De Novo Kinetochore Assembly Requires the Centromeric Histone H3 Variant

Abstract

Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G 1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin. INTRODUCTIONAccurate chromosome segregation in mitosis and meiosis is essential for the maintenance of genomic stability. Chromosomes attach to the mitotic spindle at the kinetochore, the protein complex that assembles onto centromeric DNA. Although kinetochore function is conserved, the underlying centromeric DNA is highly variable. Budding yeast contain a 125-base pair sequence-specific centromere that is sufficient for kinetochore formation (Fitzgerald-Hayes et al., 1982). In contrast, centromeres in multicellular eukaryotes are composed of megabases of highly repetitive DNA that lack sequence specificity (for review, see Sullivan et al., 2001). In these organisms, kinetochore assembly seems to be propagated by unidentified epigenetic component(s) (Karpen and Allshire, 1997;Sullivan et al., 2001).The best-characterized kinetochore is in budding yeast where Ͼ65 components have been identified that constitutively localize to the kinetochore (for reviews, see Biggins and Walczak, 2003;McAinsh et al., 2003). Most of the yeast kinetochore proteins are found in biochemically distinct complexes known as the CBF3, CTF19/COMA, MTW1, NDC80, and DAM1 complexes that seem to assemble on a single centromeric nucleosome (Meluh et al., 1998). Although the exact architecture of the kinetochore is not known, dependency relationships subdivide the kinetochore into inner, central, and outer domains. The inner kinetochore contains the CBF3 complex (Ndc10, Cep3, Skp1, and Ctf13) as well as the DNA binding proteins Mif2, Cbf1, and the yeast centromeric histone H3 variant (CenH3) Cse4. CBF3 binds directly to the centromeric DNA and is thought to nucleate kinetochore assembly because all kinetochore proteins require it for localization (Russell et al., 1999;Goshima and Yanagida, 2000;He et al., 2001;Janke et al., 2001Janke et al., , 2002. The central kinetochore contains the MTW1 (Mtw1, Dsn1, Nnf1, and Nsl1) and CTF19/COMA (Ctf19, Mcm16, Mcm19, Mcm21, Mcm22, Ctf3, Chl4, Okp1, Ame1, Iml3, Nkp1, and Nkp2) complexes. CTF19/COMA can be further divided into two subcomplexes, with Ame1...

The Composition, Functions, and Regulation of the Budding Yeast Kinetochore

“…Although dicentric chromosomes are normally unstable and lost during cell division, the galactose promoter controls the second centromere in this assay and transcription through the centromere abolishes its function. When the cells are shifted into glucose, transcription through the centromere is halted allowing a second kinetochore to form, which can subsequently be assayed for the de novo assembly of kinetochore proteins and other kinetochore functions (Tanaka et al 1999;Mythreye and Bloom 2003;Collins et al 2005). One-hybrid assays can also identify kinetochore proteins (Ortiz et al 1999).…”

Kinetochore Function and Chromosome Segregation Rely on Critical Residues in Histones H3 and H4 in Budding Yeast

“…Chromatin immunoprecipitation assays and quantitative real-time PCR Chromatin immunoprecipitation (ChIP) was performed using antibodies against Cse4 as described previously (Collins et al 2005), and samples were quantified by quantitative real-time PCR (7900HT, ABI Prism). DNA samples were amplified using a SYBR PCR mix (Applied Biosystems) at 95°f or 10 min, 40 cycles of 95°for 15 sec, and 55°for 1 min using CEN3 (SB1253 and SB1254) and PHO5-specific primers (SB3063 and SB3064).…”

Synthetic physical interactions map kinetochore regulators and regions sensitive to constitutive Cdc14 localization

“…The kinetochore consists of at least 60 unique gene products present in multiple copies that stretch from the specialized H3 histone subunit (Cse4, CENP-A in mammals) to the microtubule binding proteins of the Dam1 complex (9). Kinetochores normally assemble hierarchically from the centromere-bound proteins (10). Once assembled, the structural homeostasis of the kinetochore is regulated by proteins that are recruited to kinetochores.…”