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

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Science  26 Mar 2010:
Vol. 327, Issue 5973, pp. 1657-1661
DOI: 10.1126/science.1185100

Shelterin the Ends

The ends of linear chromosomes suffer two problems: They cannot be replicated to their termini, resulting in loss of terminal sequences; and they can be mistakenly sensed as DNA double-strand breaks, activating DNA repair pathways that can result in serious genome derangement. These problems are solved by the addition of telomeres, repeat sequences at the ends of chromosomes, which are shielded by a protein complex called shelterin. Sfeir et al. (p. 1657) show that the mouse Rap1 protein, which is part of the shelterin complex and which binds to a second shelterin protein called TRF2, helps prevent telomeres undergoing unscheduled homologous recombination. Such recombination could threaten telomere integrity by generating sequence exchanges between sister telomeres resulting in critically shortened telomeres.

Abstract

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.

The shelterin subunit TRF2 is involved in the repression of the telomeric DNA damage response (1). Deletion of TRF2 results in activation of the ataxia telangiectasia mutated (ATM) kinase and telomere fusions mediated by nonhomologous end joining (NHEJ). TRF2 also contributes to the repression of homology-directed repair (HDR), which can create undesirable telomeric sister chromatid exchanges (T-SCEs). HDR at telomeres occurs in Ku70/80-deficient cells upon deletion of either TRF2 or the two POT1 proteins (2, 3). The repression of ATM signaling, NHEJ, and HDR by TRF2 could potentially involve Rap1, which depends on TRF2 for its stable expression and recruitment to telomeres (4, 5). Telomere protection is one of the functions of the distantly related Rap1 orthologs in yeast. In Saccharomyces cerevisiae and Schizosccharomyces pombe, Rap1 contributes to the repression of NHEJ at chromosome ends, whereas Kluyveromyces lactis Rap1 represses HDR (69). Human Rap1 affects telomere-length homeostasis and has been reported to repress telomere fusions (1012). Here, we determined how Rap1 loss affects telomere function by generating mouse cells lacking a functional Rap1 gene or lacking the endogenous TRF2 and complemented with a TRF2 mutant incapable of binding Rap1.

Because the first exon of the mouse Rap1 gene immediately abuts the essential Kars lysyl–tRNA synthetase gene, we developed a conditional knockout strategy to delete exon 2 (Fig. 1, A to C). The Rap1∆ex2 allele generated by Cre recombinase treatment of Rap1F/F cells can potentially encode a Rap1 fragment that lacks the TRF2-binding domain (Fig. 1A). We verified that this truncated form of Rap1, if it were produced, would not bind chromatin or localize to telomeres (fig. S1, A to C). Immunofluorescence (IF) and immunoblotting showed that Cre-treated SV40LT-immortalized Rap1F/F mouse embryonic fibroblasts (MEFs) indeed lacked any detectable Rap1 protein, and chromatin immunoprecipitation (ChIP) showed the loss of Rap1 from telomeres (Fig. 1, D to F, and fig. S1D). The expression and localization of other shelterin components were not significantly affected (Fig. 1, D to F, and fig. S1E).

Fig. 1

Deletion of Rap1 does not affect cell and organismal viability. (A) Schematic of Rap1, the mouse Rap1 (Terf2ip) locus, the targeting construct, the floxed allele, and the ∆ex2 allele. N, Nde I; B, Bam HI; F1, F2, and R, polymerase chain reaction primers. Rap1 shRNAs are shown at the bottom. At right, Rap1∆ex2-encoded protein. (B) Genomic blot of Nde I–digested DNA from embryonic stem (ES) cells. Probe is in (A). (C) Genotyping of tail DNAs. Primers are in (A). (D) Immunoblots for Rap1 (Ab1252), TRF2 (Ab1254), and TRF1 (Ab1449) from Rap1F/F and Rap1F/+ MEFs 5 days after Hit-and-run (H&R)-Cre (first lane) or pWZL-Cre (second lane). (E) Loss of Rap1 IF signal from Cre-treated (day 5) Rap1F/F MEFs. Red, Rap1; green, telomeric fluorescence in situ hybridization (FISH); blue, DNA (DAPI, 4′,6′-diamidino-2-phenylindole). (F) Telomeric ChIPs on Cre-treated (day 5) Rap1F/F MEFs. Numbers represent ratios of percent telomeric DNA in the ChIPs [preimmune (PI) signal subtracted] on cells with (+) and without (−) Cre. (G) Proliferation of SV40LT-immortalized Rap1F/F and Rap1F/+ MEFs infected as indicated. (H) Offspring from Rap1ex2/+ and Rap1ex2/∆ex2 intercrosses.

The growth rate of the Rap1ex2/∆ex2 MEFs was similar to that of control cells, regardless of whether the cells were immortalized with SV40LT, and primary MEFs lacking wild-type Rap1 did not show a growth arrest or p53 activation (Fig. 1G and fig. S1, F and G). Furthermore, Rap1ex2/∆ex2 mice were born at the expected frequencies and were fertile (Fig. 1H). The survival of Rap1ex2/∆ex2 cells and mice indicates that Rap1 deletion does not result in major telomere dysfunction, which is known to be lethal. We further corroborated this conclusion by infecting Rap1ex2/∆ex2 MEFs with a short hairpin RNA (shRNA)–targeting exon 1 (Fig. 1A and fig. S1H), which did not induce a growth arrest or other phenotypes typical of telomere dysfunction.

In the second approach to remove Rap1 from telomeres, we used previously characterized TRF2F/−p53−/− MEFs (4) to replace the endogenous TRF2 with a mutant that does not bind to Rap1. A short predicted helix at position 290 in the previously mapped Rap1-binding region [amino acids 260 to 360 (5)] was conserved in TRF2 orthologs but not in TRF1 (fig. S2, A and B). Two mutations in this region (A289S and F290S) reduced the interaction between Rap1 and TRF2 in coimmunoprecipitation experiments (fig. S2C). To generate TRF2∆Rap1, we deleted amino acids 284 to 297 (Fig. 2A). TRF2∆Rap1 failed to bind to Rap1 in coimmunoprecipitation experiments, whereas it retained its interaction with Apollo (Fig. 2B). Wild-type TRF2 and TRF2∆Rap1 were expressed in TRF2F/−p53−/− MEFs, and the endogenous TRF2 was removed with Cre (Fig. 2C). Although TRF2∆Rap1 localized to telomeres efficiently, IF and ChIP indicated that the telomeres lacked Rap1, and the overall abundance of Rap1 in the cells was reduced (Fig. 2, C to E, and fig. S3A). Other shelterin components were affected to an extent (less than twofold; Fig. 2, D and E) that is not expected to be functionally important because heterozygous MEFs and mice lacking one copy of TRF1, TPP1, TRF2, or POT1a/b display no telomere defect. Consistent with the viability of Rap1∆ex2/∆ex2 cells, cells expressing TRF2∆Rap1 proliferated at the same rate as cells expressing wild-type TRF2 (fig. S3B).

Fig. 2

A TRF2 mutant deficient for Rap1 binding. (A) The TRF2Rap1 mutant. H, predicted helix. Amino acid residues: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; Q, Gln; R, Arg; S, Ser; and T, Thr. (B) Coimmunoprecipitation of Myc-TRF2 or Myc-TRF2Rap1 with FLAG-Rap1 or FLAG-Apollo from cotransfected 293T cells. In, 2.5% of input. (C) Immunoblots for TRF2 and Rap1 from TRF2F/-p53−/− MEFs expressing the indicated alleles at 72 and 96 hours after H&R-Cre. (D) IF-FISH to monitor TRF2, Rap1, and TIN2 at telomeres in TRF2F/−p53−/− MEFs expressing TRF2∆Rap1 or vector control at day 4 after Cre treatment. (E) Telomeric ChIP of TRF2F/−p53−/− MEFs expressing TRF2 or TRF2∆Rap1 at day 7 after Cre treatment. Duplicate dot blots were probed for telomeric DNA or the dispersed Bam HI repeats. ChIP ratios represent the percentage of telomeric DNA recovered in TRF2∆Rap1- versus TRF2-expressing cells calculated as in Fig. 1.

Rap1∆ex2/∆ex2 cells did not show telomere dysfunction–induced foci [TIFs (13)], which are telomeric DNA damage foci that report on ATM and/or ATR (ataxia telangiectasia and Rad3-related) signaling at chromosome ends, and phosphorylation of Chk1 and Chk2 was not evident (Fig. 3, A to C). Further depletion of Rap1 mRNA with an shRNA also failed to elicit a DNA damage signal in Rap1∆ex2/∆ex2 cells (Fig. 3B). Consistent with these results, TRF2∆Rap1 was equivalent to wild-type TRF2 in its ability to repress TIFs in cells lacking the endogenous TRF2 (Fig. 3D). The mutant form of TRF2 also repressed the induction of Chk-2 phosphorylation to the same extent as wild-type TRF2 (Fig. 3E). The low level of Chk2-P observed in Cre-treated TRF2- and TRF2∆Rap1-expressing cells is likely due to Cre-induced DNA damage, because the phosphorylation of Chk2 was diminished when using a version of Cre that eventually disappears from the cells due to self-deletion (fig. S4). Furthermore, telomere fusions were not induced by deletion of Rap1, and TRF2∆Rap1 had the same ability as wild-type TRF2 to repress NHEJ at telomeres (Fig. 3, F to H). However, because NHEJ of telomeres lacking TRF2 requires active DNA damage signaling (14), the lack of telomere fusions could be due to the lack of ATM or ATR activation. We therefore used a TPP1 shRNA to activate ATR kinase signaling at telomeres. This approach previously resulted in the reactivation of NHEJ at telomeres of cells lacking both TRF2 and ATM (14). Despite the telomeric ATR kinase signal elicited by the TPP1 shRNA (Fig. 3, B and D), Rap1 removal from telomeres did not induce their fusion (Fig. 3, G and H).

Fig. 3

No DNA damage signal or NHEJ at the telomeres lacking Rap1. (A) TIF assay on Rap1F/F MEFs treated with Cre and the indicated shRNA. Red, IF for 53BP1; green, telomeric FISH; blue, DNA (DAPI). (B) TIF assay quantification. Mean ± SEM of two independent experiments (n ≥ 100 nuclei each). (C) Chk2-P in Rap1-deficient MEFs. TRF2-null cells and ionizing radiation (IR)–treated cells [1 hour after 2-Gray (Gy) dose] serve as positive controls. (D) Quantification of TIF assays on TRF2F/−p53−/− cells expressing TRF2, TRF2Rap1, or vector control at day 4 after Cre treatement. Mean ± SD of three independent experiments (n ≥ 100 nuclei each). (E) Chk1 and Chk2 phosphorylation in TRF2F/−p53−/− MEFs expressing TRF2, TRF2Rap1, or vector control. Ultraviolet (1 hour after 25 J/m2 dose)– and IR (1 hour after 2-Gy dose)–treated cells serve as positive controls. (F) Metaphase chromosomes from Rap1F/F cells 5 days after Cre treatment. Red, DNA (DAPI); green, telomeric FISH. (G) Quantification of telomere fusions, detected as in (F) in Rap1F/F MEFs with the indicated Cre and shRNA treatments. Average (av) percentage of telomeres fused is given. (H) Quantification of telomere fusions in TRF2F/−p53−/− MEFs [with (+) or without (–) Cre, day 4] complemented with TRF2 or TRF2Rap1 or vector control and treated with TPP1 shRNA as indicated.

Thus, Rap1 does not appear to be required in the repression of either NHEJ or ATM kinase signaling, explaining why the deletion of Rap1 does not curb cellular or organismal viability. In addition, Rap1 was not required for the maintenance of several other features of mouse telomeres, including the maintenance of telomere length over three generations of mouse breeding and in cultured cells, the amount of single-stranded telomeric DNA, the telomeric nucleosomal organization, the methylation of telomeric H3K9, and the abundance of telomeric transcripts [TERRA (15)] (fig. S5).

HDR threatens telomere integrity because unequal T-SCEs can change telomere lengths. T-SCEs are most frequent when either TRF2 or POT1a/b are deleted from Ku-deficient cells (2, 3), although low frequency of T-SCEs have been reported for POT1a deficiency alone (16). To determine whether Rap1 was required for TRF2-mediated repression of T-SCEs, we introduced TRF2∆Rap1 into SV40LT-immortalized TRF2F/−Ku70−/− MEFs, which display frequent T-SCEs upon deletion of TRF2 with Cre (2) (Fig. 4). Whereas the telomeric exchanges were repressed by wild-type TRF2, TRF2∆Rap1 failed to block the telomeric HDR (Fig. 4, A to D). The frequency of T-SCEs was the same whether the cells expressed TRF2∆Rap1 or no TRF2. Furthermore, T-SCEs were induced by Cre-mediated deletion of Rap1 from Rap1F/FKu70−/− cells (Fig. 4E). The T-SCEs occurred despite the absence of TIFs in cells lacking both Ku70 and telomeric Rap1 (fig. S6).

Fig. 4

Rap1 is a repressor of telomere recombination. (A) Rap1 and TRF2 from TRF2F/−Ku70−/− MEFs expressing TRF2, TRF2∆Rap1, or vector control analyzed 4 days after Cre treatment. (B) Chromosome orientation (CO) FISH analysis on cells as in (A). Arrows: T-SCEs. (C) Enlarged T-SCE events in Cre-treated TRF2F/−Ku70−/− MEFs expressing TRF2Rap1. (D) Quantification of T-SCEs as assessed in (B). Bars represent the mean ± SD from three independent experiments (n > 1100 chromosome ends each). P values are based on Student’s two-tailed t test. (E) Quantification of T-SCEs as assessed in (B) in cells of the indicated Rap1 and Ku70 status. Method as in (D). Error bars: SEM (Rap1F/+Ku70−/+ and Rap1F/FKu70−/+) or SD (Rap1F/FKu70−/−). (F) The functions of shelterin components. See text for details.

These data indicate that Rap1 functions at mouse telomeres to repress HDR, which has the potential for generating shortened telomeres and can promote telomerase-independent telomere maintenance. Rap1 appears to be an adaptor protein: Its C terminus serves to anchor the protein in shelterin; its BRCT domain, when dimerized in the shelterin complex, could bind a phosphorylated target protein; and the surface charge of its Myb-type motif makes it a third potential protein-interaction domain (17). As adaptors, the Rap1 orthologs could fulfill diverse functions in different organisms, because alterations in one of the protein-interaction domains could endow Rap1 with a new binding partner and thus instigate a new function.

These results underscore the functional compartmentalization within shelterin (Fig. 4F), which contains at least four subunits dedicated to distinct functions. The replication of telomeric DNA is facilitated by TRF1 (18), and TPP1/POT1 are required for the repression of ATR signaling (1, 19). TRF2 is the predominant repressor of both ATM signaling and NHEJ, and the current data show that these functions of TRF2 do not require Rap1. Finally, our results identify a fifth component of shelterin, Rap1, as an important repressor of HDR. Repression of HDR also requires TPP1/POT1 because removal of either Rap1 or POT1a/b result in telomere recombination. In a parallel pathway, Ku70/80 inhibits HDR, but it has not been established whether this function is telomere specific (2). This separation of function revealed that telomeres can undergo HDR without being detected by the ATM and ATR kinase pathways. When HDR takes place at telomeres lacking TRF2 or POT1a/b, DNA damage signaling results in the formation of TIFs. In the case of Rap1 removal, however, the telomeres lack detectable TIFs, yet are susceptible to HDR. Thus, consistent with the telomere recombination events in yeast lacking both Mec1 and Tel1 (20), the formation of DNA damage foci at telomeres is not a prerequisite for HDR.

Supporting Online Material

www.sciencemag.org/cgi/content/full/327/5973/1657/DC1

Materials and Methods

Figs. S1 to S6

References

  • * These authors contributed equally to this work.

  • Present address: Department of Molecular Biology, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, NY 10065, USA.

References and Notes

  1. D. White, H. Takai, and the Rockefeller University Transgenics Facility are thanked for help in generating genetically modified mice. We thank L. Jovine for identifying the conserved helix in TRF2. A.S. is supported by Susan G. Komen for the Cure. G.B.C. was supported by the Women in Science Fellowship Program and The Leukemia & Lymphoma Society. Supported by NIH grants AG016642 and GM049046.
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