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Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation.

Sutani T, Sakata T, Nakato R, Masuda K, Ishibashi M, Yamashita D, Suzuki Y, Hirano T, Bando M, Shirahige K - Nat Commun (2015)

Bottom Line: Pharmacological and genetic attenuation of transcription largely rescue bulk chromosome segregation defects observed in condensin mutants.We also demonstrate that condensin is associated with and reduces unwound DNA segments generated by transcription, providing a direct link between an in vitro activity of condensin and its in vivo function.The human condensin isoform condensin I also binds to unwound DNA regions at the transcription start sites of active genes, implying that our findings uncover a fundamental feature of condensin complexes.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan.

ABSTRACT
Chromosome condensation is a hallmark of mitosis in eukaryotes and is a prerequisite for faithful segregation of genetic material to daughter cells. Here we show that condensin, which is essential for assembling condensed chromosomes, helps to preclude the detrimental effects of gene transcription on mitotic condensation. ChIP-seq profiling reveals that the fission yeast condensin preferentially binds to active protein-coding genes in a transcription-dependent manner during mitosis. Pharmacological and genetic attenuation of transcription largely rescue bulk chromosome segregation defects observed in condensin mutants. We also demonstrate that condensin is associated with and reduces unwound DNA segments generated by transcription, providing a direct link between an in vitro activity of condensin and its in vivo function. The human condensin isoform condensin I also binds to unwound DNA regions at the transcription start sites of active genes, implying that our findings uncover a fundamental feature of condensin complexes.

No MeSH data available.


Related in: MedlinePlus

Presence of ssDNA at condensin binding sites.(a) Treatment of condensin-bound DNA fragments with nuclease P1, which is specific to ssDNA/single-stranded RNA. DNA fragments purified by Cut14-PK ChIP from prometaphase cells were treated with P1 on beads and then eluted and measured by qPCR (left). P1 sensitivity was specific to condensin-bound fragments, because bulk DNA at the same sites (purified by anti-histone H3 ChIP from prometaphase cells) or cohesin-associated DNA (purified by Rad21-GFP ChIP from asynchronous cells) showed no sensitivity (middle and right, respectively). (b) RNase treatment of condensin-bound DNA fragments. RNase A or RNase H treatment, which digests single-stranded RNA or RNA within DNA:RNA hybrids, respectively, caused no reduction in qPCR measurements, precluding the possibility that the condensin-DNA association is mediated by RNA. Error bars represent s.d. (n=2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.
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f4: Presence of ssDNA at condensin binding sites.(a) Treatment of condensin-bound DNA fragments with nuclease P1, which is specific to ssDNA/single-stranded RNA. DNA fragments purified by Cut14-PK ChIP from prometaphase cells were treated with P1 on beads and then eluted and measured by qPCR (left). P1 sensitivity was specific to condensin-bound fragments, because bulk DNA at the same sites (purified by anti-histone H3 ChIP from prometaphase cells) or cohesin-associated DNA (purified by Rad21-GFP ChIP from asynchronous cells) showed no sensitivity (middle and right, respectively). (b) RNase treatment of condensin-bound DNA fragments. RNase A or RNase H treatment, which digests single-stranded RNA or RNA within DNA:RNA hybrids, respectively, caused no reduction in qPCR measurements, precluding the possibility that the condensin-DNA association is mediated by RNA. Error bars represent s.d. (n=2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.

Mentions: The Cut3-Cut14 heterodimer, the core component of the condensin complex, promotes renaturation of complementary single-stranded DNA (ssDNA) fragments and has a higher affinity for ssDNA than double-stranded DNA in vitro3536. Because transcription is accompanied by DNA unwinding37, we speculated that unwound DNA segments in transcribed genes might be targeted and restored by condensin. We tested this idea first by examining whether condensin-bound DNA contains single-stranded regions. DNA associated with condensin was isolated by ChIP, treated on the immunoprecipitation beads with nuclease P1 (which digests ssDNA or single-stranded RNA) and then eluted from the beads. qPCR quantification revealed that the majority of condensin-bound DNA is sensitive to P1 (Fig. 4a). This sensitivity to nuclease P1 was specific to condensin-bound DNA, because DNA associated with the related complex cohesin410 (purified by Rad21-GFP ChIP) showed no sensitivity to nuclease P1 (Fig. 4a). Bulk nucleosomal DNA purified by histone H3 ChIP also showed no P1 sensitivity at condensin binding sites (Fig. 4a), revealing that condensin binding was directly linked to P1-sensitive structures. Finally, treatment with RNases instead of nuclease P1 produced no reduction in the amount of recovered DNA (Fig. 4b), suggesting that P1 sensitivity was not due to digestion of RNA. Taken together, these results indicate that condensin-bound DNA contains ssDNA.


Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation.

Sutani T, Sakata T, Nakato R, Masuda K, Ishibashi M, Yamashita D, Suzuki Y, Hirano T, Bando M, Shirahige K - Nat Commun (2015)

Presence of ssDNA at condensin binding sites.(a) Treatment of condensin-bound DNA fragments with nuclease P1, which is specific to ssDNA/single-stranded RNA. DNA fragments purified by Cut14-PK ChIP from prometaphase cells were treated with P1 on beads and then eluted and measured by qPCR (left). P1 sensitivity was specific to condensin-bound fragments, because bulk DNA at the same sites (purified by anti-histone H3 ChIP from prometaphase cells) or cohesin-associated DNA (purified by Rad21-GFP ChIP from asynchronous cells) showed no sensitivity (middle and right, respectively). (b) RNase treatment of condensin-bound DNA fragments. RNase A or RNase H treatment, which digests single-stranded RNA or RNA within DNA:RNA hybrids, respectively, caused no reduction in qPCR measurements, precluding the possibility that the condensin-DNA association is mediated by RNA. Error bars represent s.d. (n=2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525155&req=5

f4: Presence of ssDNA at condensin binding sites.(a) Treatment of condensin-bound DNA fragments with nuclease P1, which is specific to ssDNA/single-stranded RNA. DNA fragments purified by Cut14-PK ChIP from prometaphase cells were treated with P1 on beads and then eluted and measured by qPCR (left). P1 sensitivity was specific to condensin-bound fragments, because bulk DNA at the same sites (purified by anti-histone H3 ChIP from prometaphase cells) or cohesin-associated DNA (purified by Rad21-GFP ChIP from asynchronous cells) showed no sensitivity (middle and right, respectively). (b) RNase treatment of condensin-bound DNA fragments. RNase A or RNase H treatment, which digests single-stranded RNA or RNA within DNA:RNA hybrids, respectively, caused no reduction in qPCR measurements, precluding the possibility that the condensin-DNA association is mediated by RNA. Error bars represent s.d. (n=2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.
Mentions: The Cut3-Cut14 heterodimer, the core component of the condensin complex, promotes renaturation of complementary single-stranded DNA (ssDNA) fragments and has a higher affinity for ssDNA than double-stranded DNA in vitro3536. Because transcription is accompanied by DNA unwinding37, we speculated that unwound DNA segments in transcribed genes might be targeted and restored by condensin. We tested this idea first by examining whether condensin-bound DNA contains single-stranded regions. DNA associated with condensin was isolated by ChIP, treated on the immunoprecipitation beads with nuclease P1 (which digests ssDNA or single-stranded RNA) and then eluted from the beads. qPCR quantification revealed that the majority of condensin-bound DNA is sensitive to P1 (Fig. 4a). This sensitivity to nuclease P1 was specific to condensin-bound DNA, because DNA associated with the related complex cohesin410 (purified by Rad21-GFP ChIP) showed no sensitivity to nuclease P1 (Fig. 4a). Bulk nucleosomal DNA purified by histone H3 ChIP also showed no P1 sensitivity at condensin binding sites (Fig. 4a), revealing that condensin binding was directly linked to P1-sensitive structures. Finally, treatment with RNases instead of nuclease P1 produced no reduction in the amount of recovered DNA (Fig. 4b), suggesting that P1 sensitivity was not due to digestion of RNA. Taken together, these results indicate that condensin-bound DNA contains ssDNA.

Bottom Line: Pharmacological and genetic attenuation of transcription largely rescue bulk chromosome segregation defects observed in condensin mutants.We also demonstrate that condensin is associated with and reduces unwound DNA segments generated by transcription, providing a direct link between an in vitro activity of condensin and its in vivo function.The human condensin isoform condensin I also binds to unwound DNA regions at the transcription start sites of active genes, implying that our findings uncover a fundamental feature of condensin complexes.

View Article: PubMed Central - PubMed

Affiliation: Research Center for Epigenetic Disease, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo 113-0032, Japan.

ABSTRACT
Chromosome condensation is a hallmark of mitosis in eukaryotes and is a prerequisite for faithful segregation of genetic material to daughter cells. Here we show that condensin, which is essential for assembling condensed chromosomes, helps to preclude the detrimental effects of gene transcription on mitotic condensation. ChIP-seq profiling reveals that the fission yeast condensin preferentially binds to active protein-coding genes in a transcription-dependent manner during mitosis. Pharmacological and genetic attenuation of transcription largely rescue bulk chromosome segregation defects observed in condensin mutants. We also demonstrate that condensin is associated with and reduces unwound DNA segments generated by transcription, providing a direct link between an in vitro activity of condensin and its in vivo function. The human condensin isoform condensin I also binds to unwound DNA regions at the transcription start sites of active genes, implying that our findings uncover a fundamental feature of condensin complexes.

No MeSH data available.


Related in: MedlinePlus