Mammalian H3.3 is a variant of the canonical histone H3.1 essential for genome reprogramming in the fertilized eggs and maintenance of chromatin structure in neuronal cells. An H3.3-specific histone chaperone, DAXX, directs the deposition of H3.3 onto pericentric and telomeric heterochromatin. H3.3 differs from H3.1 by only five amino acids, yet DAXX can distinguish the two with high precision. By a combination of structural, biochemical and cell-based targeting analyses, here we show that Ala87 and Gly90 are the principal determinants of H3.3 specificity. DAXX uses a shallow hydrophobic pocket to accommodate the small, hydrophobic Ala87 of H3.3, whereas a polar binding environment in DAXX prefers Gly90 in H3.3 over the hydrophobic Met90 in H3.1. An H3.3-H4 heterodimer is bound by the histone-binding domain of DAXX, which makes extensive contacts with both H3.3 and H4.

The H3 histone variant CENP-A is an epigenetic marker critical for the centromere identity and function. However, the precise regulation of the spatiotemporal deposition and propagation of CENP-A at centromeres during the cell cycle is still poorly understood. Here, we show that CENP-A is phosphorylated at Ser68 during early mitosis by Cdk1. Our results demonstrate that phosphorylation of Ser68 eliminates the binding of CENP-A to the assembly factor HJURP, thus preventing the premature loading of CENP-A to the centromere prior to mitotic exit. Because Cdk1 activity is at its minimum at the mitotic exit, the ratio of Cdk1/PP1α activity changes in favor of Ser68 dephosphorylation, thus making CENP-A available for centromeric deposition by HJURP. Thus, we reveal that dynamic phosphorylation of CENP-A Ser68 orchestrates the spatiotemporal assembly of newly synthesized CENP-A at active centromeres during the cell cycle.

Specific recognition of centromere-specific histone variant CENP-A-containing chromatin by CENP-N is an essential process in the assembly of the kinetochore complex at centromeres prior to mammalian cell division. However, the mechanisms of CENP-N recruitment to centromeres/kinetochores remain unknown. Here, we show that a CENP-A-specific RG loop (Arg80/Gly81) plays an essential and dual regulatory role in this process. The RG loop assists the formation of a compact "ladder-like" structure of CENP-A chromatin, concealing the loop and thus impairing its role in recruiting CENP-N. Upon G1/S-phase transition, however, centromeric chromatin switches from the compact to an open state, enabling the now exposed RG loop to recruit CENP-N prior to cell division. Our results provide the first insights into the mechanisms by which the recruitment of CENP-N is regulated by the structural transitions between compaction and relaxation of centromeric chromatin during the cell cycle.