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Stable morphology, but dynamic internal reorganisation, of interphase human chromosomes in living cells.

Müller I, Boyle S, Singer RH, Bickmore WA, Chubb JR - PLoS ONE (2010)

Bottom Line: This contrasted with the behaviour of specific loci on labelled chromosomes, which showed more progressive reorganisation, and revealed that "looping out" of chromatin from chromosome territories is a dynamic state.Chromosome structure showed tremendous resistance to inhibitors of transcription, histone deacetylation and chromatin remodelling.Together, these data indicate steric constraints determine structure, rather than innate chromosome architecture or function-driven anchoring, with interphase chromatin organisation governed primarily by opposition between needs for decondensation and the space available for this to happen.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, UK.

ABSTRACT
Despite the distinctive structure of mitotic chromosomes, it has not been possible to visualise individual chromosomes in living interphase cells, where chromosomes spend over 90% of their time. Studies of interphase chromosome structure and dynamics use fluorescence in-situ hybridisation (FISH) on fixed cells, which potentially damages structure and loses dynamic information. We have developed a new methodology, involving photoactivation of labelled histone H3 at mitosis, to visualise individual and specific human chromosomes in living interphase cells. Our data revealed bulk chromosome volume and morphology are established rapidly after mitosis, changing only incrementally after the first hour of G1. This contrasted with the behaviour of specific loci on labelled chromosomes, which showed more progressive reorganisation, and revealed that "looping out" of chromatin from chromosome territories is a dynamic state. We measured considerable heterogeneity in chromosome decondensation, even between sister chromatids, which may reflect local structural impediments to decondensation and could potentially amplify transcriptional noise. Chromosome structure showed tremendous resistance to inhibitors of transcription, histone deacetylation and chromatin remodelling. Together, these data indicate steric constraints determine structure, rather than innate chromosome architecture or function-driven anchoring, with interphase chromatin organisation governed primarily by opposition between needs for decondensation and the space available for this to happen.

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Chromosome territory structure is resistant to inhibition of HDACs, ATP synthesis and Topoisomerase II.A,B) Example images of single chromosomes after TSA treatment. Projections of 3D stacks shown 1.5 h after mitosis (before treatment) and after 1 h incubation with 10 nM TSA. Bottom cell in A was marked during activation of the mitotic chromosome. Bar 12 µm. C) Western blots to detect histone acetylation levels before and after 1 h TSA treatment using antibodies against H3Ac. Loading checked using a GFP antibody to assess H3 PA-GFP. The same extracts were loaded for all blots, but run separately. D) Data from 13 daughter chromosomes were used to calculate changes in volume, surface area, sphericity and longest axis before (blue bar) and after 1 h of TSA (red bar). Values for chromosomes before TSA treatment were set to 100% and data for treated chromosomes calculated as a proportion of this. No changes in volume, surface area, sphericity and longest axis of the chromosomes were observed with either ATP depletion by azide and 2-deoxyglucose (n = 30) (E) or topoisomerase II inhibition with etoposide (n = 7) (F). For ATP depletion, images were also captured after removal of treament (yellow).
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pone-0011560-g005: Chromosome territory structure is resistant to inhibition of HDACs, ATP synthesis and Topoisomerase II.A,B) Example images of single chromosomes after TSA treatment. Projections of 3D stacks shown 1.5 h after mitosis (before treatment) and after 1 h incubation with 10 nM TSA. Bottom cell in A was marked during activation of the mitotic chromosome. Bar 12 µm. C) Western blots to detect histone acetylation levels before and after 1 h TSA treatment using antibodies against H3Ac. Loading checked using a GFP antibody to assess H3 PA-GFP. The same extracts were loaded for all blots, but run separately. D) Data from 13 daughter chromosomes were used to calculate changes in volume, surface area, sphericity and longest axis before (blue bar) and after 1 h of TSA (red bar). Values for chromosomes before TSA treatment were set to 100% and data for treated chromosomes calculated as a proportion of this. No changes in volume, surface area, sphericity and longest axis of the chromosomes were observed with either ATP depletion by azide and 2-deoxyglucose (n = 30) (E) or topoisomerase II inhibition with etoposide (n = 7) (F). For ATP depletion, images were also captured after removal of treament (yellow).

Mentions: To disrupt potential roles for inactive chromatin in the maintenance of chromosome architecture, we treated cells with the histone deacetylase inhibitor Trichostatin A (TSA). This caused an increase in general levels of histone H3 acetylation and H3K9 acetylation (Figure 5C). Hyperacetylation of histones has been suggested to result in chromatin opening [47]. However, TSA induced no significant changes in chromosome morphology in living cells (Figure 5A,D; p values >0.2). Both sister chromosomes from one mitosis appeared to decondense after TSA treatment (Figure 5B) although these changes were lost in population averages. Effects on specific chromosomes or chromosome segments cannot be excluded [48].


Stable morphology, but dynamic internal reorganisation, of interphase human chromosomes in living cells.

Müller I, Boyle S, Singer RH, Bickmore WA, Chubb JR - PLoS ONE (2010)

Chromosome territory structure is resistant to inhibition of HDACs, ATP synthesis and Topoisomerase II.A,B) Example images of single chromosomes after TSA treatment. Projections of 3D stacks shown 1.5 h after mitosis (before treatment) and after 1 h incubation with 10 nM TSA. Bottom cell in A was marked during activation of the mitotic chromosome. Bar 12 µm. C) Western blots to detect histone acetylation levels before and after 1 h TSA treatment using antibodies against H3Ac. Loading checked using a GFP antibody to assess H3 PA-GFP. The same extracts were loaded for all blots, but run separately. D) Data from 13 daughter chromosomes were used to calculate changes in volume, surface area, sphericity and longest axis before (blue bar) and after 1 h of TSA (red bar). Values for chromosomes before TSA treatment were set to 100% and data for treated chromosomes calculated as a proportion of this. No changes in volume, surface area, sphericity and longest axis of the chromosomes were observed with either ATP depletion by azide and 2-deoxyglucose (n = 30) (E) or topoisomerase II inhibition with etoposide (n = 7) (F). For ATP depletion, images were also captured after removal of treament (yellow).
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getmorefigures.php?uid=PMC2903487&req=5

pone-0011560-g005: Chromosome territory structure is resistant to inhibition of HDACs, ATP synthesis and Topoisomerase II.A,B) Example images of single chromosomes after TSA treatment. Projections of 3D stacks shown 1.5 h after mitosis (before treatment) and after 1 h incubation with 10 nM TSA. Bottom cell in A was marked during activation of the mitotic chromosome. Bar 12 µm. C) Western blots to detect histone acetylation levels before and after 1 h TSA treatment using antibodies against H3Ac. Loading checked using a GFP antibody to assess H3 PA-GFP. The same extracts were loaded for all blots, but run separately. D) Data from 13 daughter chromosomes were used to calculate changes in volume, surface area, sphericity and longest axis before (blue bar) and after 1 h of TSA (red bar). Values for chromosomes before TSA treatment were set to 100% and data for treated chromosomes calculated as a proportion of this. No changes in volume, surface area, sphericity and longest axis of the chromosomes were observed with either ATP depletion by azide and 2-deoxyglucose (n = 30) (E) or topoisomerase II inhibition with etoposide (n = 7) (F). For ATP depletion, images were also captured after removal of treament (yellow).
Mentions: To disrupt potential roles for inactive chromatin in the maintenance of chromosome architecture, we treated cells with the histone deacetylase inhibitor Trichostatin A (TSA). This caused an increase in general levels of histone H3 acetylation and H3K9 acetylation (Figure 5C). Hyperacetylation of histones has been suggested to result in chromatin opening [47]. However, TSA induced no significant changes in chromosome morphology in living cells (Figure 5A,D; p values >0.2). Both sister chromosomes from one mitosis appeared to decondense after TSA treatment (Figure 5B) although these changes were lost in population averages. Effects on specific chromosomes or chromosome segments cannot be excluded [48].

Bottom Line: This contrasted with the behaviour of specific loci on labelled chromosomes, which showed more progressive reorganisation, and revealed that "looping out" of chromatin from chromosome territories is a dynamic state.Chromosome structure showed tremendous resistance to inhibitors of transcription, histone deacetylation and chromatin remodelling.Together, these data indicate steric constraints determine structure, rather than innate chromosome architecture or function-driven anchoring, with interphase chromatin organisation governed primarily by opposition between needs for decondensation and the space available for this to happen.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell and Developmental Biology, College of Life Sciences, University of Dundee, Dundee, UK.

ABSTRACT
Despite the distinctive structure of mitotic chromosomes, it has not been possible to visualise individual chromosomes in living interphase cells, where chromosomes spend over 90% of their time. Studies of interphase chromosome structure and dynamics use fluorescence in-situ hybridisation (FISH) on fixed cells, which potentially damages structure and loses dynamic information. We have developed a new methodology, involving photoactivation of labelled histone H3 at mitosis, to visualise individual and specific human chromosomes in living interphase cells. Our data revealed bulk chromosome volume and morphology are established rapidly after mitosis, changing only incrementally after the first hour of G1. This contrasted with the behaviour of specific loci on labelled chromosomes, which showed more progressive reorganisation, and revealed that "looping out" of chromatin from chromosome territories is a dynamic state. We measured considerable heterogeneity in chromosome decondensation, even between sister chromatids, which may reflect local structural impediments to decondensation and could potentially amplify transcriptional noise. Chromosome structure showed tremendous resistance to inhibitors of transcription, histone deacetylation and chromatin remodelling. Together, these data indicate steric constraints determine structure, rather than innate chromosome architecture or function-driven anchoring, with interphase chromatin organisation governed primarily by opposition between needs for decondensation and the space available for this to happen.

Show MeSH
Related in: MedlinePlus