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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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Photoactivation of nucleolar-associated chromatin to compare its localization in parental and daughter cells following mitosis. Photoactivation of PA-GFP-H2B stably expressed in HeLa cells. ROIs were either nucleolar-associated (I and II) or separate from nucleoli in the nucleoplasm (III and IV). These ROIs were photoactivated at 406 nm (blue arrows), and representative midplane deconvolved images are shown from cells either before (I and III) or after (II and IV) mitosis. Both postmitotic images show daughter cells 4.5 h after the onset of telophase. PA-GFP-H2B is shown in green, and nucleoli are labeled with mCherry-B23 (red). White arrows mark photoactivated chromatin that relocalizes at nucleoli in daughter cells; yellow arrowheads point at chromatin that relocalizes at the nuclear periphery in daughter cells. Scale bars, 10 μm.
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Figure 9: Photoactivation of nucleolar-associated chromatin to compare its localization in parental and daughter cells following mitosis. Photoactivation of PA-GFP-H2B stably expressed in HeLa cells. ROIs were either nucleolar-associated (I and II) or separate from nucleoli in the nucleoplasm (III and IV). These ROIs were photoactivated at 406 nm (blue arrows), and representative midplane deconvolved images are shown from cells either before (I and III) or after (II and IV) mitosis. Both postmitotic images show daughter cells 4.5 h after the onset of telophase. PA-GFP-H2B is shown in green, and nucleoli are labeled with mCherry-B23 (red). White arrows mark photoactivated chromatin that relocalizes at nucleoli in daughter cells; yellow arrowheads point at chromatin that relocalizes at the nuclear periphery in daughter cells. Scale bars, 10 μm.

Mentions: To identify nucleoli in live cells, the nucleolar marker protein B23 was fused to mCherry and transiently expressed in the HeLa PA-GFP-H2B stable cell line. A ROI either overlapping the nucleolus (Figure 9, I, blue arrow) or in the nucleoplasm separate from the nucleolus (Figure 9, III, blue arrow) was photoactivated using laser excitation at 406 nm and imaged using time-lapse microscopy. The chromatin either just above, or below, the nucleolus was also activated to some extent. To control for these additional activated regions, daughter cells were also analyzed where the activated region was separate from the nucleolus (Figure 9, IV).


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)

Photoactivation of nucleolar-associated chromatin to compare its localization in parental and daughter cells following mitosis. Photoactivation of PA-GFP-H2B stably expressed in HeLa cells. ROIs were either nucleolar-associated (I and II) or separate from nucleoli in the nucleoplasm (III and IV). These ROIs were photoactivated at 406 nm (blue arrows), and representative midplane deconvolved images are shown from cells either before (I and III) or after (II and IV) mitosis. Both postmitotic images show daughter cells 4.5 h after the onset of telophase. PA-GFP-H2B is shown in green, and nucleoli are labeled with mCherry-B23 (red). White arrows mark photoactivated chromatin that relocalizes at nucleoli in daughter cells; yellow arrowheads point at chromatin that relocalizes at the nuclear periphery in daughter cells. Scale bars, 10 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2965689&req=5

Figure 9: Photoactivation of nucleolar-associated chromatin to compare its localization in parental and daughter cells following mitosis. Photoactivation of PA-GFP-H2B stably expressed in HeLa cells. ROIs were either nucleolar-associated (I and II) or separate from nucleoli in the nucleoplasm (III and IV). These ROIs were photoactivated at 406 nm (blue arrows), and representative midplane deconvolved images are shown from cells either before (I and III) or after (II and IV) mitosis. Both postmitotic images show daughter cells 4.5 h after the onset of telophase. PA-GFP-H2B is shown in green, and nucleoli are labeled with mCherry-B23 (red). White arrows mark photoactivated chromatin that relocalizes at nucleoli in daughter cells; yellow arrowheads point at chromatin that relocalizes at the nuclear periphery in daughter cells. Scale bars, 10 μm.
Mentions: To identify nucleoli in live cells, the nucleolar marker protein B23 was fused to mCherry and transiently expressed in the HeLa PA-GFP-H2B stable cell line. A ROI either overlapping the nucleolus (Figure 9, I, blue arrow) or in the nucleoplasm separate from the nucleolus (Figure 9, III, blue arrow) was photoactivated using laser excitation at 406 nm and imaged using time-lapse microscopy. The chromatin either just above, or below, the nucleolus was also activated to some extent. To control for these additional activated regions, daughter cells were also analyzed where the activated region was separate from the nucleolus (Figure 9, IV).

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