Division of Molecular Genetics • Fukagawa Group

The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP-A. In addition to CENP-A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP-T-W-S-X complex (Nishino et al., Cell, 2012). However, it is unclear how the CENP-T-W-S-X complex associates with centromere chromatin. Here, we demonstrate that the CENP-T-W-S-X complex binds preferentially to ~100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP-T-W-S-X complex primarily binds to DNA as a (CENP-T-W-S-X)2 structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP-T-W-S-X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP-T or CENP-W, but not CENP-S or CENP-X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP-T-W-S-X complex. In summary, our work reveals the structural features and properties of the CENP-T-W-S-X complex for its localization to centromeres.

Experimental design to distinguish negative and positive supercoils (top). Negative supercoiled topoisomers slowly migrate following addition of chloroquine, whereas positive supercoiled topoisomers migrate faster following chloroquine addition. Plasmid supercoiling assays performed with CENP-T-W-S-X and histone H2A-H2B-H3-H4 complexes in the presence or absence of chloroquine (Below).　The CENP-T-W-S-X induces positive supercoils.