Replacement of histone H3 with CENP-A directs global nucleosome array condensation and loosening of nucleosome superhelical termini

Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 09/2011; 108(40):16588-93. DOI: 10.1073/pnas.1113621108
Source: PubMed


Centromere protein A (CENP-A) is a histone H3 variant that marks centromere location on the chromosome. To study the subunit structure and folding of human CENP-A-containing chromatin, we generated a set of nucleosomal arrays with canonical core histones and another set with CENP-A substituted for H3. At the level of quaternary structure and assembly, we find that CENP-A arrays are composed of octameric nucleosomes that assemble in a stepwise mechanism, recapitulating conventional array assembly with canonical histones. At intermediate structural resolution, we find that CENP-A-containing arrays are globally condensed relative to arrays with the canonical histones. At high structural resolution, using hydrogen-deuterium exchange coupled to mass spectrometry (H/DX-MS), we find that the DNA superhelical termini within each nucleosome are loosely connected to CENP-A, and we identify the key amino acid substitution that is largely responsible for this behavior. Also the C terminus of histone H2A undergoes rapid hydrogen exchange relative to canonical arrays and does so in a manner that is independent of nucleosomal array folding. These findings have implications for understanding CENP-A-containing nucleosome structure and higher-order chromatin folding at the centromere.

Download full-text


Available from: Walter Englander
  • Source
    • "In the H3.3/H4/DAXX crystal structures, H3.3 L2 is extended at the expense of a full helical turn at the C-terminus of the H3.3 α2 helix (15,16). Using an H/DX rate-mapping strategy that has proven especially informative in other proteins/complexes with available high-resolution structures (19,29), we found that the reduced H/DX protection maps precisely to L2 of H3.3, which extends into the α2 helix in the DAXX complex (Figure 3A and G). In contrast, helical residues immediately adjacent to the extended loop, 105–108 and 122–126, are stabilized by 10-1000-fold upon DAXX binding (Figure 3A–E). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Histone chaperones are a diverse class of proteins that facilitate chromatin assembly. Their ability to stabilize highly abundant histone proteins in the cellular environment prevents non-specific interactions and promotes nucleosome formation, but the various mechanisms for doing so are not well understood. We now focus on the dynamic features of the DAXX histone chaperone that have been elusive from previous structural studies. Using hydrogen/deuterium exchange coupled to mass spectrometry (H/DX-MS), we elucidate the concerted binding-folding of DAXX with histone variants H3.3/H4 and H3.2/H4 and find that high local stability at the variant-specific recognition residues rationalizes its known selectivity for H3.3. We show that the DAXX histone binding domain is largely disordered in solution and that formation of the H3.3/H4/DAXX complex induces folding and dramatic global stabilization of both histone and chaperone. Thus, DAXX uses a novel strategy as a molecular chaperone that paradoxically couples its own folding to substrate recognition and binding. Further, we propose a model for the chromatin assembly reaction it mediates, including a stepwise folding pathway that helps explain the fidelity of DAXX in associating with the H3.3 variant, despite an extensive and nearly identical binding surface on its counterparts, H3.1 and H3.2.
    Full-text · Article · Feb 2014 · Nucleic Acids Research
  • Source
    • "Results indicated that the αN helix of CENP-A contacts superhelical DNA termini of the nucleosome and is more flexible than that of H3 in folded arrays, indicating that DNA is more loosely connected to CENP-A relative to H3. The C terminal region of H2A also experiences increased exchange in CENP-A containing arrays (folded and unfolded) compared to canonical nucleosomes, indicating that H2A may adopt different conformations in CENP-A- containing and canonical nucleosomes (Panchenko et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Histone proteins are dynamically modified to mediate a variety of cellular processes including gene transcription, DNA damage repair, and apoptosis. Regulation of these processes occurs through the recruitment of non-histone proteins to chromatin by specific combinations of histone post-translational modifications (PTMs). Mass spectrometry has emerged as an essential tool to discover and quantify histone PTMs both within and between samples in an unbiased manner. Developments in mass spectrometry that allow for characterization of large histone peptides or intact protein has made it possible to determine which modifications occur simultaneously on a single histone polypeptide. A variety of techniques from biochemistry, biophysics, and chemical biology have been employed to determine the biological relevance of discovered combinatorial codes. This review first describes advancements in the field of mass spectrometry that have facilitated histone PTM analysis and then covers notable approaches to probe the biological relevance of these modifications in their nucleosomal context.
    Full-text · Article · Dec 2013 · Frontiers in Genetics
  • Source
    • "These findings are consistent with the data obtained independently from stepwise assembly of CENP-A nucleosomes not only confirming the octameric structure of CENP-A nucleosomes but also the loosening of the interaction between DNA superhelical termini and CENP-A (Conde e Silva et al. 2007; Panchenko et al. 2011). CENP-A octamers formed in vitro have also been reported to induce conventional left-handed negative supercoiling to DNA (Barnhart et al. 2011; Conde e Silva et al. 2007; Panchenko et al. 2011; Tachiwana et al. 2011; Yoda et al. 2000). It was recently demonstrated that the mutation of the putative CENP-A: CENP-A dimer interface can abrogate centromeric targeting of CENP-A in Drosophila and mammalian tissue culture cells (Bassett et al. 2012; Zhang et al. 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Centromere is a unique chromosomal locus where the kinetochore is formed to mediate faithful chromosome partitioning, thus maintaining ploidy during cell division. Centromere identity is inherited via an epigenetic mechanism involving a histone H3 variant, called centromere protein A (CENP-A) which replaces H3 in centromeric chromatin. In spite of extensive efforts in field of centromere biology during the past decade, controversy persists over the structural nature of the CENP-A-containing epigenetic mark, both at nucleosomal and chromatin levels. Here, we review recent findings and hypotheses regarding the structure of CENP-A-containing complexes.
    Full-text · Article · Jan 2013 · Chromosome Research
Show more