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• W2010842801 abstract "•An S phase-specific TRF2-RTEL1 interaction is required for t-loop disassembly•RTEL1 C4C4 and PIP-box motifs control distinct genome maintenance pathways•A unique binding site in TRF2 interacts with and recruits RTEL1 to telomeres•TRF2-RTEL1 interaction is abolished by the disease causing RTEL1R1264H mutation The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to prevent catastrophic t-loop processing by structure-specific nucleases. We show that the TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1R1264H. Conversely, we define a TRF2I124D substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1R1264H mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase. The helicase RTEL1 promotes t-loop unwinding and suppresses telomere fragility to maintain the integrity of vertebrate telomeres. An interaction between RTEL1 and PCNA is important to prevent telomere fragility, but how RTEL1 engages with the telomere to promote t-loop unwinding is unclear. Here, we establish that the shelterin protein TRF2 recruits RTEL1 to telomeres in S phase, which is required to prevent catastrophic t-loop processing by structure-specific nucleases. We show that the TRF2-RTEL1 interaction is mediated by a metal-coordinating C4C4 motif in RTEL1, which is compromised by the Hoyeraal-Hreidarsson syndrome (HHS) mutation, RTEL1R1264H. Conversely, we define a TRF2I124D substitution mutation within the TRFH domain of TRF2, which eliminates RTEL1 binding and phenocopies the RTEL1R1264H mutation, giving rise to aberrant t-loop excision, telomere length heterogeneity, and loss of the telomere as a circle. These results implicate TRF2 in the recruitment of RTEL1 to facilitate t-loop disassembly at telomeres in S phase. Vertebrate telomeres are essential nucleoprotein structures that protect chromosome ends from promiscuous DNA repair activities and nucleolytic degradation (for review, see de Lange, 2005de Lange T. Shelterin: the protein complex that shapes and safeguards human telomeres.Genes Dev. 2005; 19: 2100-2110Crossref PubMed Scopus (2271) Google Scholar). Telomeric DNA is composed of repetitive double-stranded TTAGGG sequences that extend into a 3′ single-stranded overhang on the G-rich strand (Moyzis et al., 1988Moyzis R.K. Buckingham J.M. Cram L.S. Dani M. Deaven L.L. Jones M.D. Meyne J. Ratliff R.L. Wu J.R. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes.Proc. Natl. Acad. Sci. USA. 1988; 85: 6622-6626Crossref PubMed Scopus (1867) Google Scholar, Wellinger et al., 1993Wellinger R.J. Wolf A.J. Zakian V.A. Saccharomyces telomeres acquire single-strand TG1-3 tails late in S phase.Cell. 1993; 72: 51-60Abstract Full Text PDF PubMed Scopus (340) Google Scholar). Due to the inherent nature of semiconservative DNA replication, telomeres progressively shorten with each cell division. To counter the end replication problem, telomeric repeats can be extended by telomerase, a reverse transcriptase that utilizes an associated RNA moiety (TERC) as a template to add de novo telomeric sequences to the 3′ end of the G-rich strand of the telomere (Greider and Blackburn, 1985Greider C.W. Blackburn E.H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.Cell. 1985; 43: 405-413Abstract Full Text PDF PubMed Scopus (2588) Google Scholar, Shippen-Lentz and Blackburn, 1990Shippen-Lentz D. Blackburn E.H. Functional evidence for an RNA template in telomerase.Science. 1990; 247: 546-552Crossref PubMed Scopus (323) Google Scholar). Telomere function is also critically dependent on a complex formed by six telomere-associated proteins, known as shelterin, which comprises TRF1 (telomere repeat binding factor 1), TRF2 (telomere repeat binding factor 2), POT1 (protection of telomeres 1), TIN2 (TRF1-interacting nuclear factor 2), Rap1 (repressor activator protein 1), and TPP1 (TINF2-interacting protein 2) that function to safeguard chromosome ends from the DNA damage response (DDR) and to control telomere maintenance by telomerase (for review, see Palm and de Lange, 2008Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1384) Google Scholar). Shelterin associates with telomeres by virtue of intrinsic duplex telomeric repeat binding conferred by TRF1 and TRF2 and binding of the single-stranded telomeric 3′ overhang by POT1 (Baumann and Cech, 2001Baumann P. Cech T.R. Pot1, the putative telomere end-binding protein in fission yeast and humans.Science. 2001; 292: 1171-1175Crossref PubMed Scopus (801) Google Scholar). While TRF1 promotes telomere replication and functions as a negative regulator of telomere length (Sfeir et al., 2009Sfeir A. Kosiyatrakul S.T. Hockemeyer D. MacRae S.L. Karlseder J. Schildkraut C.L. de Lange T. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication.Cell. 2009; 138: 90-103Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar), TRF2 plays a crucial role in telomere end protection (van Steensel et al., 1998van Steensel B. Smogorzewska A. de Lange T. TRF2 protects human telomeres from end-to-end fusions.Cell. 1998; 92: 401-413Abstract Full Text Full Text PDF PubMed Scopus (1440) Google Scholar). Consequently, loss of TRF1 compromises telomere replication, giving rise to telomere fragility, whereas loss of TRF2 results in loss of the 3′ overhang and extensive chromosome end-to-end fusions (Celli and de Lange, 2005Celli G.B. de Lange T. DNA processing is not required for ATM-mediated telomere damage response after TRF2 deletion.Nat. Cell Biol. 2005; 7: 712-718Crossref PubMed Scopus (467) Google Scholar, Sfeir et al., 2009Sfeir A. Kosiyatrakul S.T. Hockemeyer D. MacRae S.L. Karlseder J. Schildkraut C.L. de Lange T. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication.Cell. 2009; 138: 90-103Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Electron microscopy and stochastic optical reconstruction microscopy have revealed that telomeres can adopt lasso-like configurations called t-loops (Doksani et al., 2013Doksani Y. Wu J.Y. de Lange T. Zhuang X. Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation.Cell. 2013; 155: 345-356Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, Griffith et al., 1999Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. de Lange T. Mammalian telomeres end in a large duplex loop.Cell. 1999; 97: 503-514Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar), which are believed to form as a result of strand invasion of the 3′ single-stranded telomeric DNA into upstream duplex TTAGGG repeats (Griffith et al., 1999Griffith J.D. Comeau L. Rosenfield S. Stansel R.M. Bianchi A. Moss H. de Lange T. Mammalian telomeres end in a large duplex loop.Cell. 1999; 97: 503-514Abstract Full Text Full Text PDF PubMed Scopus (1924) Google Scholar). As the 3′ end of the telomere is buried within the t-loop, this structure has been proposed to safeguard the chromosome ends against nucleolytic attack or promiscuous DNA repair activities (for review, see O’Sullivan and Karlseder, 2010O’Sullivan R.J. Karlseder J. Telomeres: protecting chromosomes against genome instability.Nat. Rev. Mol. Cell Biol. 2010; 11: 171-181Crossref PubMed Scopus (0) Google Scholar). The amino-terminal basic domain of the shelterin component TRF2 is important for stabilizing t-loops and acts to protect the structure from unscheduled nucleolytic processing (Wang et al., 2004Wang R.C. Smogorzewska A. de Lange T. Homologous recombination generates T-loop-sized deletions at human telomeres.Cell. 2004; 119: 355-368Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). TRF2 is also essential for t-loop formation in vivo (Doksani et al., 2013Doksani Y. Wu J.Y. de Lange T. Zhuang X. Super-resolution fluorescence imaging of telomeres reveals TRF2-dependent T-loop formation.Cell. 2013; 155: 345-356Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), which likely contributes to its key role in end protection. Mechanisms must also exist to disassemble t-loops to allow telomerase access to the 3′ end and/or to avoid collisions with the replisome during S phase. RTEL1 (regulator of telomere length 1) (Ding et al., 2004Ding H. Schertzer M. Wu X. Gertsenstein M. Selig S. Kammori M. Pourvali R. Poon S. Vulto I. Chavez E. et al.Regulation of murine telomere length by Rtel: an essential gene encoding a helicase-like protein.Cell. 2004; 117: 873-886Abstract Full Text Full Text PDF PubMed Scopus (257) Google Scholar) is an essential helicase with intrinsic D-loop-disrupting activity that is co-opted to telomeres to dismantle t-loops (Uringa et al., 2012Uringa E.J. Lisaingo K. Pickett H.A. Brind’Amour J. Rohde J.H. Zelensky A. Essers J. Lansdorp P.M. RTEL1 contributes to DNA replication and repair and telomere maintenance.Mol. Biol. Cell. 2012; 23: 2782-2792Crossref PubMed Scopus (79) Google Scholar). In the absence of RTEL1, t-loops are inappropriately resolved by the SLX1-SLX4 nuclease complex, leading to catastrophic telomere length changes and loss of the telomere as double-stranded telomere circles (T-circles or TCs) (Vannier et al., 2012Vannier J.B. Pavicic-Kaltenbrunner V. Petalcorin M.I. Ding H. Boulton S.J. RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity.Cell. 2012; 149: 795-806Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). RTEL1 also executes a second function to counteract the formation of telomeric guanine quadruplex (G4) DNA structures during telomere replication, which are a major source of telomere fragility (Vannier et al., 2012Vannier J.B. Pavicic-Kaltenbrunner V. Petalcorin M.I. Ding H. Boulton S.J. RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity.Cell. 2012; 149: 795-806Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Direct binding of RTEL1 to proliferating cell nuclear antigen (PCNA) is critical for preventing telomere fragility but is dispensable for t-loop disassembly (Vannier et al., 2013Vannier J.B. Sandhu S. Petalcorin M.I. Wu X. Nabi Z. Ding H. Boulton S.J. RTEL1 is a replisome-associated helicase that promotes telomere and genome-wide replication.Science. 2013; 342: 239-242Crossref PubMed Scopus (150) Google Scholar). The importance of RTEL1 in human disease has been established with the identification of variants that confer increased risk of human brain tumors (Shete et al., 2009Shete S. Hosking F.J. Robertson L.B. Dobbins S.E. Sanson M. Malmer B. Simon M. Marie Y. Boisselier B. Delattre J.Y. et al.Genome-wide association study identifies five susceptibility loci for glioma.Nat. Genet. 2009; 41: 899-904Crossref PubMed Scopus (651) Google Scholar, Wrensch et al., 2009Wrensch M. Jenkins R.B. Chang J.S. Yeh R.F. Xiao Y. Decker P.A. Ballman K.V. Berger M. Buckner J.C. Chang S. et al.Variants in the CDKN2B and RTEL1 regions are associated with high-grade glioma susceptibility.Nat. Genet. 2009; 41: 905-908Crossref PubMed Scopus (418) Google Scholar). Mutations in RTEL1 also give rise to Hoyeraal-Hreidarsson syndrome (HHS), a severe form of the telomereopathy dyskeratosis congenita (DKC; for review, see Vannier et al., 2014Vannier J.B. Sarek G. Boulton S.J. RTEL1: functions of a disease-associated helicase.Trends Cell Biol. 2014; 24: 416-425Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). HHS is characterized by its similarity to DKC, but patients also present with additional complications including bone marrow failure, microcephaly, and immunodeficiency (Vulliamy et al., 2006Vulliamy T.J. Marrone A. Knight S.W. Walne A. Mason P.J. Dokal I. Mutations in dyskeratosis congenita: their impact on telomere length and the diversity of clinical presentation.Blood. 2006; 107: 2680-2685Crossref PubMed Scopus (269) Google Scholar). So far, 18 distinct RTEL1 mutations have been identified in HHS (for review, see Vannier et al., 2014Vannier J.B. Sarek G. Boulton S.J. RTEL1: functions of a disease-associated helicase.Trends Cell Biol. 2014; 24: 416-425Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), but how these mutations impact on the known functions of RTEL1 is not known. Here, we define the mechanism by which RTEL1 is recruited to telomeres to promote t-loop disassembly, which we show is dependent on an S phase-specific interaction with the shelterin component TRF2. We demonstrate that the TRF2 interaction site in RTEL1 maps to an uncharacterized C4C4 metal-binding motif, which is commonly mutated in HHS patients. We establish that the p.R1264H mutation within the C4C4 motif, which has a carrier frequency of 1 in 100 in the Ashkenazi Jewish population (Fedick et al., 2014Fedick A.M. Shi L. Jalas C. Treff N.R. Ekstein J. Kornreich R. Edelmann L. Mehta L. Savage S.A. Carrier screening of RTEL1 mutations in the Ashkenazi Jewish population.Clin. Genet. 2014; https://doi.org/10.1111/cge.12459Crossref Scopus (13) Google Scholar), compromises the RTEL1-TRF2 interaction, leading to catastrophic t-loop resolution accompanied by rapid changes in telomere length, telomere loss, and TC formation. Conversely, we identify a single amino acid substitution (p.I124D) within the TRFH dimerization domain of TRF2 that specifically abolishes RTEL1 binding and prevents its recruitment to telomeres. Remarkably, the TRF2 p.I124D mutation gives rise to rapid changes in telomere length, telomere loss, and TC formation and thereby phenocopies the p.R1264H mutation in RTEL1. Our results reveal that t-loops are highly dynamic and regulated structures whose assembly and disassembly is coordinated during the cell cycle. RTEL1 serves a dual function in telomere integrity; it suppresses telomere fragility by unwinding telomeric G4 DNA and also disassembles t-loops to prevent catastrophic loss of the telomere (Vannier et al., 2012Vannier J.B. Pavicic-Kaltenbrunner V. Petalcorin M.I. Ding H. Boulton S.J. RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity.Cell. 2012; 149: 795-806Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Recently, we have shown that RTEL1’s ability to suppress telomere fragility strictly depends on an interaction with PCNA. Unexpectedly, however, the RTEL1-PCNA interaction was found to be dispensable for t-loop unwinding (Vannier et al., 2013Vannier J.B. Sandhu S. Petalcorin M.I. Wu X. Nabi Z. Ding H. Boulton S.J. RTEL1 is a replisome-associated helicase that promotes telomere and genome-wide replication.Science. 2013; 342: 239-242Crossref PubMed Scopus (150) Google Scholar), suggesting the existence of a separate mechanism for recruiting RTEL1 to telomeres. We reasoned that RTEL1 could be recruited to telomeres via an interaction with one of the shelterin components, which are the major constituents of vertebrate telomeres. To test this hypothesis, we first modified a human bacterial artificial chromosome (BAC) carrying the RTEL1 genomic locus by inserting a FLAP tag (containing GFP and Flag) at the N terminus (NFLAP) of the RTEL1 coding sequence using homologous recombination (Poser et al., 2008Poser I. Sarov M. Hutchins J.R. Hériché J.K. Toyoda Y. Pozniakovsky A. Weigl D. Nitzsche A. Hegemann B. Bird A.W. et al.BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals.Nat. Methods. 2008; 5: 409-415Crossref PubMed Scopus (478) Google Scholar). Extracts from HEK293 cells stably expressing NFLAP-tagged RTEL1 at near endogenous levels were subjected to immunoprecipitation for 5 of the 6 endogenous shelterin components and then blotted for RTEL1. Although known partner shelterin components were present in immunoprecipitates of endogenous TRF1, TPP1, or POT1, NFLAP-tagged RTEL1 was not detectable (Figure S1A). However, blotting of immunoprecipitates of endogenous TRF2, and to a lesser extent Rap1, revealed an association with NFLAP-tagged RTEL1 (Figure 1A). Importantly, these interactions were resistant to benzonase treatment, indicating that the interactions between TRF2/Rap1 and RTEL1 are independent of DNA/RNA bridging. Furthermore, TRF2 immunoprecipitated RTEL1 from lysates that were mock depleted with IgG but not from extracts immunodepleted for NFLAP-tagged RTEL1 with a GFP antibody (Figure 1B), thus demonstrating the specificity of the interaction. The interaction between RTEL1 and TRF2/Rap1 was also observed by reciprocal coimmunoprecipitation. TRF2 was coprecipitated from HEK293 cells with NFLAP-tagged RTEL1, but not with irrelevant control protein GFP-ALC1 (Figure 1C). Silencing of Rap1 with lentivirus-mediated RNA interference (shRNA) did not significantly affect binding of RTEL1 to TRF2 (data not shown), which raised the possibility that RTEL1 interacts indirectly with Rap1 and directly with TRF2. To confirm direct binding of RTEL1 to TRF2 in vitro, we performed GST pull-down assays with purified full-length and truncated versions of human TRF2 and affinity purified NFLAP-RTEL1. Western blotting analysis revealed that NFLAP-RTEL1 associates with full-length TRF2 protein (GST-TRF2) and TRF2 lacking the N-terminal basic domain (GST-TRF2ΔB), but not with the basic N-terminal domain (GST-ΔB) or GST alone (Figure 1D). Taken together, these data indicate that RTEL1 directly interacts with TRF2. Since RTEL1 only transiently associates with telomeres (Uringa et al., 2012Uringa E.J. Lisaingo K. Pickett H.A. Brind’Amour J. Rohde J.H. Zelensky A. Essers J. Lansdorp P.M. RTEL1 contributes to DNA replication and repair and telomere maintenance.Mol. Biol. Cell. 2012; 23: 2782-2792Crossref PubMed Scopus (79) Google Scholar), we next examined whether the association of RTEL1 with TRF2 varied throughout the cell cycle. To this end we synchronized HEK293 NFLAP-RTEL1 cells and RTEL1v5 mouse embryonic fibroblasts (MEFs), stably expressing Myc-tagged TRF2, by double thymidine block or by incubation with thymidine followed by nocodazole treatment (Figures S1B and S1C). Although a low level of association between RTEL1 and TRF2 was detected throughout the cell cycle, the RTEL1-TRF2 interaction is significantly increased at the G1-to-S transition and peaked in S phase (Figures 1E and S1D). Similarly, we observed that the RTEL1-TRF2 interaction is significantly enhanced in S phase RTEL1v5 mouse cells as determined by proximity ligation assay PLA (Figures 1F and S1E) and is unaffected by inhibiting DNA replication (Figure S2). These results establish that the RTEL1-TRF2 interaction occurs predominantly during S phase and does not require active DNA replication. To determine the minimal TRF2 interaction site within RTEL1, we conducted a peptide array binding experiment, as previously described (Ward et al., 2010Ward J.D. Muzzini D.M. Petalcorin M.I. Martinez-Perez E. Martin J.S. Plevani P. Cassata G. Marini F. Boulton S.J. Overlapping mechanisms promote postsynaptic RAD-51 filament disassembly during meiotic double-strand break repair.Mol. Cell. 2010; 37: 259-272Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). We reasoned that the TRF2 interaction site was unlikely to map to the N-terminal helicase motif and so focused our attention on the C terminus of RTEL1, which contains three recognizable motifs: a PIP box (PCNA binding), a harmonin N-like domain (Faure et al., 2014Faure G. Revy P. Schertzer M. Londono-Vallejo A. Callebaut I. The C-terminal extension of human RTEL1, mutated in Hoyeraal-Hreidarsson syndrome, contains harmonin-N-like domains.Proteins. 2014; 82: 897-903Crossref PubMed Scopus (26) Google Scholar), and a C4C4 motif (unknown function). Binding of GST-TRF2 to a 20-mer peptide-scanning array encompassing the C-terminal region of RTEL1 identified a single TRF2-binding peptide that overlapped with the uncharacterized C4C4 motif (Figure 2A). To confirm that the C4C4 motif of RTEL1 is sufficient to confer binding to TRF2 and to determine if this interaction requires metal coordination, we generated recombinant GST and GST-C4C4 motif fusions and performed pull-downs of endogenous TRF2 from HEK293 cell extracts in the absence (−EDTA) or presence (+EDTA) of EDTA. As shown in Figure 2B (lane 2), the lower isoform of endogenous TRF2 is specifically pulled down from whole-cell extract by GST-C4C4, but not with GST alone (Figure 2B; lane 1). Addition of EDTA to the pull-down, which is predicted to abolish metal coordination of the C4C4 motif, resulted in the complete loss of TRF2 binding (Figure 2B; lane 4). These results reveal that the C4C4 motif of RTEL1 is sufficient for binding to TRF2, and this requires metal coordination. To assess the biological importance of the C4C4 domain for RTEL1 activities, we introduced C4C4 mutations by site-directed mutagenesis into the human and mouse versions of RTEL1 (see map in Figure 2C). The RTEL1R1264H (human)/RTEL1R1237H (mouse) mutation corresponds to the HHS disease-causing mutation (Ballew et al., 2013Ballew B.J. Joseph V. De S. Sarek G. Vannier J.B. Stracker T. Schrader K.A. Small T.N. O’Reilly R. Manschreck C. et al.A recessive founder mutation in regulator of telomere elongation helicase 1, RTEL1, underlies severe immunodeficiency and features of Hoyeraal Hreidarsson syndrome.PLoS Genet. 2013; 9: e1003695Crossref PubMed Scopus (88) Google Scholar) and is located between the second and third metal-coordinating pair of cysteines (Kellenberger et al., 2005Kellenberger E. Dominguez C. Fribourg S. Wasielewski E. Moras D. Poterszman A. Boelens R. Kieffer B. Solution structure of the C-terminal domain of TFIIH P44 subunit reveals a novel type of C4C4 ring domain involved in protein-protein interactions.J. Biol. Chem. 2005; 280: 20785-20792Crossref PubMed Scopus (28) Google Scholar). We also generated two cysteine-to-alanine substitutions (RTEL1C1279A/C1282A in human or RTEL1C1252A/C1255A in mouse) that are predicted to disrupt the fourth pair of cysteines coordinating a second metal ion (Kellenberger et al., 2005Kellenberger E. Dominguez C. Fribourg S. Wasielewski E. Moras D. Poterszman A. Boelens R. Kieffer B. Solution structure of the C-terminal domain of TFIIH P44 subunit reveals a novel type of C4C4 ring domain involved in protein-protein interactions.J. Biol. Chem. 2005; 280: 20785-20792Crossref PubMed Scopus (28) Google Scholar). First, we evaluated whether the mutations within the C4C4 motif affect the RTEL1-TRF2 interaction. To this end U2OS cells stably expressing empty vector, wild-type (WT) Myc-C4C4 (an equivalent to the human RTEL11234–1292 region), or two C4C4 mutants, Myc-C4C4R1264H and Myc-C4C4C1279A/C1282A, were analyzed by immunoblotting with anti-Myc antibody. The decreased steady-state abundance of the mutated human C4C4 motif suggests that the mutations might impact on its stability (Figure S3A). To account for the reduced expression, we compensated Myc-C4C4 protein levels of both mutants in immunoprecipitation experiments. Western blotting analysis revealed that endogenous TRF2 was efficiently immunoprecipitated with WT Myc-C4C4 peptide but not with either mutant (Figure S3B). We also introduced the C4C4 motif mutations into the full-length human RTEL1 and found that endogenous TRF2 interacted exclusively with WT Flag-RTEL1, whereas mutations within the C4C4 motif abolished RTEL1’s ability to bind TRF2 (Figure 2D). These results were also confirmed by an in vitro GST pull-down experiment, in which we observed robust interaction of GST-TRF2 with full-length Flag-tagged WT RTEL1, but not with RTEL1R1264H or double-mutant RTEL1C1279A/C1282A (Figure 2E). Finally, we examined the interaction of TRF2 with RTEL1 in normal BJ human fibroblasts and in patient-derived MSK-41 cells harboring inherited homozygous RTEL1R1264H HHS disease-causing mutations (Ballew et al., 2013Ballew B.J. Joseph V. De S. Sarek G. Vannier J.B. Stracker T. Schrader K.A. Small T.N. O’Reilly R. Manschreck C. et al.A recessive founder mutation in regulator of telomere elongation helicase 1, RTEL1, underlies severe immunodeficiency and features of Hoyeraal Hreidarsson syndrome.PLoS Genet. 2013; 9: e1003695Crossref PubMed Scopus (88) Google Scholar). As shown in Figure 2F, the endogenous RTEL1-TRF2 interaction is undetectable in MSK-41 cells (lane 1) but is readily detectable in normal BJ human fibroblasts (Figure 2F, lane 2). An interaction between endogenous TRF2 and Rap1, which served as a positive control, was observed by PLA assay in BJ fibroblasts, retinal-pigmented epithelial RPE1 cells, and in the patient-derived MSK-41 fibroblasts (Figure 2G). In contrast, the RTEL1-TRF2 interaction was only detectable by PLA assay in BJ fibroblasts and RPE1 cells but not in the MSK-41 fibroblasts (Figure 2H). These results establish that the interaction of RTEL1 and TRF2 is compromised by the RTEL1 p.R1264H mutation in HHS. To investigate the impact of C4C4 motif mutations on the telomere functions of RTEL1, we complemented conditional RTEL1F/F MEFs with retroviruses expressing either empty vector, full-length WT V5-RTEL1, single-mutant V5-RTEL1R1237H, or double-mutant V5-RTEL1C1252A/C1255A. Transient expression of Cre recombinase in the transduced RTEL1F/F MEFs resulted in the expected loss of the floxed RTEL1 alleles and concomitant elimination of the endogenous RTEL1 protein within 96 hr, leaving the V5-RTEL1 WT or mutants as the only source of RTEL1 protein (Figures 3A and 3B ). Conditional deletion of RTEL1 in MEFs leads to rapid accumulation of TCs as a consequence of processing of persistent t-loops by the SLX1/4 nuclease (Vannier et al., 2012Vannier J.B. Pavicic-Kaltenbrunner V. Petalcorin M.I. Ding H. Boulton S.J. RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity.Cell. 2012; 149: 795-806Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar). Cells expressing empty vector and MEFs complemented either with V5-RTEL1R1237H or V5-RTEL1C1252A/C1255A exhibited very high levels of TCs after eliminating the RTEL1 floxed alleles. In contrast, RTEL1-deficient cells expressing WT V5-RTEL1 exhibited low levels of TCs, comparable to control virus-infected cells (Figure 3C). Next, we examined the levels of telomere fragility and telomere loss in complemented RTEL1F/F MEFs by fluorescent in situ hybridization (FISH) using telomere-specific probes. As expected, inactivation of RTEL1 in RTEL1F/F MEFs, 96 hr after Cre infection, resulted in high levels of fragile telomeres, which manifest as multiple spatially distinct telomere FISH signals (Figure 3D). However, inactivation of RTEL1 in RTEL1F/F MEFs complemented with either WT V5-RTEL1, V5-RTEL1R1237H, or V5-RTEL1C1252A/C1255A did not result in significant induction of telomere fragility when compared to cells complemented with an empty vector control (Figure 3D). Strikingly, inactivation of RTEL1 in RTEL1F/F MEFs expressing V5-RTEL1R1237H or V5-RTEL1C1252A/C1255A resulted in a significant increase in telomere loss, corresponding to 6.2 ± 2.8 and 5.8 ± 3.3 telomeres that lacked a telomere FISH signal on one or both sister chromatid ends, which is comparable to the levels observed in RTEL1 null cells (7.5 ± 3.2) and significantly different from WT V5-RTEL1-complemented cells that present 2.7 ± 1.4 ends lacking detectable telomeric signals in WT V5-RTEL1-complemented cells (Figure 3E). These results establish that a functional C4C4 motif in RTEL1 is essential for suppressing TCs and telomere loss, but is dispensable for preventing telomere fragility. We next evaluated how loss of the RTEL1-TRF2 interaction might affect the DDR at telomeres. To this end we quantified the number of dysfunctional telomeres by scoring for 53BP1 colocalization with telomeres. Conditional deletion of RTEL1 in RTEL1F/F MEFs expressing V5-RTEL1R1237H or V5-RTEL1C1252A/C1255A resulted in enhanced telomere-dysfunction-induced foci (TIFs), corresponding to 7.2% ± 0.5% and 8.3% ± 1.3% foci per nucleus compared to 2.5% ± 1.6% TIFs per nucleus in the cells expressing WT V5-RTEL1 (Figures 3F and S4). Thus, disrupting the TRF2 binding site on RTEL1 causes a mild DDR that resembles the phenotype of RTEL1 deletion. To determine whether loss of the RTEL1-TRF2 interaction induces telomere recombination, we employed chromosome orientation (CO)-FISH to monitor the frequency of telomere sister chromatid exchange (T-SCEs). Inactivation of RTEL1 in RTEL1F/F MEFs expressing V5-RTEL1R1237H or V5-RTEL1C1252A/C1255A resulted in a 3-fold increase in the number of T-SCEs when compared to WT V5-RTEL1-complemented cells (Figure 3G). Collectively, these results lead us to propose that recruitment of RTEL1 to telomeres by TRF2 is important for t-loop disassembly and prevents catastrophic loss of the telomere, but is dispensable for efficient telomere replication. Given that a functional C4C4 motif and RTEL1-TRF2 interaction is required for t-loop disassembly but is dispensable for preventing telomere fragility, we wished to determine the impact of C4C4 motif mutations on genome-wide replication dynamics using the molecular combing method (Michalet et al., 1997Michalet X. Ekong R. Fougerousse F. Rousseaux S. Schurra C. Ho" @default.
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• W2010842801 title "TRF2 Recruits RTEL1 to Telomeres in S Phase to Promote T-Loop Unwinding" @default.
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• W2010842801 doi "https://doi.org/10.1016/j.molcel.2014.12.024" @default.
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