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Spatial relationship between transcription sites and chromosome territories.

Verschure PJ, van Der Kraan I, Manders EM, van Driel R - J. Cell Biol. (1999)

Bottom Line: Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19.Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains.Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

View Article: PubMed Central - PubMed

Affiliation: E.C. Slater Instituut, BioCentrum Amsterdam, University of Amsterdam, 1018 TV Amsterdam, The Netherlands. a311pjvx@chem.uva.nl

ABSTRACT
We have investigated the spatial relationship between transcription sites and chromosome territories in the interphase nucleus of human female fibroblasts. Immunolabeling of nascent RNA was combined with visualization of chromosome territories by fluorescent in situ hybridization (FISH). Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19. The other X chromosome territory, probably the inactive X chromosome, was devoid of transcription sites. A distinct substructure was observed in interphase chromosome territories. Intensely labeled subchromosomal domains are surrounded by less strongly labeled areas. The intensely labeled domains had a diameter in the range of 300-450 nm and were sometimes interconnected, forming thread-like structures. Similar large scale chromatin structures were observed in HeLa cells expressing green fluorescent protein (GFP)-tagged histone H2B. Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains. These observations support a model in which transcriptionally active chromatin in chromosome territories is markedly compartmentalized. Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

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Chromosome territories have a distinct substructure. X chromosome territories in nuclei of female primary fibroblasts were labeled by FISH, using a chromosome-specific DNA probe library. A, Single optical section through the center of a nucleus showing two labeled X chromosome territories. The image has undergone 3-D image restoration. Bar, 2.1 μm. C and E, Five consecutive optical sections (step size along z-axis is 0.2 μm) showing structural details of the two territories visible in A. The images shown have been subjected to 3-D image restoration. Territories show strongly labeled chromosomal subdomains surrounded by less intensely labeled areas. Intensely labeled chromosomal subdomains have a diameter in the range of 300–450 nm. In several cases, subchromosomal domains appear interconnected, forming thread-like structures (also see Fig. 4). Bar, 4.2 μm. B and D, Same optical sections as in C and E, respectively. Shown are unprocessed, crude images that have not undergone 3-D image restoration. Structural details that are enhanced after 3-D image restoration are visible in the unprocessed optical sections. Bar, 4.2 μm.
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Figure 3: Chromosome territories have a distinct substructure. X chromosome territories in nuclei of female primary fibroblasts were labeled by FISH, using a chromosome-specific DNA probe library. A, Single optical section through the center of a nucleus showing two labeled X chromosome territories. The image has undergone 3-D image restoration. Bar, 2.1 μm. C and E, Five consecutive optical sections (step size along z-axis is 0.2 μm) showing structural details of the two territories visible in A. The images shown have been subjected to 3-D image restoration. Territories show strongly labeled chromosomal subdomains surrounded by less intensely labeled areas. Intensely labeled chromosomal subdomains have a diameter in the range of 300–450 nm. In several cases, subchromosomal domains appear interconnected, forming thread-like structures (also see Fig. 4). Bar, 4.2 μm. B and D, Same optical sections as in C and E, respectively. Shown are unprocessed, crude images that have not undergone 3-D image restoration. Structural details that are enhanced after 3-D image restoration are visible in the unprocessed optical sections. Bar, 4.2 μm.

Mentions: A striking feature that emerges from our confocal images after FISH labeling is that chromosome territories display a distinct substructure (Fig. 3). Chromosome territories do not appear as compact objects. Rather, they show a modulated intensity distribution inside the territory. Since the chromosome-specific library-probes label the complete metaphase chromosome, it is unlikely that considerable parts of the interphase chromosome remain unlabeled (Carter et al. 1990; Telenius et al. 1992). The chromosome territories contained strongly labeled chromosomal subdomains, surrounded by less intensely labeled areas. The strongly labeled subchromosomal structures have a diameter in the range of 300–450 nm (Fig. 3 and Fig. 4). Intensely labeled parts of a chromosome often seemed interconnected, forming thread-like, folded structures. A similar, distinct substructure in nuclei stained for DNA with Sytox green was observed (Fig. 1C and Fig. D) and in nuclei of HeLa cells expressing GFP-histone H2B (see Visualization of Chromatin Using GFP-histone H2B; Fig. 5), suggesting a reticular organization of chromatin. It is tempting to suggest that, at least locally, the chromatin fiber that constitutes the chromosome can be followed in an optical section.


Spatial relationship between transcription sites and chromosome territories.

Verschure PJ, van Der Kraan I, Manders EM, van Driel R - J. Cell Biol. (1999)

Chromosome territories have a distinct substructure. X chromosome territories in nuclei of female primary fibroblasts were labeled by FISH, using a chromosome-specific DNA probe library. A, Single optical section through the center of a nucleus showing two labeled X chromosome territories. The image has undergone 3-D image restoration. Bar, 2.1 μm. C and E, Five consecutive optical sections (step size along z-axis is 0.2 μm) showing structural details of the two territories visible in A. The images shown have been subjected to 3-D image restoration. Territories show strongly labeled chromosomal subdomains surrounded by less intensely labeled areas. Intensely labeled chromosomal subdomains have a diameter in the range of 300–450 nm. In several cases, subchromosomal domains appear interconnected, forming thread-like structures (also see Fig. 4). Bar, 4.2 μm. B and D, Same optical sections as in C and E, respectively. Shown are unprocessed, crude images that have not undergone 3-D image restoration. Structural details that are enhanced after 3-D image restoration are visible in the unprocessed optical sections. Bar, 4.2 μm.
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Figure 3: Chromosome territories have a distinct substructure. X chromosome territories in nuclei of female primary fibroblasts were labeled by FISH, using a chromosome-specific DNA probe library. A, Single optical section through the center of a nucleus showing two labeled X chromosome territories. The image has undergone 3-D image restoration. Bar, 2.1 μm. C and E, Five consecutive optical sections (step size along z-axis is 0.2 μm) showing structural details of the two territories visible in A. The images shown have been subjected to 3-D image restoration. Territories show strongly labeled chromosomal subdomains surrounded by less intensely labeled areas. Intensely labeled chromosomal subdomains have a diameter in the range of 300–450 nm. In several cases, subchromosomal domains appear interconnected, forming thread-like structures (also see Fig. 4). Bar, 4.2 μm. B and D, Same optical sections as in C and E, respectively. Shown are unprocessed, crude images that have not undergone 3-D image restoration. Structural details that are enhanced after 3-D image restoration are visible in the unprocessed optical sections. Bar, 4.2 μm.
Mentions: A striking feature that emerges from our confocal images after FISH labeling is that chromosome territories display a distinct substructure (Fig. 3). Chromosome territories do not appear as compact objects. Rather, they show a modulated intensity distribution inside the territory. Since the chromosome-specific library-probes label the complete metaphase chromosome, it is unlikely that considerable parts of the interphase chromosome remain unlabeled (Carter et al. 1990; Telenius et al. 1992). The chromosome territories contained strongly labeled chromosomal subdomains, surrounded by less intensely labeled areas. The strongly labeled subchromosomal structures have a diameter in the range of 300–450 nm (Fig. 3 and Fig. 4). Intensely labeled parts of a chromosome often seemed interconnected, forming thread-like, folded structures. A similar, distinct substructure in nuclei stained for DNA with Sytox green was observed (Fig. 1C and Fig. D) and in nuclei of HeLa cells expressing GFP-histone H2B (see Visualization of Chromatin Using GFP-histone H2B; Fig. 5), suggesting a reticular organization of chromatin. It is tempting to suggest that, at least locally, the chromatin fiber that constitutes the chromosome can be followed in an optical section.

Bottom Line: Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19.Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains.Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

View Article: PubMed Central - PubMed

Affiliation: E.C. Slater Instituut, BioCentrum Amsterdam, University of Amsterdam, 1018 TV Amsterdam, The Netherlands. a311pjvx@chem.uva.nl

ABSTRACT
We have investigated the spatial relationship between transcription sites and chromosome territories in the interphase nucleus of human female fibroblasts. Immunolabeling of nascent RNA was combined with visualization of chromosome territories by fluorescent in situ hybridization (FISH). Transcription sites were found scattered throughout the territory of one of the two X chromosomes, most likely the active X chromosome, and that of both territories of chromosome 19. The other X chromosome territory, probably the inactive X chromosome, was devoid of transcription sites. A distinct substructure was observed in interphase chromosome territories. Intensely labeled subchromosomal domains are surrounded by less strongly labeled areas. The intensely labeled domains had a diameter in the range of 300-450 nm and were sometimes interconnected, forming thread-like structures. Similar large scale chromatin structures were observed in HeLa cells expressing green fluorescent protein (GFP)-tagged histone H2B. Strikingly, nascent RNA was almost exclusively found in the interchromatin areas in chromosome territories and in between strongly GFP-labeled chromatin domains. These observations support a model in which transcriptionally active chromatin in chromosome territories is markedly compartmentalized. Active loci are located predominantly at or near the surface of compact chromatin domains, depositing newly synthesized RNA directly into the interchromatin space.

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