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Dynamics of Histone H3 Deposition In Vivo Reveal a Nucleosome Gap-Filling Mechanism for H3.3 to Maintain Chromatin Integrity

Publication . Ray-Gallet, Dominique; Woolfe, Adam; Vassias, Isabelle; Pellentz, Céline; Lacoste, Nicolas; Puri, Aastha; Schultz, David C.; Pchelintsev, Nikolay A.; Adams, Peter D.; Jansen, Lars E.T.; Almouzni, Geneviève

Establishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. We propose that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. We discuss the importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity.

2011-12-23Journal 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

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

Enhancer regions show high histone H3.3 turnover that changes during differentiation

Publication . Deaton, Aimee M; Gómez-Rodríguez, Mariluz; Mieczkowski, Jakub; Tolstorukov, Michael Y; Kundu, Sharmistha; Sadreyev, Ruslan I; Jansen, Lars ET; Kingston, Robert E

The organization of DNA into chromatin is dynamic; nucleosomes are frequently displaced to facilitate the ability of regulatory proteins to access specific DNA elements. To gain insight into nucleosome dynamics, and to follow how dynamics change during differentiation, we used a technique called time-ChIP to quantitatively assess histone H3.3 turnover genome-wide during differentiation of mouse ESCs. We found that, without prior assumptions, high turnover could be used to identify regions involved in gene regulation. High turnover was seen at enhancers, as observed previously, with particularly high turnover at super-enhancers. In contrast, regions associated with the repressive Polycomb-Group showed low turnover in ESCs. Turnover correlated with DNA accessibility. Upon differentiation, numerous changes in H3.3 turnover rates were observed, the majority of which occurred at enhancers. Thus, time-ChIP measurement of histone turnover shows that active enhancers are unusually dynamic in ESCs and changes in highly dynamic nucleosomes predominate at enhancers during differentiation.

2016-06-15Journal 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