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The E3 ubiquitin ligase Rnf8 stabilizes Tpp1 to promote telomere end protection

Nature Structural & Molecular Biology volume 18, pages 1400–1407 (2011)Cite this article

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Abstract

The mammalian shelterin component TPP1 has essential roles in telomere maintenance and, together with POT1, is required for the repression of DNA damage signaling at telomeres. Here we show that in Mus musculus, the E3 ubiquitin ligase Rnf8 localizes to uncapped telomeres and promotes the accumulation of DNA damage proteins 53Bp1 and γ-H2ax. In the absence of Rnf8, Tpp1 is unstable, resulting in telomere shortening and chromosome fusions through the alternative nonhomologous end-joining (A-NHEJ) repair pathway. The Rnf8 RING-finger domain is essential for Tpp1 stability and retention at telomeres. Rnf8 physically interacts with Tpp1 to generate Ubc13-dependent Lys63 polyubiquitin chains that stabilize Tpp1 at telomeres. The conserved Tpp1 residue Lys233 is important for Rnf8-mediated Tpp1 ubiquitylation and localization to telomeres. Thus, Tpp1 is a newly identified substrate for Rnf8, indicating a previously unrecognized role for Rnf8 in telomere end protection.

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Figure 1: Rnf8 is required for the accumulation of DNA damage response factors to dysfunctional telomeres.
Figure 2: Rnf8 is required to protect telomere ends from chromosome aberrations.
Figure 3: Rnf8 is required for the accumulation of the Tpp1–Pot1a-b complex at telomeres.
Figure 4: Rnf8 is required for preventing Tpp1 from proteasome-mediated degradation.
Figure 5: Rnf8 physically interacts with and ubiquitylates Tpp1.
Figure 6: Lys233 is required for TPP1 ubiquitylation, telomere localization and genome stability.

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Acknowledgements

We are grateful to J. Karlseder (Salk Institute) for providing anti-mouse Trf1 and Trf2 antibodies, to Z. Songyang (Baylor College of Medicine) for providing anti-human TPP1 antibody and TPP1 cDNA constructs and to D. Durocher (Samuel Lunenfeld Research Institute) for providing mouse Rnf8 and Rnf8 mutant cDNAs. The linkage-specific antibodies to Lys63 and Lys48 ubiquitin conjugates were provided by V.M. Dixit (Genentech), and pLPC-hRAP1 was a gift from M. Lei (University of Michigan Medical School). We would like to thank A. Multani (MD Anderson Cancer Center) for help with chromosome analysis and I. Patanam for technical support. S.C. acknowledges generous financial support from the National Cancer Institute (RO1 CA129037) and the Michael and Betty Kadoorie Cancer Genetic Research Program. J.J. is a Pew Scholar and is supported by a grant (AU-1711) from the Welch Foundation.

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Author notes

  1. Rekha Rai and Ju-Mei Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut, USA

    Rekha Rai & Sandy Chang

  2. Department of Biochemistry and Molecular Biology, University of Texas Health Science Center, Houston, Texas, USA

    Ju-Mei Li & Jianping Jin

  3. Department of Lymphoma Research, The M.D. Anderson Cancer Center, Houston, Texas, USA

    Hong Zheng

  4. Genome Stability Research Laboratory, University of Hong Kong, Pokfulam, Hong Kong

    Gabriel Tsz-Mei Lok & Michael S-Y Huen

  5. Department of Genetics, The M.D. Anderson Cancer Center, Houston, Texas, USA

    Yu Deng

  6. Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, Connecticut, USA

    Michael S-Y Huen & Junjie Chen

  7. Department of Experimental Radiation Oncology, The M.D. Anderson Cancer Center, Houston, Texas, USA

    Junjie Chen

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Contributions

R.R., J.J. and S.C. conceived the project and designed the experiments. R.R., J.J., J.-M.L. and H.Z. conducted the experiments and generated data for the figures. Y.D. generated anti-mouse Tpp1 antibody. J.C. provided the Rnf8−/− mice and anti-human RNF8 antibody. M.S.-Y.H. and G.T.-M.L. provided human RNF8 cDNAs and shared unpublished results. R.R., J.J. and S.C. analyzed and interpreted the data. R.R. and S.C. created the figures and wrote the paper.

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Correspondence to Sandy Chang.

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Rai, R., Li, JM., Zheng, H. et al. The E3 ubiquitin ligase Rnf8 stabilizes Tpp1 to promote telomere end protection. Nat Struct Mol Biol 18, 1400–1407 (2011). https://doi.org/10.1038/nsmb.2172

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