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TRF2 Recruits RTEL1 to Telomeres in S Phase to Promote T-Loop Unwinding

Summary 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.


Figure S2
or presence of low or high doses of DNA synthesis inhibitors (aphidicolin or hydroxyurea) as indicated on the top. Whole-cell lysates were subjected to immunoprecipitation with RTEL1 antibody or rabbit normal IgG as a control. Immunocomplexes were resolved on SDS-PAGE and followed by Western blotting analysis with antibodies against Myc and RTEL1. Input (5%) is shown on the right. The asterisks indicate unspecific bands detected by rabbit polyclonal anti-RTEL1 antibody.     Figure 3F. RTEL1 F/F MEFs transduced with empty vector (ctrl), wild-type V5-RTEL1 (WT), mutant V5-RTEL1 R1237H (R/H), or double mutant V5-RTEL1 C1252A/C1255A (C/A) were infected with control (Ad-GFP) or Cre-expressing (Ad-Cre-GFP) adenovirus. At 96 hours after infection cells were processed for 53BP1 and FITC-Tel repeat fluorescence. Dashed lines indicate nucleus outlines (as determined by DAPI; not shown). Insets represent 3X magnifications of the indicated fields. Scale bar is 10 µm.  The arrays were incubated either with GST or with human full-length RTEL1 fused with GST and bound proteins were analyzed by immunoblotting with anti-GST antibody. The position of the N and C termini is shown, and the sequence of the region covering the RTEL1-binding site is indicated. (B) TRF2 TRFH recombinant proteins were incubated for two hours in the absence (-EDTA) or presence (+EDTA) of 50 mM of EDTA either with GST or GST-C4C4 fusion protein. Protein complexes were eluted and analyzed by SDS-PAGE followed by Coomassie brilliant blue staining. The input control in lanes 1 to 3 reflects 10% of the total amount of proteins used for the pull-down experiments.     Figure 6H. TRF2 F/-MEFs transduced with empty vector (ctrl), wild type Myc-TRF2 (WT) or mutant Myc-TRF2 I124D (I/D) were infected with control (Ad-GFP) or Cre-expressing (Ad-Cre-GFP) adenovirus. At 96 hours after infection, cells were processed for 53BP1 and FITC-Tel repeat fluorescence. Dashed lines indicate nucleus outlines (as determined by DAPI; not shown). Insets represent 3X magnifications of the indicated fields. Scale bar is 10 µm.

Supplemental Experimental Procedures
Cell lysis, Western blotting, and Immunoprecipitation Cells were rinsed twice with PBS, transferred to an ice-cold NET lysis buffer (50mM Tris (pH 7.2) 150 mM NaCl, 0.5% NP-40, 1x EDTA-free Complete protease inhibitor cocktail (Roche), 1x PhosSTOP phosphatase inhibitor cocktail (Roche)) and kept to lyse 10 minutes on ice. The cell lysates were then briefly vortexed and passed through a 23G syringe five times. The soluble protein fractions were collected after centrifugation at 16000 x g for 10 minutes at 4°C. Western blotting analysis was performed as described previously (Vannier et al., 2012). For protein immunoprecipitation, whole-cell extracts were precleared with protein G sepharose (Sigma-Aldrich), and one to two miligrams of precleared extract was incubated with antibodies as indicated in the text. Where specified, genomic DNA was removed by treatment with benzonase (Novagen; EMD Chemicals). Immunocomplexes were subjected to SDS-PAGE followed by immunoblotting using nitrocellulose membrane (GE Healthcare).

Peptide array studies
Peptide arrays were generated by automatic "pep-spot" synthesis and synthesized on Whatman 50 cellulose membranes using Fmoc-chemistry with the AutoSpot-Robot ASS 222 (Intavis Bioanalytical Instruments) as previously described (Horejsi et al., 2014). The binding of spot-immobilized peptides with purified recombinant GST-TRF2, GST-RTEL1, or H6-SUMO-C4C4-RING fusion proteins was carried out by incubating the membranes with 100 to 150 µg of recombinant protein as reported earlier (Thorslund et al., 2007;Ward et al., 2010).

Cell cycle synchronization
HEK 293 cells or RTEL1v5 fibroblasts expressing wild-type Myc-TRF2 were synchronized by the double-thymidine-block method as previously described (Whitfield et al., 2002), with minor modifications. Briefly, cells were treated with 2 mM thymidine (Sigma-Aldrich) for 18 hours, thymidine-free media for 9 hours to release the cells, and 2 mM thymidine was added to media for an additional 16 hours to arrest the cells at the G1 to S transition. Cells were washed twice with PBS and then released in fresh complete DMEM. Cells were analyzed at 70 minutes time intervals by immunoblotting and in situ PLA assay. For synchronization in mitosis, a thymidine-nocodazole block was used (Whitfield et al., 2002). Briefly, cells at confluence of 60% were treated with 2mM thymidine for 24 hours, washed twice in PBS, and released into complete DMEM for 3 hours. Next, cells were treated with 50 µg/mL of nocodazole (Sigma-Aldrich) for 15 hours, and the cells were washed twice with PBS and a fresh complete medium was added to the cell culture. Synchronized cells were analyzed at 90 minutes time intervals by Western blotting and in situ PLA with antibodies indicated in text.

Cell transfection and RNA interference
in non-target control.

Site-directed mutagenesis
Single amino acid substitutions were performed with the following primers: human

PNA FISH, CO-FISH, Q-FISH, and IF-FISH
Telomeric Peptide Nucleic Acid Fluorescence In Situ Hybrydysation (PNA FISH) was performed as described before (Lansdorp et al., 1996). Briefly, cells were treated with 0.2 µg/ml of colcemid for 90 minutes to arrest cells in metaphase. Trypsinized cells were incubated in 75 mM KCL, fixed with methanol:acetic acid (3:1), and spread on glass slide. To preserve chromosome architecture better the slides were rehydrated in PBS for 5 minutes, fixed in 4% formaldehyde for 5 minutes, treated with 1 mg/ml of pepsin for 10 minutes at 37°C, and fixed in 4% formaldehyde for 5 minutes. Next, slides were dehydrated in 70%, 85%, and 100% ( Slides were mounted with ProLong Gold antifade containing DAPI and images were acquired with an Olympus FLV1000 inverted microscope equipped with a 63X oil objective. Following acquisition, images were imported into ImageJ (NIH) and Adobe Photoshop CS5 for manual quantitation and processing.

Telomere circle (TC) assay
Cells grown at confluence between 70 to 80% were collected from two 10 cm dishes and extraction of genomic DNA for T-circle assay was performed as described previously (Munoz-Jordan et al., 2001). Total gDNA was digested by AluI/HinfI restriction enzymes and the TCA assay was performed as reported (Zellinger and Riha, 2007), with two essential modifications: (1) Phi29 DNA (Thermo Scientific) polymerization employed a mammalian telomere primer, and (2)  NaOH) for 10 minutes followed by neutralization in 1M NaCl/0.5M Tris-HCl (pH 7.0) for 10 minutes. DNA was crosslinked to the membrane immediately by UV crosslinking, then rinsed in 2X SSC. Telomere DNA was detected following hybridization with TTAGGG probe. Images were captured with Storm 840 scanner and quantified by ImageQuant TL software (Amersham Bioscience).

DNA combing
DNA combing was performed essentially as described in (Vannier et al., 2013).
Briefly, RTEL1 F/F mouse embryo fibroblasts expressing an empty vector, wild-type RTEL1, or mutants RTEL1 were infected with control-or Cre-expressing adenovirus. assembled from different fields of view and were processed as described in (Vannier et al., 2013). SUMO protease (10U/1mg of proteins) was added upon dialysis to cleave the SUMO-His tag. After Ulp1 protease cleaved the SUMO fusion, the samples were loaded onto the HisTrap FF nickel column, to remove His-SUMO and His-Ulp1, and subsequently cleaved proteins were recovered from the unbound flow through fraction. Next, proteins were concentrated using Amicon Ultra (Millipore) filter units and proteins were subjected on gel filtration. Fractions containing proteins were evaluated by SDS-PAGE followed by Coomassie staining. Concentration of the purified proteins were examined by Bradford assay or from the absorbance at 280 nm based on the molar absorption coefficients determined from the Edelhoch relationship.