Ring Finger Protein RNF169 Antagonizes the Ubiquitin-dependent Signaling Cascade at Sites of DNA Damage*

Background: Ubiquitylation plays important roles in DNA damage signal transduction. However, mechanistic details that drive these ubiquitin-dependent signals at DNA breaks remain to be determined. Results: RNF169 localized at DNA double-strand breaks (DSBs), inhibited DNA damage-induced ubiquitin formation, and attenuated 53BP1 accumulation. Conclusion: RNF169 antagonizes ubiquitin signaling at DNA DSBs. Significance: RNF169 represents a negative regulator of the ubiquitin-dependent DNA damage signaling cascade. Ubiquitin signals emanating from DNA double-strand breaks (DSBs) trigger the ordered assembly of DNA damage mediator and repair proteins. This highly orchestrated process is accomplished, in part, through the concerted action of the RNF8 and RNF168 E3 ligases, which have emerged as core signaling intermediates that promote DSB-associated ubiquitylation events. In this study, we report the identification of RNF169 as a negative regulator of the DNA damage signaling cascade. We found that RNF169 interacted with ubiquitin structures and relocalized to DSBs in an RNF8/RNF168-dependent manner. Moreover, ectopic expression of RNF169 attenuated ubiquitin signaling and compromised 53BP1 accumulation at DNA damage sites, suggesting that RNF169 antagonizes RNF168 functions at DSBs. Our study unveils RNF169 as a component in DNA damage signal transduction and adds to the complexity of regulatory ubiquitylation in genome stability maintenance.

[1]  Jiri Bartek,et al.  RNF8 Ubiquitylates Histones at DNA Double-Strand Breaks and Promotes Assembly of Repair Proteins , 2007, Cell.

[2]  N. Mailand,et al.  HERC2 coordinates ubiquitin-dependent assembly of DNA repair factors on damaged chromosomes , 2010, Nature Cell Biology.

[3]  Junjie Chen,et al.  Critical Roles of Ring Finger Protein RNF8 in Replication Stress Responses* , 2011, The Journal of Biological Chemistry.

[4]  Edward S. Miller,et al.  RIDDLE immunodeficiency syndrome is linked to defects in 53BP1-mediated DNA damage signaling , 2007, Proceedings of the National Academy of Sciences.

[5]  Bernhard Kuster,et al.  Requirement of ATM-dependent monoubiquitylation of histone H2B for timely repair of DNA double-strand breaks. , 2011, Molecular cell.

[6]  Junjie Chen,et al.  Regulation of chromatin architecture by the PWWP domain-containing DNA damage-responsive factor EXPAND1/MUM1. , 2010, Molecular cell.

[7]  Edward S. Miller,et al.  The RIDDLE Syndrome Protein Mediates a Ubiquitin-Dependent Signaling Cascade at Sites of DNA Damage , 2009, Cell.

[8]  R. Greenberg,et al.  ATM-Dependent Chromatin Changes Silence Transcription In cis to DNA Double-Strand Breaks , 2010, Cell.

[9]  R. Greenberg,et al.  The ubiquitin landscape at DNA double-strand breaks , 2009, The Journal of cell biology.

[10]  Junjie Chen,et al.  Assembly of checkpoint and repair machineries at DNA damage sites. , 2010, Trends in biochemical sciences.

[11]  Laurence Pelletier,et al.  Orchestration of the DNA-Damage Response by the RNF8 Ubiquitin Ligase , 2007, Science.

[12]  Michael B. Yaffe,et al.  RNF8 Transduces the DNA-Damage Signal via Histone Ubiquitylation and Checkpoint Protein Assembly , 2007, Cell.

[13]  C. Doil,et al.  Homozygous deficiency of ubiquitin-ligase ring-finger protein RNF168 mimics the radiosensitivity syndrome of ataxia-telangiectasia , 2011, Cell Death and Differentiation.

[14]  S. Confalonieri,et al.  UMI, a Novel RNF168 Ubiquitin Binding Domain Involved in the DNA Damage Signaling Pathway , 2010, Molecular and Cellular Biology.

[15]  Frédérick A. Mallette,et al.  RNF8‐ and RNF168‐dependent degradation of KDM4A/JMJD2A triggers 53BP1 recruitment to DNA damage sites , 2012, The EMBO journal.

[16]  Shreya Paliwal,et al.  The ubiquitin-selective segregase VCP/p97 orchestrates the response to DNA double-strand breaks , 2011, Nature Cell Biology.

[17]  A. Gingras,et al.  Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1 , 2010, Nature.

[18]  N. Dantuma,et al.  The AAA-ATPase VCP/p97 promotes 53BP1 recruitment by removing L3MBTL1 from DNA double-strand breaks , 2011, Nature Structural &Molecular Biology.

[19]  D. Durocher,et al.  Genomic Instability, Defective Spermatogenesis, Immunodeficiency, and Cancer in a Mouse Model of the RIDDLE Syndrome , 2011, PLoS genetics.

[20]  Junjie Chen,et al.  The E3 ligase RNF8 regulates KU80 removal and NHEJ repair , 2012, Nature Structural &Molecular Biology.

[21]  S. Elledge,et al.  Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage , 2007, Proceedings of the National Academy of Sciences.

[22]  D. Durocher,et al.  The ubiquitous role of ubiquitin in the DNA damage response , 2010, DNA Repair.

[23]  N. Mailand,et al.  A new non‐catalytic role for ubiquitin ligase RNF8 in unfolding higher‐order chromatin structure , 2012, The EMBO journal.

[24]  D. Durocher,et al.  Regulatory ubiquitylation in response to DNA double-strand breaks. , 2009, DNA repair.

[25]  S. Gasser,et al.  Crosstalk between histone modifications during the DNA damage response. , 2009, Trends in cell biology.

[26]  D. Durocher,et al.  The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination , 2009, Proceedings of the National Academy of Sciences.

[27]  Yu Deng,et al.  The E3 ubiquitin ligase Rnf8 stabilizes Tpp1 to promote telomere end protection , 2011, Nature Structural &Molecular Biology.

[28]  J. Ellenberg,et al.  RNF168 Binds and Amplifies Ubiquitin Conjugates on Damaged Chromosomes to Allow Accumulation of Repair Proteins , 2009, Cell.

[29]  S. Jackson,et al.  Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. , 2011, Genes & development.

[30]  H. Walden,et al.  Ubiquitin signalling in DNA replication and repair , 2010, Nature Reviews Molecular Cell Biology.