The human CENP-A centromeric nucleosome-associated complex

Abstract

The basic element for chromosome inheritance, the centromere, is epigenetically determined in mammals. The prime candidate for specifying centromere identity is the array of nucleosomes assembled with CENP-A, the centromere-specific histone H3 variant. Here, we show that CENP-A nucleosomes directly recruit a proximal CENP-A nucleosome associated complex (NAC) comprised of three new human centromere proteins (CENP-M, CENP-N and CENP-T), along with CENP-U(50), CENP-C and CENP-H. Assembly of the CENP-A NAC at centromeres is dependent on CENP-M, CENP-N and CENP-T. Facilitates chromatin transcription (FACT) and nucleophosmin-1 (previously implicated in transcriptional chromatin remodelling and as a multifunctional nuclear chaperone, respectively) are absent from histone H3-containing nucleosomes, but are stably recruited to CENP-A nucleosomes independent of CENP-A NAC. Seven new CENP-A-nucleosome distal (CAD) centromere components (CENP-K, CENP-L, CENP-O, CENP-P, CENP-Q, CENP-R and CENP-S) are identified as assembling on the CENP-A NAC. The CENP-A NAC is essential, as disruption of the complex causes errors of chromosome alignment and segregation that preclude cell survival despite continued centromere-derived mitotic checkpoint signalling.

Condensin Regulates the Stiffness of Vertebrate Centromeres

“…1) CENP-H and CENP-A (markers for the inner kinetochore) localize as discrete spots that are not distorted in kinetochores undergoing excursions (Figure 2, a and b). CENP-A is a modified histone H3 specific for inner kinetochore chromatin (Warburton et al, 1997;Marshall et al, 2008), and CENP-H purifies with CENP-A containing mononucleosomes in vitro (Foltz et al, 2006). 2) Although we cannot exclude that levels of some kinetochore proteins may differ in condensin-depleted kinetochores, loss of condensin had no detectable effect on the absolute number of CENP-H molecules per kinetochore (29 in SMC2 ON ; 31 in SMC2 OFF ) measured by quantitative fluorescence (Figure 2e) relative to the amount of Ndc80 at budding yeast kinetochores (Joglekar et al, 2006).…”

CENP-O Class Proteins Form a Stable Complex and Are Required for Proper Kinetochore Function

“…In recent years, many kinetochore proteins have been identified in vertebrate cells by various approaches (Cheeseman et al, 2004;Obuse et al, 2004a,b;Minoshima et al, 2005;Foltz et al, 2006;Izuta et al, 2006;McAinsh et al, 2006;Meraldi et al, 2006;Okada et al, 2006). Characterization of the cellular functions of each of these proteins and the protein-protein network within cells will lead to an understanding of how kinetochores assemble and function.…”

Keeping kinetochores on track

“…This approach identified seven novel orthologous kinetochore proteins in mammals, three novel proteins in D. melanogaster and two novel proteins in plants. These results implied that most kinetochore proteins are conserved in eukaryotes, a finding that was confirmed by biochemical and genetic studies identifying those same proteins as bona fide kinetochore proteins (Cheeseman et al, 2004;Foltz et al, 2006;Obuse et al, 2004;Okada et al, 2006;Schittenhelm et al, 2007). The overall conclusion of these studies was that the core of eukaryotic kinetochores is composed of two large conserved protein networks: on one side the KMN network, which consists of Knl-1, the hetero-tetrameric MIND/Mis12 subcomplex, and the hetero-tetrameric-NDC80 subcomplex, and on the other side the CCAN network (Constitutive Centromere Associated Network, which is also called CENP-A NAC/CAD or CENP-H/I complex), which consists of 15 subunits.…”

“…Importantly, the CCAN network is most likely directly controlling microtubule dynamics: at least one CCAN subunit, CENP-Q, can efficiently bind microtubules in vitro and our live cell imaging of GFP-CENP-I, another CCAN subunit, indicates that it preferentially accumulates on the sister-kinetochore bound to growing microtubules, a behaviour that is only known for a few microtubule-binding proteins . Interestingly, the CCAN network directly binds to the centromeric CENP-A nucleosomes and contributes to the assembly of the centromeric nucleosomes, suggesting that it acts as a link between centromeric DNA and the microtubule plus-ends (Carroll et al, 2009;Foltz et al, 2006;Okada et al, 2006). One critical challenge for the future will be to determine the molecular mechanisms by which the CCAN network controls the turnover rate of kinetochore-microtubules, and the precise function of the individual components of this large protein network in this process.…”