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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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Feedback control of cohesin on REC8 promoter. (A) Rec8 activates its own promoter. Yeast cells were induced to undergo meiosis, and samples were prepared for Northern blot as for Figure 3D. PREC8-GFP::LEU2 (HY2157); PREC8-GFP::LEU2 rec8Δ (HY2229). (B) Quantification of Northern blots. Wild type, filled circles; rec8Δ, open circles. (C) GPF protein level by immunoblot. Cells were induced to undergo synchronous meiosis as for A, and protein extracts were prepared for immunoblot. (D) GFP production in live cells. Aliquots were withdrawn 6 h after induction of meiosis. GFP intensity was measured by fluorescence microscopy as for Figure 6D. PCLB2-SCC3, HY2285. (E) Ectopic production of Rec8 in Scc2-depleted cells. To induce PCUP1-REC8 (HY2122) expression, 100 μM CuSO4 was added to the culture medium after induction of meiosis. (F) ChIP of Rec8 at centromere III. Chromosome III SGD coordinates are shown on the x-axis. Ratio of immunoprecipitation to input is shown on the y-axis. Rec8'3HA, filled circles; PCUP1-Rec8'3HA, PCLB2-SCC2, open circles. (G) Rec8 localization on meiotic chromosomes. Yeast cells were induced for synchronous meiosis and nuclear spreads were prepared for immunofluorescence as for Figure 1A. Bar, 2 μm.
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Figure 7: Feedback control of cohesin on REC8 promoter. (A) Rec8 activates its own promoter. Yeast cells were induced to undergo meiosis, and samples were prepared for Northern blot as for Figure 3D. PREC8-GFP::LEU2 (HY2157); PREC8-GFP::LEU2 rec8Δ (HY2229). (B) Quantification of Northern blots. Wild type, filled circles; rec8Δ, open circles. (C) GPF protein level by immunoblot. Cells were induced to undergo synchronous meiosis as for A, and protein extracts were prepared for immunoblot. (D) GFP production in live cells. Aliquots were withdrawn 6 h after induction of meiosis. GFP intensity was measured by fluorescence microscopy as for Figure 6D. PCLB2-SCC3, HY2285. (E) Ectopic production of Rec8 in Scc2-depleted cells. To induce PCUP1-REC8 (HY2122) expression, 100 μM CuSO4 was added to the culture medium after induction of meiosis. (F) ChIP of Rec8 at centromere III. Chromosome III SGD coordinates are shown on the x-axis. Ratio of immunoprecipitation to input is shown on the y-axis. Rec8'3HA, filled circles; PCUP1-Rec8'3HA, PCLB2-SCC2, open circles. (G) Rec8 localization on meiotic chromosomes. Yeast cells were induced for synchronous meiosis and nuclear spreads were prepared for immunofluorescence as for Figure 1A. Bar, 2 μm.

Mentions: If a cohesin-associated region acts as a transcriptional regulatory sequence, Rec8-associated cohesin could be the trans-acting factor that activates the REC8 promoter. To test this hypothesis, we assayed the transcriptional activity of PREC8-GFP::LEU2 in rec8Δ cells (Figure 7A). As predicted, GFP transcription was decreased by 75% in the absence of Rec8 during meiosis (Figure 7, A and B). Accordingly, the level of GFP protein decreased to one-fifth of the level in the wild type (Figure 7, C and D), suggesting a positive feedback control by Rec8 of its own promoter. As in Scc2-depleted cells, a low basal level of GFP expression remained in rec8Δ cells (Figure 7, A and B), demonstrating that the REC8 promoter remained minimally active in the absence of meiotic cohesin. Because REC8 expression was also low in an scc3 mutant in meiosis (Lin et al., 2011), and because PREC8-MCD1 was expressed in the absence of Rec8 and suppressed rec8Δ phenotype in REC8 promoter activation (Figure 4A), our data suggest that cohesin acts as a transcriptional activator at the REC8 promoter.


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)

Feedback control of cohesin on REC8 promoter. (A) Rec8 activates its own promoter. Yeast cells were induced to undergo meiosis, and samples were prepared for Northern blot as for Figure 3D. PREC8-GFP::LEU2 (HY2157); PREC8-GFP::LEU2 rec8Δ (HY2229). (B) Quantification of Northern blots. Wild type, filled circles; rec8Δ, open circles. (C) GPF protein level by immunoblot. Cells were induced to undergo synchronous meiosis as for A, and protein extracts were prepared for immunoblot. (D) GFP production in live cells. Aliquots were withdrawn 6 h after induction of meiosis. GFP intensity was measured by fluorescence microscopy as for Figure 6D. PCLB2-SCC3, HY2285. (E) Ectopic production of Rec8 in Scc2-depleted cells. To induce PCUP1-REC8 (HY2122) expression, 100 μM CuSO4 was added to the culture medium after induction of meiosis. (F) ChIP of Rec8 at centromere III. Chromosome III SGD coordinates are shown on the x-axis. Ratio of immunoprecipitation to input is shown on the y-axis. Rec8'3HA, filled circles; PCUP1-Rec8'3HA, PCLB2-SCC2, open circles. (G) Rec8 localization on meiotic chromosomes. Yeast cells were induced for synchronous meiosis and nuclear spreads were prepared for immunofluorescence as for Figure 1A. Bar, 2 μm.
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Figure 7: Feedback control of cohesin on REC8 promoter. (A) Rec8 activates its own promoter. Yeast cells were induced to undergo meiosis, and samples were prepared for Northern blot as for Figure 3D. PREC8-GFP::LEU2 (HY2157); PREC8-GFP::LEU2 rec8Δ (HY2229). (B) Quantification of Northern blots. Wild type, filled circles; rec8Δ, open circles. (C) GPF protein level by immunoblot. Cells were induced to undergo synchronous meiosis as for A, and protein extracts were prepared for immunoblot. (D) GFP production in live cells. Aliquots were withdrawn 6 h after induction of meiosis. GFP intensity was measured by fluorescence microscopy as for Figure 6D. PCLB2-SCC3, HY2285. (E) Ectopic production of Rec8 in Scc2-depleted cells. To induce PCUP1-REC8 (HY2122) expression, 100 μM CuSO4 was added to the culture medium after induction of meiosis. (F) ChIP of Rec8 at centromere III. Chromosome III SGD coordinates are shown on the x-axis. Ratio of immunoprecipitation to input is shown on the y-axis. Rec8'3HA, filled circles; PCUP1-Rec8'3HA, PCLB2-SCC2, open circles. (G) Rec8 localization on meiotic chromosomes. Yeast cells were induced for synchronous meiosis and nuclear spreads were prepared for immunofluorescence as for Figure 1A. Bar, 2 μm.
Mentions: If a cohesin-associated region acts as a transcriptional regulatory sequence, Rec8-associated cohesin could be the trans-acting factor that activates the REC8 promoter. To test this hypothesis, we assayed the transcriptional activity of PREC8-GFP::LEU2 in rec8Δ cells (Figure 7A). As predicted, GFP transcription was decreased by 75% in the absence of Rec8 during meiosis (Figure 7, A and B). Accordingly, the level of GFP protein decreased to one-fifth of the level in the wild type (Figure 7, C and D), suggesting a positive feedback control by Rec8 of its own promoter. As in Scc2-depleted cells, a low basal level of GFP expression remained in rec8Δ cells (Figure 7, A and B), demonstrating that the REC8 promoter remained minimally active in the absence of meiotic cohesin. Because REC8 expression was also low in an scc3 mutant in meiosis (Lin et al., 2011), and because PREC8-MCD1 was expressed in the absence of Rec8 and suppressed rec8Δ phenotype in REC8 promoter activation (Figure 4A), our data suggest that cohesin acts as a transcriptional activator at the REC8 promoter.

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