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Title: A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin

Abstract

Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.

Authors:
 [1];  [2];  [3];  [1];  [1];  [3];  [2];  [4];  [5]
  1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
  2. Yale School of Medicine, New Haven, CT (United States)
  3. Netherlands Cancer Inst., Amsterdam (Netherlands)
  4. Georgetown Univ. Medical Center, Washington, DC (United States)
  5. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
National Institutes of Health (NIH); USDOE
OSTI Identifier:
1379517
Alternate Identifier(s):
OSTI ID: 1379518
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Genes & Development
Additional Journal Information:
Journal Volume: 30; Journal Issue: 14; Journal ID: ISSN 0890-9369
Publisher:
Cold Springs Harbor Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Janssen, Aniek, Breuer, Gregory A., Brinkman, Eva K., van der Meulen, Annelot I., Borden, Sean V., van Steensel, Bas, Bindra, Ranjit S., LaRocque, Jeannine R., and Karpen, Gary H. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin. United States: N. p., 2016. Web. doi:10.1101/gad.283028.116.
Janssen, Aniek, Breuer, Gregory A., Brinkman, Eva K., van der Meulen, Annelot I., Borden, Sean V., van Steensel, Bas, Bindra, Ranjit S., LaRocque, Jeannine R., & Karpen, Gary H. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin. United States. doi:10.1101/gad.283028.116.
Janssen, Aniek, Breuer, Gregory A., Brinkman, Eva K., van der Meulen, Annelot I., Borden, Sean V., van Steensel, Bas, Bindra, Ranjit S., LaRocque, Jeannine R., and Karpen, Gary H. Fri . "A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin". United States. doi:10.1101/gad.283028.116. https://www.osti.gov/servlets/purl/1379517.
@article{osti_1379517,
title = {A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin},
author = {Janssen, Aniek and Breuer, Gregory A. and Brinkman, Eva K. and van der Meulen, Annelot I. and Borden, Sean V. and van Steensel, Bas and Bindra, Ranjit S. and LaRocque, Jeannine R. and Karpen, Gary H.},
abstractNote = {Repair of DNA double-strand breaks (DSBs) must be properly orchestrated in diverse chromatin regions to maintain genome stability. The choice between two main DSB repair pathways, nonhomologous end-joining (NHEJ) and homologous recombination (HR), is regulated by the cell cycle as well as chromatin context. Pericentromeric heterochromatin forms a distinct nuclear domain that is enriched for repetitive DNA sequences that pose significant challenges for genome stability. Heterochromatic DSBs display specialized temporal and spatial dynamics that differ from euchromatic DSBs. Although HR is thought to be the main pathway used to repair heterochromatic DSBs, direct tests of this hypothesis are lacking. Here, we developed an in vivo single DSB system for both heterochromatic and euchromatic loci in Drosophila melanogaster. Live imaging of single DSBs in larval imaginal discs recapitulates the spatio-temporal dynamics observed for irradiation (IR)-induced breaks in cell culture. Importantly, live imaging and sequence analysis of repair products reveal that DSBs in euchromatin and heterochromatin are repaired with similar kinetics, employ both NHEJ and HR, and can use homologous chromosomes as an HR template. This direct analysis reveals important insights into heterochromatin DSB repair in animal tissues and provides a foundation for further explorations of repair mechanisms in different chromatin domains.},
doi = {10.1101/gad.283028.116},
journal = {Genes & Development},
number = 14,
volume = 30,
place = {United States},
year = {2016},
month = {7}
}

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Cited by: 25 works
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Works referenced in this record:

Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks
journal, March 2014

  • Aymard, François; Bugler, Beatrix; Schmidt, Christine K.
  • Nature Structural & Molecular Biology, Vol. 21, Issue 4
  • DOI: 10.1038/nsmb.2796

ATM and Artemis promote homologous recombination of radiation-induced DNA double-strand breaks in G2
journal, September 2009

  • Beucher, Andrea; Birraux, Julie; Tchouandong, Leopoldine
  • The EMBO Journal, Vol. 28, Issue 21
  • DOI: 10.1038/emboj.2009.276

Easy quantitative assessment of genome editing by sequence trace decomposition
journal, October 2014

  • Brinkman, Eva K.; Chen, Tao; Amendola, Mario
  • Nucleic Acids Research, Vol. 42, Issue 22
  • DOI: 10.1093/nar/gku936

Histone modifications predispose genome regions to breakage and translocation
journal, June 2015

  • Burman, Bharat; Zhang, Zhuzhu Z.; Pegoraro, Gianluca
  • Genes & Development, Vol. 29, Issue 13
  • DOI: 10.1101/gad.262170.115

Homologous-recombination-deficient tumours are dependent on Polθ-mediated repair
journal, February 2015

  • Ceccaldi, Raphael; Liu, Jessica C.; Amunugama, Ravindra
  • Nature, Vol. 518, Issue 7538
  • DOI: 10.1038/nature14184

Dual Roles for DNA Polymerase Theta in Alternative End-Joining Repair of Double-Strand Breaks in Drosophila
journal, July 2010


Age-Associated Loss of Lamin-B Leads to Systemic Inflammation and Gut Hyperplasia
journal, November 2014


Double-Strand Breaks in Heterochromatin Move Outside of a Dynamic HP1a Domain to Complete Recombinational Repair
journal, March 2011


Nuclear dynamics of radiation-induced foci in euchromatin and heterochromatin
journal, October 2013

  • Chiolo, Irene; Tang, Jonathan; Georgescu, Walter
  • Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Vol. 750, Issue 1-2
  • DOI: 10.1016/j.mrfmmm.2013.08.001

Repair of Double-Strand Breaks by End Joining
journal, May 2013

  • Chiruvella, K. K.; Liang, Z.; Wilson, T. E.
  • Cold Spring Harbor Perspectives in Biology, Vol. 5, Issue 5
  • DOI: 10.1101/cshperspect.a012757

Rapid and Tunable Control of Protein Stability in Caenorhabditis elegans Using a Small Molecule
journal, August 2013


The DNA Damage Response: Making It Safe to Play with Knives
journal, October 2010


Formaldehyde Fixation of Drosophila Tissues onto Slides for Whole-Mount FISH
journal, May 2012


Chromatin Movement in the Maintenance of Genome Stability
journal, March 2013


Double-Strand Break Repair Assays Determine Pathway Choice and Structure of Gene Conversion Events in Drosophila melanogaster
journal, December 2013

  • Do, Anthony T.; Brooks, Joseph T.; Le Neveu, Margot K.
  • G3: Genes|Genomes|Genetics, Vol. 4, Issue 3
  • DOI: 10.1534/g3.113.010074

Recognition of Double Strand Breaks by a Mutator Protein (MU2) in Drosophila melanogaster
journal, May 2009


The HP1 protein family: getting a grip on chromatin
journal, April 2000


Alterations of Global Histone H3K9 and H3K27 Methylation Levels in Bladder Cancer
journal, January 2014

  • Ellinger, Jörg; Bachmann, Anne; Göke, Friederike
  • Urologia Internationalis, Vol. 93, Issue 1
  • DOI: 10.1159/000355467

Mammalian Su ( var ) Genes in Chromatin Control
journal, November 2010


The Heterochromatic Barrier to DNA Double Strand Break Repair: How to Get the Entry Visa
journal, September 2012

  • Goodarzi, Aaron A.; Jeggo, Penny A.
  • International Journal of Molecular Sciences, Vol. 13, Issue 12
  • DOI: 10.3390/ijms130911844

ATM Signaling Facilitates Repair of DNA Double-Strand Breaks Associated with Heterochromatin
journal, July 2008


The Release 6 reference sequence of the Drosophila melanogaster genome
journal, January 2015

  • Hoskins, Roger A.; Carlson, Joseph W.; Wan, Kenneth H.
  • Genome Research, Vol. 25, Issue 3
  • DOI: 10.1101/gr.185579.114

DNA double-strand breaks in heterochromatin elicit fast repair protein recruitment, histone H2AX phosphorylation and relocation to euchromatin
journal, April 2011

  • Jakob, Burkhard; Splinter, Jörn; Conrad, Sandro
  • Nucleic Acids Research, Vol. 39, Issue 15, p. 6489-6499
  • DOI: 10.1093/nar/gkr230

Genetic instability: tipping the balance
journal, December 2012


Genetic manipulation of genomes with rare-cutting endonucleases
journal, June 1996


Multiple-Pathway Analysis of Double-Strand Break Repair Mutations in Drosophila
journal, January 2007


Comprehensive analysis of the chromatin landscape in Drosophila melanogaster
journal, December 2010

  • Kharchenko, Peter V.; Alekseyenko, Artyom A.; Schwartz, Yuri B.
  • Nature, Vol. 471, Issue 7339
  • DOI: 10.1038/nature09725

Nuclear position dictates DNA repair pathway choice
journal, November 2014

  • Lemaître, Charlène; Grabarz, Anastazja; Tsouroula, Katerina
  • Genes & Development, Vol. 28, Issue 22
  • DOI: 10.1101/gad.248369.114

Live Imaging of Drosophila Larval Neuroblasts
journal, January 2014

  • Lerit, Dorothy A.; Plevock, Karen M.; Rusan, Nasser M.
  • Journal of Visualized Experiments, Issue 89
  • DOI: 10.3791/51756

Homology-directed repair is a major double-strand break repair pathway in mammalian cells
journal, April 1998

  • Liang, F.; Han, M.; Romanienko, P. J.
  • Proceedings of the National Academy of Sciences, Vol. 95, Issue 9
  • DOI: 10.1073/pnas.95.9.5172

γH2AX: a sensitive molecular marker of DNA damage and repair
journal, February 2010

  • Mah, L-J; El-Osta, A.; Karagiannis, T. C.
  • Leukemia, Vol. 24, Issue 4
  • DOI: 10.1038/leu.2010.6

Homologous pairing and chromosome dynamics in meiosis and mitosis
journal, March 2004

  • McKee, Bruce D.
  • Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, Vol. 1677, Issue 1-3
  • DOI: 10.1016/j.bbaexp.2003.11.017

Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis
journal, April 2014

  • Nagel, Z. D.; Margulies, C. M.; Chaim, I. A.
  • Proceedings of the National Academy of Sciences, Vol. 111, Issue 18
  • DOI: 10.1073/pnas.1401182111

53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair
journal, January 2010

  • Noon, Angela T.; Shibata, Atsushi; Rief, Nicole
  • Nature Cell Biology, Vol. 12, Issue 2
  • DOI: 10.1038/ncb2017

H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability
journal, December 2006

  • Peng, Jamy C.; Karpen, Gary H.
  • Nature Cell Biology, Vol. 9, Issue 1
  • DOI: 10.1038/ncb1514

Heterochromatic Genome Stability Requires Regulators of Histone H3 K9 Methylation
journal, March 2009


Loss of the Suv39h Histone Methyltransferases Impairs Mammalian Heterochromatin and Genome Stability
journal, November 2001


Differential Usage of Alternative Pathways of Double-Strand Break Repair in Drosophila
journal, November 2005


Chromatin Remodeling at DNA Double-Strand Breaks
journal, March 2013


piRNA Production Requires Heterochromatin Formation in Drosophila
journal, August 2011


Plasticity in patterns of histone modifications and chromosomal proteins in Drosophila heterochromatin
journal, December 2010

  • Riddle, N. C.; Minoda, A.; Kharchenko, P. V.
  • Genome Research, Vol. 21, Issue 2
  • DOI: 10.1101/gr.110098.110

Megabase Chromatin Domains Involved in DNA Double-Strand Breaks in Vivo
journal, September 1999

  • Rogakou, Emmy P.; Boon, Chye; Redon, Christophe
  • The Journal of Cell Biology, Vol. 146, Issue 5
  • DOI: 10.1083/jcb.146.5.905

Heterochromatic breaks move to the nuclear periphery to continue recombinational repair
journal, October 2015

  • Ryu, Taehyun; Spatola, Brett; Delabaere, Laetitia
  • Nature Cell Biology, Vol. 17, Issue 11
  • DOI: 10.1038/ncb3258

Chromatin organization is a major influence on regional mutation rates in human cancer cells
journal, July 2012

  • Schuster-Böckler, Benjamin; Lehner, Ben
  • Nature, Vol. 488, Issue 7412
  • DOI: 10.1038/nature11273

Development of a novel method to create double-strand break repair fingerprints using next-generation sequencing
journal, February 2015


MDC1 Directly Binds Phosphorylated Histone H2AX to Regulate Cellular Responses to DNA Double-Strand Breaks
journal, December 2005


Crosstalk between chromatin state and DNA damage response in cellular senescence and cancer
journal, September 2012

  • Sulli, Gabriele; Di Micco, Raffaella; di Fagagna, Fabrizio d'Adda
  • Nature Reviews Cancer, Vol. 12, Issue 10
  • DOI: 10.1038/nrc3344

MiMIC: a highly versatile transposon insertion resource for engineering Drosophila melanogaster genes
journal, July 2011

  • Venken, Koen J. T.; Schulze, Karen L.; Haelterman, Nele A.
  • Nature Methods, Vol. 8, Issue 9
  • DOI: 10.1038/nmeth.1662

Heterochromatin and Gene Expression in Drosophila
journal, December 1995


Epigenetic regulation of gene expression by Drosophila Myb and E2F2-RBF via the Myb-MuvB/dREAM complex
journal, March 2008

  • Wen, H.; Andrejka, L.; Ashton, J.
  • Genes & Development, Vol. 22, Issue 5
  • DOI: 10.1101/gad.1626308

DNA damage and decisions: CtIP coordinates DNA repair and cell cycle checkpoints
journal, July 2010


Fly-FUCCI: A Versatile Tool for Studying Cell Proliferation in Complex Tissues
journal, April 2014


    Works referencing / citing this record:

    And yet, it moves: nuclear and chromatin dynamics of a heterochromatic double-strand break
    journal, August 2017

    • Caridi, P. Christopher; Delabaere, Laetitia; Zapotoczny, Grzegorz
    • Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 372, Issue 1731
    • DOI: 10.1098/rstb.2016.0291

    And yet, it moves: nuclear and chromatin dynamics of a heterochromatic double-strand break
    journal, August 2017

    • Caridi, P. Christopher; Delabaere, Laetitia; Zapotoczny, Grzegorz
    • Philosophical Transactions of the Royal Society B: Biological Sciences, Vol. 372, Issue 1731
    • DOI: 10.1098/rstb.2016.0291