TRF1 negotiates TTAGGG repeat-associated replication problems by recruiting the BLM helicase and the TPP1/POT1 repressor of ATR signaling

Abstract

The semiconservative replication of telomeres is facilitated by the shelterin component TRF1. Without TRF1, replication forks stall in the telomeric repeats, leading to ATR kinase signaling upon S-phase progression, fragile metaphase telomeres that resemble the common fragile sites (CFSs), and the association of sister telomeres. In contrast, TRF1 does not contribute significantly to the end protection functions of shelterin. We addressed the mechanism of TRF1 action using mouse conditional knockouts of BLM, TRF1, TPP1, and Rap1 in combination with expression of TRF1 and TIN2 mutants. The data establish that TRF1 binds BLM to facilitate lagging but not leading strand telomeric DNA synthesis. As the template for lagging strand telomeric DNA synthesis is the TTAGGG repeat strand, TRF1-bound BLM is likely required to remove secondary structures formed by these sequences. In addition, the data establish that TRF1 deploys TIN2 and the TPP1/POT1 heterodimers in shelterin to prevent ATR during telomere replication and repress the accompanying sister telomere associations. Thus, TRF1 uses two distinct mechanisms to promote replication of telomeric DNA and circumvent the consequences of replication stress. These data are relevant to the expression of CFSs and provide insights into TIN2, which is compromised in dyskeratosis congenita (DC) and related disorders.

Keywords: BLM; G quadruplex; TRF1; replication; shelterin; telomere.

Figure 1.

BLM cooperates with TRF1 to prevent fragile lagging end telomeres. (A) Cre-mediated deletion of TRF1 and BLM from MEFs of the indicated genotypes monitored by immunoblotting. (*) Nonspecific band. (B) Metaphase telomeric FISH (green) showing fragile telomeres (arrows) and sister telomere associations (asterisks) in MEFs as in A. (Red) DNA DAPI stain. (C) Quantification of fragile telomeres in cells shown in B. Fragile telomeres were scored in three experiments on >2000 long arm telomeres per experiment. Each symbol indicates the percentage of fragile telomeres in an analyzed metaphase. Error bars show SDs. Statistical significance ([*] P < 0.05; [ns] not significant) from one-way ANOVA with Tukey’s correction for multiple comparisons. (D) CO-FISH examples of lagging end fragile telomeres (arrows) in TRF1^F/+BLM^F/F cells (±Cre). (Green) Lagging end telomeres; (red) leading end telomeres; (blue) DAPI. (E) Quantification of leading end (red) and lagging end (green) fragile telomeres (as shown in D). Data are means of three independent experiments ± SDs. More than 1500 long arm telomeres were scored per experiment. Statistics are as in C.

Figure 2.

TRF1 binds BLM to prevent fragile lagging end telomeres. (A) Alignment of human and mouse TRF1 hinge domain sequences. (Green box) Basic patches implicated in BLM binding. (B) Schematic of the mouse TRF1 alleles used in coimmunoprecipitations (co-IPs) with human BLM. Deletions of the individual basic patches (as shown in A) are indicated by Δ. (C) Anti-Myc co-IPs of human BLM with mouse TRF1 alleles shown in B from cotransfected 293T cells. Immunoblots were probed with anti-Myc (top) and anti-BLM (bottom) antibodies. BLM is partially degraded in these experiments. (D) Immunoblot for Myc-TRF1 and Myc-TRF1ΔBLM (TRF1ΔDouble in B and C) in TRF1^F/FCre-ER^T2 cells with or without 4-hydroxytamoxifen (4OHT) treatment. (E) Examples of metaphase TTAGGG FISH (green) images of 4OHT-treated TRF1^F/FCre-ER^T2 MEFs complemented with the indicated Myc-TRF1 constructs. (Arrows) Fragile telomeres; (red) DAPI. (F) Frequency of fragile telomeres (plotted for individual metaphases) in TRF1^F/FCre-ER^T2 cells as in E. More than 1500 long arm telomeres were scored in each of four independent experiments. Error bars represent SDs. Statistics are as in Figure 1C. (G) CO-FISH showing leading end (red) and lagging end (green) fragile telomeres in 4OHT-treated TRF1^F/FCre-ER^T2 cells with or without TRF1ΔBLM. (Blue) DAPI. (H) Quantification of leading and lagging end fragile telomeres in TRF1^F/FCre-ER^T2 cells as in G. Data are means ± SDs of three independent experiments. Statistics are as in Figure 1C.

Figure 3.

BLM-independent functions of TRF1. (A) TIF assay on the indicated cells with and without Cre. IF for 53BP1 (red) combined with telomeric TTAGGG FISH (green). (Blue) DAPI. (B) Percentages of nuclei (cells as in A) showing more than five 53BP1 TIFs. Data are means from three independent experiments ± SDs. At least 100 cells were scored in each experiment. (C) IF-FISH as in A on 4OHT-treated TRF1^F/F Cre-ER^T2 cells with full-length TRF1 or TRF1ΔBLM. (D) Quantification of TIF-positive (more than five) nuclei as in C. Data in B and D are means of three independent experiments ± SDs (n ≥ 100 nuclei per experiment). (E) Percentages of sister telomere associations (illustrated in Fig. 1B) in the indicated cells with or without Cre. (F) Quantification of sister telomere associations in TRF1^F/FCre-ER^T2 cells transduced with the indicated constructs. Data in E and F are plotted as percentages of long arm sister telomere associations in each metaphase analyzed in at least three independent experiments (>2000 telomeres per experiment). Statistics are as in Figure 1C.

Figure 4.

Tethering of TIN2 to TRF2 represses TIFs and sister telomere associations upon TRF1 deletion. (A) Schematic of Flag-HA₂-TIN2 and Flag-HA₂-TIN2^RCT with TRF1-, TPP1-, and TRF2-binding sites shown. (B) Immunoblotting for TIN2 and TIN2^RCT in TRF1^F/FCre-ER^T2 cells ±4OHT. (C) TIF analysis (as in Fig. 3A) on the TRF1^F/FCre-ER^T2 cells shown in B. (D) Quantification of cells with more than five 53BP1 TIFs per nucleus as shown in C. See Figure 3B. (E) Sister telomere associations (asterisks) detected with telomeric FISH (green) in the cells described in B. (Red) DAPI. (F) Percentages of sister telomere associations (as in E) in each analyzed metaphase. Scoring and statistics are as in Figures 3E and 1C, respectively.

Figure 5.

Repression of sister telomere associations requires TRF1 and TPP1. (A) Immunoblotting for TIN2^RCT in TRF1^F/F53BP1^−/− and TRF1^F/FTPP1^F/F53BP1^−/− cells ±Cre. (B) IF for γH2AX (red) combined with telomeric FISH (green) in cells as in A. (Blue) DAPI. (C) Percentage of cells with more than five γ-H2AX TIFs per nucleus as shown in B. Data are means of three independent experiments ± SDs (>100 nuclei per experiment). (D) Sister telomere associations (asterisks) detected as in Figure 4E in the cells described in A. (Red) DAPI. (E) Percentages of sister telomere associations (as in D) in each analyzed metaphase. Scoring and statistics are as in Figures 3E and 1C, respectively. (F) Summary of telomere fragility and nonsister telomere fusions/associations in TRF1^F/F53BP1^−/− and TRF1^F/FTPP1^F/F53BP1^−/− cells. Means of three independent experiments ± SDs. Fragile telomeres were scored on >1500 long arm telomeres per experiment.

Figure 6.

Proposed mechanism for TRF1-mediated telomere protection during telomere replication. TRF1 recruits the BLM helicase to dismantle G quadruplexes in the lagging strand template, thereby preventing replication fork stalling and persistence of single-stranded (ss) gaps that lead to the formation of the fragile telomere phenotype specifically in lagging end telomeres. How TRF1 prevents fragile leading end telomeres is not known. TRF1 is proposed to repress ATR signaling in a BLM-independent manner using TIN2-bound TPP1/POT1 to prevent RPA accumulation on the G-rich telomeric DNA exposed when lagging strand DNA synthesis is impaired. The shown mechanism does not explain how TRF1 prevents ATR activation by the single-stranded C-rich telomeric DNA. Exposed C-rich telomeric DNA (the leading strand template) will be adjacent to a 3′ double-stranded–single-stranded transition, which is a suboptimal substrate for ATR activation. The position of the TRF1 complexes engaged in the proposed functions (drawn ahead of and behind the replication fork) is not known. The TRF1 complexes are likely connected to TRF2/Rap1 (not drawn) through their shared binding partner, TIN2. The stalled replisome is not depicted.