Close
Close

preLights is supported by…

Related posts

Related preLists

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

Preprint posted on 1 September 2020 https://www.biorxiv.org/content/10.1101/2020.09.01.277103v1

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

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

  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

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

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

Read preprint (No Ratings Yet)

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.

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

Also in the biochemistry category:

Structural basis of respiratory complexes adaptation to cold temperatures

Young-Cheul Shin, Pedro Latorre-Muro, Amina Djurabekova, et al.



Selected by Pamela Ornelas

Lens Placode Modulates Extracellular Matrix Formation During Early Eye Development

Cecília G. De Magalhães, Ales Cvekl, Ruy G. Jaeger, et al.



Selected by Bhaval Parmar

Generalized Biomolecular Modeling and Design with RoseTTAFold All-Atom

Rohith Krishna, Jue Wang, Woody Ahern, et al.



Selected by Saanjbati Adhikari

Also in the cell biology category:

Fetal brain response to maternal inflammation requires microglia

Bridget Elaine LaMonica Ostrem, Nuria Dominguez Iturza, Jeffrey Stogsdill, et al.



Selected by Manuel Lessi

Alteration of long and short-term hematopoietic stem cell ratio causes myeloid-biased hematopoiesis

Katsuyuki Nishi, Taro Sakamaki, Akiomi Nagasaka, et al.



Selected by Manuel Vicente, Anaisa Ferreira

Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments

Martina Begnis, Julien Duc, Sandra Offner, et al.



Selected by Silvia Carvalho

Also in the molecular biology category:

Fetal brain response to maternal inflammation requires microglia

Bridget Elaine LaMonica Ostrem, Nuria Dominguez Iturza, Jeffrey Stogsdill, et al.



Selected by Manuel Lessi

Nanos2+ cells give rise to germline and somatic lineages in the sea anemone Nematostella vectensis

Andreas Denner, Julia Steger, Alexander Ries, et al.



Selected by Isabella Cisneros

Plant plasmodesmata bridges form through ER-driven incomplete cytokinesis

Ziqiang P. Li, Hortense Moreau, Jules D. Petit, et al.

AND

Plasmodesmata act as unconventional membrane contact sites regulating inter-cellular molecular exchange in plants

Jessica Pérez-Sancho, Marija Smokvarska, Marie Glavier, et al.



Selected by Gwendolyn K. Kirschner

preLists in the biochemistry category:

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

Preprint Peer Review – Biochemistry Course at UFRJ, Brazil

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 biochemistry 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

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:

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:

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
Close