Specification of kinetochore-forming chromatin by the histone H3 variant CENP-A

The mechanisms that specify precisely where mammalian kinetochores form wit hin arrays of centromeric heterochromatin remain largely unknown. Localizat ion of CENP-A exclusively beneath kinetochore plates suggests that this dis tinctive histone might direct kinetochore formation by altering the structu re of heterochromatin within a sub-region of the centromere. To test this h ypothesis, we experimentally mistargeted CENP-A to non-centromeric regions of chromatin and determined whether other centromere-kinetochore components were recruited. CENP-A-containing non-centromeric chromatin assembles a su bset of centromere-kinetochore components, including CENP-C, hSMC1, and HZw int-1 by a mechanism that requires the unique CENP-A N-terminal tail. The s equence-specific DNA-binding protein CENP-B and the microtubule-associated proteins CENP-E and HZW10 were not recruited, and neocentromeric activity w as not detected. Experimental mistargeting of CENP-A to inactive centromere s or to acentric double-minute chromosomes was also not sufficient to assem ble complete kinetochore activity. The recruitment of centromere-kinetochor e proteins to chromatin appears to be a unique function of CENP-A, as the m istargeting of other components was not sufficient for assembly of the same complex. Our results indicate at least two distinct steps in kinetochore a ssembly: (1) precise targeting of CENP-A, which is sufficient to assemble c omponents of a centromere-prekinetochore scaffold; and (2) targeting of kin etochore microtubule-associated proteins by an additional mechanism present only at active centromeres.