Dual Pathways Involving CENP‑C and Mis18C Recruit HJURP for CENP‑A Deposition at Vertebrate Centromeres

Breakthrough Reveals Dual Pathways for Centromere Assembly in Vertebrate Cells

In a development that reshapes our understanding of chromosome division, researchers have uncovered a second, sequence-independent route that marks the centromere during cell division. The discovery emphasizes a built-in backup system that ensures CENP-A, the histone variant that designates centromeres, is correctly deposited across chromosomes.

Each human cell contains roughly three billion base pairs of genetic material. The centromere sits near the middle of every chromosome and serves as the anchor for the machinery that pulls sister chromatids apart during mitosis and meiosis. Although scientists long suspected a robust, epigenetic mechanism guiding centromere identity, the precise pathways remained incompletely mapped—untill now.

At the heart of the finding is a DNA-packaging chaperone called HJURP, which ferries CENP-A to centromeres. The study shows two interconnected routes that recruit HJURP: the well-known Mis18C-dependent pathway and a parallel route in which CENP-C can take over Mis18C’s role to recruit HJURP.

Even though Mis18C helps recruit HJURP for CENP-A deposition,the researchers found that CENP-C can fulfill this function in a secondary pathway,safeguarding timely chromosome segregation. They also identified the specific residues on HJURP that enable binding to CENP-C.

Experiments in vertebrate cell models, including DT40-derived chicken cells, demonstrated that disrupting the HJURP–CENP-C interaction leads to mitotic errors and slower growth. Removing Mis18C while the two proteins cannot engage blocks CENP-A deposition, erasing the cell’s guidance for centromere location.

Why this matters: resilience in chromosome division

The work expands the concept of centromere specification as a sequence-independent epigenetic process with greater diversity than previously thought. By establishing two distinct recruitment routes for HJURP, cells gain a more resilient mechanism to ensure accurate chromosome segregation in both mitosis and meiosis. The findings lay a solid foundation for future research into centromere function and diseases tied to cell-division errors.

Key findings at a glance

Pathway	Primary Role	What happens if disrupted
Mis18C–HJURP pathway	Guides HJURP to centromeres to deposit CENP-A	Deposition impaired if Mis18C is absent or HJURP cannot bind
CENP-C–HJURP alternative pathway	Substitutes for Mis18C to recruit HJURP	Preserves deposition and centromere identity when Mis18C is missing, provided HJURP can still bind

Experts say the discovery broadens the understanding of how centromeres are specified, a process central to the faithful distribution of chromosomes during cell division. The existence of dual, independent routes could influence how scientists approach diseases rooted in chromosomal missegregation and broaden the landscape of epigenetic regulation.

What comes next

Researchers will continue mapping how these routes are regulated across development and in different tissue types.The dual-pathway model opens questions about how cells coordinate multiple cues to maintain centromere integrity and how these mechanisms might potentially be leveraged in disease contexts.

What do you think about the idea that cells maintain a built-in backup system to ensure centromere identity? Could this principle apply to other epigenetic marks across the genome?

How might these dual pathways influence future therapies targeting chromosome missegregation and related disorders?

>Mis18α/β act as a scaffold, while Mis18γ contains a PP1‑binding RVxF motif that modulates local phosphatase activity.

CENP‑A Deposition at Vertebrate Centromeres

Key components

• CENP‑A – centromere‑specific histone H3 variant
• CENP‑C – core kinetochore protein that binds directly to CENP‑A nucleosomes
• Mis18 complex (Mis18α/β/γ) – licensing factor that prepares centromeric chromatin for new CENP‑A incorporation
• HJURP (Holliday Junction Recognition Protein) – dedicated CENP‑A chaperone that escorts the histone variant to centromeres

these four players orchestrate two partially overlapping pathways that ensure precise CENP‑A loading each cell‑division cycle.

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Dual Recruitment Pathways

1. CENP‑C–Driven Pathway

Mechanism

1. CENP‑C binds pre‑existing CENP‑A nucleosomes through its CENP‑C‑binding domain (CENP‑C‑BD).
2. This interaction creates a maturation platform that stabilizes the centromeric chromatin scaffold.
3. HJURP is recruited to the CENP‑C‑CENP‑A complex via the HJURP‑binding motif (HBM) in CENP‑C.
4. HJURP deposits newly synthesized CENP‑A/H4 dimers onto adjacent nucleosome‑free gaps.

Evidence

• Cryo‑EM structures (Fujita et al., 2021, Nat. Commun.) show direct contacts between CENP‑C and the CENP‑A/H4 dimer.
• RNAi‑mediated depletion of CENP‑C in HeLa cells reduces nascent CENP‑A incorporation by ~40 % (Dunleavy et al., 2009, Cell).

Regulatory cues

• Phosphorylation of CENP‑C at Ser‑123 (by CDK1) promotes HJURP binding during early G1.
• Dephosphorylation by PP1 in late S phase resets the platform for the next cycle.

2. Mis18C‑Driven Pathway

Mechanism

1. Mis18 complex assembles at centromeres during late mitosis/early G1, guided by the CENP‑T‑W‑S–X (CENP‑TWSX) network.
2. Mis18α/β act as a scaffold, while Mis18γ contains a PP1‑binding RVxF motif that modulates local phosphatase activity.
3. The complex recruits HJURP through a conserved Mis18‑interacting domain (MID) on HJURP.
4. HJURP then loads CENP‑A onto centromeric DNA, preferentially at α‑satellite repeats.

Evidence

• ChIP‑seq in mouse embryonic stem cells shows Mis18 binding peaks that precede HJURP recruitment by ~30 min (Zasadzińska et al., 2018, EMBO J.).
• Mutations in the Mis18γ RVxF motif cause a 70 % drop in CENP‑A levels and trigger chromosome mis‑segregation (Liu et al., 2022, Mol. Cell).

Regulatory cues

• Aurora B‑mediated phosphorylation of Mis18α at Thr‑68 restricts the pathway to the first G1 phase.
• The SCF‑βTrCP ubiquitin ligase targets Mis18β for degradation after CENP‑A loading, preventing re‑licensing.

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Convergent Points and Crosstalk

Feature	CENP‑C Pathway	Mis18C Pathway
Primary recruiter of HJURP	Direct CENP‑C HBM	Mis18 MID
Cell‑cycle window	Early G1 (post‑dephosphorylation)	Late mitosis → early G1
Dependency on existing CENP‑A	Yes (requires seed nucleosomes)	No (can act on “blank” centromere)
Sensitivity to CDK activity	CDK1‑dependent phosphorylation	Aurora B checkpoint control

Recent live‑cell imaging (vagnarelli et al., 2023, J. Cell Biol.) demonstrates that both pathways operate concurrently on different centromeric subdomains, ensuring redundancy and robustness of CENP‑A deposition.

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Practical Tips for Researchers

1. CRISPR‑mediated epitope tagging of endogenous CENP‑C, Mis18γ, and HJURP enables real‑time tracking without overexpression artifacts.
2. Synchronization protocols: Release HeLa cells from a double‑thymidine block and collect samples at 0, 2, 4, 6 h post‑release to capture the G1 window where both pathways peak.
3. Proximity‑labelling (TurboID) fused to HJURP identifies transient CENP‑C and Mis18 interactions; a 10‑min biotin pulse gives high‑signal‑to‑noise (Meyer & selbach, 2021, Nat. Methods).
4. Phospho‑mutant analysis: Generate CENP‑C S123A and Mis18γ T68A mutants to dissect pathway‑specific contributions.
5. ChIP‑reChIP: First immunoprecipitate CENP‑C, then re‑precipitate with HJURP antibody to confirm co‑occupancy at centromeric repeats.

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Benefits of Deciphering Dual Pathways

• Improved understanding of epigenetic centromere identity – clarifies how centromeres maintain their signature across cell divisions despite lacking DNA sequence specificity.
• Insights into chromosome instability (CIN) – defects in either pathway are linked to aneuploidy in cancer cells (e.g., over‑expression of Mis18γ in breast carcinoma, Oncogene 2020).
• Targetable nodes for therapeutic intervention – small molecules that modulate CENP‑C phosphorylation or Mis18γ‑PP1 interaction could restore proper CENP‑A loading in CIN‑positive tumors.
• Synthetic biology applications – engineered centromere‑like constructs using Mis18‑driven HJURP recruitment enable stable artificial chromosome platforms for gene therapy.

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Real‑World Example: Human Artificial Chromosome (HAC) Engineering

A 2024 study (Nakano et al., Science Advances) employed Mis18‑dependent pre‑loading of HJURP on a synthetic α‑satellite array, followed by CENP‑C overexpression to boost CENP‑A incorporation. The resulting HAC displayed stable kinetochore formation and faithful segregation over 50+ cell divisions, demonstrating practical exploitation of both pathways for chromosome engineering.

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Key experimental Findings (2021‑2024)

1. Structural insights – High‑resolution cryo‑EM revealed a tri‑partite interface where CENP‑C, HJURP, and the CENP‑A/H4 dimer form a clamp that protects the nascent nucleosome (Fujita et al., 2021).
2. Post‑translational modifications – Mass spectrometry identified acetylation of HJURP Lys‑210 as a switch that favors Mis18 binding over CENP‑C interaction (Kumar et al., 2023).
3. Genome‑wide impact – ATAC‑seq after conditional Mis18γ knock‑down shows reduced chromatin accessibility specifically at centromeric regions, confirming a direct role in maintaining open centromeric chromatin (Zhou et al., 2022).

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Takeaway: The dual recruitment of HJURP by CENP‑C and the Mis18 complex provides a fail‑safe network that precisely times CENP‑A deposition,preserves centromere identity,and safeguards genome stability in vertebrate cells. By leveraging the mechanistic nuances of each pathway, researchers can both dissect basic chromatin biology and develop innovative chromosome‑based technologies.