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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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R-Loops form in abundance at Pol III-transcribed genes but they do not significantly impact the association of condensin.A. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). B. As in A. Cells were grown in minimal medium for a minimum of 18 hours to promote the over-expression of RnhA driven by the nmt promoter. C. Genomic DNA was extracted from rnh1+rnh201+ and rnh1Δrnh201Δ cells in preparation for the DRIP procedure. Equal amount of genomic DNA were spotted on a nylon membrane and incubated with 2 µg/mL of purified S9.6 antibody. The amount of S9.6 bound to the DNA was revealed using chemiluminescence. D. DRIP-qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). E. Cells of the indicated genotypes were grown in minimum medium lacking thiamine for a minimum of 18 hours to drive the over-expression of RnhA. ChIP-qPCR was then performed (mean ± standard deviation from 3 biological replicates).
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pgen-1004794-g003: R-Loops form in abundance at Pol III-transcribed genes but they do not significantly impact the association of condensin.A. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). B. As in A. Cells were grown in minimal medium for a minimum of 18 hours to promote the over-expression of RnhA driven by the nmt promoter. C. Genomic DNA was extracted from rnh1+rnh201+ and rnh1Δrnh201Δ cells in preparation for the DRIP procedure. Equal amount of genomic DNA were spotted on a nylon membrane and incubated with 2 µg/mL of purified S9.6 antibody. The amount of S9.6 bound to the DNA was revealed using chemiluminescence. D. DRIP-qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). E. Cells of the indicated genotypes were grown in minimum medium lacking thiamine for a minimum of 18 hours to drive the over-expression of RnhA. ChIP-qPCR was then performed (mean ± standard deviation from 3 biological replicates).

Mentions: To establish whether or not R-Loops form at Pol III-transcribed genes in fission yeast, we first monitored by ChIP the chromatin association of RNase H1, one of the endogenous enzymes known to disassemble R-Loops. More specifically, we introduced at the endogenous locus a point mutation (D129N) in the fission yeast RNase H1 (Rnh1), because the same mutation was shown to weaken the catalytic activity of human RNase H1 [29]. Consistent with this, the D129N mutation did stabilize the interaction of Rnh1 with Pol III-transcribed genes (Figure 3A). Furthermore, the interaction of Rnh1D129N with Pol III-transcribed genes was lost upon over-expression in vivo of RnhA, the RNase H1 enzyme from E.coli (Figure 3B). Upon over-expression, RnhA itself did not stably associate with Pol III-transcribed genes (Figure S4), showing that the loss of Rnh1D129N from Pol III-transcribed genes upon over-expression of RnhA cannot be explained by its mere replacement by bacterial RnhA. Finally, Figure S5 shows that the association of Rnh1D129N with the rDNA repeats increased significantly in the absence of topoisomerase I (top1Δ), consistent with the observations reported previously that lack of Top1 triggers the accumulation of R-Loops at rDNA in budding yeast [30]. This confirmed that Rnh1D129N was able to detect significant changes in R-Loop accumulation. Taken together, these data show that ChIP with Rnh1D129N is a reliable way to identify R-Loop forming regions in fission yeast.


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)

R-Loops form in abundance at Pol III-transcribed genes but they do not significantly impact the association of condensin.A. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). B. As in A. Cells were grown in minimal medium for a minimum of 18 hours to promote the over-expression of RnhA driven by the nmt promoter. C. Genomic DNA was extracted from rnh1+rnh201+ and rnh1Δrnh201Δ cells in preparation for the DRIP procedure. Equal amount of genomic DNA were spotted on a nylon membrane and incubated with 2 µg/mL of purified S9.6 antibody. The amount of S9.6 bound to the DNA was revealed using chemiluminescence. D. DRIP-qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). E. Cells of the indicated genotypes were grown in minimum medium lacking thiamine for a minimum of 18 hours to drive the over-expression of RnhA. ChIP-qPCR was then performed (mean ± standard deviation from 3 biological replicates).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004794-g003: R-Loops form in abundance at Pol III-transcribed genes but they do not significantly impact the association of condensin.A. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). B. As in A. Cells were grown in minimal medium for a minimum of 18 hours to promote the over-expression of RnhA driven by the nmt promoter. C. Genomic DNA was extracted from rnh1+rnh201+ and rnh1Δrnh201Δ cells in preparation for the DRIP procedure. Equal amount of genomic DNA were spotted on a nylon membrane and incubated with 2 µg/mL of purified S9.6 antibody. The amount of S9.6 bound to the DNA was revealed using chemiluminescence. D. DRIP-qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). E. Cells of the indicated genotypes were grown in minimum medium lacking thiamine for a minimum of 18 hours to drive the over-expression of RnhA. ChIP-qPCR was then performed (mean ± standard deviation from 3 biological replicates).
Mentions: To establish whether or not R-Loops form at Pol III-transcribed genes in fission yeast, we first monitored by ChIP the chromatin association of RNase H1, one of the endogenous enzymes known to disassemble R-Loops. More specifically, we introduced at the endogenous locus a point mutation (D129N) in the fission yeast RNase H1 (Rnh1), because the same mutation was shown to weaken the catalytic activity of human RNase H1 [29]. Consistent with this, the D129N mutation did stabilize the interaction of Rnh1 with Pol III-transcribed genes (Figure 3A). Furthermore, the interaction of Rnh1D129N with Pol III-transcribed genes was lost upon over-expression in vivo of RnhA, the RNase H1 enzyme from E.coli (Figure 3B). Upon over-expression, RnhA itself did not stably associate with Pol III-transcribed genes (Figure S4), showing that the loss of Rnh1D129N from Pol III-transcribed genes upon over-expression of RnhA cannot be explained by its mere replacement by bacterial RnhA. Finally, Figure S5 shows that the association of Rnh1D129N with the rDNA repeats increased significantly in the absence of topoisomerase I (top1Δ), consistent with the observations reported previously that lack of Top1 triggers the accumulation of R-Loops at rDNA in budding yeast [30]. This confirmed that Rnh1D129N was able to detect significant changes in R-Loop accumulation. Taken together, these data show that ChIP with Rnh1D129N is a reliable way to identify R-Loop forming regions in fission yeast.

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