Abstract
During cell division, segregation of sister chromatids to daughter cells is achieved by the poleward pulling force of microtubules, which attach to the chromatids by means of a multiprotein complex, the kinetochore. Kinetochores assemble at the centromeric DNA organized by specialized centromeric nucleosomes. In contrast to other eukaryotes, which typically have large repetitive centromeric regions, budding yeast CEN DNA is defined by a 125 bp sequence and assembles a single centromeric nucleosome. In budding yeast, as well as in other eukaryotes, the Cse4 histone variant (known in vertebrates as CENP-A) is believed to substitute for histone H3 at the centromeric nucleosome. However, the exact composition of the CEN nucleosome remains a subject of debate. We report the use of a novel ChIP approach to reveal the composition of the centromeric nucleosome and its localization on CEN DNA in budding yeast. Surprisingly, we observed a strong interaction of H3, as well as Cse4, H4, H2A, and H2B, but not histone chaperone Scm3 (HJURP in human) with the centromeric DNA. H3 localizes to centromeric DNA at all stages of the cell cycle. Using a sequential ChIP approach, we could demonstrate the co-occupancy of H3 and Cse4 at the CEN DNA. Our results favor a H3-Cse4 heterotypic octamer at the budding yeast centromere. Whether or not our model is correct, any future model will have to account for the stable association of histone H3 with the centromeric DNA.
Highlights
During eukaryotic cell division sister chromatids, containing identical copies of genetic information, are pulled apart and driven towards opposite spindle poles by the microtubules of the mitotic spindle, which attach to the centromeric DNA sequences of the sisters via kinetochore protein complexes
During cell division, replicated DNA molecules are pulled to daughter cells by microtubules, which originate at the spindle poles and attach to a multiprotein complex, the kinetochore
It is believed that the centromeric nucleosome is devoid of histone H3 and instead contains its variant, termed CENP-A in vertebrates or Cse4 in budding yeast
Summary
During eukaryotic cell division sister chromatids, containing identical copies of genetic information, are pulled apart and driven towards opposite spindle poles by the microtubules of the mitotic spindle, which attach to the centromeric DNA sequences of the sisters via kinetochore protein complexes. It is imperative for proper chromosomal segregation that each chromosome assembles the kinetochore only at one site. Centromeric nucleosomes were reported to have histone H3 substituted by a histone variant, CENP-A, called Cse in budding yeast [2]. Budding yeast [22] and fission yeast [14,23,24] appear to be an exception, in several organisms CENP-A is loaded on the DNA outside of S phase, in anaphase of mitosis or the following G1 [25,26], when it is proposed to replace histone H3
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