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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

The dissociation of DNA loops from the nuclear scaffold in BAF53 knockdown cells. (A) Nonfixed nuclei on coverslips were submitted to the maximum fluorescent halo technique (MFHT) for DNA loop-size measurements. Immunostaining with an anti-lamin antibody delimitated the matrix from DNA loops. Scale bar, 5 μm. (B) The mean of the halo length of each cell was plotted versus the standard deviation of the halo length of the cell. t-test; P < 0.001.
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f4-molce-38-9-789: The dissociation of DNA loops from the nuclear scaffold in BAF53 knockdown cells. (A) Nonfixed nuclei on coverslips were submitted to the maximum fluorescent halo technique (MFHT) for DNA loop-size measurements. Immunostaining with an anti-lamin antibody delimitated the matrix from DNA loops. Scale bar, 5 μm. (B) The mean of the halo length of each cell was plotted versus the standard deviation of the halo length of the cell. t-test; P < 0.001.

Mentions: According to the multi-loop subcompartment model, a chromosomal subdomain is a cluster of DNA loops (Munkel et al., 1999). Because the dissociation of DNA loops from the nuclear scaffold would result in the disintegration of the chromosomal subdomain, we investigated whether BAF53 knockdown affects the attachment of the DNA loop to the nuclear scaffold. We measured the chromatin loop sizes using the maximum fluorescent halo technique (Vogelstein et al., 1980). Nuclei were extracted with 2 M NaCl, and then were exposed for 1 min to short-wave ultraviolet (UV) light in the presence of ethidium bromide to observe the DNA halo. The nuclear scaffold was distinguished from the halo by immunostaining with anti-lamin A/C antibody (Fig. 4).


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

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

The dissociation of DNA loops from the nuclear scaffold in BAF53 knockdown cells. (A) Nonfixed nuclei on coverslips were submitted to the maximum fluorescent halo technique (MFHT) for DNA loop-size measurements. Immunostaining with an anti-lamin antibody delimitated the matrix from DNA loops. Scale bar, 5 μm. (B) The mean of the halo length of each cell was plotted versus the standard deviation of the halo length of the cell. t-test; P < 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588722&req=5

f4-molce-38-9-789: The dissociation of DNA loops from the nuclear scaffold in BAF53 knockdown cells. (A) Nonfixed nuclei on coverslips were submitted to the maximum fluorescent halo technique (MFHT) for DNA loop-size measurements. Immunostaining with an anti-lamin antibody delimitated the matrix from DNA loops. Scale bar, 5 μm. (B) The mean of the halo length of each cell was plotted versus the standard deviation of the halo length of the cell. t-test; P < 0.001.
Mentions: According to the multi-loop subcompartment model, a chromosomal subdomain is a cluster of DNA loops (Munkel et al., 1999). Because the dissociation of DNA loops from the nuclear scaffold would result in the disintegration of the chromosomal subdomain, we investigated whether BAF53 knockdown affects the attachment of the DNA loop to the nuclear scaffold. We measured the chromatin loop sizes using the maximum fluorescent halo technique (Vogelstein et al., 1980). Nuclei were extracted with 2 M NaCl, and then were exposed for 1 min to short-wave ultraviolet (UV) light in the presence of ethidium bromide to observe the DNA halo. The nuclear scaffold was distinguished from the halo by immunostaining with anti-lamin A/C antibody (Fig. 4).

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