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
Read preprintSign up to customise the site to your preferences and to receive alerts
Register hereAlso in the biochemistry category:
Triglyceride metabolism controls inflammation and APOE4-associated disease states in microglia
Gustavo Stelzer, Marcus Oliveira
Impaired 26S proteasome causes learning and memory deficiency and induces neuroinflammation mediated by NF-κB in mice
Gustavo Stelzer, Marcus Oliveira
Notch3 is a genetic modifier of NODAL signalling for patterning asymmetry during mouse heart looping
Bhaval Parmar
Also in the cell biology category:
Germplasm stability in zebrafish requires maternal Tdrd6a and Tdrd6c
Justin Gutkowski
Leukocytes use endothelial membrane tunnels to extravasate the vasculature
Felipe Del Valle Batalla
Platelet-derived LPA16:0 inhibits adult neurogenesis and stress resilience in anxiety disorder
Harvey Roweth
Also in the molecular biology category:
Germplasm stability in zebrafish requires maternal Tdrd6a and Tdrd6c
Justin Gutkowski
Green synthesized silver nanoparticles from Moringa: Potential for preventative treatment of SARS-CoV-2 contaminated water
Safieh Shah, Benjamin Dominik Maier
Non-disruptive inducible labeling of ER-membrane contact sites using the Lamin B Receptor
Jonathan Townson
preListsbiochemistry category:
in theBSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
Peer Review in Biomedical Sciences
Communication of scientific knowledge has changed dramatically in recent decades and the public perception of scientific discoveries depends on the peer review process of articles published in scientific journals. Preprints are key vehicles for the dissemination of scientific discoveries, but they are still not properly recognized by the scientific community since peer review is very limited. On the other hand, peer review is very heterogeneous and a fundamental aspect to improve it is to train young scientists on how to think critically and how to evaluate scientific knowledge in a professional way. Thus, this course aims to: i) train students on how to perform peer review of scientific manuscripts in a professional manner; ii) develop students' critical thinking; iii) contribute to the appreciation of preprints as important vehicles for the dissemination of scientific knowledge without restrictions; iv) contribute to the development of students' curricula, as their opinions will be published and indexed on the preLights platform. The evaluations will be based on qualitative analyses of the oral presentations of preprints in the field of biomedical sciences deposited in the bioRxiv server, of the critical reports written by the students, as well as of the participation of the students during the preprints discussions.
List by | Marcus Oliveira et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
20th “Genetics Workshops in Hungary”, Szeged (25th, September)
In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf
List by | Nándor Lipták |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
List by | Osvaldo Contreras |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
List by | Pablo Ranea Robles |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |
Also in the cell biology category:
November in preprints – the CellBio edition
This is the first community-driven preList! A group of preLighters, with expertise in different areas of cell biology, have worked together to create this preprint reading lists for researchers with an interest in cell biology. Categories include: 1) cancer cell biology 2) cell cycle and division 3) cell migration and cytoskeleton 4) cell organelles and organisation 5) cell signalling and mechanosensing 6) genetics/gene expression
List by | Felipe Del Valle Batalla et al. |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
List by | Dey Lab |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
List by | Joseph Jose Thottacherry |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
List by | Dey Lab, Amanda Haage |
Also in the molecular biology category:
2024 Hypothalamus GRC
This 2024 Hypothalamus GRC (Gordon Research Conference) preList offers an overview of cutting-edge research focused on the hypothalamus, a critical brain region involved in regulating homeostasis, behavior, and neuroendocrine functions. The studies included cover a range of topics, including neural circuits, molecular mechanisms, and the role of the hypothalamus in health and disease. This collection highlights some of the latest advances in understanding hypothalamic function, with potential implications for treating disorders such as obesity, stress, and metabolic diseases.
List by | Nathalie Krauth |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
List by | Alex Eve, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
List by | Alex Eve |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |