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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 non-homologous 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 absence of DNA damage foci and underscore the functional compartmentalization within shelterin.
The shelterin subunit TRF2 is a main player in the repression of the telomeric DNA damage response (1). Deletion of TRF2 results in activation of the ATM kinase and rampant telomere fusions mediated by NHEJ. TRF2 also contributes to the repression of HDR, which can create undesirable telomeric sister chromatid exchanges (T-SCEs). HDR at telomeres is unleashed 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 its interacting partner 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 (6-9). Human Rap1 affects telomere length homeostasis and a tethering experiment has suggested that Rap1 can repress telomere fusions (10-12). Here we use two complementary approaches to determine how Rap1 loss affects telomere function. Gene targeting was used to generate mouse cells lacking a functional Rap1 gene. In addition, we generated mouse cells devoid of the endogenous TRF2 that were complemented with a TRF2 mutant incapable of binding Rap1. The two approaches yielded the same results.
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. 1A-C). The Rap1Δex2 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. S1A-C). IF and immunoblotting showed that Cre-treated SV40LT-immortalized Rap1F/F MEFs indeed lacked any detectable Rap1 protein and ChIP showed the loss of Rap1 from telomeres (Fig. 1D-F, fig. S1D). As the expression and localization of other shelterin components were not significantly affected (Fig. 1D-F, fig. S1E), the phenotypes of Rap1Δex2/Δex2 MEFs should inform specifically on the telomeric function of Rap1.
The growth rate of the Rap1Δex2/Δex2 MEFs was similar to 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, fig. S1F, G). Furthermore, Rap1Δex2/Δex2 mice were born at the expected frequencies and were fertile (Fig. 1H). The survival of Rap1Δex2/Δex2 cells and mice argues that Rap1 deletion does not result in major telomere dysfunction, which is known to be lethal. This conclusion was further corroborated by infecting Rap1Δex2/Δex2 MEFs with an shRNA targeting exon 1 (Fig. 1A, fig. S1H), which did not induce a growth arrest or other phenotypes typical of telomere dysfunction (see Fig. 3, data not shown).
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. In order to identify amino acids critical for Rap1 interaction, we searched for highly conserved, TRF2-specific motifs in the previously mapped Rap1 binding region (aa 260-360; (5)). A short predicted helix at position 290 was conserved in TRF2 orthologs but not in TRF1 (fig. S2A,B). Two mutations in this region (A289S and F290S) reduced the interaction between Rap1 and TRF2 in co-IP experiments (fig. S2C). To generate TRF2ΔRap1, amino acids 284-297 were deleted (Fig. 2A). TRF2ΔRap1 failed to bind to Rap1 in co-IP experiments whereas it retained its interaction with the TRF2-interacting protein Apollo (Fig. 2B). Wild type TRF2 and TRF2ΔRap1 were expressed in TRF2F/-p53-/- MEFs and the endogenous TRF2 was removed with Cre (Fig. 2C). Although TRF2ΔRap1 localized to telomeres efficiently, IF and ChIP indicated that the telomeres lacked Rap1 and the overall level of Rap1 in the cells was reduced (Fig. 2C-E, fig. S3A). Other shelterin components were affected to an extent (<2-fold; Fig. 2D,E) that is not expected to be functionally significant as heterozygous MEFs and mice lacking one copy of TRF1, TPP1, TRF2, or POT1a/b display no telomere defect. Consistent with the viability of Rap1Δex2/Δex2 cells, cells expressing TRF2ΔRap1 proliferated at the same rate as cells expressing wild type TRF2 (fig. S3B).
Telomeres lacking Rap1 were examined for the hallmarks of telomere dysfunction. Rap1Δex2/Δex2 cells did not show Telomere Dysfunction-Induced Foci (TIFs; (13)), which are telomeric DNA damage foci that report on ATM and/or ATR signaling at chromosome ends, and phosphorylation of Chk1 and Chk2 was not evident (Fig. 3A-C). Further depletion of Rap1 mRNA with an shRNA also failed to elicit a DNA damage signal in Rap1Δex2/Δex2 cells (Fig. 3B). Consistent with these results, TRF2ΔRap1 was equal 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 TRF2ΔRap1-expressing cells is likely due to Cre-induced DNA damage, since 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 TRF2ΔRap1 had the same ability as wild type TRF2 to repress NHEJ at telomeres (Fig. 3F-H). However, as NHEJ of telomeres lacking TRF2 requires active DNA damage signaling (14), the lack of telomere fusions could be due to the lack of ATM signaling. 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 TRF2/ATM deficient cells (14). Despite the telomeric ATR kinase signal elicited by the TPP1 shRNA, (Fig. 3B,D), Rap1 removal from telomeres did not induce their fusion (Fig. 3G,H).
These data argue against a requirement for Rap1 in either the repression of NHEJ or ATM kinase signaling and explain why the deletion of Rap1 did 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 status of H3K9 in telomeric chromatin, and the level of telomeric UUAGGG RNAs (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 levels 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 TRF2ΔRap1 into SV40LT-immortalized TRF2F/-Ku70-/- MEFs which display frequent T-SCEs upon deletion of TRF2 with Cre ((2) and Fig. 4). Whereas the telomeric exchanges were repressed by wild type TRF2, TRF2ΔRap1 failed to block the telomeric HDR (Fig. 4A-C). The frequency of T-SCEs was the same whether the cells expressed TRF2ΔRap1 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 absence of TIFs in cells lacking both Ku70 and telomeric Rap1 (fig. S6).
These data indicate that Rap1 functions at mouse telomeres to repress HDR. Repression of recombination is important since unequal exchanges can curb the viability of daughter cells that inherit shortened telomeres and uncontrolled HDR promotes telomerase-independent telomere maintenance. The mechanism by which Rap1 affects HDR remains to be elucidated. Rap1 has the domain structure of an adaptor protein, combining three protein-protein interaction modules in one polypeptide. Its C-terminus serves to anchor the protein in shelterin and the N-terminal BRCT domain, when dimerized in the shelterin complex, could bind a phosphorylated target protein. The third potential protein interaction module in Rap1 is its Myb-type motif. Myb motifs often bind DNA, but the surface charge of the Myb domain in mammalian Rap1 makes it more suitable to bind to another protein (17). This view of Rap1 as an adaptor explains how the Rap1 orthologs can fulfill diverse functions in different organisms as alterations in one of the protein-interaction domains could endow Rap1 with a new interacting partner and thus instigate a new function.
These results further underscore the remarkable compartmentalization within shelterin (Fig. 4F). Shelterin contains at least four proteins 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 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 since 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). The extensive separation of function within shelterin permitted the observation 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.
Devon White, Hiro Takai, and the RU Transgenics Facility are thanked for help in generating genetically modified mice. We thank Luca Jovine for assistance in identifying the Rap1-binding region in TRF2. AS is supported by a postdoctoral fellowship from Susan G. Komen for the Cure. CGB was supported by the Women & Science Fellowship Program and The Leukemia & Lymphoma Society. Supported by NIH grants AG016642 and GM049046.