Cohesin plays a dual role in gene regulation and sister-chromatid cohesion during meiosis in Saccharomyces cerevisiae

Abstract

Sister-chromatid cohesion mediated by cohesin ensures proper chromosome segregation during cell division. Cohesin is also required for postreplicative DNA double-strand break repair and gene expression. The molecular mechanisms of these diverse cohesin functions remain to be elucidated. Here we report that the cohesin subunits Scc3 and Smc1 are both required for the production of the meiosis-specific subunit Rec8 in the budding yeast Saccharomyces cerevisiae. Using a genetic approach, we depleted Scc3 and Smc1 independently in cells that were undergoing meiosis. Both Scc3- and Smc1-depleted cells were inducible for meiosis, but the REC8 promoter was only marginally activated, leading to reduced levels of REC8 transcription and protein production. In contrast, the expression of MCD1, the mitotic counterpart of REC8, was not subject to Scc3 regulation in vegetative cells. We provide genetic evidence to show that sister-chromatid cohesion is not necessary for activation of REC8 gene expression. Cohesin appears to positively regulate the expression of a variety of genes during yeast meiosis. Our results suggest that the cohesin complex plays a dual role in gene regulation and sister-chromatid cohesion during meiotic differentiation in yeast.

Figure 1.—

Requirement for Scc3 in sister-chromatid cohesion during yeast meiosis. (A) Protein levels of Scc3 during yeast meiosis. Yeast cells were induced for synchronous meiosis, and aliquots were withdrawn at indicated times. Total protein extracts were prepared by the TCA method for Western blots, which were probed by anti-HA (12CA5) and anti-β-tubulin antibodies. The level of Tub2 (β-tubulin) served as a loading control. Note that Scc3 was largely depleted in meiosis in P_CLB2SCC3 cells. MT, mitosis. Protein extracts were prepared from cells grown asynchronously in YPD medium. Wild-type (WT), strain 3072; P_CLB2SCC3, strain 3200. (B and C) Assay of sister-chromatid cohesion in strains 3078C, 3206, HY2130, and HY1472. Yeast aliquots were withdrawn at indicted time points and fixed for fluorescence microscopy. An array of tetO was inserted at the URA3 locus, ∼35 kb from centromere V. Expression of tetR-GFP generated a GFP signal that could be visualized as a dot by fluorescence microscopy. Cohesed sister chromatids formed only one GFP dot. At least 100 cells were counted at each time point.

Figure 2.—

Reduced Rec8 protein level in Scc3-depleted cells. Yeast cells were induced to undergo synchronous meiosis as in Figure 1. (A) Chromosome association of Rec8 in wild-type (2824) and P_CLB2SCC3 (HY2294) cells. Yeast aliquots were collected 6 hr after induction of meiosis, and surface nuclear spreads were prepared for immunofluorescence with an anti-HA antibody. Red, DNA; green, Rec8. (B) Rec8 protein level in wild-type and P_CLB2SCC3 cells in meiosis. Yeast aliquots were collected at indicated times and prepared for Western blot as in A. An anti-Dmc1-specific antibody was used to detect the level of Dmc1. (C) Chromosome association of Scc3 in wild-type (3072) and rec8Δ (HY1495) cells. Surface yeast nuclear spreads were prepared as in A. Note that Scc3 remains chromosome-bound in rec8Δ cells. Red, DNA; green, Scc3. Bar, 2 μm. (D) Scc3 protein level in wild-type and rec8Δ cells. Western blots were prepared as in B. Note that the level of Scc3 remains normal in rec8Δ cells.

Figure 3.—

Requirement for Scc3 for Rec8 but not for Smc3 production in yeast meiosis. Yeast cells were induced for synchronous meiosis, aliquots were withdrawn at indicated times, and protein extracts were prepared for Western blots probed by anti-V5, anti-HA, and anti-β-tubulin antibodies. (A) Protein level of Smc3 in wild-type (HY1510C) and P_CLB2SCC3 (HY1566) cells. (B) Chromosome localization of Smc3 during yeast meiosis. Yeast cells were collected 6 hr after induction of meiosis and prepared for surface nuclear spread as in Figure 2A. Tub1 (α-Tubulin) was detected by a specific antibody (YOL135). Red, Smc3; green, Tub1; blue, DNA. (C) Protein levels of Rec8 in spo11-Y135F (HY1499) and P_CLB2SCC3 spo11-Y135F (HY1483) cells.

Figure 4.—

Scc3 regulates REC8 promoter activity during yeast meiosis. (A) mRNA levels of IME1, REC8, SCC3, and ACT1 in wild-type (NH144), P_CLB2SCC3 (3200), and rec8Δ (HY1495) cells. Yeast cells were induced to undergo synchronous meiosis, and aliquots were withdrawn at the indicated times and prepared for Northern blots probed by gene-specific probes. (B) RT-PCR analysis of IME1, REC8, and ACT1 transcripts. Yeast aliquots were withdrawn 6 hr after induction of meiosis; total mRNA was extracted, reversed to cDNA, and amplified by gene-specific primers. (Right) Quantitative analysis with an average of two independent experiments shown. Error bars show standard deviation. (C) ChIP of Rbp3 in wild-type (HY3000) and P_CLB2SCC3 (HY3003) cells during yeast meiosis. Yeast cells were induced to undergo synchronous meiosis; aliquots were withdrawn 6 hr after induction and prepared for ChIP analysis. (Left) A representative gel image. (Right) Quantitative analysis of Rpb3 binding at the REC8 and DMC1 genes from two independent experiments. (D) A heterologous reporter assay of REC8 promoter activity (HY2106 and HY2108). Plasmid pHG105 was digested with MluI and transformed into the REC8 locus. Yeast cells were induced to undergo synchronous meiosis, and aliquots were withdrawn for Northern blots as shown in A. Gene-specific probes were used to detect the mRNA levels of IME1, GFP, and ACT1.

Figure 5.—

Ectopic expression of REC8 in meiotic cells. (A) The expression level of P_CUP1REC8 in wild-type (HY1417C) and P_CLB2SCC3 (HY1417) cells during meiosis. Yeast cells were induced for synchronous meiosis, and aliquots were withdrawn at indicated times for Northern blots as shown in Figure 4A. Note that P_CUP1REC8 is expressed in P_CLB2SCC3 cells. (Right) A semiquantitative measurement of REC8 transcripts over those of ACT1. (B) Protein level of Rec8 in wild-type and P_CLB2SCC3 cells. Western blots were prepared as in Figure 1A to reveal the levels of Rec8-3HA and β-tubulin. (C) Chromosome association of Rec8 in wild-type and P_CLB2SCC3 cells. Yeast surface nuclear spreads were prepared for immunofluorescence as in Figure 2A. Note that Rec8 is produced but does not bind to chromosomes in P_CLB2SCC3 cells. Red, DNA; green, Rec8. Bar, 2 μm.

Figure 6.—

Scc3 is not required for MCD1 expression in vegetative cells. (A) A diagram showing the experimental procedure. (B) Yeast budding index showing cell progression. Yeast aliquots were withdrawn at indicated times after G1 release, fixed, and examined by phase-contrast microscopy. (C) mRNA levels of SCC3, MCD1, and ACT1 after release from α-factor arrest. Yeast aliquots were withdrawn at indicated times and prepared for Northern blots probed by gene-specific probes as shown in Figure 4A. Note that MCD1 is expressed only after release from α-factor arrest. (D) Protein levels of Scc3 and Mcd1. Yeast aliquots were withdrawn at indicated times and prepared for Western blots probed by anti-HA, anti-Mcd1, and anti-β-tubulin antibodies. Note that Mcd1 remains at a normal level in Scc3-depleted vegetative cells.

Figure 7.—

Activation of REC8 promoter requires Smc1 but not sister-chromatid cohesion. (A) Depletion of Smc1 during meiosis (2821 and HY1875). Yeast cells were induced to undergo synchronous meiosis, and aliquots were withdrawn at the indicated times for Western blots as in Figure 1A. (B) Transcriptional level of REC8 during meiosis (NH144 and HY1875). Total RNA was extracted and probed with gene-specific probes as in Figure 4A. (C) Protein level of Rec8 in wild-type (HY1503C) and P_CLB2SMC1 (HY1868) cells in meiosis. Yeast protein extracts were prepared for Western blots, which detected the levels of Rec8 and Dmc1 as shown in Figure 2B. The level of β-tubulin served as a loading control. (D) A heterologous reporter assay of REC8 promoter activity in wild-type (HY2460) and P_CLB2SMC1 (HY2460-1) cells in meiosis. P_REC8GFP was placed at the URA3 locus by transformation. Yeast cells were induced to undergo synchronous meiosis, and aliquots were withdrawn at the indicated times and prepared for Western blots probed by anti-GFP (Ab290) and anti-β-tubulin antibodies. (E) Rec8 protein level in wild-type (HY2740) and P_SCC1CDC6 (HY2741) cells during meiosis. Representative time points are shown. (F) Chromosome localization of Rec8 in wild-type and P_SCC1CDC6 cells during meiosis. Yeast cells were collected 6 hr after induction of meiosis and prepared for surface nuclear spread as in Figure 2A. Note that chromosomes still formed rod-shaped structures in the absence of sister chromatids. Red, DNA; green, Rec8. Bar, 2 μm.