The histone chaperone HJURP directs the formation of functional centromeres by assembling the histone variant CENP-A into chromatin, Barnhart et al. reveal.
CENP-A is a specialized version of histone H3 that marks the position of centromeres. A protein called HJURP helps deliver CENP-A to centromeric chromatin, but whether HJURP simply protects CENP-A until the histone is incorporated or whether the protein actively assembles CENP-A into nucleosomes is unclear.
Barnhart et al. targeted HJURP to noncentromeric chromatin by tagging it with the Lac repressor protein LacI and expressing this fusion protein in cells carrying an array of LacI-binding DNA sequences on one of their chromosomes. LacI-HJURP bound to this array and stably incorporated CENP-A into the underlying chromatin. Moreover, additional centromeric and kinetochore proteins were also recruited to the array, allowing it to stably attach to spindle microtubules during mitosis.
A short N-terminal fragment of HJURP fused to LacI was sufficient to assemble these ectopic kinetochores. This same fragment—which contains the Scm3 domain conserved in yeast homologues of HJURP—could also assemble CENP-A into DNA-bound nucleosomes in vitro. Centromeric nucleosomes have been proposed to differ in size and structure from regular nucleosomes, but the CENP-A nucleosomes formed by HJURP in vitro were octameric and contained negatively supercoiled DNA, just like canonical nucleosomes.
Barnhart et al. found that HJURP normally requires the centromeric protein complex Mis18 to localize to centromeres. Senior author Daniel Foltz now wants to investigate how the Mis18 complex associates with centromeres and how it recruits HJURP and CENP-A.