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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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Activation of REC8 promoter by forced localization of cohesin. (A) Generating a functional tetR-SCC3 fusion allele. Yeast strains (NH144, 3200, and HY2636) were sporulated, and tetrads were dissected for determination of spore viability. We used the CLB2 promoter to replace the endogenous SCC3 promoter to generate PCLB2-SCC3. (B) A schematic diagram showing forced localization of Scc3, and therefore cohesin, to the 10× copies of tetO sequence located 5′ upstream of the REC8 promoter. The distance between the first tetO and the REC8 promoter is ∼2 kb. (C) Immunoblot showing the production of GFP. Cells were induced to undergo synchronous meiosis, and protein extracts were prepared for immunoblotting. Strains used: HY2685 (scc2 tetO), HY2684 (scc2 tetO/tetR-Scc3), and HY2692 (scc2 tetR-Scc3). (D) Quantification of PREC8-GFP production. Yeast cells were induced to undergo synchronous meiosis, and the fluorescence intensities of GFP and RFP were determined as for Figure 5C. Strains used: WT, HY3100; scc2 PREC8-GFP::LEU2, HY3044; scc2 tetR-SCC3 PREC8-GFP::LEU2, HY3045; scc2 PREC8-GFP::DCC1, HY3046; and scc2 tetR-SCC3 PREC8-GFP::DCC1, HY3047. All strains harbor one copy of PDMC1-mAPPLE (RFP) and 10 copies of tetO inserted at the LEU2 locus. (E) A model depicts Scc2 and cohesin action in transcriptional activation during meiosis. Scc2, red squares; cohesin, green ovals. Black ovals represent an unknown transcription factor. Chromosomes are shown as black and gray lines.
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Figure 8: Activation of REC8 promoter by forced localization of cohesin. (A) Generating a functional tetR-SCC3 fusion allele. Yeast strains (NH144, 3200, and HY2636) were sporulated, and tetrads were dissected for determination of spore viability. We used the CLB2 promoter to replace the endogenous SCC3 promoter to generate PCLB2-SCC3. (B) A schematic diagram showing forced localization of Scc3, and therefore cohesin, to the 10× copies of tetO sequence located 5′ upstream of the REC8 promoter. The distance between the first tetO and the REC8 promoter is ∼2 kb. (C) Immunoblot showing the production of GFP. Cells were induced to undergo synchronous meiosis, and protein extracts were prepared for immunoblotting. Strains used: HY2685 (scc2 tetO), HY2684 (scc2 tetO/tetR-Scc3), and HY2692 (scc2 tetR-Scc3). (D) Quantification of PREC8-GFP production. Yeast cells were induced to undergo synchronous meiosis, and the fluorescence intensities of GFP and RFP were determined as for Figure 5C. Strains used: WT, HY3100; scc2 PREC8-GFP::LEU2, HY3044; scc2 tetR-SCC3 PREC8-GFP::LEU2, HY3045; scc2 PREC8-GFP::DCC1, HY3046; and scc2 tetR-SCC3 PREC8-GFP::DCC1, HY3047. All strains harbor one copy of PDMC1-mAPPLE (RFP) and 10 copies of tetO inserted at the LEU2 locus. (E) A model depicts Scc2 and cohesin action in transcriptional activation during meiosis. Scc2, red squares; cohesin, green ovals. Black ovals represent an unknown transcription factor. Chromosomes are shown as black and gray lines.

Mentions: To determine whether chromosomal loading of cohesin is sufficient for gene activation, we reasoned that forced localization of cohesin to the chromosome would increase gene expression in Scc2-depleted cells. We constructed a functional tetR-SCC3 fusion allele because it suppressed a meiosis-specific Scc3-depletion allele (Figure 8A). Using the specific interaction between tetO and tetR, we tethered Scc3, and therefore cohesin, to the 10xtetO sequence, which was positioned <2 kb 5′ upstream of the REC8 promoter (Figure 8B). By assaying the production of Rec8-3HA, we found that forced localization of cohesin to the 5′ upstream sequence of the endogenous REC8 promoter increased Rec8 production (Supplemental Figure 5A). Similarly, forced localization of cohesin to the 5′ upstream sequence of the PREC8-GFP construct positioned at the LEU2 locus significantly increased GFP production in Scc2-depleted cells (Figure 8, C and D). Forced localization of Scc3 alone, however, was not sufficient to activate the REC8 promoter in Smc1-depleted cells (Supplemental Figure 5, B and C). In addition, when PREC8-GFP was positioned at the DCC1 locus that is ∼6 kb downstream of the 10xtetO sequence, its expression level did not increase in Scc2-depleted cells even in the presence of tetR-Scc3 (Figure 8D). Therefore activation of the REC8 promoter depends on a functional cohesin complex positioned adjacent to its 5′ upstream sequence. These results provide direct evidence that cohesin binding to the chromosome is both necessary and sufficient for activation of the REC8 promoter 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)

Activation of REC8 promoter by forced localization of cohesin. (A) Generating a functional tetR-SCC3 fusion allele. Yeast strains (NH144, 3200, and HY2636) were sporulated, and tetrads were dissected for determination of spore viability. We used the CLB2 promoter to replace the endogenous SCC3 promoter to generate PCLB2-SCC3. (B) A schematic diagram showing forced localization of Scc3, and therefore cohesin, to the 10× copies of tetO sequence located 5′ upstream of the REC8 promoter. The distance between the first tetO and the REC8 promoter is ∼2 kb. (C) Immunoblot showing the production of GFP. Cells were induced to undergo synchronous meiosis, and protein extracts were prepared for immunoblotting. Strains used: HY2685 (scc2 tetO), HY2684 (scc2 tetO/tetR-Scc3), and HY2692 (scc2 tetR-Scc3). (D) Quantification of PREC8-GFP production. Yeast cells were induced to undergo synchronous meiosis, and the fluorescence intensities of GFP and RFP were determined as for Figure 5C. Strains used: WT, HY3100; scc2 PREC8-GFP::LEU2, HY3044; scc2 tetR-SCC3 PREC8-GFP::LEU2, HY3045; scc2 PREC8-GFP::DCC1, HY3046; and scc2 tetR-SCC3 PREC8-GFP::DCC1, HY3047. All strains harbor one copy of PDMC1-mAPPLE (RFP) and 10 copies of tetO inserted at the LEU2 locus. (E) A model depicts Scc2 and cohesin action in transcriptional activation during meiosis. Scc2, red squares; cohesin, green ovals. Black ovals represent an unknown transcription factor. Chromosomes are shown as black and gray lines.
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Figure 8: Activation of REC8 promoter by forced localization of cohesin. (A) Generating a functional tetR-SCC3 fusion allele. Yeast strains (NH144, 3200, and HY2636) were sporulated, and tetrads were dissected for determination of spore viability. We used the CLB2 promoter to replace the endogenous SCC3 promoter to generate PCLB2-SCC3. (B) A schematic diagram showing forced localization of Scc3, and therefore cohesin, to the 10× copies of tetO sequence located 5′ upstream of the REC8 promoter. The distance between the first tetO and the REC8 promoter is ∼2 kb. (C) Immunoblot showing the production of GFP. Cells were induced to undergo synchronous meiosis, and protein extracts were prepared for immunoblotting. Strains used: HY2685 (scc2 tetO), HY2684 (scc2 tetO/tetR-Scc3), and HY2692 (scc2 tetR-Scc3). (D) Quantification of PREC8-GFP production. Yeast cells were induced to undergo synchronous meiosis, and the fluorescence intensities of GFP and RFP were determined as for Figure 5C. Strains used: WT, HY3100; scc2 PREC8-GFP::LEU2, HY3044; scc2 tetR-SCC3 PREC8-GFP::LEU2, HY3045; scc2 PREC8-GFP::DCC1, HY3046; and scc2 tetR-SCC3 PREC8-GFP::DCC1, HY3047. All strains harbor one copy of PDMC1-mAPPLE (RFP) and 10 copies of tetO inserted at the LEU2 locus. (E) A model depicts Scc2 and cohesin action in transcriptional activation during meiosis. Scc2, red squares; cohesin, green ovals. Black ovals represent an unknown transcription factor. Chromosomes are shown as black and gray lines.
Mentions: To determine whether chromosomal loading of cohesin is sufficient for gene activation, we reasoned that forced localization of cohesin to the chromosome would increase gene expression in Scc2-depleted cells. We constructed a functional tetR-SCC3 fusion allele because it suppressed a meiosis-specific Scc3-depletion allele (Figure 8A). Using the specific interaction between tetO and tetR, we tethered Scc3, and therefore cohesin, to the 10xtetO sequence, which was positioned <2 kb 5′ upstream of the REC8 promoter (Figure 8B). By assaying the production of Rec8-3HA, we found that forced localization of cohesin to the 5′ upstream sequence of the endogenous REC8 promoter increased Rec8 production (Supplemental Figure 5A). Similarly, forced localization of cohesin to the 5′ upstream sequence of the PREC8-GFP construct positioned at the LEU2 locus significantly increased GFP production in Scc2-depleted cells (Figure 8, C and D). Forced localization of Scc3 alone, however, was not sufficient to activate the REC8 promoter in Smc1-depleted cells (Supplemental Figure 5, B and C). In addition, when PREC8-GFP was positioned at the DCC1 locus that is ∼6 kb downstream of the 10xtetO sequence, its expression level did not increase in Scc2-depleted cells even in the presence of tetR-Scc3 (Figure 8D). Therefore activation of the REC8 promoter depends on a functional cohesin complex positioned adjacent to its 5′ upstream sequence. These results provide direct evidence that cohesin binding to the chromosome is both necessary and sufficient for activation of the REC8 promoter 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