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Review
. 2007 Oct 9;104(41):15974-81.
doi: 10.1073/pnas.0707648104. Epub 2007 Sep 24.

Structure, dynamics, and evolution of centromeric nucleosomes

Yamini Dalal  1 , Takehito Furuyama, Danielle Vermaak, Steven Henikoff
Affiliations
Review

Structure, dynamics, and evolution of centromeric nucleosomes

Yamini Dalal et al. Proc Natl Acad Sci U S A. .

Abstract

Centromeres are defining features of eukaryotic chromosomes, providing sites of attachment for segregation during mitosis and meiosis. The fundamental unit of centromere structure is the centromeric nucleosome, which differs from the conventional nucleosome by the presence of a centromere-specific histone variant (CenH3) in place of canonical H3. We have shown that the CenH3 nucleosome core found in interphase Drosophila cells is a heterotypic tetramer, a "hemisome" consisting of one molecule each of CenH3, H4, H2A, and H2B, rather than the octamer of canonical histones that is found in bulk nucleosomes. The surprising discovery of hemisomes at centromeres calls for a reevaluation of evidence that has long been interpreted in terms of a more conventional nucleosome. We describe how the hemisome structure of centromeric nucleosomes can account for enigmatic properties of centromeres, including kinetochore accessibility, epigenetic inheritance, rapid turnover of misincorporated CenH3, and transcriptional quiescence of pericentric heterochromatin. Structural differences mediated by loop 1 are proposed to account for the formation of stable tetramers containing CenH3 rather than stable octamers containing H3. Asymmetric CenH3 hemisomes might interrupt the global condensation of octameric H3 arrays and present an asymmetric surface for kinetochore formation. We suggest that this simple mechanism for differentiation between centromeric and packaging nucleosomes evolved from an archaea-like ancestor at the dawn of eukaryotic evolution.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
CenH3 nucleosomes are hemisomes in vivo (12). (A) Western blots of D. melanogaster S2 cell chromatin after cross-linking with dimethylsuberimidate. Anti-H3 cross-linked products include multiple dimeric (2) and trimeric (3) species, tetramers (4) and octamers (8) (Left), whereas an anti-CenH3 antibody detects only CenH3/H4 dimers (2) and CenH3/H4/H2A/H2B tetramers (4). M, markers. (B) EM shows the “beads-on-a-string” conformation of CenH3- immunoprecipitated chromatin, with extended linkers. (C) CenH3-immunoprecipitated nucleosomes display a tight distribution of heights determined by atomic force microscopy, averaging half that of bulk chromatin (H4 IP). DNA provides an internal marker. (Adapted from ref. with permission.)
Fig. 2.
Fig. 2.
Mislocalization of overexpressed and mutant Cid to euchromatin. (A) Overexpressed Cid-GFP localizes to both centromeres (arrowheads) and euchromatin in interphase cells and is incorporated into metaphase chromosomes in Drosophila Kc cells but not into the heterochromatic chromocenter (Left) (60). H3.3 is found in chromosome arms and rDNA (arrow) but is undetectable in heterochromatin and centromeres (Right). (B) GFP fusions with Cid from D. melanogaster and Drosophila pseudoobscura localize to D. melanogaster centromeres, whereas a GFP fusion with Cid from D. bipectinata shows a euchromatic distribution (80). (C) Swaps between segments of melanogaster (blue) and bipectinata (orange) Cid show that the 15-aa loop 1 segment alone is responsible for targeting (80). Single amino acid substitutions to glycine or alanine cause melanogaster Cid-GFP to display a euchromatic distribution (orange letters), where uppercase letters indicate mislocalization when expressed under both the Cid endogenous promoter and an induced heat shock promoter, and lowercase letters indicate when mislocalization occurred only with the induced heat shock promoter. [Reproduced with permission from ref. (A Left), ref. (A Right), and ref. (B).]
Fig. 3.
Fig. 3.
A structural change proposed to underlie the transition from wild-type to mutant Cid. (A) Half-nucleosome showing the juxtaposition of H3 loop 1 (magenta), H4α2 (green), and H2Bα2 (blue) and the bending of H4α2 around the subdomain created by H3 loop 1 + H4 loop 2 [Protein Data Bank (PDB) ID code 1KX5] (6). (B) Space-fill model showing top and bottom views of the interactions between H3 loop 1 residues whose CenH3 counterparts are critical for targeting to centromeres (yellow) and H4α2 + loop 2 (green). (C) Alignment of α2 and α3 helices comprising the three four-helix bundles that hold together conventional octameric nucleosomes. The three-dimensional structure of the nucleosome shows that H2Bα2 and H4α3 (Middle) are misaligned relative to alignments of H4α2 with H2Bα3 (Top) and H3α2 with H3α3 (Bottom) (6). It is proposed that H2Bα2 and H4α3 become well aligned in CenH3 nucleosomes, thus strengthening the four-helix bundle in the middle of the CenH3 core. (D) Aligning the dimeric histone from M. kandleri (PDB ID code 1F1E) with H3/H4 at the N-terminal residues of H4α2 [from yeast Asf1-H3/H4 (PDB ID code 2HUE] shows that the corresponding helix in this tetrameric archaeal histone is straight, whereas H3/H4 bends tightly around the H3 loop1 + H4 loop 2 subdomain.
Fig. 4.
Fig. 4.
Model for the kinetochore. CenH3 hemisomes (red/gray disks) are separated by extended linker DNAs and so are decondensed relative to surrounding heterochromatin (blue disks). Asymmetric CenH3 nucleosomes assemble in random orientations [CenH3/H4 (red) and H2A/H2B (gray)]. Only one unit of a CenH3-rich block is shown. During mitotic condensation, heterochromatin packs tightly as a result of its homogeneity. Intervening blocks of CenH3 chromatin cannot pack into this crystal-like structure because of its smaller size, long linkers, and heterogeneity in its relative orientation, resulting in extruded loops of uncondensed CenH3 nucleosomes that serve as the foundation for kinetochore formation. The flanking gray cones represent pericentric regions flanking the primary constriction.
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