An epigenetic mark generated by the incorporation of CENP-A into centromeric nucleosomes

Abstract

Mammalian centromeres are defined epigenetically. Although the physical nature of the epigenetic mark is unknown, nucleosomes in which CENP-A replaces histone H3 are at the foundation of centromeric chromatin. Hydrogen/deuterium exchange MS is now used to show that assembly into nucleosomes imposes stringent conformational constraints, reducing solvent accessibility in almost all histone regions by >3 orders of magnitude. Despite this, nucleosomes assembled with CENP-A are substantially more conformationally rigid than those assembled with histone H3 independent of DNA template. Substitution of the CENP-A centromere targeting domain into histone H3 to convert it into a centromere-targeted histone that can functionally replace CENP-A in centromere maintenance generates the same more rigid nucleosome, as does CENP-A. Thus, the targeting information directing CENP-A deposition at the centromere produces a structurally distinct nucleosome, supporting a CENP-A-driven self-assembly mechanism that mediates maintenance of centromere identity.

Fig. 1.

Reduced H/D exchange upon assembly of histones into nucleosomes. (A) Experimental scheme for examining the solvent accessibility to nucleosomes assembled with αI-satellite DNA from human centromeres and with CENP-A in place of histone H3. (B) Composition of the five different nucleosomes reconstituted and examined by H/D exchange in this study. Either nucleosomes containing CENP-A (C–G) or histone H3.1 (H–L) were analyzed by H/D exchange. Horizontal blocks represent peptides from CENP-A-containing nucleosomes (C) or from H3-containing nucleosomes (H) monitored for H/D exchange over a time course from 10² to 10⁶ s. Within each block the color-coded percentage of deuteration is represented with early time points at the top progressing to the latest time point at the bottom. Peptides are placed beneath schematics showing the location of the α-helices of each histone. Multiple charge states were detected for a subset of peptides, each represented by its own block. Peptides highlighted in C and H from the nucleosomes are enlarged (D–G Left and I–L Left) and compared with data collected from the corresponding subnucleosomal heterotetramers (D–G Right and I–L Right). The time course for the heterotetramer experiment extends over a range from 10¹ to 10⁴ s, and these data are from published experiments (19).

Fig. 2.

Centromeric nucleosomes containing CENP-A are more protected from H/D exchange relative to those containing histone H3. (A) Comparison of peptides from the corresponding position in the α2-helices from histone H3 and CENP-A. (B) Comparison of H/D exchange for an identical peptide from histone H4 (amino acids 61–84) in nucleosomes (assembled with either CENP-A or H3) or their respective subnucleosomal heterotetramers [(CENP-A/H4)₂ or (H3/H4)₂] (19).

Fig. 3.

The rigidified nucleosome structure generated by the incorporation of CENP-A does not require centromeric DNA. (A) The peptides (the same region as in Fig. 2A) are shown here from experiments in which the CENP-A-containing and H3-containing nucleosomes are now each assembled with rDNA. (B) Comparison of H/D exchange from an identical peptide from histone H4 from CENP-A-containing and H3-containing nucleosomes assembled with α-satellite DNA or rDNA.

Fig. 4.

The CATD confers rigidity to the nucleosome. (A) Diagram of the H3^CATD-containing nucleosome. (B) Negligible exchange is observed in the ₁₀₅FEDAYL₁₁₀ peptide from the CATD in the H3^CATD nucleosome. (C) H/D exchange data corresponding to the FEDAYL peptide from CENP-A and H3^CATD or FEDTNL from the H3-containing nucleosomes are plotted as described in Fig. 1. (D–F) Models for self-directed assembly of CENP-A nucleosomes (D), higher-order chromatin organization at the centromere driven by self-association of CENP-A-containing nucleosomes (E), and recruitment of the CENP-A^NAC (18) via direct interaction with CENP-A-containing nucleosomes (F). See Discussion for details.