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The CENP-A nucleosome: a battle between Dr Jekyll and Mr Hyde.

Bui M, Walkiewicz MP, Dimitriadis EK, Dalal Y - Nucleus (2013)

Bottom Line: The structure of the centromere-specific histone centromeric protein A (CENP-A) nucleosome has been a hot topic of debate for the past five years.Structures proposed include octamers, hexamers, homotypic and heterotypic tetramers.This controversy has led to the proposal that CENP-A nucleosomes undergo cell-cycle dependent transitions between the multiple states previously documented to exist in vivo and in vitro.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD USA.

ABSTRACT
The structure of the centromere-specific histone centromeric protein A (CENP-A) nucleosome has been a hot topic of debate for the past five years. Structures proposed include octamers, hexamers, homotypic and heterotypic tetramers. This controversy has led to the proposal that CENP-A nucleosomes undergo cell-cycle dependent transitions between the multiple states previously documented to exist in vivo and in vitro. In recent work from our laboratory, we sought to test this hypothesis. We discovered that CENP-A nucleosomes undergo unique oscillations in human cells, a finding mirrored in a parallel study performed in budding yeast. This review provides additional insights into the potential mechanisms for the interconversion of CENP-A nucleosomal species, and speculates on a biological role for oscillations in vivo.

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Figure 1. Comparing in vitro reconstituted CENP-A nucleosomes to CENP-A nucleosome purified from human cells using AFM. (A) AFM micrographs showing in vitro reconstituted bulk nucleosomes onto plasmids containing α-satellite DNA derived from human centromeres (left), in vitro reconstituted chromatin digested for 1 min with MNase (middle), and IP’ed in vitro reconstituted CENP-A chromatin digested with MNase (right). Scale bar = 10 nm. (B) Graphical representation of AFM measurements comparing in vitro (listed above and Table 1) and in vivo samples from Bui et al., 2012. Dotted red line designates nucleosomes of mean octameric heights.
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Figure 1: Figure 1. Comparing in vitro reconstituted CENP-A nucleosomes to CENP-A nucleosome purified from human cells using AFM. (A) AFM micrographs showing in vitro reconstituted bulk nucleosomes onto plasmids containing α-satellite DNA derived from human centromeres (left), in vitro reconstituted chromatin digested for 1 min with MNase (middle), and IP’ed in vitro reconstituted CENP-A chromatin digested with MNase (right). Scale bar = 10 nm. (B) Graphical representation of AFM measurements comparing in vitro (listed above and Table 1) and in vivo samples from Bui et al., 2012. Dotted red line designates nucleosomes of mean octameric heights.

Mentions: We first confirmed the default “ground” state of CENP-A nucleosomes, reconstituted in vitro in the absence of any biological processes or chaperones, is octameric. Following standard salt dialysis of histones purified from human cells22 combined with plasmid DNA containing α-satellite arrays, we observed that bulk histones form chromatin arrays packaged with nucleosomes whose sizes were consistent with those of canonical octamers (Fig. 1 and Table 1). Next, to mimic conditions typically used for in vivo purifications, we gently digested these bulk in vitro arrays with Micrococcal Nuclease (MNase) and immuno-precipitated (IP’ed) CENP-A-containing nucleosomes from the reconstituted mix. Analysis of such in vitro reconstituted CENP-A particles indicated that their sizes corresponded closely to those of canonical octameric nucleosomes (Fig. 1 and Table 1). These in vitro data are consistent with previous reports suggesting that the octamer is the default structure for CENP-A nucleosomes assembled by salt dialysis.4,5 This experiment also suggests that there is no inherent instability within octameric CENP-A nucleosomes, at least when subjected to experimental conditions such as nuclease digestion, IP or AFM analyses. The logical conclusion from these results is that alternative species observed for native CENP-A nucleosomes in vivo are not likely to be generated from technical artifacts arising during purification or analysis.


The CENP-A nucleosome: a battle between Dr Jekyll and Mr Hyde.

Bui M, Walkiewicz MP, Dimitriadis EK, Dalal Y - Nucleus (2013)

Figure 1. Comparing in vitro reconstituted CENP-A nucleosomes to CENP-A nucleosome purified from human cells using AFM. (A) AFM micrographs showing in vitro reconstituted bulk nucleosomes onto plasmids containing α-satellite DNA derived from human centromeres (left), in vitro reconstituted chromatin digested for 1 min with MNase (middle), and IP’ed in vitro reconstituted CENP-A chromatin digested with MNase (right). Scale bar = 10 nm. (B) Graphical representation of AFM measurements comparing in vitro (listed above and Table 1) and in vivo samples from Bui et al., 2012. Dotted red line designates nucleosomes of mean octameric heights.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585026&req=5

Figure 1: Figure 1. Comparing in vitro reconstituted CENP-A nucleosomes to CENP-A nucleosome purified from human cells using AFM. (A) AFM micrographs showing in vitro reconstituted bulk nucleosomes onto plasmids containing α-satellite DNA derived from human centromeres (left), in vitro reconstituted chromatin digested for 1 min with MNase (middle), and IP’ed in vitro reconstituted CENP-A chromatin digested with MNase (right). Scale bar = 10 nm. (B) Graphical representation of AFM measurements comparing in vitro (listed above and Table 1) and in vivo samples from Bui et al., 2012. Dotted red line designates nucleosomes of mean octameric heights.
Mentions: We first confirmed the default “ground” state of CENP-A nucleosomes, reconstituted in vitro in the absence of any biological processes or chaperones, is octameric. Following standard salt dialysis of histones purified from human cells22 combined with plasmid DNA containing α-satellite arrays, we observed that bulk histones form chromatin arrays packaged with nucleosomes whose sizes were consistent with those of canonical octamers (Fig. 1 and Table 1). Next, to mimic conditions typically used for in vivo purifications, we gently digested these bulk in vitro arrays with Micrococcal Nuclease (MNase) and immuno-precipitated (IP’ed) CENP-A-containing nucleosomes from the reconstituted mix. Analysis of such in vitro reconstituted CENP-A particles indicated that their sizes corresponded closely to those of canonical octameric nucleosomes (Fig. 1 and Table 1). These in vitro data are consistent with previous reports suggesting that the octamer is the default structure for CENP-A nucleosomes assembled by salt dialysis.4,5 This experiment also suggests that there is no inherent instability within octameric CENP-A nucleosomes, at least when subjected to experimental conditions such as nuclease digestion, IP or AFM analyses. The logical conclusion from these results is that alternative species observed for native CENP-A nucleosomes in vivo are not likely to be generated from technical artifacts arising during purification or analysis.

Bottom Line: The structure of the centromere-specific histone centromeric protein A (CENP-A) nucleosome has been a hot topic of debate for the past five years.Structures proposed include octamers, hexamers, homotypic and heterotypic tetramers.This controversy has led to the proposal that CENP-A nucleosomes undergo cell-cycle dependent transitions between the multiple states previously documented to exist in vivo and in vitro.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD USA.

ABSTRACT
The structure of the centromere-specific histone centromeric protein A (CENP-A) nucleosome has been a hot topic of debate for the past five years. Structures proposed include octamers, hexamers, homotypic and heterotypic tetramers. This controversy has led to the proposal that CENP-A nucleosomes undergo cell-cycle dependent transitions between the multiple states previously documented to exist in vivo and in vitro. In recent work from our laboratory, we sought to test this hypothesis. We discovered that CENP-A nucleosomes undergo unique oscillations in human cells, a finding mirrored in a parallel study performed in budding yeast. This review provides additional insights into the potential mechanisms for the interconversion of CENP-A nucleosomal species, and speculates on a biological role for oscillations in vivo.

Show MeSH
Related in: MedlinePlus