The CENP-H–I complex is required for the efficient incorporation of newly synthesized CENP-A into centromeres

Abstract

In vertebrates, centromeres lack defined sequences and are thought to be propagated by epigenetic mechanisms involving the incorporation of specialized nucleosomes containing the histone H3 variant centromere protein (CENP)-A. However, the precise mechanisms that target CENP-A to centromeres remain poorly understood. Here, we isolated a multi-subunit complex, which includes the established inner kinetochore components CENP-H and CENP-I, and nine other proteins, from both human and chicken cells. Our analysis of these proteins demonstrates that the CENP-H-I complex can be divided into three functional sub-complexes, each of which is required for faithful chromosome segregation. Interestingly, newly expressed CENP-A is not efficiently incorporated into centromeres in knockout mutants of a subclass of CENP-H-I complex proteins, indicating that the CENP-H-I complex may function, in part, as a marker directing CENP-A deposition to centromeres.

Hierarchical Inactivation of a Synthetic Human Kinetochore by a Chromatin Modifier

“…CENP-A is upstream of almost all other known components in the kinetochore assembly pathway. However, that pathway is multiplex, as recent studies in chicken and Drosophila show that inner kinetochore proteins CENP-H and -C are required for normal CENP-A loading or retention (Okada et al, 2006;Goshima et al, 2007;Erhardt et al, 2008).…”

“…CENP-A is upstream of almost all other known components in the kinetochore assembly pathway. However, that pathway is multiplex, as recent studies in chicken and Drosophila show that inner kinetochore proteins CENP-H and -C are required for normal CENP-A loading or retention (Okada et al, 2006;Goshima et al, 2007;Erhardt et al, 2008).Our work was inspired by an approach first developed a number of years ago in which cloned fragments of human centromeric DNA were used to form human artificial chromosomes (HACs) in HT1080 fibrosarcoma cells (Harrington et al, 1997;Ikeno et al, 1998). Originally, HAC formation was only achieved with regular repeated arrays of ␣-satellite DNA containing CENP-B boxes Ohzeki et al, 2002;Okada et al, 2007).…”

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.…”