IMMUNOLOCALIZATION OF CENP-A SUGGESTS A DISTINCT NUCLEOSOME STRUCTUREAT THE INNER KINETOCHORE PLATE OF ACTIVE CENTROMERES

The trilaminar kinetochore directs the segregation of chromosomes in m itosis and meiosis. Despite its importance, the molecular architecture of this structure remains poorly understood [1]. The best known compo nent of the kinetochore plates is CENP-C, a protein that is required f or kinetochore assembly [2], but whose molecular role in kinetochore s tructure and function is unknown. Here we have raised for the first ti me monospecific antisera to CENP-A [3], a 17 kD centromere-specific hi stone variant that is 62% identical to the carboxy-terminal domain of histone H3 [4,5] and that resembles the yeast centromeric component CS E4 [6]. We have found by simultaneous immunofluorescence with centrome re antigens of known ultrastructural location that CENP-A is concentra ted in the region of the inner kinetochore plate at active centromeres . Because CENP A was previously shown to co-purify with nucleosomes [7 ], our data suggest a specific nucleosomal substructure for the kineto chore. In human cells, these kinetochore-specific nucleosomes are enri ched in alpha-satellite DNA [8]. However, the association of CENP-A wi th neocentromeres lacking detectable alpha-satellite DNA, and the lack of CENP-A association with alpha-satellite-rich inactive centromeres of dicentric chromosomes together suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence reco gnition. We speculate that CENP-A binding could be a consequence of ep igenetic tagging of mammalian centromeres.