CENP-A chromatin prevents replication stress at centromeres to avoid structural aneuploidy
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
Categories: biochemistry, cell biology, molecular biology
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
- 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).
- 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.
- 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.
- 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.
- 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
- https://doi.org/10.1083/jcb.202005099
- https://doi.org/10.1073/pnas.1615133114
- https://doi.org/10.1016/j.celrep.2016.10.084
- https://doi.org/10.21769/BioProtoc.2792
- https://doi.org/10.1016/j.cell.2019.08.055
- https://doi.org/10.1126/science.aan6490
- https://doi.org/10.1091/mbc.e20-06-0379
- https://dx.doi.org/10.1016%2Fj.devcel.2015.05.012
- https://doi.org/10.1016/j.devcel.2019.07.016
- https://doi.org/10.1007/s12035-018-1246-y
doi: https://doi.org/10.1242/prelights.25699
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