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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

Binding of human condensin I complex to the TSS of active genes transcribed by RNAP2 and RNAP3.(a,b) ChIP-seq profiles of condensin I (using an antibody against NCAPG), RNAP2 (using 8WG16 monoclonal antibody, a) and RNAP3 (using monoclonal antibody against RPC32 subunit, b) in HeLa cells under the indicated conditions. M, cells arrested in prometaphase by nocodazole; Asy, asynchronous cells. Where indicated, the NCAPG subunit was depleted by siRNA treatment (Supplementary Fig. 8a). The upper boxes show the positions of protein-coding genes, and the lower boxes in b indicate the positions of tRNA genes (those within ORFs are shown in purple; others are shown in orange). The four sets of graphs in a and b show ChIP-seq profiles. The y axes of the ChIP-seq profiles indicate the normalized read intensity. Red indicates regions where enrichment determined by ChIP was statistically significant. (c) Distance of condensin I peaks from TSSs. More than 70% of NCAPG-binding sites are localized within ±5 kb of a TSS. (d) Level of condensin I binding during mitosis correlates with level of transcription during interphase. All RefSeq genes were ranked and divided into four equal groups based on their expression levels (high to low) in asynchronous cells, and averaged condensin I binding profiles around the TSS for each group were calculated. (e) Averaged mitotic NCAPG ChIP-seq profiles around tRNA genes. The profile for tRNA genes associated with RNAP3 in asynchronous cells is shown in purple.
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f6: Binding of human condensin I complex to the TSS of active genes transcribed by RNAP2 and RNAP3.(a,b) ChIP-seq profiles of condensin I (using an antibody against NCAPG), RNAP2 (using 8WG16 monoclonal antibody, a) and RNAP3 (using monoclonal antibody against RPC32 subunit, b) in HeLa cells under the indicated conditions. M, cells arrested in prometaphase by nocodazole; Asy, asynchronous cells. Where indicated, the NCAPG subunit was depleted by siRNA treatment (Supplementary Fig. 8a). The upper boxes show the positions of protein-coding genes, and the lower boxes in b indicate the positions of tRNA genes (those within ORFs are shown in purple; others are shown in orange). The four sets of graphs in a and b show ChIP-seq profiles. The y axes of the ChIP-seq profiles indicate the normalized read intensity. Red indicates regions where enrichment determined by ChIP was statistically significant. (c) Distance of condensin I peaks from TSSs. More than 70% of NCAPG-binding sites are localized within ±5 kb of a TSS. (d) Level of condensin I binding during mitosis correlates with level of transcription during interphase. All RefSeq genes were ranked and divided into four equal groups based on their expression levels (high to low) in asynchronous cells, and averaged condensin I binding profiles around the TSS for each group were calculated. (e) Averaged mitotic NCAPG ChIP-seq profiles around tRNA genes. The profile for tRNA genes associated with RNAP3 in asynchronous cells is shown in purple.

Mentions: Our findings in fission yeast prompted us to investigate the chromosomal localization of condensin in human cells. Most metazoan species possess two types of condensin complex, condensin I and II, with condensin I being more closely related to condensin in yeast9. Moreover, unlike condensin II and similar to fission yeast condensin, condensin I is associated with chromatin only during M phase2841. We thus focused on condensin I. We first determined its genome-wide binding profile in prometaphase HeLa cells by ChIP-seq2223 using an antibody against NCAPG (also known as CAP-G), a subunit specific to condensin I (ref. 42). The chromosome-wide distribution of NCAPG indicates that condensin I was enriched around the centromeres (Supplementary Fig. 7a), as reported in refs 15, 41. In addition, the condensin I profile showed a total of 7,825 distinct binding peaks along the chromosome arms (Fig. 6a). Close inspection revealed that most peaks overlapped with the transcription start sites (TSSs) of a subset of genes. More than 70% of detected peaks were located within ±5 kb of the TSS of an RNAP2-driven gene (Fig. 6c and Supplementary Fig. 7b). It is noteworthy that such TSS-proximal regions account for only 9.1% of the genome. The obtained condensin I binding profile was successfully validated by ChIP-qPCR. This binding was (i) more prominent at detected peaks than at sites distant from the peaks; (ii) more noticeable in mitotic cells than in cells at G1, which is consistent with the mitosis-specific association of condensin I with chromatin41; and (iii) diminished in NCAPG-depleted cells (Supplementary Fig. 8a–c), thereby verifying the specificity of the antibody used. Accumulation of condensin I at TSSs has also been reported recently in chicken DT40 cells15. In human cells, RNAP2 is enriched at the TSS of many genes because of the so-called RNAP2 promoter proximal pausing43. We found that condensin I peaks in prometaphase cells were associated with RNAP2 peaks in asynchronous cells, and the degree of condensin I binding at these peaks correlated nicely with the expression level of the corresponding genes in interphase cells (Fig. 6a,d). Therefore, as in fission yeast, human condensin I is localized at actively transcribed parts of the genome. Mitotic transcription is, however, repressed more strongly in human cells than in fission yeast44, as confirmed by RNAP2 ChIP-seq and RT–qPCR analysis in mitotic cells (Fig. 6a and Supplementary Fig. 8d). Moreover, transcription inhibitors caused no dissociation of condensin I from human mitotic chromosomes (Supplementary Fig. 8e). In addition, mitotic depletion of the TATA-binding protein (TBP), an essential component of the pre-initiation complex that is required for transcription initiation32, did not alter condensin I binding to TSSs (Supplementary Fig. 8f,g). Therefore, the binding of human condensin I at TSS regions is likely to be dependent on the chromatin structure or modifications specific to active genes, rather than transcription per se (see below).


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)

Binding of human condensin I complex to the TSS of active genes transcribed by RNAP2 and RNAP3.(a,b) ChIP-seq profiles of condensin I (using an antibody against NCAPG), RNAP2 (using 8WG16 monoclonal antibody, a) and RNAP3 (using monoclonal antibody against RPC32 subunit, b) in HeLa cells under the indicated conditions. M, cells arrested in prometaphase by nocodazole; Asy, asynchronous cells. Where indicated, the NCAPG subunit was depleted by siRNA treatment (Supplementary Fig. 8a). The upper boxes show the positions of protein-coding genes, and the lower boxes in b indicate the positions of tRNA genes (those within ORFs are shown in purple; others are shown in orange). The four sets of graphs in a and b show ChIP-seq profiles. The y axes of the ChIP-seq profiles indicate the normalized read intensity. Red indicates regions where enrichment determined by ChIP was statistically significant. (c) Distance of condensin I peaks from TSSs. More than 70% of NCAPG-binding sites are localized within ±5 kb of a TSS. (d) Level of condensin I binding during mitosis correlates with level of transcription during interphase. All RefSeq genes were ranked and divided into four equal groups based on their expression levels (high to low) in asynchronous cells, and averaged condensin I binding profiles around the TSS for each group were calculated. (e) Averaged mitotic NCAPG ChIP-seq profiles around tRNA genes. The profile for tRNA genes associated with RNAP3 in asynchronous cells is shown in purple.
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Related In: Results  -  Collection

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f6: Binding of human condensin I complex to the TSS of active genes transcribed by RNAP2 and RNAP3.(a,b) ChIP-seq profiles of condensin I (using an antibody against NCAPG), RNAP2 (using 8WG16 monoclonal antibody, a) and RNAP3 (using monoclonal antibody against RPC32 subunit, b) in HeLa cells under the indicated conditions. M, cells arrested in prometaphase by nocodazole; Asy, asynchronous cells. Where indicated, the NCAPG subunit was depleted by siRNA treatment (Supplementary Fig. 8a). The upper boxes show the positions of protein-coding genes, and the lower boxes in b indicate the positions of tRNA genes (those within ORFs are shown in purple; others are shown in orange). The four sets of graphs in a and b show ChIP-seq profiles. The y axes of the ChIP-seq profiles indicate the normalized read intensity. Red indicates regions where enrichment determined by ChIP was statistically significant. (c) Distance of condensin I peaks from TSSs. More than 70% of NCAPG-binding sites are localized within ±5 kb of a TSS. (d) Level of condensin I binding during mitosis correlates with level of transcription during interphase. All RefSeq genes were ranked and divided into four equal groups based on their expression levels (high to low) in asynchronous cells, and averaged condensin I binding profiles around the TSS for each group were calculated. (e) Averaged mitotic NCAPG ChIP-seq profiles around tRNA genes. The profile for tRNA genes associated with RNAP3 in asynchronous cells is shown in purple.
Mentions: Our findings in fission yeast prompted us to investigate the chromosomal localization of condensin in human cells. Most metazoan species possess two types of condensin complex, condensin I and II, with condensin I being more closely related to condensin in yeast9. Moreover, unlike condensin II and similar to fission yeast condensin, condensin I is associated with chromatin only during M phase2841. We thus focused on condensin I. We first determined its genome-wide binding profile in prometaphase HeLa cells by ChIP-seq2223 using an antibody against NCAPG (also known as CAP-G), a subunit specific to condensin I (ref. 42). The chromosome-wide distribution of NCAPG indicates that condensin I was enriched around the centromeres (Supplementary Fig. 7a), as reported in refs 15, 41. In addition, the condensin I profile showed a total of 7,825 distinct binding peaks along the chromosome arms (Fig. 6a). Close inspection revealed that most peaks overlapped with the transcription start sites (TSSs) of a subset of genes. More than 70% of detected peaks were located within ±5 kb of the TSS of an RNAP2-driven gene (Fig. 6c and Supplementary Fig. 7b). It is noteworthy that such TSS-proximal regions account for only 9.1% of the genome. The obtained condensin I binding profile was successfully validated by ChIP-qPCR. This binding was (i) more prominent at detected peaks than at sites distant from the peaks; (ii) more noticeable in mitotic cells than in cells at G1, which is consistent with the mitosis-specific association of condensin I with chromatin41; and (iii) diminished in NCAPG-depleted cells (Supplementary Fig. 8a–c), thereby verifying the specificity of the antibody used. Accumulation of condensin I at TSSs has also been reported recently in chicken DT40 cells15. In human cells, RNAP2 is enriched at the TSS of many genes because of the so-called RNAP2 promoter proximal pausing43. We found that condensin I peaks in prometaphase cells were associated with RNAP2 peaks in asynchronous cells, and the degree of condensin I binding at these peaks correlated nicely with the expression level of the corresponding genes in interphase cells (Fig. 6a,d). Therefore, as in fission yeast, human condensin I is localized at actively transcribed parts of the genome. Mitotic transcription is, however, repressed more strongly in human cells than in fission yeast44, as confirmed by RNAP2 ChIP-seq and RT–qPCR analysis in mitotic cells (Fig. 6a and Supplementary Fig. 8d). Moreover, transcription inhibitors caused no dissociation of condensin I from human mitotic chromosomes (Supplementary Fig. 8e). In addition, mitotic depletion of the TATA-binding protein (TBP), an essential component of the pre-initiation complex that is required for transcription initiation32, did not alter condensin I binding to TSSs (Supplementary Fig. 8f,g). Therefore, the binding of human condensin I at TSS regions is likely to be dependent on the chromatin structure or modifications specific to active genes, rather than transcription per se (see below).

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