Propagation of centromeric chromatin requires exit from mitosis

TLDR

Centromeres ensure chromosomal inheritance by nucleating kinetochore assembly, and in humans their identity is epigenetically determined, with CENP‑A histone H3 variant as a key candidate mark. The study investigates how CENP‑A is inherited and assembled during the cell cycle. Using a covalent fluorescent pulse‑chase labeling approach with SNAP tagging, the authors tracked CENP‑A dynamics across mitosis and interphase. They found that mature CENP‑A is quantitatively partitioned to sister centromeres during S phase and remains stably associated through multiple divisions, that loading of nascent CENP‑A onto replicated centromere DNA requires passage through mitosis but not microtubule attachment, and that assembly and stabilization of new CENP‑A nucleosomes occurs exclusively in the subsequent G1 phase.

Abstract

Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A-containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.