Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N

Department of Biochemistry, Stanford University School of Medicine, Beckman Center, Palo Alto, CA 94503-5307, USA.
Nature Cell Biology (Impact Factor: 19.68). 07/2009; 11(7):896-902. DOI: 10.1038/ncb1899
Source: PubMed


Centromeres are specialized chromosomal domains that direct kinetochore assembly during mitosis. CENP-A (centromere protein A), a histone H3-variant present exclusively in centromeric nucleosomes, is thought to function as an epigenetic mark that specifies centromere identity. Here we identify the essential centromere protein CENP-N as the first protein to selectively bind CENP-A nucleosomes but not H3 nucleosomes. CENP-N bound CENP-A nucleosomes in a DNA sequence-independent manner, but did not bind soluble CENP-A-H4 tetramers. Mutations in CENP-N that reduced its affinity for CENP-A nucleosomes caused defects in CENP-N localization and had dominant effects on the recruitment of CENP-H, CENP-I and CENP-K to centromeres. Depletion of CENP-N using siRNA (short interfering RNA) led to similar centromere assembly defects and resulted in reduced assembly of nascent CENP-A into centromeric chromatin. These data suggest that CENP-N interprets the information encoded within CENP-A nucleosomes and recruits other proteins to centromeric chromatin that are required for centromere function and propagation.

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Available from: Lars Jansen, Feb 24, 2014
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    • "Because reducing H4K20me1 levels at centromeres causes mislocalization of CENP-H and CENP-T, this modification might help render CENP-A chromatin competent for kinetochore assembly (Figure 6). Other histone modifications of CENP-A nucleosomes or of centromeric nucleosomes containing canonical H3 might also be involved in formation of functional centromeric chromatin, possibly by influencing interactions with other kinetochore proteins such as CENP-C or CENP-N, which bind to CENP-A nucleosomes concentrated in centromeres (Carroll et al., 2009; Guse et al., 2011; Kato et al., 2013). "
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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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    • "Recently, it has been demonstrated that CENP-C and CENP-T, which were described as components of the NAC, are crucial for the formation of the kinetochore in both human and Drosophila cells [58]–[61]. Moreover, CENP-C and CENP-N were shown to bind directly to CENP-A, but not to H3, -containing nucleosomes [62], [63]. CENP-N is a key subunit of the CCAN that is required for the loading of all other CCAN subunits onto kinetochores [15], [17], [62]. "
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    ABSTRACT: The viral E3 ubiquitin ligase ICP0 protein has the unique property to temporarily localize at interphase and mitotic centromeres early after infection of cells by the herpes simplex virus type 1 (HSV-1). As a consequence ICP0 induces the proteasomal degradation of several centromeric proteins (CENPs), namely CENP-A, the centromeric histone H3 variant, CENP-B and CENP-C. Following ICP0-induced centromere modification cells trigger a specific response to centromeres called interphase Centromere Damage Response (iCDR). The biological significance of the iCDR is unknown; so is the degree of centromere structural damage induced by ICP0. Interphase centromeres are complex structures made of proximal and distal protein layers closely associated to CENP-A-containing centromeric chromatin. Using several cell lines constitutively expressing GFP-tagged CENPs, we investigated the extent of the centromere destabilization induced by ICP0. We show that ICP0 provokes the disappearance from centromeres, and the proteasomal degradation of several CENPs from the NAC (CENP-A nucleosome associated) and CAD (CENP-A Distal) complexes. We then investigated the nucleosomal occupancy of the centromeric chromatin in ICP0-expressing cells by micrococcal nuclease (MNase) digestion analysis. ICP0 expression either following infection or in cell lines constitutively expressing ICP0 provokes significant modifications of the centromeric chromatin structure resulting in higher MNase accessibility. Finally, using human artificial chromosomes (HACs), we established that ICP0-induced iCDR could also target exogenous centromeres. These results demonstrate that, in addition to the protein complexes, ICP0 also destabilizes the centromeric chromatin resulting in the complete breakdown of the centromere architecture, which consequently induces iCDR.
    PLoS ONE 09/2012; 7(9):e44227. DOI:10.1371/journal.pone.0044227 · 3.23 Impact Factor
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    • "Kinetochore localization is determined by CENP-A which is recognized by CENP-N and CENP-C [14]–[16]. CENP-L binds to the C-terminal region of CENP-N in vitro [14] and CENP-K kinetochore localisation depends on the presence of CENP-N and -C [5], [8], [14], [52]. We therefore asked, if these kinetochore proteins, being directly or closely linked to CENP-A, are able to recruit single CENP-PORQU proteins or the whole complex to an ectopic chromatin site in human cell nuclei in vivo. "
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    ABSTRACT: Kinetochores are multi-protein megadalton assemblies that are required for attachment of microtubules to centromeres and, in turn, the segregation of chromosomes in mitosis. Kinetochore assembly is a cell cycle regulated multi-step process. The initial step occurs during interphase and involves loading of the 15-subunit constitutive centromere associated complex (CCAN), which contains a 5-subunit (CENP-P/O/R/Q/U) sub-complex. Here we show using a fluorescent three-hybrid (F3H) assay and fluorescence resonance energy transfer (FRET) in living mammalian cells that CENP-P/O/R/Q/U subunits exist in a tightly packed arrangement that involves multifold protein-protein interactions. This sub-complex is, however, not pre-assembled in the cytoplasm, but rather assembled on kinetochores through the step-wise recruitment of CENP-O/P heterodimers and the CENP-P, -O, -R, -Q and -U single protein units. SNAP-tag experiments and immuno-staining indicate that these loading events occur during S-phase in a manner similar to the nucleosome binding components of the CCAN, CENP-T/W/N. Furthermore, CENP-P/O/R/Q/U binding to the CCAN is largely mediated through interactions with the CENP-N binding protein CENP-L as well as CENP-K. Once assembled, CENP-P/O/R/Q/U exchanges slowly with the free nucleoplasmic pool indicating a low off-rate for individual CENP-P/O/R/Q/U subunits. Surprisingly, we then find that during late S-phase, following the kinetochore-binding step, both CENP-Q and -U but not -R undergo oligomerization. We propose that CENP-P/O/R/Q/U self-assembles on kinetochores with varying stoichiometry and undergoes a pre-mitotic maturation step that could be important for kinetochores switching into the correct conformation necessary for microtubule-attachment.
    PLoS ONE 09/2012; 7(9):e44717. DOI:10.1371/journal.pone.0044717 · 3.23 Impact Factor
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