (Bean et al., 2005; de Bruin et al., 2006; Eser et al., 2011). For comparison, we also monitored a gene expressed at the earlier M/G1 boundary (SIC1). We first conducted single-time-course experiments for eight different strains, in which we measured transcript levels at numerous time points in synchronous cultures (Figure 3). Then we analyzed the most informative time points in multiple independent trials (Figure 4).


Functional overlap among distinct G1/S inhibitory pathways allows robust G1 arrest by yeast mating pheromones.

Pope PA, Pryciak PM - Mol. Biol. Cell (2013)

Effects of Far1, Whi5, and Stb1 on G1/S mRNA levels. The effects of Whi5 and Stb1 on G1/S transcript levels were measured in FAR1 cln2∆ (left) and far1∆ cln2∆ (right) backgrounds. PGAL1-CDC20 strains were arrested in M phase and released with or without α factor. At 30-min intervals, mRNA levels were measured by RT-qPCR (see Materials and Methods). Five G1/S transcripts (CLN1, YOX1, RNR1, POL1, and CDC21) and one M/G1 transcript (SIC1) were monitored. mRNA levels at each time point were plotted relative to the levels present in the M-phase–arrested cultures (t = 0). The drop in G1/S transcript levels from M phase (t = 0) to G1 (t = 30 min) was unexpected because these genes are not believed to be active during mitosis; this behavior might reflect imperfect synchronization in M phase, or it might indicate that maximal repression of these genes requires nuclear localization of Swi6 and DNA binding by SBF/MBF, which are inhibited by high Cdk activity in M phase (Sidorova et al., 1995; Koch et al., 1996; Queralt and Igual, 2003; Geymonat et al., 2004). See Figure 4 for further analyses.
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Related In: Results  -  Collection

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Figure 3: Effects of Far1, Whi5, and Stb1 on G1/S mRNA levels. The effects of Whi5 and Stb1 on G1/S transcript levels were measured in FAR1 cln2∆ (left) and far1∆ cln2∆ (right) backgrounds. PGAL1-CDC20 strains were arrested in M phase and released with or without α factor. At 30-min intervals, mRNA levels were measured by RT-qPCR (see Materials and Methods). Five G1/S transcripts (CLN1, YOX1, RNR1, POL1, and CDC21) and one M/G1 transcript (SIC1) were monitored. mRNA levels at each time point were plotted relative to the levels present in the M-phase–arrested cultures (t = 0). The drop in G1/S transcript levels from M phase (t = 0) to G1 (t = 30 min) was unexpected because these genes are not believed to be active during mitosis; this behavior might reflect imperfect synchronization in M phase, or it might indicate that maximal repression of these genes requires nuclear localization of Swi6 and DNA binding by SBF/MBF, which are inhibited by high Cdk activity in M phase (Sidorova et al., 1995; Koch et al., 1996; Queralt and Igual, 2003; Geymonat et al., 2004). See Figure 4 for further analyses.
Mentions: We considered two possible explanations for why removing the transcriptional repressors did not fully eliminate G1 arrest in far1∆ cln2∆ cells: 1) pheromone signaling might still be able to inhibit G1/S transcription even without the repressors; and 2) the G1/S transcripts could be fully derepressed, but pheromone signaling might exert nontranscriptional effects that inhibit exit from G1. To test these possibilities, we analyzed G1/S transcript levels via real-time quantitative PCR (RT-qPCR). We chose five representative genes (CLN1, YOX1, POL1, RNR1, and CDC21) that are induced by SBF and/or MBF at the G1/S transition (Bean et al., 2005; de Bruin et al., 2006; Eser et al., 2011). For comparison, we also monitored a gene expressed at the earlier M/G1 boundary (SIC1). We first conducted single-time-course experiments for eight different strains, in which we measured transcript levels at numerous time points in synchronous cultures (Figure 3). Then we analyzed the most informative time points in multiple independent trials (Figure 4).

Bottom Line: In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1.Deleting SIC1 alone strongly disrupts Far1-independent G1 arrest, revealing that inhibition of B-type cyclin-Cdk activity can empower weak arrest pathways.Overall our findings illustrate how multiple distinct G1/S-braking mechanisms help to prevent premature cell cycle commitment and ensure a robust signal-induced G1 arrest.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605.

ABSTRACT
In budding yeast, mating pheromones arrest the cell cycle in G1 phase via a pheromone-activated Cdk-inhibitor (CKI) protein, Far1. Alternate pathways must also exist, however, because deleting the cyclin CLN2 restores pheromone arrest to far1 cells. Here we probe whether these alternate pathways require the G1/S transcriptional repressors Whi5 and Stb1 or the CKI protein Sic1, whose metazoan analogues (Rb or p27) antagonize cell cycle entry. Removing Whi5 and Stb1 allows partial escape from G1 arrest in far1 cln2 cells, along with partial derepression of G1/S genes, which implies a repressor-independent route for inhibiting G1/S transcription. This route likely involves pheromone-induced degradation of Tec1, a transcriptional activator of the cyclin CLN1, because Tec1 stabilization also causes partial G1 escape in far1 cln2 cells, and this is additive with Whi5/Stb1 removal. Deleting SIC1 alone strongly disrupts Far1-independent G1 arrest, revealing that inhibition of B-type cyclin-Cdk activity can empower weak arrest pathways. Of interest, although far1 cln2 sic1 cells escaped G1 arrest, they lost viability during pheromone exposure, indicating that G1 exit is deleterious if the arrest signal remains active. Overall our findings illustrate how multiple distinct G1/S-braking mechanisms help to prevent premature cell cycle commitment and ensure a robust signal-induced G1 arrest.

Show MeSH
Related in: MedlinePlus