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The centromere, responsible for chromosome segregation during mitosis, is epigenetically defined by CENP-A containing chromatin. The amount of centromeric CENP-A has direct implications for both the architecture and epigenetic inheritance of centromeres. Using complementary strategies, we determined that typical human centromeres contain ∼400 molecules of CENP-A, which is controlled by a mass-action mechanism. This number, despite representing only ∼4% of all centromeric nucleosomes, forms a ∼50-fold enrichment to the overall genome. In addition, although pre-assembled CENP-A is randomly segregated during cell division, this amount of CENP-A is sufficient to prevent stochastic loss of centromere function and identity. Finally, we produced a statistical map of CENP-A occupancy at a human neocentromere and identified nucleosome positions that feature CENP-A in a majority of cells. In summary, we present a quantitative view of the centromere that provides a mechanistic framework for both robust epigenetic inheritance of centromeres and the paucity of neocentromere formation.DOI: http://dx.doi.org/10.7554/eLife.02137.001., (Copyright © 2014, Bodor et al.)

Active centromeres are marked by nucleosomes assembled with CENP-A, a centromere-specific histone H3 variant. The CENP-A centromere targeting domain (CATD), comprised of loop 1 and the alpha2 helix within the histone fold, is sufficient to target histone H3 to centromeres and to generate the same conformational rigidity to the initial subnucleosomal heterotetramer with histone H4 as does CENP-A. We now show in human cells and in yeast that depletion of CENP-A is lethal, but recruitment of normal levels of kinetochore proteins, centromere-generated mitotic checkpoint signaling, chromosome segregation, and viability can be rescued by histone H3 carrying the CATD. These data offer direct support for centromere identity maintained by a unique nucleosome that serves to distinguish the centromere from the rest of the chromosome.

Centromere protein F (CENP-F) (or mitosin) accumulates to become an abundant nuclear protein in G2, assembles at kinetochores in late G2, remains kinetochore-bound until anaphase, and is degraded at the end of mitosis. Here we show that the absence of nuclear CENP-F does not affect cell cycle progression in S and G2. In a subset of CENP-F depleted cells, kinetochore assembly fails completely, thereby provoking massive chromosome mis-segregation. In contrast, the majority of CENP-F depleted cells exhibit a strong mitotic delay with reduced tension between kinetochores of aligned, bi-oriented sister chromatids and decreased stability of kinetochore microtubules. These latter kinetochores generate mitotic checkpoint signaling when unattached, recruiting maximum levels of Mad2. Use of YFP-marked Mad1 reveals that throughout the mitotic delay some aligned, CENP-F depleted kinetochores continuously recruit Mad1. Others rebind YFP-Mad1 intermittently so as to produce ‘twinkling’, demonstrating cycles of mitotic checkpoint reactivation and silencing and a crucial role for CENP-F in efficient assembly of a stable microtubule–kinetochore interface. [ABSTRACT FROM AUTHOR]

While the physical nature of the epigenetic mark defining the location of the centromere on the chromosome is not known, nucleosomes in which CENP-A replaces histone H3 are at the foundation of the centromeric chromatin. Hydrogen/deuterium exchange mass spectrometry is now used to show that assembly into nucleosomes imposes stringent conformational constraints, reducing solvent accessibility in almost all histone regions by >L3 orders of magnitude. Nucleosomes assembled with CENP-A are substantially more conformationally rigid than those assembled with histone H3, independent of DNA template. Substitution of the CATD, comprised of the loop 1 and α2 helix of the CENP-A histone fold domain, into histone H3 generates the same more rigid nucleosome as does CENP-A. In human cells, while depletion of CENPA is lethal, recruitment of normal levels of kinetochore proteins, centromere-generated mitotic checkpoint signaling, normal chromosome segregation and viability can be rescued by this chimeric H3 containing the CATD. The CATD, itself, is confined to the structured "core" of the nucleosome demonstrating that much (if not all) of the essential features of CENP-A are within the rigid core of the nucleosome. These data support a model of centromere identity maintained by a unique nucleosome structure that serves to distinguish the centromere from the rest of the chromosome. [ABSTRACT FROM AUTHOR]