Loss of Rap1 Induces Telomere Recombination in the Absence of NHEJ or a DNA Damage Signal

The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA.
Science (Impact Factor: 33.61). 03/2010; 327(5973):1657-61. DOI: 10.1126/science.1185100
Source: PubMed


Shelterin is an essential telomeric protein complex that prevents DNA damage signaling and DNA repair at mammalian chromosome ends. Here we report on the role of the TRF2-interacting factor Rap1, a conserved shelterin subunit of unknown function. We removed Rap1 from mouse telomeres either through gene deletion or by replacing TRF2 with a mutant that does not bind Rap1. Rap1 was dispensable for the essential functions of TRF2--repression of ATM kinase signaling and nonhomologous end joining (NHEJ)--and mice lacking telomeric Rap1 were viable and fertile. However, Rap1 was critical for the repression of homology-directed repair (HDR), which can alter telomere length. The data reveal that HDR at telomeres can take place in the absence of DNA damage foci and underscore the functional compartmentalization within shelterin.

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    • "The only telomere deprotection phenotype in Rap1-deficient mouse cells is a propensity for telomere-telomere recombination when Ku70/80 are also absent (Sfeir et al., 2010). "
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    ABSTRACT: The conserved protein Rap1 functions at telomeres in fungi, protozoa, and vertebrates. Like yeast Rap1, human Rap1 has been implicated in telomere length regulation and repression of nonhomologous end-joining (NHEJ) at telomeres. However, mouse telomeres lacking Rap1 do not succumb to NHEJ. To determine the functions of human Rap1, we generated several transcription activator-like effector nuclease (TALEN)-mediated human cell lines lacking Rap1. Loss of Rap1 did not affect the other components of shelterin, the modification of telomeric histones, the subnuclear position of telomeres, or the 3' telomeric overhang. Telomeres lacking Rap1 did not show a DNA damage response, NHEJ, or consistent changes in their length, indicating that Rap1 does not have an important function in protection or length regulation of human telomeres. As human Rap1, like its mouse and unicellular orthologs, affects gene expression, we propose that the conservation of Rap1 reflects its role in transcriptional regulation rather than a function at telomeres. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
    Cell Reports 11/2014; 9(4):1273-80. DOI:10.1016/j.celrep.2014.10.014 · 8.36 Impact Factor
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    • "The hypothesis of exercise-induced shelterin instability is reinforced by the simultaneous decrease in TERF2IP mRNA, the protein of which forms part of the shelterin complex and is recruited to telomeres via interaction with TRF2 (also known as TERF2) [65]. TERF2IP deficiency reduces telomere stability and increases recombination [66]. Equivocal results have identified TERF2IP as both a negative regulator [64] and a positive regulator of telomere length [65]. "
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    ABSTRACT: Telomeres are specialized nucleoprotein structures that protect chromosomal ends from degradation. These structures progressively shorten during cellular division and can signal replicative senescence below a critical length. Telomere length is predominantly maintained by the enzyme telomerase. Significant decreases in telomere length and telomerase activity are associated with a host of chronic diseases; conversely their maintenance underpins the optimal function of the adaptive immune system. Habitual physical activity is associated with longer leukocyte telomere length; however, the precise mechanisms are unclear. Potential hypotheses include regulation of telomeric gene transcription and/or microRNAs (miRNAs). We investigated the acute exercise-induced response of telomeric genes and miRNAs in twenty-two healthy males (mean age = 24.1±1.55 years). Participants undertook 30 minutes of treadmill running at 80% of peak oxygen uptake. Blood samples were taken before exercise, immediately post-exercise and 60 minutes post-exercise. Total RNA from white blood cells was submitted to miRNA arrays and telomere extension mRNA array. Results were individually validated in white blood cells and sorted T cell lymphocyte subsets using quantitative real-time PCR (qPCR). Telomerase reverse transcriptase (TERT) mRNA (P = 0.001) and sirtuin-6 (SIRT6) (P<0.05) mRNA expression were upregulated in white blood cells after exercise. Fifty-six miRNAs were also differentially regulated post-exercise (FDR <0.05). In silico analysis identified four miRNAs (miR-186, miR-181, miR-15a and miR-96) that potentially targeted telomeric gene mRNA. The four miRNAs exhibited significant upregulation 60 minutes post-exercise (P<0.001). Telomeric repeat binding factor 2, interacting protein (TERF2IP) was identified as a potential binding target for miR-186 and miR-96 and demonstrated concomitant downregulation (P<0.01) at the corresponding time point. Intense cardiorespiratory exercise was sufficient to differentially regulate key telomeric genes and miRNAs in white blood cells. These results may provide a mechanistic insight into telomere homeostasis and improved immune function and physical health.
    PLoS ONE 04/2014; 9(4):e92088. DOI:10.1371/journal.pone.0092088 · 3.23 Impact Factor
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    • "Immunofluorescence-Telomere FISH The immunofluorescence (IF)-FISH method was performed based on an established protocol (Sfeir et al., 2010). Briefly, cells were grown on gelatintreated coverslips and fixed with 2% paraformaldehyde for 10 min at room temperature. "
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    ABSTRACT: Telomere length homeostasis is essential for genomic stability and unlimited self-renewal of embryonic stem cells (ESCs). We show that telomere-associated protein Rif1 is required to maintain telomere length homeostasis by negatively regulating Zscan4 expression, a critical factor for telomere elongation by recombination. Depletion of Rif1 results in terminal hyperrecombination, telomere length heterogeneity, and chromosomal fusions. Reduction of Zscan4 by shRNA significantly rescues telomere recombination defects of Rif1-depleted ESCs and associated embryonic lethality. Further, Rif1 negatively modulates Zscan4 expression by maintaining H3K9me3 levels at subtelomeric regions. Mechanistically, Rif1 interacts and stabilizes H3K9 methylation complex. Thus, Rif1 regulates telomere length homeostasis of ESCs by mediating heterochromatic silencing.
    Developmental Cell 04/2014; 29(1):7-19. DOI:10.1016/j.devcel.2014.03.004 · 9.71 Impact Factor
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