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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

Expansion of the interchromatin compartment space by BAF53 knockdown. (A) Representative confocal midsections of the nuclei of HeLa cells (H2B-GFP) after treatment with the hyper-osmolar medium. Note that the formation of hypercondensed chromatin (HCC) is unchanged, but the interchromatin compartment space is expanded in BAF53-knockdown cell nuclei. Pixels with gray values below the assigned thresholds (TH = 40) are highlighted in blue. (B) Distributions of the occupancies of blue 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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f2-molce-38-9-789: Expansion of the interchromatin compartment space by BAF53 knockdown. (A) Representative confocal midsections of the nuclei of HeLa cells (H2B-GFP) after treatment with the hyper-osmolar medium. Note that the formation of hypercondensed chromatin (HCC) is unchanged, but the interchromatin compartment space is expanded in BAF53-knockdown cell nuclei. Pixels with gray values below the assigned thresholds (TH = 40) are highlighted in blue. (B) Distributions of the occupancies of blue 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: It has been known that the placement of cells in hyper-osmolar medium leads to the formation of hypercondensed chromatin (HCC). This approach was employed to highlight chromosomal subdomains successfully (Albiez et al., 2006; Cremer et al., 2000). Under hyper-osmolar conditions, hypercondensation of chromatin allowed clear visualization of chromosomal subdomains of H2B-GFP-tagged chromatin (Fig. 2). Bead images with similar signal intensity and size were found throughout the nucleoplasm, nuclear periphery, and perinucleolar regions. Previously, it was shown that structural patterns of hypercondensed chromatin reflect a higher-order chromatin and interchromatin domain topology (Albiez et al., 2006). Since the protein scaffolds that play a pivotal role in the formation of chromosomal subdomains are expected to be a primary determinant of the higher-order chromatin and interchromatin domain topology, this result suggests that chromatin coalesces with the underlying protein scaffold into a highly condensed mass during the hypercondensation process, and this process is delimited by the domain structure. Placement of BAF53 knockdown cells into hyper-osmolar medium also led to hypercondensation of chromatin, resulting in bead images similar to those of the control cells. This finding implied that condensation of chromatin under hyper-osmolar conditions is apparently unaltered after BAF53 knockdown. Significantly, however, the interchromatin-lacunas space widened after BAF53 knockdown. This change is more clearly demonstrated by assigning thresholds and highlighting pixels with gray values below the assigned thresholds in blue. The widening of the interchromatin-lacunas space argues that the even distribution of the protein scaffold that contributes to the formation of hypercondensed chromatin bundles, as well as chromosomal subdomains, is at least in part distorted or disrupted by BAF53 knockdown.


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

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

Expansion of the interchromatin compartment space by BAF53 knockdown. (A) Representative confocal midsections of the nuclei of HeLa cells (H2B-GFP) after treatment with the hyper-osmolar medium. Note that the formation of hypercondensed chromatin (HCC) is unchanged, but the interchromatin compartment space is expanded in BAF53-knockdown cell nuclei. Pixels with gray values below the assigned thresholds (TH = 40) are highlighted in blue. (B) Distributions of the occupancies of blue 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4588722&req=5

f2-molce-38-9-789: Expansion of the interchromatin compartment space by BAF53 knockdown. (A) Representative confocal midsections of the nuclei of HeLa cells (H2B-GFP) after treatment with the hyper-osmolar medium. Note that the formation of hypercondensed chromatin (HCC) is unchanged, but the interchromatin compartment space is expanded in BAF53-knockdown cell nuclei. Pixels with gray values below the assigned thresholds (TH = 40) are highlighted in blue. (B) Distributions of the occupancies of blue 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: It has been known that the placement of cells in hyper-osmolar medium leads to the formation of hypercondensed chromatin (HCC). This approach was employed to highlight chromosomal subdomains successfully (Albiez et al., 2006; Cremer et al., 2000). Under hyper-osmolar conditions, hypercondensation of chromatin allowed clear visualization of chromosomal subdomains of H2B-GFP-tagged chromatin (Fig. 2). Bead images with similar signal intensity and size were found throughout the nucleoplasm, nuclear periphery, and perinucleolar regions. Previously, it was shown that structural patterns of hypercondensed chromatin reflect a higher-order chromatin and interchromatin domain topology (Albiez et al., 2006). Since the protein scaffolds that play a pivotal role in the formation of chromosomal subdomains are expected to be a primary determinant of the higher-order chromatin and interchromatin domain topology, this result suggests that chromatin coalesces with the underlying protein scaffold into a highly condensed mass during the hypercondensation process, and this process is delimited by the domain structure. Placement of BAF53 knockdown cells into hyper-osmolar medium also led to hypercondensation of chromatin, resulting in bead images similar to those of the control cells. This finding implied that condensation of chromatin under hyper-osmolar conditions is apparently unaltered after BAF53 knockdown. Significantly, however, the interchromatin-lacunas space widened after BAF53 knockdown. This change is more clearly demonstrated by assigning thresholds and highlighting pixels with gray values below the assigned thresholds in blue. The widening of the interchromatin-lacunas space argues that the even distribution of the protein scaffold that contributes to the formation of hypercondensed chromatin bundles, as well as chromosomal subdomains, is at least in part distorted or disrupted by 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