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Scc2 regulates gene expression by recruiting cohesin to the chromosome as a transcriptional activator during yeast meiosis.

Lin W, Jin H, Liu X, Hampton K, Yu HG - Mol. Biol. Cell (2011)

Bottom Line: Rec8-associated meiotic cohesin is required for the full activation of the REC8 promoter, revealing that cohesin has a positive feedback on transcriptional regulation.Finally, we provide evidence that chromosomal binding of cohesin is sufficient for target-gene activation during meiosis.Our data support a noncanonical role for cohesin as a transcriptional activator during cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA.

ABSTRACT
To tether sister chromatids, a protein-loading complex, including Scc2, recruits cohesin to the chromosome at discrete loci. Cohesin facilitates the formation of a higher-order chromosome structure that could also influence gene expression. How cohesin directly regulates transcription remains to be further elucidated. We report that in budding yeast Scc2 is required for sister-chromatid cohesion during meiosis for two reasons. First, Scc2 is required for activating the expression of REC8, which encodes a meiosis-specific cohesin subunit; second, Scc2 is necessary for recruiting meiotic cohesin to the chromosome to generate sister-chromatid cohesion. Using a heterologous reporter assay, we have found that Scc2 increases the activity of its target promoters by recruiting cohesin to establish an upstream cohesin-associated region in a position-dependent manner. Rec8-associated meiotic cohesin is required for the full activation of the REC8 promoter, revealing that cohesin has a positive feedback on transcriptional regulation. Finally, we provide evidence that chromosomal binding of cohesin is sufficient for target-gene activation during meiosis. Our data support a noncanonical role for cohesin as a transcriptional activator during cell differentiation.

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Scc2 is required for REC8 gene expression. (A) Rec8 protein level in wild-type (HY1503C) and PCLB2-SCC2 (HY1586) cells. Yeast cells were induced to undergo synchronous meiosis, then subjected to immunoblot as for Figure 2A. Rec8'3HA was detected by an anti-HA antibody (16B12). (B) Smc3 protein level during meiosis. Note that the levels of Smc3 remain normal in the absence of Scc2 in meiosis. SMC3-3HA, HY1750C; PCLB2-SCC2 SMC3-3HA, HY1750. (C) Rec8 protein level in spo11Δ cells during meiosis. Note that, in the double mutant PCLB2-SCC2 spo11Δ (HY1975), only residual Rec8-3HA was detected. (D) Northern blot showing the levels of IME1, REC8, SCC2, and ACT1 transcripts during meiosis. Wild-type (NH144) and PCLB2-SCC2 (HY1279) cells were induced to undergo synchronous meiosis; aliquots were withdrawn at indicated time points. Total RNA was extracted and prepared for Northern blots. Gene-specific probes sequentially probed the same blots. The level of ACT1 served as a loading control. (E) Quantification of Northern blots. The ratio of the gene of interest to ACT1 is shown. Wild type, filled circles; PCLB2-SCC2, open circles. (F) RT-PCR assay of transcripts. Aliquots were withdrawn 6 h after induction of meiosis. Total mRNA was extracted and reverse transcribed to cDNA. A semiquantitative PCR was used to amplify target cDNA with gene-specific primers. (G) ChIP of Pol II from WT (NH144) and PCLB2-SCC2 (HY1279) cells during meiosis. Yeast cells were induced to undergo synchronous meiosis, and ChIP was performed with an anti-Pol II CTD antibody (8WT16). Semiquantitative PCR was used to determine Pol II enrichment at the REC8 locus. Two representative time points are shown.
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Figure 3: Scc2 is required for REC8 gene expression. (A) Rec8 protein level in wild-type (HY1503C) and PCLB2-SCC2 (HY1586) cells. Yeast cells were induced to undergo synchronous meiosis, then subjected to immunoblot as for Figure 2A. Rec8'3HA was detected by an anti-HA antibody (16B12). (B) Smc3 protein level during meiosis. Note that the levels of Smc3 remain normal in the absence of Scc2 in meiosis. SMC3-3HA, HY1750C; PCLB2-SCC2 SMC3-3HA, HY1750. (C) Rec8 protein level in spo11Δ cells during meiosis. Note that, in the double mutant PCLB2-SCC2 spo11Δ (HY1975), only residual Rec8-3HA was detected. (D) Northern blot showing the levels of IME1, REC8, SCC2, and ACT1 transcripts during meiosis. Wild-type (NH144) and PCLB2-SCC2 (HY1279) cells were induced to undergo synchronous meiosis; aliquots were withdrawn at indicated time points. Total RNA was extracted and prepared for Northern blots. Gene-specific probes sequentially probed the same blots. The level of ACT1 served as a loading control. (E) Quantification of Northern blots. The ratio of the gene of interest to ACT1 is shown. Wild type, filled circles; PCLB2-SCC2, open circles. (F) RT-PCR assay of transcripts. Aliquots were withdrawn 6 h after induction of meiosis. Total mRNA was extracted and reverse transcribed to cDNA. A semiquantitative PCR was used to amplify target cDNA with gene-specific primers. (G) ChIP of Pol II from WT (NH144) and PCLB2-SCC2 (HY1279) cells during meiosis. Yeast cells were induced to undergo synchronous meiosis, and ChIP was performed with an anti-Pol II CTD antibody (8WT16). Semiquantitative PCR was used to determine Pol II enrichment at the REC8 locus. Two representative time points are shown.

Mentions: We next examined the effect of Scc2 depletion on the accumulation of the meiotic cohesin subunit Rec8, of which the encoding gene is induced for expression only after yeast cells commit to meiosis (Chu et al., 1998). Immunoblot analysis revealed that Rec8 protein levels were dramatically lower in Scc2-depleted cells than in cells showing the normal pattern of accumulation (Figure 3A). In wild-type cells, for comparison, Rec8 was detected 2 h after induction of meiosis, peaked around 6 h, and then was degraded before the completion of meiosis at ∼12 h (Figure 3A). In contrast, Rec8 was present at a much lower level in Scc2-depleted cells; only a trace amount could be detected by immunoblot (Figure 3A, right). Compared with those of the wild type, the protein levels of other cohesin subunits, for example Smc3, were not dramatically changed in Scc2-depleted meiotic cells (Figure 3B). Together, our data suggest that Scc2 is specifically required for maintaining normal Rec8 protein levels during meiosis.


Scc2 regulates gene expression by recruiting cohesin to the chromosome as a transcriptional activator during yeast meiosis.

Lin W, Jin H, Liu X, Hampton K, Yu HG - Mol. Biol. Cell (2011)

Scc2 is required for REC8 gene expression. (A) Rec8 protein level in wild-type (HY1503C) and PCLB2-SCC2 (HY1586) cells. Yeast cells were induced to undergo synchronous meiosis, then subjected to immunoblot as for Figure 2A. Rec8'3HA was detected by an anti-HA antibody (16B12). (B) Smc3 protein level during meiosis. Note that the levels of Smc3 remain normal in the absence of Scc2 in meiosis. SMC3-3HA, HY1750C; PCLB2-SCC2 SMC3-3HA, HY1750. (C) Rec8 protein level in spo11Δ cells during meiosis. Note that, in the double mutant PCLB2-SCC2 spo11Δ (HY1975), only residual Rec8-3HA was detected. (D) Northern blot showing the levels of IME1, REC8, SCC2, and ACT1 transcripts during meiosis. Wild-type (NH144) and PCLB2-SCC2 (HY1279) cells were induced to undergo synchronous meiosis; aliquots were withdrawn at indicated time points. Total RNA was extracted and prepared for Northern blots. Gene-specific probes sequentially probed the same blots. The level of ACT1 served as a loading control. (E) Quantification of Northern blots. The ratio of the gene of interest to ACT1 is shown. Wild type, filled circles; PCLB2-SCC2, open circles. (F) RT-PCR assay of transcripts. Aliquots were withdrawn 6 h after induction of meiosis. Total mRNA was extracted and reverse transcribed to cDNA. A semiquantitative PCR was used to amplify target cDNA with gene-specific primers. (G) ChIP of Pol II from WT (NH144) and PCLB2-SCC2 (HY1279) cells during meiosis. Yeast cells were induced to undergo synchronous meiosis, and ChIP was performed with an anti-Pol II CTD antibody (8WT16). Semiquantitative PCR was used to determine Pol II enrichment at the REC8 locus. Two representative time points are shown.
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Figure 3: Scc2 is required for REC8 gene expression. (A) Rec8 protein level in wild-type (HY1503C) and PCLB2-SCC2 (HY1586) cells. Yeast cells were induced to undergo synchronous meiosis, then subjected to immunoblot as for Figure 2A. Rec8'3HA was detected by an anti-HA antibody (16B12). (B) Smc3 protein level during meiosis. Note that the levels of Smc3 remain normal in the absence of Scc2 in meiosis. SMC3-3HA, HY1750C; PCLB2-SCC2 SMC3-3HA, HY1750. (C) Rec8 protein level in spo11Δ cells during meiosis. Note that, in the double mutant PCLB2-SCC2 spo11Δ (HY1975), only residual Rec8-3HA was detected. (D) Northern blot showing the levels of IME1, REC8, SCC2, and ACT1 transcripts during meiosis. Wild-type (NH144) and PCLB2-SCC2 (HY1279) cells were induced to undergo synchronous meiosis; aliquots were withdrawn at indicated time points. Total RNA was extracted and prepared for Northern blots. Gene-specific probes sequentially probed the same blots. The level of ACT1 served as a loading control. (E) Quantification of Northern blots. The ratio of the gene of interest to ACT1 is shown. Wild type, filled circles; PCLB2-SCC2, open circles. (F) RT-PCR assay of transcripts. Aliquots were withdrawn 6 h after induction of meiosis. Total mRNA was extracted and reverse transcribed to cDNA. A semiquantitative PCR was used to amplify target cDNA with gene-specific primers. (G) ChIP of Pol II from WT (NH144) and PCLB2-SCC2 (HY1279) cells during meiosis. Yeast cells were induced to undergo synchronous meiosis, and ChIP was performed with an anti-Pol II CTD antibody (8WT16). Semiquantitative PCR was used to determine Pol II enrichment at the REC8 locus. Two representative time points are shown.
Mentions: We next examined the effect of Scc2 depletion on the accumulation of the meiotic cohesin subunit Rec8, of which the encoding gene is induced for expression only after yeast cells commit to meiosis (Chu et al., 1998). Immunoblot analysis revealed that Rec8 protein levels were dramatically lower in Scc2-depleted cells than in cells showing the normal pattern of accumulation (Figure 3A). In wild-type cells, for comparison, Rec8 was detected 2 h after induction of meiosis, peaked around 6 h, and then was degraded before the completion of meiosis at ∼12 h (Figure 3A). In contrast, Rec8 was present at a much lower level in Scc2-depleted cells; only a trace amount could be detected by immunoblot (Figure 3A, right). Compared with those of the wild type, the protein levels of other cohesin subunits, for example Smc3, were not dramatically changed in Scc2-depleted meiotic cells (Figure 3B). Together, our data suggest that Scc2 is specifically required for maintaining normal Rec8 protein levels during meiosis.

Bottom Line: Rec8-associated meiotic cohesin is required for the full activation of the REC8 promoter, revealing that cohesin has a positive feedback on transcriptional regulation.Finally, we provide evidence that chromosomal binding of cohesin is sufficient for target-gene activation during meiosis.Our data support a noncanonical role for cohesin as a transcriptional activator during cell differentiation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, FL 32306-4370, USA.

ABSTRACT
To tether sister chromatids, a protein-loading complex, including Scc2, recruits cohesin to the chromosome at discrete loci. Cohesin facilitates the formation of a higher-order chromosome structure that could also influence gene expression. How cohesin directly regulates transcription remains to be further elucidated. We report that in budding yeast Scc2 is required for sister-chromatid cohesion during meiosis for two reasons. First, Scc2 is required for activating the expression of REC8, which encodes a meiosis-specific cohesin subunit; second, Scc2 is necessary for recruiting meiotic cohesin to the chromosome to generate sister-chromatid cohesion. Using a heterologous reporter assay, we have found that Scc2 increases the activity of its target promoters by recruiting cohesin to establish an upstream cohesin-associated region in a position-dependent manner. Rec8-associated meiotic cohesin is required for the full activation of the REC8 promoter, revealing that cohesin has a positive feedback on transcriptional regulation. Finally, we provide evidence that chromosomal binding of cohesin is sufficient for target-gene activation during meiosis. Our data support a noncanonical role for cohesin as a transcriptional activator during cell differentiation.

Show MeSH
Related in: MedlinePlus