Figure 2. Recombination factors are recruited at TRF1 depleted telomeres.

(A) Table listing chromatin remodellers identified. The corresponding number of unique peptide isolated is indicated for each factor of interest. Same as in Figure 1, light to darker colours indicate increasing relative protein abundance. (B) Validation of chromatin remodeller factors by ChIP-dot blot analysis in wt (+GFP) and TRF1^-/- (+CRE) conditions using ChIP grade antibodies against chosen factors after chromatin preparation from MEFs. The blot was revealed with a DIG-Tel-C-rich probe. ChIP signals were normalised to DNA input and GFP control. Data are represented as telomeric enrichment of proteins relative to GFP ± SEM of at least three independent biological replicates. P values, two-tailed student t-test (*, p<0.05; **, p<0.01; ****, p<0.0001). (C) Representative image of Immunofluorescence showing co-localisation of Telomeres (red) with PML (green) in MEFs nuclei (DAPI) treated with GFP and CRE. Data from two independent biological replicates are represented as number of Telomeres-PML co-localising foci divided by the total number of PML present per nucleus (n = 300 nuclei) with a violin plot, where the median is underlined in red and quartiles in white. P values, two-tailed student t-test (****, p<0.0001). Source data are provided as a Source Data File. (D) Representative images of the chromosome-oriented CO-FISH assay with denaturation, used to score for telomeric T-SCEs in TRF1^F/F MEFs infected with GFP or CRE. Telomeres are labelled with TelPNA-C-rich-Cy3 (red) and TelLNA-G-rich-FAM (green), while chromosomes are counterstained with DAPI (blue). Scale bar, 10 µm. Enlarged intersections show the difference between a chromosome with No T-SCE (top) and a chromosome with T-SCE (bottom). T-SCE images show double T-SCEs (left) and single chromatid events (right). Scale bar, 2 µm. For quantification, T-SCE was considered positive when involved in a reciprocal exchange of telomere signal with its sister chromatid (both telomeres yellow) and for asymmetrical exchanges at single chromatid (one telomere yellow). Data are indicated as % of T-SCE per sister telomere (n = > 3000 chromsome ends) and are represented with a violin plot, where the median is underlined in red and quartiles in white. P value, two-tailed student t-test (****, p<0.0001).

Figure 2—figure supplement 1. Validation of telomeric ChIP with ALU probe and effect of APH on telomeric chromatin and APBs.

(A) Control for Figure 2B, dot-blot for validation of chromatin remodellers factor specifically recruited at TRF1 depleted telomeres. The blot was revealed with a DIG-Alu probe (left panel). ChIP signals were normalised to DNA input (top, right panel) and GFP control and data are represented as relative Alu enrichment ± SEM of 3 independent biological replicates. P values, two-tailed student t-test. Source data are provided as a Source Data File. (B) Detection of chromatin remodeler factors by ChIP-dot blot analysis in wt (+DMSO) and Aphidicolin treated cells (+Aph) conditions using ChIP grade antibodies in MEFs chromatin (left panel). The blot was revealed with a DIG-Tel-C-rich probe. ChIP signals were normalised to DNA input (top middle panel) and GFP control. IgG and TRF2 antibodies were used respectively as negative and positive control for the ChIP experiment (top right panel). Data are represented as telomeric enrichment of proteins relative to DMSO ± SEM of 2 independent biological replicates. P values, two-tailed student t-test (**, p<0.01). (C) Representative image of Immunofluorescence showing co-localisation of Telomeres (red) with PML (green) in MEFs nuclei (DAPI) treated with DMSO or Aphidicolin. Data are represented as number of Telomeres-PML co-localising foci divided by the total number of PML present per nucleus (n > 100 nuclei) and three independent biological replicates are shown in a violin plot, where the median is underlined in red and quartiles in white (left graph). Number of total PML foci per nucleus from three independent biological replicates are shown in a violin plot, where the median is underlined in red and quartiles in white (right graph). P values, two-tailed student t-test (*, p<0.05; ****, p<0.0001). Source data are provided as a Source Data File.

Figure 2—figure supplement 2. TRF1 loss and APH replication stress induce different types of telomeric recombination.

(A) Time course quantification of the different classes of T-SCEs using denaturing CO-FISH. TRF1^F/F MEFs were collected 4 days and 7 days post-infection with CRE-adenovirus or GFP- (control). The different types of exchanges were classified into three different categories: all exchanges (single + double); double exchanges (reciprocal, both chromatids); single exchanges (asymmetrical, single chromatid). Violin plots are representing as % of T-SCE per chromosome ends (n = at least 3000 events were scored), where the median is underlined in red and quartiles in white. P value, two-tailed student t-test (****, p<0.0001; ***, p<0.001; n.s. = non significant). (B) Quantification of telomeric T-SCEs in wt MEFs treated with DMSO or Aphidicolin. Telomeric exchanges are classified as double exchanges (reciprocal, both chromatids-left graph) and single exchanges (asymmetrical, single chromatid-right graph). Violin plots represent % of T-SCE per chromosome ends (n = at least 1500 events were scored), where the median is underlined in red and quartiles in white. P value, two-tailed student t-test (*, p<0.05).