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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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Resistance of chromosome territory volume and morphology to transcription inhibition.A,B) Two examples of single chromosomes (green) in the interphase nucleus (red) before (1.5 h after cell division, left picture) and after treatment for 1 h with 1 µM actD (right picture). Bar 12 µm. C) Typical disruption of nucleolar components upon transcription inhibition by 1 h treatment with actD. Antibody staining (red) against 3 different nucleolar proteins (coilin, pKi67 and fibrillarin). Nuclei were stained using DAPI (blue). Bar 10 µm. D) Volume, surface area, sphericity and longest axis of single chromosomes (n = 14) before (blue bar) and after 1 h actD treatment (red bar). The values of the chromosomes before the treatment were set independently to 100% and data for treated chromosomes was calculated relative to these.
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pone-0011560-g004: Resistance of chromosome territory volume and morphology to transcription inhibition.A,B) Two examples of single chromosomes (green) in the interphase nucleus (red) before (1.5 h after cell division, left picture) and after treatment for 1 h with 1 µM actD (right picture). Bar 12 µm. C) Typical disruption of nucleolar components upon transcription inhibition by 1 h treatment with actD. Antibody staining (red) against 3 different nucleolar proteins (coilin, pKi67 and fibrillarin). Nuclei were stained using DAPI (blue). Bar 10 µm. D) Volume, surface area, sphericity and longest axis of single chromosomes (n = 14) before (blue bar) and after 1 h actD treatment (red bar). The values of the chromosomes before the treatment were set independently to 100% and data for treated chromosomes was calculated relative to these.

Mentions: To address to what extent transcription stabilises chromosome structure we used the transcriptional inhibitor actinomyocinD (actD) [45]. 1 µM actD strongly inhibited the ability of HT-1080 cells to transcribe, as assessed by cellular incorporation of the uridine analogue 5-fluorouridine into nascent RNA (Figure S1A). Effects of actD on nuclear structure were observed using immunofluorescence with nucleolar antibodies (Figure 4C) which showed typical disruption of nucleolar structure by drug treatment [46]. If transcription maintained chromosome structure, a transcription inhibitor would be expected to alter chromosome volume and morphology, leading to a more condensed state. The overall structure of the dispersed chromosome in Figure 4A did not change after being exposed to actD, even as the nucleolus diminished in size. A few chromosomes did show changes. The upper chromosome in Figure 4B initially showed a diffuse extension before retraction after inhibition of transcription. However, averaged over all the labelled chromosomes, inhibition of transcription led to no significant quantitative changes of volume, surface area, sphericity or longest axes of randomly labelled chromosomes (Figure 4D, p values all >0.35). Examples where transcription did appear to maintain chromosome structure may reflect particularly gene dense chromosomes or chromosome segments [36].


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)

Resistance of chromosome territory volume and morphology to transcription inhibition.A,B) Two examples of single chromosomes (green) in the interphase nucleus (red) before (1.5 h after cell division, left picture) and after treatment for 1 h with 1 µM actD (right picture). Bar 12 µm. C) Typical disruption of nucleolar components upon transcription inhibition by 1 h treatment with actD. Antibody staining (red) against 3 different nucleolar proteins (coilin, pKi67 and fibrillarin). Nuclei were stained using DAPI (blue). Bar 10 µm. D) Volume, surface area, sphericity and longest axis of single chromosomes (n = 14) before (blue bar) and after 1 h actD treatment (red bar). The values of the chromosomes before the treatment were set independently to 100% and data for treated chromosomes was calculated relative to these.
© Copyright Policy
Related In: Results  -  Collection

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pone-0011560-g004: Resistance of chromosome territory volume and morphology to transcription inhibition.A,B) Two examples of single chromosomes (green) in the interphase nucleus (red) before (1.5 h after cell division, left picture) and after treatment for 1 h with 1 µM actD (right picture). Bar 12 µm. C) Typical disruption of nucleolar components upon transcription inhibition by 1 h treatment with actD. Antibody staining (red) against 3 different nucleolar proteins (coilin, pKi67 and fibrillarin). Nuclei were stained using DAPI (blue). Bar 10 µm. D) Volume, surface area, sphericity and longest axis of single chromosomes (n = 14) before (blue bar) and after 1 h actD treatment (red bar). The values of the chromosomes before the treatment were set independently to 100% and data for treated chromosomes was calculated relative to these.
Mentions: To address to what extent transcription stabilises chromosome structure we used the transcriptional inhibitor actinomyocinD (actD) [45]. 1 µM actD strongly inhibited the ability of HT-1080 cells to transcribe, as assessed by cellular incorporation of the uridine analogue 5-fluorouridine into nascent RNA (Figure S1A). Effects of actD on nuclear structure were observed using immunofluorescence with nucleolar antibodies (Figure 4C) which showed typical disruption of nucleolar structure by drug treatment [46]. If transcription maintained chromosome structure, a transcription inhibitor would be expected to alter chromosome volume and morphology, leading to a more condensed state. The overall structure of the dispersed chromosome in Figure 4A did not change after being exposed to actD, even as the nucleolus diminished in size. A few chromosomes did show changes. The upper chromosome in Figure 4B initially showed a diffuse extension before retraction after inhibition of transcription. However, averaged over all the labelled chromosomes, inhibition of transcription led to no significant quantitative changes of volume, surface area, sphericity or longest axes of randomly labelled chromosomes (Figure 4D, p values all >0.35). Examples where transcription did appear to maintain chromosome structure may reflect particularly gene dense chromosomes or chromosome segments [36].

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