CENP-A-containing Nucleosomes: Easier Disassembly versus Exclusive Centromeric Localization

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Abstract

CENP-A is a histone variant that replaces conventional H3 in nucleosomes of functional centromeres. We report here, from reconstitutions of CENP-A- and H3-containing nucleosomes on linear DNA fragments and the comparison of their electrophoretic mobility, that CENP-A induces some positioning of its own and some unwrapping at the entry–exit relative to canonical nucleosomes on both 5 S DNA and the α-satellite sequence on which it is normally loaded. This steady-state unwrapping was quantified to 7(±2) bp by nucleosome reconstitutions on a series of DNA minicircles, followed by their relaxation with topoisomerase I. The unwrapping was found to ease nucleosome invasion by exonuclease III, to hinder the binding of a linker histone, and to promote the release of an H2A-H2B dimer by nucleosome assembly protein 1 (NAP-1). The (CENP-A-H4)2 tetramer was also more readily destabilized with heparin than the (H3-H4)2 tetramer, suggesting that CENP-A has evolved to confer its nucleosome a specific ability to disassemble. This dual relative instability is proposed to facilitate the progressive clearance of CENP-A nucleosomes that assemble promiscuously in euchromatin, especially as is seen following CENP-A transient over-expression.

Introduction

Centromeres represent a specific chromosome region in eukaryotic cells that defines the site of kinetochore formation, thereby directing spindle attachment and chromosome segregation during mitosis.1 Maintenance of centromere identity for millions of years appears to result from a subtle evolutionary interplay between genetic features (the rapidly diverging and highly repetitive hundreds to thousands of kbp-long centromeric DNA;2 e.g. the 171 bp repeated α-satellite in human), and epigenetic features.3 The main candidate for the centromeric epigenetic mark is CENP-A, a histone H3 variant that replaces H3 in centromeric nucleosomes4., 5. and has diverged significantly from species to species, in contrast to canonical H3, which has remained nearly invariant. The C-terminal two-thirds of CENP-A (the histone fold) is relatively homologous (62% identical) to H3, but its N-terminal one-third is unique.

An early question raised about CENP-A is how it targets to the centromere. Although CENP-A and the CENP-A orthologs in different organisms (also referred to as CENH3s3) are produced throughout the cell cycle, like the main H3 variant enriched in transcriptionally active chromatin, H3.3, but unlike the major H3 (H3.1/2) that is synthesized exclusively during DNA replication,6., 7., 8., 9., 10. they incorporate into centromeres only during embryonic anaphase in Drosophila,11 or early G1 in humans cells.12 Replacing CENP-A residues within either the L1 loop or the α2 helix with the corresponding H3 residues results in chimeric histones that fail to target to centromeres,13., 14. while replacement of both the L1 loop and the α2 helix of H3 with the corresponding features from CENP-A, to give the H3CATD chimera (Figure 1(a)), is sufficient for targeting (CATD stands for CENP-A targeting domain).15 In addition to being targeted, H3CATD and its yeast version were found to functionally replace CENP-A in human cells or its counterpart (Cse4p) in budding yeast, with no adverse consequence on kinetochore formation, chromosome segregation or cell viability.16 Interestingly, the functionality of the CATD correlates with its property to compact the (CENP-A/H3CATD-H4)2 tetramers, as observed by gel filtration, and to slow the hydrogen/deuterium exchange rate in portions of CENP-A/H3CATD and H4 within the tetramers, relative to tetramers made with H3.1/2 and H3.3.15 Remarkably, this latter property was preserved upon tetramer association with H2A-H2B dimers to form nucleosomes on α-satellite and 5 S DNAs.17

Over-expression of CENP-A or its orthologs results in accumulation of the variant at non-centromeric chromosomal sites and in rare cases leads to the formation of neocentromeres lacking repetitive DNA and significant sequence homology to α-satellite DNA.18., 19., 20. Proteolysis, mediated, at least in part, by the ubiquitin-proteasome pathway, regulates the level of the transiently over-expressed variant in the soluble pool in Saccharomyces cerevisiae,21 and clears it progressively from the chromosome arms in Drosophila,22 in contrast to centromere-localized subunits, which were resistant.21., 22. Perhaps relevant to the targeting problem is the purification from Drosophila cells of a soluble pre-assembly complex containing the CENP-A ortholog (CID), H4 and the generic assembly factor RbAp48 (p48).23 The occurrence of this simple complex is consistent with the ability of p48 to bind CID in vitro (in addition to H4), but not H3.23., 24., 25. This finding is in addition to p48 co-existence with several other proteins in various chromatin remodeling and modifying complexes, and in histone chaperones CAF-1 and HIRA (involved in nucleosome assembly during replication and transcription, respectively).8 An at least indirect involvement of p48, in conjunction with p46, was demonstrated in human cells by the observation that their simultaneous, but not individual, RNAi knockdown abolished targeting.26 A similar simple pre-assembly complex may not exist in other species, however, since p48 was absent from a CENP-A nucleosome-associated complex (NAC) purified from human centromeric chromatin, although it was present along with p46 as a CAF-1 component in the H3.1 equivalent complex.27

Using single nucleosomes assembled on a series of DNA minicircles, followed by their relaxation with topoisomerase I (the so-called “DNA minicircle approach”28., 29.), three conformational states have been identified between which conventional nucleosomes can thermally fluctuate.30., 31. The first state, we referred to previously as the “closed negative” state, introduces a topological deformation in linking number relative to naked DNA between −1.4 and −1.7 (ΔLkn(i) in equation (3) in Materials and Methods), and has DNAs entering and exiting the nucleosome negatively crossed. This form corresponds to the canonical conformation with ∼147 bp of wrapped DNA, and usually is energetically favored. The second state, where the entry–exit DNAs are uncrossed (we termed this previously the “open” state (ΔLkn = −0.7 to −1)), concomitantly involves the unwrapping of ∼21 bp through breakage of the most distal H3 αN/DNA-binding sites at superhelix locations (SHL) ± 6.5.32 The third, “closed positive”, state has ΔLkn = −0.4 to −0.6 with positively crossed entry–exit DNAs, and is energetically the least favored, as expected from the DNA left-handed wrapping around the histones. Modeling has defined the topological and energetic (see above) parameters of these conformational states and their dependence on effectors of nucleosome dynamics, such as the modification status of the histone tails,30 or the DNA sequence.31 Remarkably, nucleosomes in chromatin fibers showed very similar values for these parameters, as observed in recent single-molecule experiments.33

In this work, we investigated the intrinsic properties of CENP-A nucleosomes, with respect to their structure, dynamics and stability, in comparison with those of conventional (H3) nucleosomes, in the hope that they could explain some of the peculiarities of centromeric chromatin mentioned above. When reconstituted on linear DNA fragments and subjected to electrophoresis, CENP-A nucleosomes were retarded relative to H3 nucleosomes, which suggested some unwrapping at their entry–exit. Consistently, the “DNA minicircle approach” revealed that CENP-A nucleosomes prefer the open, uncrossed conformation, in contrast to H3 nucleosomes, which prefer the closed negative conformation (see above). The resulting 7(±2) bp steady-state unwrapping was sufficient to compromise the binding of a linker histone and to promote dissociation of a H2A-H2B dimer by nucleosome assembly protein 1 (NAP-1). Once dimers are removed, the (CENP-A-H4)2 tetramer is also more easily released with heparin than the (H3-H4)2 tetramer. We conclude that, at least in the absence of the above-mentioned additional CENP-ANAC components that are recruited by CENP-A nucleosomes to functional centromeres, CENP-A nucleosomes are easier to disassemble than canonical nucleosomes. The potential physiological relevance of this result is discussed.

Section snippets

CENP-A-dependent DNA unwrapping and phasing of nucleosomes

The sequences in the domains of interest of the histone H3 family used in the present work are compared in Figure 1(a). Here, we focus on the SHL ± 6.5 and SHL ± 2.5 (incidentally ± 1.5) which are shown, for the positive ones, in the upper gyre of the crystallized nucleosome core particle.32 These histones were mixed with stoichiometric amounts of H4, H2A and H2B to form the octamers that are displayed in Figure 1(b).

Octamers were used to reconstitute mononucleosomes on linear ∼350 bp 5 S,34 and

CENP-A versus the α-satellite sequence

It is plausible that an interplay may occur during evolution of CENP-A and the corresponding centromeric DNA sequence (see Introduction). Along this line, a direct influence of the DNA sequence on centromeric chromatin is shown by the recent observation that the length of the α-satellite arrays and their density in CENP-B boxes had a strong impact on nucleation, spreading and maintenance of functional centromeres in human artificial chromosomes.44 We then asked whether our in vitro

DNAs

DNA fragments of the pBR series, 351 bp, 353 bp, 354 bp, 356 bp, 358 bp, 360 bp and 363 bp long originate from plasmid pBR322,30 and the 357 bp fragment of the 5 S series originates from Lytechinus variegatus 5 S rDNA.31 The α-satellite DNA fragments, 346 bp, 348 bp, 350 bp, 352 bp, 354 bp, 356 bp, and 358 bp long were obtained from human DNA by PCR and cloning in plasmid pUC18, to give recombinant plasmids pNCS-α(346-358).

Histones

H5 and native octamers (Figure 1(b)) originated from chicken erythrocyte

Acknowledgements

N. C. e S. thanks the French Ministry of Research (MENRT), the Foundation for Medical Research (FRM) and the Association for Cancer Research (ARC) for three successive fellowships. A. P. thanks V. Ramakrishnan (University of Cambridge, UK), and F. Livolant (University of Orsay, France) and K. Luger (Colorado State University, USA) for the gifts of expression vectors for cH3 and tailless xH3, respectively, S. Leuba (University of Pittsburg, USA) for the gift of yNAP-1, and C. Lavelle (Institut

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