Regarding the fundamental unit of centromere specifying chromatin, we report nuclease digestion experiments that demonstrate a remarkable similarity in the behavior of octameric CENP-A-containing nucleosomes reconstituted with recombinant components and the form present at functional human centromeres. We conclude that the predominant form of CENP-A particles at functional centromeres is an octamer with loose terminal DNA based on several key findings: 1) the smallest CENP-A containing particle protects ~110 bp from MNase digestion, which is ~30–50 bp longer than what could be accommodated by tetrameric models, 2) three size classes of CENP-A particles all map to the same nucleosome positions on the complex DNA of neocentromeres, and 3) CENP-A nucleosomes at normal centromeres share the same apparent dyad axis positioning as their conventional counterparts containing H3 on the 171 bp α-satellite DNA repeat sequence.
Our findings do not exclude the possibility that a minor population of CENP-A-containing particles with special stoichiometry exists, nor do they exclude the possibility that other forms exist at particular steps during a cell cycle coupled program of CENP-A nucleosome maturation and propagation18
. Mutation at the CENP-A–CENP-A interface abrogates CENP-A accumulation at centromeres42,43
, suggesting a particle with two copies of CENP-A is required at least transiently in this program. Atomic force microscopy (AFM) measurements of CENP-A-containing particles that were isolated from phases outside of S-phase are shorter than conventional nucleosomes, but are of similar height at S-phase22
. These findings were interpreted as evidence for hemisomes as the predominant form through the majority of the cell cycle22
. The use of AFM-based height measurements to differentiate between hemisomes and octameric nucleosomes from isolated CENP-A-containing particles may not be as straightforward as it originally seemed, since reconstituted, recombinant CENP-A-containing octameric nucleosomes are substantially shorter than their canonical counterparts containing conventional H344
. Further, and to this point, in addition to the neocentromere-harboring cell lines derived from healthy tissue, our studies also include the same tumor-derived cell type as used in the AFM study22
, HeLa (Supplementary Fig. 2e,f
). Under our culturing conditions ~70% of the HeLa cell population is outside of S-phase (Supplementary Fig. 2f
). We observe DNA fragment lengths consistent with an octameric CENP-A nucleosomes in HeLa (Supplementary Fig. 2e
) with no evidence of the biphasic behavior predicted by a model where there are long periods of the cell cycle where CENP-A forms radically different particles (e.g. a hemisome and octameric nucleosome switching model22
). Therefore, since sub-octameric forms are not highly populated in the genome, we conclude that such minor species would be present at very low levels or only very transiently during the cell cycle.
Our findings also uncovered remarkable coupling of the propensity of the CENP-A nucleosome to unwrap its terminal DNA with its strongly phased position within the 171 bp monomer unit of centromeric α-satellite DNA. We further conclude that CENP-B binding to the CENP-B box generates asymmetric unwrapping of CENP-A nucleosome terminal DNA. Nucleosomes, CENP-A-containing or bulk nucleosomes, are not positioned evenly between the sites of CENP-B boxes within α-satellite monomers. Rather, the site for the CENP-B box is immediately adjacent 5’ of the entry–exit site. Thus, this places the 3’ end of the CENP-B box very near to the nucleosome (). CENP-B binding induces a ~60° bend in the DNA with the strongest kink induced 4 bp from the 3’ end of the CENP-B box45
. We think it is very likely that this property of CENP-B contributes strongly to several chromatin features we observe on α-satellite DNA: 1) the general phasing observed for bulk nucleosomes, 2) the enhanced phasing we see for CENP-A nucleosomes, and 3) the asymmetric unwrapping of nucleosome terminal DNA that is exquisitely specific to CENP-A-containing nucleosomes that are bounded by CENP-B boxes. To the latter feature, it appears that CENP-A has evolved in a manner that is poised to have its nucleosomal termini unwrapped. It is enticing to speculate that the physical relationship between CENP-A, CENP-B, and α-satellite DNA is a product of co-evolution. Whether at established centromere locations of highly repetitive DNA or at new centromere locations lacking repeats, however, CENP-A marks centromere location as part of an octameric nucleosome with loose termini.