Jcb_201412011 1..11

Genetic inheritance at cell division relies upon a region of the chromosome called the centromere. In many eukaryotes, centromeres—including new ones (i.e., neocentromeres) that must be established if the original centromere is lost—are not defined by a particular DNA sequence but rather by the presence of nucleosomes containing a histone H3 variant, CENP-A (Black and Cleveland, 2011). CENP-A is targeted to centromeres once per cell cycle via a self-propagation mechanism wherein the existing pool of CENP-A nucleosomes is at the top of a hierarchy of protein–protein interactions that culminate in the local assembly of newly expressed CENP-A (Westhorpe and Straight, 2015). CENP-A nucleosomes are also at the top of the hierarchy for recruitment of proteins required for kinetochore function during chromosome segregation. To dissect the roles of CENP-A in centromere maintenance, gain-of-function histone H3 chimeras containing various regions unique to CENP-A have been particularly helpful (Black et al., 2004, 2007; Carroll et al., 2009, 2010; Foltz et al., 2009; Fachinetti et al., 2013). Gene replacement with these chimeras in budding yeast (Black et al., 2007), fission yeast (Fachinetti et al., 2013), and human cells (Fachinetti et al., 2013) has shown that the CENP-A targeting domain (CATD; all organisms tested; Black et al., 2007; Fachinetti et al., 2013), the N terminus (budding and fission yeasts; Black et al., 2007; Fachinetti et al., 2013), the C terminus (budding yeast; Black et al., 2007), or either terminus (human cells; Fachinetti et al., 2013) are essential for viability. Such gene replacement experiments yield information about the requirements for regions of CENP-A at existing centromeres where a group of 16 proteins, termed the constitutive centromereassociated network (CCAN; Cheeseman, 2014), is constitutively engaged in a network of interactions on or near CENP-A nucleosomes. Whether or not the required sequence determinants of CENP-A are the same during centromere establishment as once a centromere is fully formed remains unknown. The centromere—defined by the presence of nucleosomes containing the histone H3 variant, CENP-A— is the chromosomal locus required for the accurate segregation of chromosomes during cell division. Although the sequence determinants of human CENP-A required to maintain a centromere were reported, those that are required for early steps in establishing a new centromere are unknown. In this paper, we used gain-of-function histone H3 chimeras containing various regions unique to CENP-A to investigate early events in centromere establishment. We targeted histone H3 chimeras to chromosomally integrated Lac operator sequences by fusing each of the chimeras to the Lac repressor. Using this approach, we found surprising contributions from a small portion of the N-terminal tail and the CENP-A targeting domain in the initial recruitment of two essential constitutive centromere proteins, CENP-C and CENP-T. Our results indicate that the regions of CENP-A required for early events in centromere establishment differ from those that are required for maintaining centromere identity. Both tails and the centromere targeting domain of CENP-A are required for centromere establishment