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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

Transcription-dependent condensin binding at RNAP2-driven active genes.(a) ChIP-seq profiles of condensin (Cut14-PK), RNAP2 (detected by monoclonal antibody 8WG16, recognizing the largest subunit Rpb1) and all RNA polymerases (RNAPs, detected using the PK epitope on the common subunit Rpb5), as well as a poly(A)-selected RNA-seq profile. All profiles are from prometaphase cells. Annotated ORFs (cyan) and other transcripts (non-coding RNA and tRNA, magenta) are shown at the bottom. chr I, chromosome I. (b) Genome-wide correlation plot of Cut14-PK and RNAP2 ChIP-seq results. Purple, green and blue correspond to centromeres (cen), rDNA and tRNA gene loci, respectively. (c) Transcription-dependent condensin binding. Cut14-PK cells in prometaphase were treated with the transcription inhibitor 1,10-phenanthroline (Ph; 60 or 120 μg ml−1) or thiolutin (Th; 20 μg ml−1) for 30 min and analysed by anti-PK or anti-RNAP2 ChIP-qPCR. The translation inhibitor cycloheximide (CHX; 100 μg ml-1) was used as a negative control. Error bars represent s.d. (n=2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.
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f2: Transcription-dependent condensin binding at RNAP2-driven active genes.(a) ChIP-seq profiles of condensin (Cut14-PK), RNAP2 (detected by monoclonal antibody 8WG16, recognizing the largest subunit Rpb1) and all RNA polymerases (RNAPs, detected using the PK epitope on the common subunit Rpb5), as well as a poly(A)-selected RNA-seq profile. All profiles are from prometaphase cells. Annotated ORFs (cyan) and other transcripts (non-coding RNA and tRNA, magenta) are shown at the bottom. chr I, chromosome I. (b) Genome-wide correlation plot of Cut14-PK and RNAP2 ChIP-seq results. Purple, green and blue correspond to centromeres (cen), rDNA and tRNA gene loci, respectively. (c) Transcription-dependent condensin binding. Cut14-PK cells in prometaphase were treated with the transcription inhibitor 1,10-phenanthroline (Ph; 60 or 120 μg ml−1) or thiolutin (Th; 20 μg ml−1) for 30 min and analysed by anti-PK or anti-RNAP2 ChIP-qPCR. The translation inhibitor cycloheximide (CHX; 100 μg ml-1) was used as a negative control. Error bars represent s.d. (n=2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.

Mentions: ChIP-seq profiles indicated that condensin was enriched on a subset of genes, with notable accumulation around transcription termination sites (TTSs; Fig. 1d). Genes that show expression peaks in the M–G1 phases20 were significantly enriched for condensin binding (for example, ecm33+, eng1+cdc22+; P=3.3 × 10–11; Fisher's exact test), indicating that condensin was localized on mitotically transcribed genes. Indeed, ChIP-seq analysis of RNAP2 revealed that condensin was perfectly co-localized with RNAP2 in mitotic cells (Fig. 2a). With the exception of centromere and rDNA regions, the co-localization was verified in a genome-wide correlation plot (Fig. 2b). Moreover, the RNAP2 binding detected by ChIP-seq reflected actively transcribing polymerase, because RNA-seq analysis revealed the presence of mRNAs of the corresponding genes (Fig. 2a). Reverse transcription–qPCR (RT–qPCR) and immunostaining for active RNAP2 also confirmed the presence of active transcription in fission yeast mitotic cells (Supplementary Fig. 2a,b). Consistent with previous work using the ChIP-chip method19, the current analysis also detected moderate accumulation of condensin at many tRNA genes (Fig. 2b). The current ChIP-seq analysis therefore successfully extended the previous study, providing condensin distribution profiles with increased sensitivity and accuracy.


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)

Transcription-dependent condensin binding at RNAP2-driven active genes.(a) ChIP-seq profiles of condensin (Cut14-PK), RNAP2 (detected by monoclonal antibody 8WG16, recognizing the largest subunit Rpb1) and all RNA polymerases (RNAPs, detected using the PK epitope on the common subunit Rpb5), as well as a poly(A)-selected RNA-seq profile. All profiles are from prometaphase cells. Annotated ORFs (cyan) and other transcripts (non-coding RNA and tRNA, magenta) are shown at the bottom. chr I, chromosome I. (b) Genome-wide correlation plot of Cut14-PK and RNAP2 ChIP-seq results. Purple, green and blue correspond to centromeres (cen), rDNA and tRNA gene loci, respectively. (c) Transcription-dependent condensin binding. Cut14-PK cells in prometaphase were treated with the transcription inhibitor 1,10-phenanthroline (Ph; 60 or 120 μg ml−1) or thiolutin (Th; 20 μg ml−1) for 30 min and analysed by anti-PK or anti-RNAP2 ChIP-qPCR. The translation inhibitor cycloheximide (CHX; 100 μg ml-1) was used as a negative control. 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

f2: Transcription-dependent condensin binding at RNAP2-driven active genes.(a) ChIP-seq profiles of condensin (Cut14-PK), RNAP2 (detected by monoclonal antibody 8WG16, recognizing the largest subunit Rpb1) and all RNA polymerases (RNAPs, detected using the PK epitope on the common subunit Rpb5), as well as a poly(A)-selected RNA-seq profile. All profiles are from prometaphase cells. Annotated ORFs (cyan) and other transcripts (non-coding RNA and tRNA, magenta) are shown at the bottom. chr I, chromosome I. (b) Genome-wide correlation plot of Cut14-PK and RNAP2 ChIP-seq results. Purple, green and blue correspond to centromeres (cen), rDNA and tRNA gene loci, respectively. (c) Transcription-dependent condensin binding. Cut14-PK cells in prometaphase were treated with the transcription inhibitor 1,10-phenanthroline (Ph; 60 or 120 μg ml−1) or thiolutin (Th; 20 μg ml−1) for 30 min and analysed by anti-PK or anti-RNAP2 ChIP-qPCR. The translation inhibitor cycloheximide (CHX; 100 μg ml-1) was used as a negative control. Error bars represent s.d. (n=2, technical replicates in qPCR). cnt, central core regions of centromeres 1 and 3.
Mentions: ChIP-seq profiles indicated that condensin was enriched on a subset of genes, with notable accumulation around transcription termination sites (TTSs; Fig. 1d). Genes that show expression peaks in the M–G1 phases20 were significantly enriched for condensin binding (for example, ecm33+, eng1+cdc22+; P=3.3 × 10–11; Fisher's exact test), indicating that condensin was localized on mitotically transcribed genes. Indeed, ChIP-seq analysis of RNAP2 revealed that condensin was perfectly co-localized with RNAP2 in mitotic cells (Fig. 2a). With the exception of centromere and rDNA regions, the co-localization was verified in a genome-wide correlation plot (Fig. 2b). Moreover, the RNAP2 binding detected by ChIP-seq reflected actively transcribing polymerase, because RNA-seq analysis revealed the presence of mRNAs of the corresponding genes (Fig. 2a). Reverse transcription–qPCR (RT–qPCR) and immunostaining for active RNAP2 also confirmed the presence of active transcription in fission yeast mitotic cells (Supplementary Fig. 2a,b). Consistent with previous work using the ChIP-chip method19, the current analysis also detected moderate accumulation of condensin at many tRNA genes (Fig. 2b). The current ChIP-seq analysis therefore successfully extended the previous study, providing condensin distribution profiles with increased sensitivity and accuracy.

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