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CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy

Simona Giunta, Solène Hervé, Ryan R. White, Therese Wilhelm, Marie Dumont, Andrea Scelfo, Riccardo Gamba, Cheng Kit Wong, Giulia Rancati, Agata Smogorzewska, Hironori Funabiki, Daniele Fachinetti

Posted on: 10 December 2020 , updated on: 3 March 2021

Preprint posted on 1 September 2020

Article now published in PNAS at https://www.pnas.org/content/118/10/e2015634118

CENP-A at the heart of centromere DNA integrity.

Selected by Sree Rama Chaitanya

Context

Sister chromatids are attached during mitosis at chromosome regions called centromeres. Maintaining the integrity of centromeres is central to chromosome segregation and genome stability. But in cancerous cells, centromere integrity is undermined concomitantly by chromosomal translocations and recombinogenic events1-2. However, what mechanisms govern centromere DNA integrity is not known.

Centromeres hold an evolutionary conundrum: they are defined not solely by the presence of repetitive genetic sequences but epigenetically through the seeded Histone 3 variant – CENP-A1. Building on their earlier work that long-term loss of CENP-A promotes recombination at repetitive α-satellite DNA (present at human centromeres)2, the team hypothesized that CENP-A could be at the nexus of centromere fragility. Therefore, in the current work, the authors set out to investigate the molecular mechanisms of CENP-A in maintaining centromere DNA integrity.

 Key findings

  1. The authors used an auxin-inducible degron (AID) system in immortalized, non-transformed, diploid retinal pigment epithelial cells (hTERT-RPE-1) to rapidly deplete endogenous CENP-A3, thereby removing CENP-A containing nucleosomes (fig.1). They depleted CENP-A at different stages of the cell cycle and evaluated recombination events at centromeres using centromeric chromosome-orientation fluorescence in situ hybridization (Cen-CO-FISH4, fig.2). They demonstrate that the absence of CENP-A impinges on centromere integrity when cells replicate their DNA during S-phases (fig.3).

    (1) Schematic of the inducible degradation system to deplete endogenous CENP-A after addition of Auxin in RPE-1 cells, (2) Schematic illustration of the Cen-CO-FISH centromeric DNA probes. Hybridization by unidirectional PNA probes differentially labels the forward or reverse strands of each sister chromatid. Each black arrow symbolizes a homogenous higher-order repeat in the α-satellite array, (3) Representative Cen-CO-FISH on metaphase chromosomes after CENP-A depletion in the previous S-phase. (Right) Schematic of the resulted Cen-CO-FISH staining patterns in normal and abnormal centromeres, with visible sister-chromatid exchange due to recombination and cross over events. Taken directly from Giunta S et. al., 2020 under a CC-BY 4.0 international license.
  2. The authors then gauged the replication fork dynamics in CENP-A depleted cells, as replication stress can trigger recombination events. To investigate this, the authors labeled DNA with nucleotide analogs CldU and IdU, and carried out a single-molecule analysis of DNA fibers. They found that lack of CENP-A reduces replication fork speed of centromere DNA (and possibly at other late replicated regions), 7hrs after thymidine release. Intriguingly, they report an increase in active forks at centromeres, suggesting dormant origin firing.
  3. RNA-DNA hybrids (also called R-loops) are a major cause of replication-transcription conflicts (and replication stress)5. Moreover, centromeres are transcriptionally promiscuous in all phases of the cell cycle. Interestingly, in mitosis, centromere-specific R-loops elicit a stress response6. Therefore, the authors hypothesized the role of R-loops in inducing replication stress in CENP-A depleted cells. They found increased R-loops in CENP-A depleted cells at late S-phase (a time when centromeres are replicated) using R-loop binding S9.6 antibody-based immunofluorescence and immunoprecipitation techniques. This increase in R-loops was corroborated with 5-fluorouridine, RNA polymerase II (RNAPII), ATR, and γH2AX levels that represent nascent transcript levels, transcriptional activity, replication stress, and DNA damage, respectively. Moreover, stably expressed RNase H (that resolves R-loops) was able to reduce γH2AX levels and centromere instability (assayed by Cen-CO-FISH) in CENP-A depleted cells. Thus, they demonstrate that loss of CENP-A leads to aberrant R-loop mediated damage at centromeres.
  4. The authors then investigated further the consequences of replication stress on centromere fragility in CENP-A depleted cells. They demonstrate that CENP-A depleted cells form aberrant replication in mitosis (possibly leading to error-prone mitotic DNA synthesis) by measuring EdU incorporated at centromeres. They also demonstrated an increased prevalence of anaphase bridges and centromeric DNA breaks in CENP-A depleted cells. Interestingly, they do not observe any differences in ultra-fine bridges in CENP-A depleted cells. Thus, they suggest that CENP-A depleted cells manifest under-replicated DNA due to prominent replication defects.
  5. Furthermore, they elegantly demonstrate centromere breakage and acrocentric or metacentric whole-arm chromosomal translocations (by multi-color FISH) within two cell cycles of CENP-A depletion. However, while mitotic defects independent of centromere dysfunction (by PLK4 inhibitor centrinone) induced chromosome rearrangements, loss of CENP-A caused structural aneuploidy, specifically at centromeric regions. Thus, they suggest that chromosome translocations at centromeres in CENP-A deficient cells are dependent on replication stress and not solely on chromosome segregation defects.

Conclusion and perspective

R-loops are major endogenous sources of replication stress that further drives genome instability, a hallmark of cancer. In the past two decades, many investigators described how RNA/DNA binding factors, transcription and replication dynamics, cis-regulatory elements, and DNA topological constraints contribute to R-loop formation5.

However, little is known about how R-loops are regulated epigenetically. In these lines, some investigators reported the role of modifications of DNA, RNA, and histones in regulating R-loop distribution5. The current study adds to this and demonstrates how a histone variant CENP-A mitigates R-loops formation at centromeres. While an earlier study demonstrates that centromere R-loops support chromosome segregation in mitosis6, here, the authors report that centromere R-loops in S-phase cause centromere fragility and ensuing chromosome translocations (for a recent work in yeast7).

Centromere R-loops raise an interesting perspective on a long-standing question in the R-loop field – what makes an R-loop good, bad, or ugly?  One possible answer could be in the local chromatin milieu and the time (including which phase of the cell cycle) at which an R-loop forms or stays unresolved.

Acknowledgments

Thanks to Simona Giunta and all the authors of this work for their support to comment on this preLight.

References

  1. https://doi.org/10.1083/jcb.202005099
  2. https://doi.org/10.1073/pnas.1615133114
  3. https://doi.org/10.1016/j.celrep.2016.10.084
  4. https://doi.org/10.21769/BioProtoc.2792
  5. https://doi.org/10.1016/j.cell.2019.08.055
  6. https://doi.org/10.1126/science.aan6490
  7. https://doi.org/10.1091/mbc.e20-06-0379
  8. https://dx.doi.org/10.1016%2Fj.devcel.2015.05.012
  9. https://doi.org/10.1016/j.devcel.2019.07.016
  10. https://doi.org/10.1007/s12035-018-1246-y

 

Tags: cenp-a, centromere, chromatin, r loops

doi: https://doi.org/10.1242/prelights.25699

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Author's response

Simona Giunta (SC) and others shared

1. The authors suggest that the loss of CENP-A induces transcriptional activity and R-loops at centromeres. However, an earlier work suggests that RNAPII and histone chaperone (FACT) supports CENP-A establishment8. How do the authors reconcile their model in light of this evidence?

SC and others: Many pieces of evidence converge to the fact that transcription facilitates centromeric CENP-A incorporation, and our work does not disprove (but neither test) this. It is important to note that loss of CENP-A per se did not induce transcription in our system, as no increase in transcripts was detected at the time of CENP-A depletion (G1/S), but only concomitantly with DNA replication. It is therefore a case of both ongoing transcriptions and replication converging to generate R-loops at centromeres.

2. The authors report that loss of CENP-A leads to aberrant centromeres in the G1-phase (Fig1I). This could be relevant to how CENP-A regulates R-loops in post-mitotic cells (for example9). Moreover, R-loops seem to play a role in neurodegenerative diseases10. What are the authors’ perspectives on this?

SC and others: An immediate perspective of this work will be to study the physiological relevance of our findings as some previous studies report a natural decrease of CENP-A during both cellular senescence and organismal aging. Understanding the impact of CENP-A depletion and, in turn, centromere fragility in cellular homeostasis is certainly relevant to human physiology and disease.

3. Does RNase H rescue the chromosomal translocations in cells deficient in CENP-A? This might suggest if R-loops play a direct role in chromosomal translocations. What do the authors think about this model or experiment?

SC and others: This is a good question which we did not directly test, but our current results under these experimental settings do point towards this model.

4. Do the authors predict any trans-acting factors like RNA/DNA helicases or nucleases that regulate CENP-A mediated R-loops? This could be relevant for combinatorial therapies in cancers with mutations in CENP-A. It would be interesting to hear the authors comment on this.

SC and others: Absolutely, this is an interesting possibility that we are planning to explore. Understanding how these replication intermediates are processed and whether there could be synergies or synthetic lethality that can be exploited for therapy – it is very far fetched given our current limited understanding of the overall mechanism, but an exciting future prospective.

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