While CENP-A is the epigenetic determinant of the centromeric function, the role of CENP-B, a centromeric protein binding a specific DNA sequence, the CENP-B-box, remains elusive. In the few mammalian species analyzed so far, the CENP-B box is contained in the major satellite repeat that is present at all centromeres, with the exception of the Y chromosome. We previously demonstrated that, in the genus Equus, numerous centromeres lack any satellite repeat.

We propose that the uncoupling between CENP-B and CENP-A may have played a role in the extensive evolutionary reshuffling of equid centromeres. This study provides new insights into the complexity of centromere organization in a largely biodiverse world where the majority of mammalian species still have to be studied.

In four Equus species, CENP-B is expressed but does not bind the majority of satellite-based centromeres, or the satellite-free ones, while it is localized at several ancestral, now-inactive, centromeres. Centromeres lacking CENP-B are functional and recruit normal amounts of CENP-A and CENP-C. The absence of CENP-B is related to the lack of CENP-B boxes rather than to peculiar features of the protein itself. CENP-B boxes are present in a previously undescribed repeat which is not the major satellite bound by CENP-A. Comparative sequence analysis suggests that this satellite was centromeric in the equid ancestor, lost centromeric function during evolution, and gave rise to a shorter CENP-A bound repeat not containing the CENP-B box but enriched in dyad symmetries. Conclusions: We propose that the uncoupling between CENP-B and CENP-A may have played a role in the extensive evolutionary reshuffling of equid centromeres. This study provides new insights into the complexity of centromere organization in a largely biodiverse world where the majority of mammalian species still have to be studied.