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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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Lack of Top1 further increases the association of condensin with Pol III-transcribed genes when Swd2.2 and Sen1 are missing.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 of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). D. Model. Lack of Swd2.2 and Sen1 increases gene transcription at Pol III-transcribed genes. According to the twin supercoiled domain model, this results in more positive supercoils downstream of the polymerase and compensatory negative supercoils upstream of the polymerase. Negative supercoils favor the formation of R-Loops (reviewed in [41]). Positive supercoils result in nucleosome eviction. This topological stress also facilitates the recruitment of condensin, either directly or indirectly.
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pgen-1004794-g005: Lack of Top1 further increases the association of condensin with Pol III-transcribed genes when Swd2.2 and Sen1 are missing.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 of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). D. Model. Lack of Swd2.2 and Sen1 increases gene transcription at Pol III-transcribed genes. According to the twin supercoiled domain model, this results in more positive supercoils downstream of the polymerase and compensatory negative supercoils upstream of the polymerase. Negative supercoils favor the formation of R-Loops (reviewed in [41]). Positive supercoils result in nucleosome eviction. This topological stress also facilitates the recruitment of condensin, either directly or indirectly.

Mentions: Based on these results, we envisaged two possible models to explain the increased localization of condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1: either the accumulation of Top1 and/or Top2 helps to recruit and/or stabilize condensin, or topological stress facilitates the association of condensin at Pol III-transcribed genes. We previously identified the deletion of Top1 (top1Δ) as a suppressor of cut3-477[22], suggesting that the accumulation of Top1 that results from lack of Swd2.2 and Sen1 is unlikely to facilitate the association of condensin with chromatin. Figures 5A&B show that the triple deletion swd2.2Δsen1Δtop1Δ was a better suppressor of cut3-477 than the double deletion swd2.2Δsen1Δ. This genetic evidence suggested that failure to monitor topological stress in top1Δ cells might facilitate the association/function of condensin. In support of this, ChIP analysis showed that there was a small but significant increase in the association of condensin at most Pol III-transcribed genes in cells deleted for Swd2.2, Sen1 and Top1 (swd2.2Δsen1Δtop1Δ cells) (Figure 5C). Taken together, these data support the following model: the absence of Swd2.2 and Sen1 increases the transcriptional activity at Pol III-transcribed genes and this might contribute to enhance local topological constraints. These constraints, either directly or indirectly, contribute to recruit or maintain condensin at Pol III-transcribed genes (Figure 5D).


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)

Lack of Top1 further increases the association of condensin with Pol III-transcribed genes when Swd2.2 and Sen1 are missing.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 of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). D. Model. Lack of Swd2.2 and Sen1 increases gene transcription at Pol III-transcribed genes. According to the twin supercoiled domain model, this results in more positive supercoils downstream of the polymerase and compensatory negative supercoils upstream of the polymerase. Negative supercoils favor the formation of R-Loops (reviewed in [41]). Positive supercoils result in nucleosome eviction. This topological stress also facilitates the recruitment of condensin, either directly or indirectly.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004794-g005: Lack of Top1 further increases the association of condensin with Pol III-transcribed genes when Swd2.2 and Sen1 are missing.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 of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). D. Model. Lack of Swd2.2 and Sen1 increases gene transcription at Pol III-transcribed genes. According to the twin supercoiled domain model, this results in more positive supercoils downstream of the polymerase and compensatory negative supercoils upstream of the polymerase. Negative supercoils favor the formation of R-Loops (reviewed in [41]). Positive supercoils result in nucleosome eviction. This topological stress also facilitates the recruitment of condensin, either directly or indirectly.
Mentions: Based on these results, we envisaged two possible models to explain the increased localization of condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1: either the accumulation of Top1 and/or Top2 helps to recruit and/or stabilize condensin, or topological stress facilitates the association of condensin at Pol III-transcribed genes. We previously identified the deletion of Top1 (top1Δ) as a suppressor of cut3-477[22], suggesting that the accumulation of Top1 that results from lack of Swd2.2 and Sen1 is unlikely to facilitate the association of condensin with chromatin. Figures 5A&B show that the triple deletion swd2.2Δsen1Δtop1Δ was a better suppressor of cut3-477 than the double deletion swd2.2Δsen1Δ. This genetic evidence suggested that failure to monitor topological stress in top1Δ cells might facilitate the association/function of condensin. In support of this, ChIP analysis showed that there was a small but significant increase in the association of condensin at most Pol III-transcribed genes in cells deleted for Swd2.2, Sen1 and Top1 (swd2.2Δsen1Δtop1Δ cells) (Figure 5C). Taken together, these data support the following model: the absence of Swd2.2 and Sen1 increases the transcriptional activity at Pol III-transcribed genes and this might contribute to enhance local topological constraints. These constraints, either directly or indirectly, contribute to recruit or maintain condensin at Pol III-transcribed genes (Figure 5D).

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