Cell-Cycle-Dependent Structural Transitions in the Human CENP-A Nucleosome In Vivo

Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, NIH, Bethesda, MD 20892, USA.
Cell (Impact Factor: 32.24). 07/2012; 150(2):317-26. DOI: 10.1016/j.cell.2012.05.035
Source: PubMed


In eukaryotes, DNA is packaged into chromatin by canonical histone proteins. The specialized histone H3 variant CENP-A provides an epigenetic and structural basis for chromosome segregation by replacing H3 at centromeres. Unlike exclusively octameric canonical H3 nucleosomes, CENP-A nucleosomes have been shown to exist as octamers, hexamers, and tetramers. An intriguing possibility reconciling these observations is that CENP-A nucleosomes cycle between octamers and tetramers in vivo. We tested this hypothesis by tracking CENP-A nucleosomal components, structure, chromatin folding, and covalent modifications across the human cell cycle. We report that CENP-A nucleosomes alter from tetramers to octamers before replication and revert to tetramers after replication. These structural transitions are accompanied by reversible chaperone binding, chromatin fiber folding changes, and previously undescribed modifications within the histone fold domains of CENP-A and H4. Our results reveal a cyclical nature to CENP-A nucleosome structure and have implications for the maintenance of epigenetic memory after centromere replication.

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Available from: Minh Bui, Oct 13, 2015
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    • "Using atomic force microscopy (AFM), they suggested that CENP-A nucleosomes are half the height of canonical nucleosomes (Dimitriadis et al., 2010). More recently, studies in human cells (Bui et al., 2012) and budding yeast (Shivaraju et al., 2012) proposed that the CENP-A-containing nucleosomes are dynamic, oscillating between octameric and tetrameric forms during cell-cycle progression. "
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    ABSTRACT: Since discovery of the centromere-specific histone H3 variant CENP-A, centromeres have come to be defined as chromatin structures that establish the assembly site for the complex kinetochore machinery. In most organisms, centromere activity is defined epigenetically, rather than by specific DNA sequences. In this review, we describe selected classic work and recent progress in studies of centromeric chromatin with a focus on vertebrates. We consider possible roles for repetitive DNA sequences found at most centromeres, chromatin factors and modifications that assemble and activate CENP-A chromatin for kinetochore assembly, plus the use of artificial chromosomes and kinetochores to study centromere function.
    Developmental Cell 09/2014; 30(5):496-508. DOI:10.1016/j.devcel.2014.08.016 · 9.71 Impact Factor
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    • "In budding yeast, it has also been suggested that both H3 and Cse4 histones co-exist in a single nucleosome on CEN DNA, forming an octameric hybrid (heterotypic) nucleosome together with two copies each of H4, H2A and H2B (27). Recent work attempted to reconcile these conflicting results by suggesting that CENP-A nucleosomes undergoes structural transitions in a cell-cycle-specific manner (28,29); however, this was disputed by subsequent reports (25,26,30). As such, the structure of centromeric nucleosomes in vivo throughout various stages of the cell cycle remains controversial. "
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    ABSTRACT: The assembly of centromeric nucleosomes is mediated by histone variant-specific chaperones. In budding yeast, the centromere-specific histone H3 variant is Cse4, and the histone chaperone Scm3 functions as a Cse4-specific nucleosome assembly factor. Here, we show that Scm3 exhibits specificity for Cse4–H4, but also interacts with major-type H3–H4 and H2A–H2B. Previously published structures of the Scm3 histone complex demonstrate that Scm3 binds only one copy of Cse4–H4. Consistent with this, we show that Scm3 deposits Cse4–H4 through a dimer intermediate onto deoxyribonucleic acid (DNA) to form a (Cse4–H4)2–DNA complex (tetrasome). Scm3-bound Cse4–H4 does not form a tetramer in the absence of DNA. Moreover, we demonstrate that Cse4 and H3 are structurally compatible to be incorporated in the same nucleosome to form heterotypic particles. Our data shed light on the mechanism of Scm3-mediated nucleosome assembly at the centromere.
    Nucleic Acids Research 03/2014; 42(9). DOI:10.1093/nar/gku205 · 9.11 Impact Factor
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    • "These studies combined with our results suggest that H3(T118ph) could function with H3(K122) acetylation to destabilize the nucleosome structure and form altosomes to regulate transcription. Recent reports suggest that acetylation of the centromeric H3 homolog CENP-A at K124 in the dyad is correlated with conversion of octameric nucleosomes in the centromere to a four-histone hemisome structure, which is hypothesized to play a role in regulation of DNA replication (46). Taken in aggregate, these studies hint at major roles for the dyad region in regulating aspects of nucleosome structure. "
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    ABSTRACT: Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA-histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure.
    Nucleic Acids Research 02/2014; 42(8). DOI:10.1093/nar/gku150 · 9.11 Impact Factor
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