Abstract
In budding yeast, a single cenH3 (Cse4) nucleosome occupies the ∼120-bp functional centromere, however conflicting structural models for the particle have been proposed. To resolve this controversy, we have applied H4S47C-anchored cleavage mapping, which reveals the precise position of histone H4 in every nucleosome in the genome. We find that cleavage patterns at centromeres are unique within the genome and are incompatible with symmetrical structures, including octameric nucleosomes and (Cse4/H4)2 tetrasomes. Centromere cleavage patterns are compatible with a precisely positioned core structure, one in which each of the 16 yeast centromeres is occupied by oppositely oriented Cse4/H4/H2A/H2B hemisomes in two rotational phases within the population. Centromere-specific hemisomes are also inferred from distances observed between closely-spaced H4 cleavages, as predicted from structural modeling. Our results indicate that the orientation and rotational position of the stable hemisome at each yeast centromere is not specified by the functional centromere sequence. DOI: http://dx.doi.org/10.7554/eLife.01861.001.
Highlights
Centromeres are the genetic loci that organize the proteinaceous kinetochore, which attaches to spindle microtubules to pull the chromosomes to the poles in both mitosis and meiosis
In the original description of H4S47C-anchored cleavage mapping, nucleosome centers were determined as clusters of cleavages around the dyad axes of highly occupied and phased nucleosomes throughout the budding yeast genome (Brogaard et al, 2012b). It appeared that cleavages at centromeres were different from cleavages at other nucleosomes, the empirical model used to interpret cleavage patterns did not permit further inferences concerning the structure and composition of the particle over centromeric DNA
Cleavages at the two closely spaced H4S47C residues can potentially result in
Summary
Centromeres are the genetic loci that organize the proteinaceous kinetochore, which attaches to spindle microtubules to pull the chromosomes to the poles in both mitosis and meiosis. There is general agreement in the centromere field that the central determinant of centromere identity and propagation is the special centromeric nucleosome containing the cenH3 (CENP-A in mammals and Cse in budding yeast) histone variant (Quenet and Dalal, 2012). In vitro and in vivo studies have led to proposals for several mutually exclusive models, including conventional octameric (cenH3/H4/H2B/H2A) nucleosomes (‘octasomes’) (Camahort et al, 2009), cenH3/H4/H2B/H2A half-nucleosomes (‘hemisomes’) (Dalal et al, 2007), (cenH3/H4) tetrasomes (Aravamudhan et al, 2013), mixed (cenH3/H3/H42/H2B2/H2A2) octasomes (Lochmann and Ivanov, 2012) and (cenH3/H4/Scm3) hexasomes (Mizuguchi et al, 2007), where Scm is a cenH3-specific histone chaperone. Evidence for each of these conflicting models has been presented for budding yeast, where the centromere is genetically defined by an ∼120-bp functional sequence on each of the 16 chromosomes
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