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.

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Available from: Walter Englander, Jul 25, 2015
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    • "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). "
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    • "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. "
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