Centromeres are specialised chromosome loci where kinetochores, which bind microtubules and are important for chromosome segregation, are assembled. Centromere formation is triggered by deposition of CENP-A, a histone H3 variant, into chromatin and requires the Mis18 complex and the CENP-A chaperone HJURP. However, artificial kinetochores can be generated at non-centromeric sites (neocentromeres) by ectopic tethering of centromeric proteins CENP-C and CENP-I. How CENP-A is recruited to these neocentromeres, though, is not known. In this study (Cao et al., 2024), Fukagawa and co-workers use their previously established auxin-inducible degron (AID) system to knock out centromere machinery and evaluate the impact on artificial kinetochore formation. The authors find that HJURP is essential for kinetochore assembly, and that this involves direct binding to CENP-C via its C-terminus. Indeed, expression of the CENP-C C-terminus alone is sufficient to recruit CENP-A to neocentromeres, even in the absence of the Mis18 complex proteins Knl2 or Mis18α. CENP-I is also dispensable here, but conversely, CENP-I fails to recruit CENP-A in the absence of CENP-C. Together, these findings highlight a distinct mechanism for artificial kinetochore formation, whereby recruitment of CENP-A to neocentromeres requires CENP-C, CENP-I and HJURP, but not the Mis18 complex, providing new insights into mechanisms of CENP-A incorporation into chromatin.
