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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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Preservation of the spatial distribution of nascent RNA, DNA, and acetylated histone H4 in human primary fibroblasts during the FISH procedure. Optical sections were obtained before (A, C, and E) and after (B, D, and F) carrying out the FISH protocol. A and B, Optical sections of nuclei in which transcription sites were immunofluorescently labeled. The distribution of nascent RNA did not change significantly during the FISH procedure. C and D, Optical sections of nuclei labeled with the fluorescent DNA stain Sytox green. The pattern of DNA staining did not change significantly after in situ hybridization. E and F, Optical sections of nuclei in which acetylated histone H4 was fluorescently labeled. The distribution did not change significantly during the chromosome painting procedure. The diffuse, low intensity labeling observed before FISH was diminished after the procedure, resulting in a somewhat more pronounced granular labeling. Images shown have been subjected to 3-D image restoration. Bar, 1.75 μm.
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Figure 1: Preservation of the spatial distribution of nascent RNA, DNA, and acetylated histone H4 in human primary fibroblasts during the FISH procedure. Optical sections were obtained before (A, C, and E) and after (B, D, and F) carrying out the FISH protocol. A and B, Optical sections of nuclei in which transcription sites were immunofluorescently labeled. The distribution of nascent RNA did not change significantly during the FISH procedure. C and D, Optical sections of nuclei labeled with the fluorescent DNA stain Sytox green. The pattern of DNA staining did not change significantly after in situ hybridization. E and F, Optical sections of nuclei in which acetylated histone H4 was fluorescently labeled. The distribution did not change significantly during the chromosome painting procedure. The diffuse, low intensity labeling observed before FISH was diminished after the procedure, resulting in a somewhat more pronounced granular labeling. Images shown have been subjected to 3-D image restoration. Bar, 1.75 μm.

Mentions: Visual inspection shows that the spatial distribution of nascent RNA did not change in any recognizable way due to the FISH procedure (Fig. 1A and Fig. B). Staining of DNA with Sytox green with or without chromosome painting showed that the FISH procedure also did not induce major changes in DNA distribution (Fig. 1C and Fig. D). After the FISH procedure, the DNA staining pattern seemed slightly more blurred and the overall DNA labeling appeared more intense. Fig. 1E and Fig. F, show immunofluorescent labeling of histone H4 acetylated at lysine 8, before and after FISH labeling. The punctate distribution of acetylated H4 throughout the nucleoplasm did not significantly change after chromosome painting. Only the diffuse, low intensity component in the labeling was somewhat diminished after FISH, making the intense punctate granular labeling more prominent. This may be due to extraction of some histone protein during FISH labeling. These results show that the FISH procedure does not result in major rearrangements in DNA, chromatin, and nascent RNA.


Spatial relationship between transcription sites and chromosome territories.

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

Preservation of the spatial distribution of nascent RNA, DNA, and acetylated histone H4 in human primary fibroblasts during the FISH procedure. Optical sections were obtained before (A, C, and E) and after (B, D, and F) carrying out the FISH protocol. A and B, Optical sections of nuclei in which transcription sites were immunofluorescently labeled. The distribution of nascent RNA did not change significantly during the FISH procedure. C and D, Optical sections of nuclei labeled with the fluorescent DNA stain Sytox green. The pattern of DNA staining did not change significantly after in situ hybridization. E and F, Optical sections of nuclei in which acetylated histone H4 was fluorescently labeled. The distribution did not change significantly during the chromosome painting procedure. The diffuse, low intensity labeling observed before FISH was diminished after the procedure, resulting in a somewhat more pronounced granular labeling. Images shown have been subjected to 3-D image restoration. Bar, 1.75 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2164981&req=5

Figure 1: Preservation of the spatial distribution of nascent RNA, DNA, and acetylated histone H4 in human primary fibroblasts during the FISH procedure. Optical sections were obtained before (A, C, and E) and after (B, D, and F) carrying out the FISH protocol. A and B, Optical sections of nuclei in which transcription sites were immunofluorescently labeled. The distribution of nascent RNA did not change significantly during the FISH procedure. C and D, Optical sections of nuclei labeled with the fluorescent DNA stain Sytox green. The pattern of DNA staining did not change significantly after in situ hybridization. E and F, Optical sections of nuclei in which acetylated histone H4 was fluorescently labeled. The distribution did not change significantly during the chromosome painting procedure. The diffuse, low intensity labeling observed before FISH was diminished after the procedure, resulting in a somewhat more pronounced granular labeling. Images shown have been subjected to 3-D image restoration. Bar, 1.75 μm.
Mentions: Visual inspection shows that the spatial distribution of nascent RNA did not change in any recognizable way due to the FISH procedure (Fig. 1A and Fig. B). Staining of DNA with Sytox green with or without chromosome painting showed that the FISH procedure also did not induce major changes in DNA distribution (Fig. 1C and Fig. D). After the FISH procedure, the DNA staining pattern seemed slightly more blurred and the overall DNA labeling appeared more intense. Fig. 1E and Fig. F, show immunofluorescent labeling of histone H4 acetylated at lysine 8, before and after FISH labeling. The punctate distribution of acetylated H4 throughout the nucleoplasm did not significantly change after chromosome painting. Only the diffuse, low intensity component in the labeling was somewhat diminished after FISH, making the intense punctate granular labeling more prominent. This may be due to extraction of some histone protein during FISH labeling. These results show that the FISH procedure does not result in major rearrangements in DNA, chromatin, and nascent RNA.

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.

Show MeSH