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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 Swd2.2 and Sen1 results in local topological stress at Pol III-transcribed genes.A. 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). B. Western blot analysis of the stability of Top1-3flag. Tubulin is used as a loading control. 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. Western blot analysis of the stability of Top2-GFP. Tubulin is used as a loading control. E. ChIP qPCR of histone H3 in 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; Wilcoxon - Mann Whitney).
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pgen-1004794-g004: Lack of Swd2.2 and Sen1 results in local topological stress at Pol III-transcribed genes.A. 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). B. Western blot analysis of the stability of Top1-3flag. Tubulin is used as a loading control. 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. Western blot analysis of the stability of Top2-GFP. Tubulin is used as a loading control. E. ChIP qPCR of histone H3 in 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; Wilcoxon - Mann Whitney).

Mentions: Because Xenopus condensin shows greater affinity in vitro for positively supercoiled DNA [21], we speculated that the cue facilitating the accumulation of condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1 could be local topological constraints. Consistent with an increase in topological stress in swd2.2Δsen1Δ cells, ChIP analysis detected strong accumulation of topoisomerase I (Top1) at most loci (Figure 4A), although the protein levels of Top1 remained unaffected (Figure 4B). We also detected enhanced accumulation of topoisomerase II (Top2), mostly at Pol III-transcribed genes (Figure 4C), when the protein levels of Top2 remained unaffected (Figure 4D). Transcription-associated topological stress was recently shown to destabilize nucleosomes [19]. At some but not all Pol III-transcribed genes that we tested, we detected a significant reduction in the recruitment of histone H3 (Figure 4E) in swd2.2Δsen1Δ cells, which is consistent with the local depletion of nucleosomes. The concomitant accumulation of Top1 and Top2 and the depletion of nucleosomes suggest that topological stress is greater at Pol III-transcribed genes in swd2.2Δsen1Δ cells. We speculate that the increased transcription of Pol III-transcribed genes in swd2.2Δsen1Δ cells could contribute at least in part to this enhanced topological stress.


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 Swd2.2 and Sen1 results in local topological stress at Pol III-transcribed genes.A. 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). B. Western blot analysis of the stability of Top1-3flag. Tubulin is used as a loading control. 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. Western blot analysis of the stability of Top2-GFP. Tubulin is used as a loading control. E. ChIP qPCR of histone H3 in 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; Wilcoxon - Mann Whitney).
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pgen-1004794-g004: Lack of Swd2.2 and Sen1 results in local topological stress at Pol III-transcribed genes.A. 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). B. Western blot analysis of the stability of Top1-3flag. Tubulin is used as a loading control. 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. Western blot analysis of the stability of Top2-GFP. Tubulin is used as a loading control. E. ChIP qPCR of histone H3 in 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; Wilcoxon - Mann Whitney).
Mentions: Because Xenopus condensin shows greater affinity in vitro for positively supercoiled DNA [21], we speculated that the cue facilitating the accumulation of condensin at Pol III-transcribed genes in the absence of Swd2.2 and Sen1 could be local topological constraints. Consistent with an increase in topological stress in swd2.2Δsen1Δ cells, ChIP analysis detected strong accumulation of topoisomerase I (Top1) at most loci (Figure 4A), although the protein levels of Top1 remained unaffected (Figure 4B). We also detected enhanced accumulation of topoisomerase II (Top2), mostly at Pol III-transcribed genes (Figure 4C), when the protein levels of Top2 remained unaffected (Figure 4D). Transcription-associated topological stress was recently shown to destabilize nucleosomes [19]. At some but not all Pol III-transcribed genes that we tested, we detected a significant reduction in the recruitment of histone H3 (Figure 4E) in swd2.2Δsen1Δ cells, which is consistent with the local depletion of nucleosomes. The concomitant accumulation of Top1 and Top2 and the depletion of nucleosomes suggest that topological stress is greater at Pol III-transcribed genes in swd2.2Δsen1Δ cells. We speculate that the increased transcription of Pol III-transcribed genes in swd2.2Δsen1Δ cells could contribute at least in part to this enhanced topological stress.

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