Article

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: 9.81). 09/2011; 108(40):16588-93. DOI: 10.1073/pnas.1113621108
Source: PubMed

ABSTRACT 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

Full-text

Available from: Walter Englander, Jul 25, 2015
0 Followers
 · 
112 Views
  • 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.
    Chromosome Research 01/2013; 21(1). DOI:10.1007/s10577-012-9335-7 · 2.69 Impact Factor
  • Source
    • "The octamer structure is not the only source of distinction between CENP-A and H3. Recent studies of DNA wrapping topology by H/DX-MS showed that residues causing structural deformation are found in the αN part of CENP-A sequence adjacent to the DNA entry–exit site of the nucleosome (Panchenko et al. 2011). In canonical chromatin, this site is known to be recognized by diverse functional proteins , e.g., for transcriptional control by stabilization of nucleosomes, inhibition of nucleosome sliding, and compaction of chromatin in mitosis (Zlatanova et al. 2008). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Chromosome segregation is the one of the great problems in biology with complexities spanning from biophysics and polymer dynamics to epigenetics. Here, we summarize the current knowledge and highlight gaps in understanding of the mechanisms controlling epigenetic regulation of chromosome segregation.
    Chromosoma 10/2012; 121(6). DOI:10.1007/s00412-012-0386-5 · 3.26 Impact Factor
  • Source
    • "Protein fragmentation, MS, and data analysis steps were performed similarly to those described for nucleosome H/DX (Panchenko et al., 2011). In brief, H/DX samples were thawed on ice and injected onto an immobilized pepsin column at an initial flow rate of 50 μl/min for 3 min followed by 150 μl/min for another 3 min. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Centromeres are defined by the presence of chromatin containing the histone H3 variant, CENP-A, whose assembly into nucleosomes requires the chromatin assembly factor HJURP. We find that whereas surface-exposed residues in the CENP-A targeting domain (CATD) are the primary sequence determinants for HJURP recognition, buried CATD residues that generate rigidity with H4 are also required for efficient incorporation into centromeres. HJURP contact points adjacent to the CATD on the CENP-A surface are not used for binding specificity but rather to transmit stability broadly throughout the histone fold domains of both CENP-A and H4. Furthermore, an intact CENP-A/CENP-A interface is a requirement for stable chromatin incorporation immediately upon HJURP-mediated assembly. These data offer insight into the mechanism by which HJURP discriminates CENP-A from bulk histone complexes and chaperones CENP-A/H4 for a substantial portion of the cell cycle prior to mediating chromatin assembly at the centromere.
    Developmental Cell 03/2012; 22(4):749-62. DOI:10.1016/j.devcel.2012.02.001 · 10.37 Impact Factor
Show more