Determinants of centromeric nucleosome conformation

Centromeric chromatin is defined by the presence of the histone H3 variant CENP-A, which forms a specialized nucleosome required for kinetochore assembly. Compared to canonical H3 nucleosomes, CENP-A nucleosomes exhibit an open DNA conformation that leaves an additional 13 base pairs of DNA accessible at the entry and exit sites. While the CENP-A N-helix has previously been implicated in promoting this enhanced DNA breathing, the contributions of the intrinsically disordered N-terminal tail and adjacent latch regions of CENP-A in nucleosome conformation remain unknown. The intrinsically disordered N-terminal regions of histone H3 are known to facilitate interactions with DNA to stabilize overall nucleosome conformation. Here, we systematically tested the contribution of each N-terminal histone region to maintaining H3 histones by utilizing a combination of MNase digestion assays and coarse-grained molecular dynamics simulations of H3/CENP-A chimera histone nucleosomes containing targeted swaps of the tail, latch, and N-helix regions. Removal or substitution of individual H3 with CENP-A N-terminal regions increased DNA accessibility and nucleosome unwrapping. While any single CENP-A N-terminal region was sufficient to open the canonical nucleosomal DNA conformation, replacement of any single CENP-A N-terminal region with its H3 counterpart was insufficient to restore the wrapped DNA conformation characteristic of canonical H3 nucleosomes. Instead, progressive incorporation of multiple H3-derived regions produced increasingly closed DNA conformations, demonstrating that the H3 tail, latch, and N-helices act cooperatively to stabilize wrapped nucleosomal DNA. Taken together, these findings demonstrate that the more restricted DNA breathing of canonical nucleosomes arises from coordinated contributions across multiple N-terminal regions and suggest that the multi-region redundancy in the conformational flexibility of the centromeric nucleosome could emphasize the importance of retaining flexibility in the centromeric nucleosome, even upon post-translational modification and binding to structural proteins. SIGNIFICANCEThe centromere is marked by nucleosomes containing CENP-A, which adopt a more open and accessible DNA conformation than canonical nucleosomes. However, the molecular determinants underlying this difference remain unclear. Previous structural investigations of the centromeric nucleosome have placed less emphasis on the intrinsically disordered N-terminal regions of CENP-A. Here, we systematically dissect the contributions of the N-terminal tail, latch, and N-helix via MNase digestion assays and molecular dynamics simulations on nucleosomes containing H3/CENP-A chimeras. We demonstrate that no individual H3-derived region is sufficient to impart a closed conformation to the nucleosomal DNA. Instead, multiple regions act together to stabilize DNA, revealing that nucleosome conformation is controlled by concerted histone-DNA interactions.