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The Actin-Related Protein BAF53 Is Essential for Chromosomal Subdomain Integrity.

Lee K, Kim JH, Kwon H - Mol. Cells (2015)

Bottom Line: Because the integrity of chromosomal subdomains is a deciding factor of the volume of a chromosome territory, we examined here the effect of BAF53 knockdown on chromosomal subdomains.In addition, the size of DNA loops measured by the maximum fluorescent halo technique increased and became irregular after BAF53 knockdown, indicating DNA loops were released from the residual nuclear structure.These data can be accounted for by the model that BAF53 is prerequisite for maintaining the structural integrity of chromosomal subdomains.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology and Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, Yongin 449-791, Korea.

ABSTRACT
A chromosome territory is composed of chromosomal subdomains. The internal structure of chromosomal subdomains provides a structural framework for many genomic activities such as replication and DNA repair, and thus is key to determining the basis of their mechanisms. However, the internal structure and regulating proteins of a chromosomal subdomain remains elusive. Previously, we showed that the chromosome territory expanded after BAF53 knockdown. Because the integrity of chromosomal subdomains is a deciding factor of the volume of a chromosome territory, we examined here the effect of BAF53 knockdown on chromosomal subdomains. We found that BAF53 knockdown led to the disintegration of histone H2B-GFP-visualized chromosomal subdomains and BrdU-labeled replication foci. In addition, the size of DNA loops measured by the maximum fluorescent halo technique increased and became irregular after BAF53 knockdown, indicating DNA loops were released from the residual nuclear structure. These data can be accounted for by the model that BAF53 is prerequisite for maintaining the structural integrity of chromosomal subdomains.

No MeSH data available.


Related in: MedlinePlus

Disintegration of the chromosomal subdomains by BAF53 knockdown. HeLa cells stably expressing GFP-tagged histone H2B were transfected with siRNA-BAF53. (A) The expression levels of H2B-GFP and BAF53 were determined by immunoblotting. (B) The expression level of H2B-GFP of a cell was measured by integrating the signals from all of the pixels of the midsection image of a fixed nucleus revealing GFP-tagged histone H2B (t-test; P > 0.5). Error bars, mean ± SD (n = 10). (C) Representative images of the midsection of a fixed nucleus revealing GFP-tagged histone H2B. Pixels with gray values above the assigned thresholds (TH = 40 and TH = 60) are highlighted in red. (D) Distributions of the occupancies of red pixels in the control and BAF53-knockdown cell nuclei. Boxes represent median and quartile values, and bars define the 5th and 95th percentiles (n = 30). t-test; *P < 0.001.
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f1-molce-38-9-789: Disintegration of the chromosomal subdomains by BAF53 knockdown. HeLa cells stably expressing GFP-tagged histone H2B were transfected with siRNA-BAF53. (A) The expression levels of H2B-GFP and BAF53 were determined by immunoblotting. (B) The expression level of H2B-GFP of a cell was measured by integrating the signals from all of the pixels of the midsection image of a fixed nucleus revealing GFP-tagged histone H2B (t-test; P > 0.5). Error bars, mean ± SD (n = 10). (C) Representative images of the midsection of a fixed nucleus revealing GFP-tagged histone H2B. Pixels with gray values above the assigned thresholds (TH = 40 and TH = 60) are highlighted in red. (D) Distributions of the occupancies of red pixels in the control and BAF53-knockdown cell nuclei. Boxes represent median and quartile values, and bars define the 5th and 95th percentiles (n = 30). t-test; *P < 0.001.

Mentions: We generated HeLa cells expressing histone H2B-GFP, which allows the visualization of chromosomal subdomains as bead images (Kanda et al., 1998). The midsection of a fixed nucleus reveals that bead images are evenly distributed throughout the nucleoplasm, nuclear periphery and perinucleolar regions, although individual beads cannot be clearly discerned from each other (Fig. 1). The less clear delineation of each bead image is probably due to out-of-focus light from the densely packed neighboring chromatin domains. To test whether BAF53 is required for the maintenance of chromosomal subdomains, we suppressed the expression of BAF53 by siRNA interference (Fig. 1A). We employed the same population of HeLa cells stably expressing histone H2B-GFP for the control and BAF53-knockdown groups, and the expression levels of H2B-GFP were in fact comparable for the both groups (Fig. 1B). After BAF53 knockdown, the nucleoplasm became more or less vacant of bead images, while the bead images at the nuclear periphery and perinucleolar regions became more conspicuous (Fig. 1C). To visualize this change more clearly, we assigned a series of threshold values to the images and highlighted pixels with gray values above the assigned thresholds in red. Pixels highlighted in red started to decrease with the low threshold, TH = 40, and decreased precipitously with the higher threshold, TH = 60, in BAF53 knockdown cells (Fig. 1D). By contrast, the number of red pixels decreased slightly with both thresholds in control cells. It is noteworthy that the red pixels that disappeared after BAF53 knockdown were preferentially located in the nucleoplasm, while the red pixels at the nuclear periphery remained relatively robust. Considering that the hallmark of bead images is repeated, stronger signals from locally overlapping segments of chromatin fibers arising from higher-order folding or clustering of chromatin loops, the decrease in the number of red pixels observed in BAF53 knockdown cells can be attributed to an unfolding of beads, leaving diffused fluorescence signals below the assigned thresholds. In addition, this finding suggests that beads in the nucleoplasm are preferentially unfolded after BAF53 knockdown.


The Actin-Related Protein BAF53 Is Essential for Chromosomal Subdomain Integrity.

Lee K, Kim JH, Kwon H - Mol. Cells (2015)

Disintegration of the chromosomal subdomains by BAF53 knockdown. HeLa cells stably expressing GFP-tagged histone H2B were transfected with siRNA-BAF53. (A) The expression levels of H2B-GFP and BAF53 were determined by immunoblotting. (B) The expression level of H2B-GFP of a cell was measured by integrating the signals from all of the pixels of the midsection image of a fixed nucleus revealing GFP-tagged histone H2B (t-test; P > 0.5). Error bars, mean ± SD (n = 10). (C) Representative images of the midsection of a fixed nucleus revealing GFP-tagged histone H2B. Pixels with gray values above the assigned thresholds (TH = 40 and TH = 60) are highlighted in red. (D) Distributions of the occupancies of red pixels in the control and BAF53-knockdown cell nuclei. Boxes represent median and quartile values, and bars define the 5th and 95th percentiles (n = 30). t-test; *P < 0.001.
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f1-molce-38-9-789: Disintegration of the chromosomal subdomains by BAF53 knockdown. HeLa cells stably expressing GFP-tagged histone H2B were transfected with siRNA-BAF53. (A) The expression levels of H2B-GFP and BAF53 were determined by immunoblotting. (B) The expression level of H2B-GFP of a cell was measured by integrating the signals from all of the pixels of the midsection image of a fixed nucleus revealing GFP-tagged histone H2B (t-test; P > 0.5). Error bars, mean ± SD (n = 10). (C) Representative images of the midsection of a fixed nucleus revealing GFP-tagged histone H2B. Pixels with gray values above the assigned thresholds (TH = 40 and TH = 60) are highlighted in red. (D) Distributions of the occupancies of red pixels in the control and BAF53-knockdown cell nuclei. Boxes represent median and quartile values, and bars define the 5th and 95th percentiles (n = 30). t-test; *P < 0.001.
Mentions: We generated HeLa cells expressing histone H2B-GFP, which allows the visualization of chromosomal subdomains as bead images (Kanda et al., 1998). The midsection of a fixed nucleus reveals that bead images are evenly distributed throughout the nucleoplasm, nuclear periphery and perinucleolar regions, although individual beads cannot be clearly discerned from each other (Fig. 1). The less clear delineation of each bead image is probably due to out-of-focus light from the densely packed neighboring chromatin domains. To test whether BAF53 is required for the maintenance of chromosomal subdomains, we suppressed the expression of BAF53 by siRNA interference (Fig. 1A). We employed the same population of HeLa cells stably expressing histone H2B-GFP for the control and BAF53-knockdown groups, and the expression levels of H2B-GFP were in fact comparable for the both groups (Fig. 1B). After BAF53 knockdown, the nucleoplasm became more or less vacant of bead images, while the bead images at the nuclear periphery and perinucleolar regions became more conspicuous (Fig. 1C). To visualize this change more clearly, we assigned a series of threshold values to the images and highlighted pixels with gray values above the assigned thresholds in red. Pixels highlighted in red started to decrease with the low threshold, TH = 40, and decreased precipitously with the higher threshold, TH = 60, in BAF53 knockdown cells (Fig. 1D). By contrast, the number of red pixels decreased slightly with both thresholds in control cells. It is noteworthy that the red pixels that disappeared after BAF53 knockdown were preferentially located in the nucleoplasm, while the red pixels at the nuclear periphery remained relatively robust. Considering that the hallmark of bead images is repeated, stronger signals from locally overlapping segments of chromatin fibers arising from higher-order folding or clustering of chromatin loops, the decrease in the number of red pixels observed in BAF53 knockdown cells can be attributed to an unfolding of beads, leaving diffused fluorescence signals below the assigned thresholds. In addition, this finding suggests that beads in the nucleoplasm are preferentially unfolded after BAF53 knockdown.

Bottom Line: Because the integrity of chromosomal subdomains is a deciding factor of the volume of a chromosome territory, we examined here the effect of BAF53 knockdown on chromosomal subdomains.In addition, the size of DNA loops measured by the maximum fluorescent halo technique increased and became irregular after BAF53 knockdown, indicating DNA loops were released from the residual nuclear structure.These data can be accounted for by the model that BAF53 is prerequisite for maintaining the structural integrity of chromosomal subdomains.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology and Protein Research Center for Bio-Industry, Hankuk University of Foreign Studies, Yongin 449-791, Korea.

ABSTRACT
A chromosome territory is composed of chromosomal subdomains. The internal structure of chromosomal subdomains provides a structural framework for many genomic activities such as replication and DNA repair, and thus is key to determining the basis of their mechanisms. However, the internal structure and regulating proteins of a chromosomal subdomain remains elusive. Previously, we showed that the chromosome territory expanded after BAF53 knockdown. Because the integrity of chromosomal subdomains is a deciding factor of the volume of a chromosome territory, we examined here the effect of BAF53 knockdown on chromosomal subdomains. We found that BAF53 knockdown led to the disintegration of histone H2B-GFP-visualized chromosomal subdomains and BrdU-labeled replication foci. In addition, the size of DNA loops measured by the maximum fluorescent halo technique increased and became irregular after BAF53 knockdown, indicating DNA loops were released from the residual nuclear structure. These data can be accounted for by the model that BAF53 is prerequisite for maintaining the structural integrity of chromosomal subdomains.

No MeSH data available.


Related in: MedlinePlus