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High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

van Koningsbruggen S, Gierlinski M, Schofield P, Martin D, Barton GJ, Ariyurek Y, den Dunnen JT, Lamond AI - Mol. Biol. Cell (2010)

Bottom Line: We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy.The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner.Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.

ABSTRACT
The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

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Comparative genome hybridization for nucleolar versus total genomic DNA. (A) Schematic showing of the preparation of nucleolar-associated (red) and total genomic (red plus green) DNA used for generating FISH probes for comparative genome hybridization analysis of metaphase chromosome spreads and interphase nuclei. (B) FISH analyses of interphase (top panels) and metaphase (bottom panels) cells. Probes labeled either total genomic DNA (panels I and VI), nucleolar-associated DNA (panels II and VII) or rDNA (blue staining, shown as a merge with DAPI in IX). Total DNA was counterstained with DAPI (V and X), nucleoli labeled using an anti-nucleolin antibody (IV). Merged images of genomic and nucleolar probes are shown in III and VIII. In interphase panels, red arrows show nucleoli. In metaphase panels, yellow arrows show rDNA repeat unit clusters, the purple arrow shows the Y chromosome, white arrowheads show centromeres, blue arrowheads show interstitial loci on nonacrocentric chromosomes, and green arrowheads show telomeres. Scale bars, 10 μm.
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Figure 1: Comparative genome hybridization for nucleolar versus total genomic DNA. (A) Schematic showing of the preparation of nucleolar-associated (red) and total genomic (red plus green) DNA used for generating FISH probes for comparative genome hybridization analysis of metaphase chromosome spreads and interphase nuclei. (B) FISH analyses of interphase (top panels) and metaphase (bottom panels) cells. Probes labeled either total genomic DNA (panels I and VI), nucleolar-associated DNA (panels II and VII) or rDNA (blue staining, shown as a merge with DAPI in IX). Total DNA was counterstained with DAPI (V and X), nucleoli labeled using an anti-nucleolin antibody (IV). Merged images of genomic and nucleolar probes are shown in III and VIII. In interphase panels, red arrows show nucleoli. In metaphase panels, yellow arrows show rDNA repeat unit clusters, the purple arrow shows the Y chromosome, white arrowheads show centromeres, blue arrowheads show interstitial loci on nonacrocentric chromosomes, and green arrowheads show telomeres. Scale bars, 10 μm.

Mentions: Fluorescence CGH was used to address whether nucleolar-associated chromatin corresponds to regions from acrocentric chromosomes only or whether it also includes loci from other chromosomes. FISH probes were prepared using either total nuclear DNA (genomic), or DNA isolated from purified nucleoli derived from the human fibrosarcoma cell line HT1080 (shown schematically in Figure 1A, with DAPI-stained images of purified nucleoli in Supplementary Figure S1A). DNA blot analysis confirmed that the nucleolar FISH probe was enriched for rDNA sequences relative to the genomic probe (Figure S1B).


High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli.

van Koningsbruggen S, Gierlinski M, Schofield P, Martin D, Barton GJ, Ariyurek Y, den Dunnen JT, Lamond AI - Mol. Biol. Cell (2010)

Comparative genome hybridization for nucleolar versus total genomic DNA. (A) Schematic showing of the preparation of nucleolar-associated (red) and total genomic (red plus green) DNA used for generating FISH probes for comparative genome hybridization analysis of metaphase chromosome spreads and interphase nuclei. (B) FISH analyses of interphase (top panels) and metaphase (bottom panels) cells. Probes labeled either total genomic DNA (panels I and VI), nucleolar-associated DNA (panels II and VII) or rDNA (blue staining, shown as a merge with DAPI in IX). Total DNA was counterstained with DAPI (V and X), nucleoli labeled using an anti-nucleolin antibody (IV). Merged images of genomic and nucleolar probes are shown in III and VIII. In interphase panels, red arrows show nucleoli. In metaphase panels, yellow arrows show rDNA repeat unit clusters, the purple arrow shows the Y chromosome, white arrowheads show centromeres, blue arrowheads show interstitial loci on nonacrocentric chromosomes, and green arrowheads show telomeres. Scale bars, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965689&req=5

Figure 1: Comparative genome hybridization for nucleolar versus total genomic DNA. (A) Schematic showing of the preparation of nucleolar-associated (red) and total genomic (red plus green) DNA used for generating FISH probes for comparative genome hybridization analysis of metaphase chromosome spreads and interphase nuclei. (B) FISH analyses of interphase (top panels) and metaphase (bottom panels) cells. Probes labeled either total genomic DNA (panels I and VI), nucleolar-associated DNA (panels II and VII) or rDNA (blue staining, shown as a merge with DAPI in IX). Total DNA was counterstained with DAPI (V and X), nucleoli labeled using an anti-nucleolin antibody (IV). Merged images of genomic and nucleolar probes are shown in III and VIII. In interphase panels, red arrows show nucleoli. In metaphase panels, yellow arrows show rDNA repeat unit clusters, the purple arrow shows the Y chromosome, white arrowheads show centromeres, blue arrowheads show interstitial loci on nonacrocentric chromosomes, and green arrowheads show telomeres. Scale bars, 10 μm.
Mentions: Fluorescence CGH was used to address whether nucleolar-associated chromatin corresponds to regions from acrocentric chromosomes only or whether it also includes loci from other chromosomes. FISH probes were prepared using either total nuclear DNA (genomic), or DNA isolated from purified nucleoli derived from the human fibrosarcoma cell line HT1080 (shown schematically in Figure 1A, with DAPI-stained images of purified nucleoli in Supplementary Figure S1A). DNA blot analysis confirmed that the nucleolar FISH probe was enriched for rDNA sequences relative to the genomic probe (Figure S1B).

Bottom Line: We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy.The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner.Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom.

ABSTRACT
The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

Show MeSH
Related in: MedlinePlus