Centromeres are epigenetically maintained by nucleosomes containing the histone H3 variant CENP-A, which in proliferating cells is replenished each G1 phase by the chaperone HJURP and the Mis18 complex. Swartz et al. (2019) showed that quiescent cells are not passive custodians of this mark: G0-arrested human cells and prophase I-arrested starfish oocytes continuously exchange CENP-A through HJURP/Mis18-dependent, Plk1-independent deposition, presumably driven by RNA polymerase II transcription that evicts centromeric nucleosomes, and loss of this exchange compromises chromosome segregation after cell-cycle reentry. The unresolved problem is to explain how centromere identity is actively maintained in non-dividing cells: (i) the molecular mechanism by which centromeric transcription (or other forces) evicts and replaces nucleosomes during quiescence, including the required remodelers, chaperones, and the initiation-versus-elongation question; (ii) the homeostatic control of the soluble CENP-A pool, in particular whether HJURP abundance is limiting and which proteostatic pathways recognize and remove excess soluble CENP-A; and (iii) whether gradual CENP-A exchange is a general property of all quiescent but proliferation-capable cell types, and what signals cause its loss during terminal differentiation. Later work has sharpened but not resolved this core: mouse oocytes were shown to maintain CENP-A for months without new assembly (Das et al. 2023), quiescence entry/exit dynamics of the centromere protein network were mapped (Marescal et al. 2026), Plk1-dependent G1 licensing was mechanistically solved (Conti et al. 2024), and SUMO (small ubiquitin-like modifier)/p97- and ubiquitin-mediated proteostasis of centromeric CENP-A and Mis18β was uncovered, while the quiescence-specific eviction mechanism, soluble-pool regulation, in vivo lineage generality, and the proposed proliferative-potential biomarker all remain open.

Background

The centromere is the chromosomal locus on which the kinetochore assembles; the kinetochore is the protein structure that attaches chromosomes to spindle microtubules so that they are segregated accurately during cell division. In most eukaryotes the position of the centromere is specified epigenetically, not by DNA sequence, by nucleosomes containing CENP-A, a variant of histone H3 (the protein around which DNA is wrapped in the nucleosome, the basic unit of chromatin). In proliferating human cells, CENP-A nucleosomes are partitioned to daughter strands when DNA is replicated and then replenished during the G1 phase of the cell cycle (Jansen et al. 2007). This deposition is carried out by the CENP-A-specific chaperone HJURP (Foltz et al. 2009) together with the Mis18 protein complex, and it is promoted by the Polo-like kinase 1 (Plk1) while cyclin-dependent kinases restrict deposition to once per cycle. Because centromeric CENP-A nucleosomes are exceptionally stable in vivo, the prevailing model held that centromere identity is maintained indefinitely by the intrinsic immobility of CENP-A once deposited.

Quiescence is a reversible proliferative arrest: quiescent (G0) cells do not divide but retain the capacity to re-enter the cell cycle, sometimes after years — as with oocytes (egg precursor cells) arrested at prophase I of meiosis until ovulation, hepatocytes, and tissue-resident stem cells. This contrasts with terminal differentiation, where cells exit the division program permanently. How an epigenetic mark like CENP-A survives long non-dividing periods was therefore unclear.

Swartz et al. (2019) overturned the static picture for quiescent cells. Using pulse-chase labeling of endogenous CENP-A (a HaloTag self-labeling tag strategy that distinguishes old from newly deposited CENP-A molecules), they showed that G0-arrested human RPE-1 cells (a retinal-pigment-epithelial cell line widely used for quiescence studies) and prophase I-arrested starfish oocytes gradually and continuously incorporate new CENP-A at centromeres, keeping total levels constant through balanced loss and replacement. Quiescent deposition requires HJURP and the Mis18 complex but, unlike G1 deposition, does not require Plk1 (which could not be detected in G0 cells). Inducible knockout of HJURP or of Mis18β (a subunit of the Mis18 complex) during quiescence gradually reduced centromeric CENP-A and increased chromosome mis-segregation after the cells re-entered division. In oocytes, inhibiting RNA polymerase II transcription (which transcribes protein-coding and many non-coding genes) reduced new CENP-A deposition by about half, leading to the proposal that transcription provides the destabilizing force that evicts centromeric nucleosomes and creates sites for redeposition. The CENP-A protein itself is long-lived, but the soluble (non-chromatin) pool appears to be regulated post-translationally: ectopic CENP-A expression downregulates endogenous CENP-A, consistent with a limiting amount of HJURP destabilizing excess soluble CENP-A. In contrast, terminally differentiated cells (C2C12 myotubes, muscle cells formed by irreversible differentiation in culture, and post-mitotic mouse tissues) fail to maintain CENP-A. The authors speculated that centromeric CENP-A levels might serve as a biomarker of a cell's future proliferative potential.

Several adjacent results frame what is at stake. In cycling cells, the transcription elongation factor and histone chaperone Spt6 protects CENP-A from loss during transcription (Bobkov et al. 2020), and in fission yeast the Mis6/CENP-I kinetochore subcomplex blocks RNA polymerase II invasion into the core centromere (Hirai et al. 2022) — evidence that transcription and CENP-A maintenance are in active tension. Yet in early Drosophila embryos CENP-A (there named CID) incorporation occurs without RNA polymerase II-mediated transcription (Ghosh and Lehner 2022), and a 2025 study of cycling human cells reports that CENP-A deposition is likewise transcription-independent (Watanabe et al. 2025), so the transcription-dependence itself may be system-specific. Reviews of CENP-A stability and of histone supply and degradation (Mitra et al. 2020; Hogan and Foltz 2021; Pukało et al. 2025) treat the control of CENP-A amounts and its turnover as outstanding questions.

Problem Statement

How is centromere identity actively maintained in non-dividing cells? Swartz et al. (2019) established that quiescent, proliferation-capable cells gradually exchange centromeric CENP-A through HJURP/Mis18-dependent, Plk1-independent deposition, and left open the mechanism and scope of this process. The source question comprises three jointly linked sub-goals:

(1) The eviction–replacement mechanism. Identify the molecular machinery by which RNA polymerase II-mediated transcription at centromeres (or alternative forces, such as DNA-break-associated turnover) evicts centromeric nucleosomes in quiescent cells and creates sites for new CENP-A deposition: which chromatin remodelers (ATP-driven enzymes that reposition or remove nucleosomes) or chaperones are required, whether elongation or initiation is the critical step when transcription is the driving force, and how eviction and redeposition are coordinated so that centromeric CENP-A levels remain at steady state rather than being diluted or overaccumulated.

(2) Homeostatic control of the CENP-A supply. Determine whether HJURP abundance is the limiting parameter for the soluble CENP-A pool, by what machinery excess soluble CENP-A is recognized, removed, or stabilized, and which proteostatic or chaperone-dependent pathways set the size of the soluble pool during quiescence — including whether the SUMO (small ubiquitin-like modifier)/p97 and ubiquitin-ligase pathways uncovered for centromere-bound CENP-A and Mis18β in cycling cells also govern the quiescent soluble pool.

(3) Lineage generality and loss signals. Establish whether gradual CENP-A exchange is a general property of all quiescent but proliferation-capable cell types or is restricted to particular lineages — reconciling the active exchange in G0 human cells and starfish oocytes with the passive long-term persistence of CENP-A in mouse oocytes — and identify the signals that determine the loss of centromere maintenance during terminal differentiation. Swartz et al. (2019) further speculated that the presence and levels of centromeric CENP-A might provide a biomarker of a cell's future proliferative potential; that proposal remains untested and is outside the scope of this statement.

These sub-goals share a common objective: a mechanistic account of how the balanced loss and replenishment of centromeric CENP-A is executed and controlled outside the cell cycle, explaining steady-state maintenance, its regulation, and its occurrence (or absence) across quiescent lineages.

References

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