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RNA processing factors Swd2.2 and Sen1 antagonize RNA Pol III-dependent transcription and the localization of condensin at Pol III genes.

Legros P, Malapert A, Niinuma S, Bernard P, Vanoosthuyse V - PLoS Genet. (2014)

Bottom Line: Genetic studies have indicated that the association of condensin with chromatin is intimately linked to gene transcription, but what transcription-associated feature(s) direct(s) the accumulation of condensin remains unclear.We provide evidence that this topological stress contributes to recruit and/or stabilize condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1.Our data challenge the idea that a processive RNA polymerase hinders the binding of condensin and suggest that transcription-associated topological stress could in some circumstances facilitate the association of condensin.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Université Lyon 01, UMR5239, LBMC; Ecole Normale Supérieure de Lyon, Lyon, France.

ABSTRACT
Condensin-mediated chromosome condensation is essential for genome stability upon cell division. Genetic studies have indicated that the association of condensin with chromatin is intimately linked to gene transcription, but what transcription-associated feature(s) direct(s) the accumulation of condensin remains unclear. Here we show in fission yeast that condensin becomes strikingly enriched at RNA Pol III-transcribed genes when Swd2.2 and Sen1, two factors involved in the transcription process, are simultaneously deleted. Sen1 is an ATP-dependent helicase whose orthologue in Saccharomyces cerevisiae contributes both to terminate transcription of some RNA Pol II transcripts and to antagonize the formation of DNA:RNA hybrids in the genome. Using two independent mapping techniques, we show that DNA:RNA hybrids form in abundance at Pol III-transcribed genes in fission yeast but we demonstrate that they are unlikely to faciliate the recruitment of condensin. Instead, we show that Sen1 forms a stable and abundant complex with RNA Pol III and that Swd2.2 and Sen1 antagonize both the interaction of RNA Pol III with chromatin and RNA Pol III-dependent transcription. When Swd2.2 and Sen1 are lacking, the increased concentration of RNA Pol III and condensin at Pol III-transcribed genes is accompanied by the accumulation of topoisomerase I and II and by local nucleosome depletion, suggesting that Pol III-transcribed genes suffer topological stress. We provide evidence that this topological stress contributes to recruit and/or stabilize condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1. Our data challenge the idea that a processive RNA polymerase hinders the binding of condensin and suggest that transcription-associated topological stress could in some circumstances facilitate the association of condensin.

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The double deletion of Swd2.2 and Sen1 facilitates the localization of condensin at Pol III-transcribed genes.A. Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures. B. Chromosome segregation in anaphase was monitored in the indicated strains after growing cells for one generation at 34°C. For each genotype, a minimum of 6 independent experiments was performed in which a minimum of 100 anaphase cells were scored (***<0.001; **<0.01 Wilcoxon - Mann Whitney). Anaphases were scored as defective when chromatin was detected lagging between the two main DNA masses C. ChIP-qPCR analysis of the amount of GFP-tagged Cut3 cross-linked to chromatin in cell populations of the indicated genotypes grown at 30°C (mean ± standard deviation from 6 biological replicates (NS: not significant, *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). The primers used in this study are shown on Table S1. D. Mitotic indexes of the cell populations used in C. Cells were fixed with cold methanol and processed for immuno-fluorescence using an anti-tubulin antibody. Cells with a spindle were counted as mitotic.
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pgen-1004794-g001: The double deletion of Swd2.2 and Sen1 facilitates the localization of condensin at Pol III-transcribed genes.A. Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures. B. Chromosome segregation in anaphase was monitored in the indicated strains after growing cells for one generation at 34°C. For each genotype, a minimum of 6 independent experiments was performed in which a minimum of 100 anaphase cells were scored (***<0.001; **<0.01 Wilcoxon - Mann Whitney). Anaphases were scored as defective when chromatin was detected lagging between the two main DNA masses C. ChIP-qPCR analysis of the amount of GFP-tagged Cut3 cross-linked to chromatin in cell populations of the indicated genotypes grown at 30°C (mean ± standard deviation from 6 biological replicates (NS: not significant, *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). The primers used in this study are shown on Table S1. D. Mitotic indexes of the cell populations used in C. Cells were fixed with cold methanol and processed for immuno-fluorescence using an anti-tubulin antibody. Cells with a spindle were counted as mitotic.

Mentions: On their own, the deletions of swd2.2 (swd2.2Δ) and sen1 (sen1Δ) partly restored growth of cut3-477 cells at the restrictive temperature (Figure 1A) and reduced the proportion of anaphase cells displaying chromosome segregation defects (Figure 1B). Combining both deletions (sen1Δswd2.2Δ) resulted in a stronger suppressor effect (Figure 1AB). The double mutant sen1Δswd2.2Δ also suppressed the other condensin mutant cut14-208 (Figure S1). Strikingly, Chromatin Immunoprecipitation (ChIP) analysis in cycling cell populations showed that the localization of condensin was altered at specific loci when Swd2.2 and Sen1 were both missing: its recruitment increased significantly at genes transcribed by RNA Pol III (Gln.04, Met.07, Ser.13, Pro.09, Tyr.04, Gly.05, 5S rRNA, Arg.04 on Figure 1C), whereas it was significantly reduced at the rDNA arrays (18S&Rfb2). The binding of condensin remained unaffected at kinetochores (cnt1) or at highly transcribed Pol II genes (Act1, Adh1, Fba1 and SPAC27E2.11c). The sequences of all the primers used in this study are available on Table S1. The mitotic indexes of both cell populations (swd2.2+sen1+ and swd2.2Δsen1Δ) were comparable (Figure 1D), ruling out that the changes in the association of condensin are due to indirect, cell-cycle defects. These data established that Sen1 and Swd2.2 act to limit the localization of condensin at Pol III-transcribed genes. The reasons why the association of condensin at the rDNA arrays is reduced in the absence of Swd2.2 and Sen1 will be explained elsewhere.


RNA processing factors Swd2.2 and Sen1 antagonize RNA Pol III-dependent transcription and the localization of condensin at Pol III genes.

Legros P, Malapert A, Niinuma S, Bernard P, Vanoosthuyse V - PLoS Genet. (2014)

The double deletion of Swd2.2 and Sen1 facilitates the localization of condensin at Pol III-transcribed genes.A. Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures. B. Chromosome segregation in anaphase was monitored in the indicated strains after growing cells for one generation at 34°C. For each genotype, a minimum of 6 independent experiments was performed in which a minimum of 100 anaphase cells were scored (***<0.001; **<0.01 Wilcoxon - Mann Whitney). Anaphases were scored as defective when chromatin was detected lagging between the two main DNA masses C. ChIP-qPCR analysis of the amount of GFP-tagged Cut3 cross-linked to chromatin in cell populations of the indicated genotypes grown at 30°C (mean ± standard deviation from 6 biological replicates (NS: not significant, *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). The primers used in this study are shown on Table S1. D. Mitotic indexes of the cell populations used in C. Cells were fixed with cold methanol and processed for immuno-fluorescence using an anti-tubulin antibody. Cells with a spindle were counted as mitotic.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230746&req=5

pgen-1004794-g001: The double deletion of Swd2.2 and Sen1 facilitates the localization of condensin at Pol III-transcribed genes.A. Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures. B. Chromosome segregation in anaphase was monitored in the indicated strains after growing cells for one generation at 34°C. For each genotype, a minimum of 6 independent experiments was performed in which a minimum of 100 anaphase cells were scored (***<0.001; **<0.01 Wilcoxon - Mann Whitney). Anaphases were scored as defective when chromatin was detected lagging between the two main DNA masses C. ChIP-qPCR analysis of the amount of GFP-tagged Cut3 cross-linked to chromatin in cell populations of the indicated genotypes grown at 30°C (mean ± standard deviation from 6 biological replicates (NS: not significant, *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). The primers used in this study are shown on Table S1. D. Mitotic indexes of the cell populations used in C. Cells were fixed with cold methanol and processed for immuno-fluorescence using an anti-tubulin antibody. Cells with a spindle were counted as mitotic.
Mentions: On their own, the deletions of swd2.2 (swd2.2Δ) and sen1 (sen1Δ) partly restored growth of cut3-477 cells at the restrictive temperature (Figure 1A) and reduced the proportion of anaphase cells displaying chromosome segregation defects (Figure 1B). Combining both deletions (sen1Δswd2.2Δ) resulted in a stronger suppressor effect (Figure 1AB). The double mutant sen1Δswd2.2Δ also suppressed the other condensin mutant cut14-208 (Figure S1). Strikingly, Chromatin Immunoprecipitation (ChIP) analysis in cycling cell populations showed that the localization of condensin was altered at specific loci when Swd2.2 and Sen1 were both missing: its recruitment increased significantly at genes transcribed by RNA Pol III (Gln.04, Met.07, Ser.13, Pro.09, Tyr.04, Gly.05, 5S rRNA, Arg.04 on Figure 1C), whereas it was significantly reduced at the rDNA arrays (18S&Rfb2). The binding of condensin remained unaffected at kinetochores (cnt1) or at highly transcribed Pol II genes (Act1, Adh1, Fba1 and SPAC27E2.11c). The sequences of all the primers used in this study are available on Table S1. The mitotic indexes of both cell populations (swd2.2+sen1+ and swd2.2Δsen1Δ) were comparable (Figure 1D), ruling out that the changes in the association of condensin are due to indirect, cell-cycle defects. These data established that Sen1 and Swd2.2 act to limit the localization of condensin at Pol III-transcribed genes. The reasons why the association of condensin at the rDNA arrays is reduced in the absence of Swd2.2 and Sen1 will be explained elsewhere.

Bottom Line: Genetic studies have indicated that the association of condensin with chromatin is intimately linked to gene transcription, but what transcription-associated feature(s) direct(s) the accumulation of condensin remains unclear.We provide evidence that this topological stress contributes to recruit and/or stabilize condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1.Our data challenge the idea that a processive RNA polymerase hinders the binding of condensin and suggest that transcription-associated topological stress could in some circumstances facilitate the association of condensin.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Université Lyon 01, UMR5239, LBMC; Ecole Normale Supérieure de Lyon, Lyon, France.

ABSTRACT
Condensin-mediated chromosome condensation is essential for genome stability upon cell division. Genetic studies have indicated that the association of condensin with chromatin is intimately linked to gene transcription, but what transcription-associated feature(s) direct(s) the accumulation of condensin remains unclear. Here we show in fission yeast that condensin becomes strikingly enriched at RNA Pol III-transcribed genes when Swd2.2 and Sen1, two factors involved in the transcription process, are simultaneously deleted. Sen1 is an ATP-dependent helicase whose orthologue in Saccharomyces cerevisiae contributes both to terminate transcription of some RNA Pol II transcripts and to antagonize the formation of DNA:RNA hybrids in the genome. Using two independent mapping techniques, we show that DNA:RNA hybrids form in abundance at Pol III-transcribed genes in fission yeast but we demonstrate that they are unlikely to faciliate the recruitment of condensin. Instead, we show that Sen1 forms a stable and abundant complex with RNA Pol III and that Swd2.2 and Sen1 antagonize both the interaction of RNA Pol III with chromatin and RNA Pol III-dependent transcription. When Swd2.2 and Sen1 are lacking, the increased concentration of RNA Pol III and condensin at Pol III-transcribed genes is accompanied by the accumulation of topoisomerase I and II and by local nucleosome depletion, suggesting that Pol III-transcribed genes suffer topological stress. We provide evidence that this topological stress contributes to recruit and/or stabilize condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1. Our data challenge the idea that a processive RNA polymerase hinders the binding of condensin and suggest that transcription-associated topological stress could in some circumstances facilitate the association of condensin.

Show MeSH
Related in: MedlinePlus