Centromeres are maintained by fastening CENP-A to DNA and directing an arginine anchor-dependent nucleosome transition

Abstract

Maintaining centromere identity relies upon the persistence of the epigenetic mark provided by the histone H3 variant, centromere protein A (CENP-A), but the molecular mechanisms that underlie its remarkable stability remain unclear. Here, we define the contributions of each of the three candidate CENP-A nucleosome-binding domains (two on CENP-C and one on CENP-N) to CENP-A stability using gene replacement and rapid protein degradation. Surprisingly, the most conserved domain, the CENP-C motif, is dispensable. Instead, the stability is conferred by the unfolded central domain of CENP-C and the folded N-terminal domain of CENP-N that becomes rigidified 1,000-fold upon crossbridging CENP-A and its adjacent nucleosomal DNA. Disrupting the ‘arginine anchor' on CENP-C for the nucleosomal acidic patch disrupts the CENP-A nucleosome structural transition and removes CENP-A nucleosomes from centromeres. CENP-A nucleosome retention at centromeres requires a core centromeric nucleosome complex where CENP-C clamps down a stable nucleosome conformation and CENP-N fastens CENP-A to the DNA.

Structural mechanisms of centromeric nucleosome recognition by the kinetochore protein CENP-N

“…Our data suggest that the CENP-A–interacting residues of CENP-N coevolve with CENP-A, which could reflect a common strategy for maintaining CENP-A specificity (17). Previous studies (7, 23) and our results (Fig. 4B and fig.…”

“…S2, A and D) show that CENP-N and fragments of CENP-C that include one of its two CENP-A nucleosome binding domains [fig. S2E, central domain (7, 23), or CENP-C motif (this work)] can simultaneously bind to the same face of the CENP-A nucleosome. Furthermore, the kinetochore protein CENP-L has been shown to mediate interactions between CENP-C and CENP-N (9, 23–25).…”

“…Two regions of CENP-C, the central region and the CENP-C motif, bind the CENP-A nucleosome by interacting with the C-terminal tail of CENP-A and the acidic patch on histones H2A and H2B in the centromeric nucleosome (5, 6). The CENP-N N-terminal region (residues 1 to 286 in humans) is responsible for recognition of the CENP-A nucleosome (7, 8), whereas its C-terminal region (residues 287 to 339) interacts with the CCAN through CENP-L (9, 10). The recruitment of CENP-N to the centromere requires recognition of the L1 loop of CENP-A (11).…”

Centromere inheritance through the germline

“…Two of these proteins, CENP-C and CENP-N, contact CENP-A nucleosomes directly (Carroll et al 2009; Guse et al 2011; Kato et al 2013), and CENP-C can reshape CENP-A nucleosomes and plays an important role in retaining CENP-A at the centromere (Falk et al 2015; Falk et al 2016). Indeed, a single point mutation of the nucleosome interaction surface of CENP-C retains binding to CENP-A but eliminates structural stability and hinders its ability to retain CENP-A nucleosomes at the centromere (Guo et al 2017). CENP-C and CENP-S have also been shown to be important for resisting unfolding of centromeric chromatin in low ionic strength solutions (Vargiu et al 2017).…”

“…However, since CENP-C protein was not detected in Drosophila or Xenopus sperm (Milks et al 2009; Raychaudhuri et al 2012), the mechanism of retaining CENP-A nucleosomes through the genome wide histone-protamine exchange in sperm is still unclear. CENP-N crosslinks CENP-A to nucleosomal DNA and also contributes strongly to CENP-A stability at centromeres (Guo et al 2017), and it is not yet clear whether or not it is present on sperm chromatin.…”