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The quantitative architecture of centromeric chromatin

Publication . Bodor, Dani L; Mata, João F; Sergeev, Mikhail; David, Ana Filipa; Salimian, Kevan J; Panchenko, Tanya; Cleveland, Don W; Black, Ben E; Shah, Jagesh V; Jansen, Lars ET

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.

2014-07-15Journal article

Open access

A Dual Inhibitory Mechanism Sufficient to Maintain Cell-Cycle-Restricted CENP-A Assembly

Publication . Stankovic, Ana; Guo, Lucie Y.; Mata, João F.; Bodor, Dani L.; Cao, Xing-Jun; Bailey, Aaron O.; Shabanowitz, Jeffrey; Hunt, Donald F.; Garcia, Benjamin A.; Black, Ben E.; Jansen, Lars E.T.

Chromatin featuring the H3 variant CENP-A at the centromere is critical for its mitotic function and epigenetic maintenance. Assembly of centromeric chromatin is restricted to G1 phase through inhibitory action of Cdk1/2 kinases in other phases of the cell cycle. Here, we identify the two key targets sufficient to maintain cell-cycle control of CENP-A assembly. We uncovered a single phosphorylation site in the licensing factor M18BP1 and a cyclin A binding site in the CENP-A chaperone, HJURP, that mediated specific inhibitory phosphorylation. Simultaneous expression of mutant proteins lacking these residues results in complete uncoupling from the cell cycle. Consequently, CENP-A assembly is fully recapitulated under high Cdk activities, indistinguishable from G1 assembly. We find that Cdk-mediated inhibition is exerted by sequestering active factors away from the centromere. Finally, we show that displacement of M18BP1 from the centromere is critical for the assembly mechanism of CENP-A.

2017-01-19Journal article

Open access

Assembly in G1 phase and long-term stability are unique intrinsic features of CENP-A nucleosomes

Publication . Bodor, D. L.; Valente, L. P.; Mata, J. F.; Black, B. E.; Jansen, L. E. T.

Centromeres are the site of kinetochore formation during mitosis. Centromere protein A (CENP-A), the centromere-specific histone H3 variant, is essential for the epigenetic maintenance of centromere position. Previously we showed that newly synthesized CENP-A is targeted to centromeres exclusively during early G1 phase and is subsequently maintained across mitotic divisions. Using SNAP-based fluorescent pulse labeling, we now demonstrate that cell cycle-restricted chromatin assembly at centromeres is unique to CENP-A nucleosomes and does not involve assembly of other H3 variants. Strikingly, stable retention is restricted to the CENP-A/H4 core of the nucleosome, which we find to outlast general chromatin across several cell divisions. We further show that cell cycle timing of CENP-A assembly is independent of centromeric DNA sequences and instead is mediated by the CENP-A targeting domain. Unexpectedly, this domain also induces stable transmission of centromeric nucleosomes, independent of the CENP-A deposition factor HJURP. This demonstrates that intrinsic properties of the CENP-A protein direct its cell cycle-restricted assembly and induces quantitative mitotic transmission of the CENP-A/H4 nucleosome core, ensuring long-term stability and epigenetic maintenance of centromere position.

2013-04-01Journal article

Open access

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant

Publication . Earnshaw, W. C.; Allshire, R. C.; Black, B. E.; Bloom, K.; Brinkley, B. R.; Brown, W.; Cheeseman, I. M.; Choo, K. H. A.; Copenhaver, G. P.; DeLuca, J. G.; Desai, A.; Diekmann, S.; Erhardt, S.; Fitzgerald-Hayes, M.; Foltz, D.; Fukagawa, T.; Gassmann, R.; Gerlich, D. W.; Glover, D. M.; Gorbsky, G. J.; Harrison, S. C.; Heun, P.; Hirota, T.; Jansen, L. E. T.; Karpen, G.; Kops, G. J. P. L.; Lampson, M. A.; Lens, S. M.; Losada, A.; Luger, K.; Maiato, H.; Maddox, P. S.; Margolis, R. L.; Masumoto, H.; McAinsh, A. D.; Mellone, B. G.; Meraldi, P.; Musacchio, A.; Oegema, K.; O’Neill, R. J.; Salmon, E. D.; Scott, K. C.; Straight, A. F.; Stukenberg, P. T.; Sullivan, B. A.; Sullivan, K. F.; Sunkel, C. E.; Swedlow, J. R.; Walczak, C. E.; Warburton, P. E.; Westermann, S.; Willard, H. F.; Wordeman, L.; Yanagida, M.; Yen, T. J.; Yoda, K.; Cleveland, D. W.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

2013-04-12Journal article

Open access