Journal: Genes & Development

Article Title: Break-induced replication promotes fragile telomere formation

doi: 10.1101/gad.328575.119

Figure Lengend Snippet: BIR mediates fragile telomeres formation in cells with telomeric DSBs. (A) Western blot analysis of Myc-FokI-ERT2-TRF1 expression in wild-type MEFs infected with empty vector, nuclease-dead (DA), or wild-type (WT) Myc-FokI-ERT2-TRF1. γ-Tubulin serves as the loading control. (B) Immunofluorescence (IF)-FISH analysis of MEFs expressing DA or WT Myc-FokI-ERT2-TRF1. Telomeres were detected by FISH with an Alexa 488-[TTAGGG]3 probe. The FokI-ERT2-TRF1 alleles were detected with anti-Myc antibodies and Alexa 647 secondary antibodies and 53BP1 with anti-53BP1 antibodies and Alexa 555 secondary antibodies. DNA was stained with DAPI. Images were false colored for presentation purposes. (C) Quantification of q arm fragile telomeres detected by FISH in cells expressing DA or WT Myc-FokI-ERT2-TRF1 after shRNA knockdown of Luc or POLD3 (96 h). (D) Western blot to monitor POLD3 knockdown with shRNA (96 h) in cells expressing DA or WT Myc-FokI-ERT2-TRF1. γ-Tubulin serves as the loading control. (E) Quantification of the TIF response in cells expressing DA or WT Myc-FokI-ERT2-TRF1 after shRNA knockdown of POLD3. Cells were induced with 4-OHT for 24 h before fixation. Data are means ± SD of three independent experiments of >50 nuclei each. (F) Quantification of q arm fragile telomere frequency in cells expressing DA or WT Myc-FokI-ERT2-TRF1 by FISH and CO-FISH. Cells were treated with 4-OHT for 24 h before harvesting for metaphase analysis. (G) Quantification of q arm fragile telomeres detected by CO-FISH in cells expressing DA or WT Myc-FokI-ERT2-TRF1 after shRNA knockdown of Luc or POLD3 (96 h). For all fragile telomere analyses, data are means ± SD of three independent experiments of ∼2000 telomeres analyzed per experiment. All P-values in this figure were derived from two-tailed unpaired t-test. (***) P ≤ 0.001, (**) P ≤ 0.01, (*) P ≤ 0.05, (n.s.) P > 0.05.

Article Snippet: The following antibodies were used for IF: Myc (Cell Signaling 9B11), 53BP1 (Abcam ab175933), goat antirabbit A555 (Thermo Fisher A32732), and goat antimouse A647 (Thermo Fisher A21237).

Techniques: Western Blot, Expressing, Infection, Plasmid Preparation, Immunofluorescence, Staining, shRNA, Derivative Assay, Two Tailed Test

Journal: Genes & Development

Article Title: Break-induced replication promotes fragile telomere formation

doi: 10.1101/gad.328575.119

Figure Lengend Snippet: SLX1/SLX4 contribute to fragile telomere formation in Blm-deficient cells. (A) Western blot analysis of BLM in BlmF/F MEFs ± Cre (96 h). γ-Tubulin serves as the loading control. (B) Telomere FISH on metaphase spreads of BlmF/F MEFs ± Cre (96 h) with Cy3-[CCCTAA]3 probes (green) and DAPI staining (red). Fragile telomeres are marked by an asterisk. (C) Knockdown of ZRANB3, SMARCAL1, and HTLF with shRNAs (6 d) in BlmF/F MEFs verified by Western blotting. Cells infected with an shRNA targeting Luciferase (shLuc) were used as the control. γ-Tubulin serves as the loading control and an asterisk marks a nonspecific band detected by the HLTF antibody. (D) Quantification of fragile telomeres detected by FISH (q arms only) in BlmF/F MEFs ± Cre (96 h) with shRNAs targeting Luc, ZRANB3, SMARCAL1, or HTLF as described in C. (E) Quantification of q arm fragile telomeres detected by FISH in BlmF/F MEFs ± Cre (96 h) after CRISPR/Cas9 targeting of Slx4 with three different sgRNAs. Control cells were infected with an sgRNA targeting Luciferase (sgLuc). The relative level of SLX4 mRNA normalized to GAPDH was determined by RT-qPCR and compared with the sgLuc sample (set to 100). (F) Western blot analysis of SLX1 after CRISPR/Cas9 targeting of Slx1 with three different sgRNAs. γ-Tubulin serves as the loading control. (G) Quantification of q arm fragile telomeres detected by FISH in BlmF/F MEFs ± Cre (96 h) after CRISPR/Cas9 targeting of Slx1 with three different sgRNAs as in F. (H) Western blot analysis of the expression of FLAG-SLX4 and various mutants in BlmF/F MEFs with γ-Tubulin as the loading control. (I) Quantification of q arm fragile telomeres detected by FISH in BlmF/F MEFs + Cre (96 h) expressing empty vector (−), sgRNA-resistant WT FLAG-SLX4 or various mutants with CRISPR/Cas9 targeting of Luc or Slx4. (J) PLA foci (red) of TRF1 and γH2AX detected in BlmF/F MEFs ± Cre (96 h). (K) Quantification of PLA foci as in J in BlmF/F MEFs ± Cre (96 h) with CRISPR/Cas9 targeting of Luc, Slx4, or Slx1. Data are means ± SD of four independent experiments of >100 nuclei each. P-values were from paired two-tailed t-tests. (*) P ≤ 0.05. (L) PLA foci (red) of FLAG-TRF1 and 53BP1 detected in BlmF/F MEFs ± Cre (96 h). (M) Quantification of FLAG-TRF1/53BP1 PLA foci in BlmF/F MEFs ± Cre (96 h) with CRISPR/Cas9 targeting of Luc, Slx4, or Slx1. Data are means ± SD of three independent experiments of >100 nuclei each. For the fragile telomere analyses in D, E, G, and I, data are means ± SD from three independent experiments with ∼2000 telomeres analyzed per experiment. All P-values except for the ones in K were derived from unpaired two-tailed t-tests. (***) P ≤ 0.001, (**) P ≤ 0.01, (*) P ≤ 0.05, (n.s.) P > 0.05.

Article Snippet: The following antibodies were used for IF: Myc (Cell Signaling 9B11), 53BP1 (Abcam ab175933), goat antirabbit A555 (Thermo Fisher A32732), and goat antimouse A647 (Thermo Fisher A21237).

Techniques: Western Blot, Staining, Infection, shRNA, Luciferase, CRISPR, Quantitative RT-PCR, Expressing, Plasmid Preparation, Two Tailed Test, Derivative Assay

Journal: Genes & Development

Article Title: Break-induced replication promotes fragile telomere formation

doi: 10.1101/gad.328575.119

Figure Lengend Snippet: BIR mediates fragile telomeres formation in cells with telomeric DSBs. (A) Western blot analysis of Myc-FokI-ERT2-TRF1 expression in wild-type MEFs infected with empty vector, nuclease-dead (DA), or wild-type (WT) Myc-FokI-ERT2-TRF1. γ-Tubulin serves as the loading control. (B) Immunofluorescence (IF)-FISH analysis of MEFs expressing DA or WT Myc-FokI-ERT2-TRF1. Telomeres were detected by FISH with an Alexa 488-[TTAGGG]3 probe. The FokI-ERT2-TRF1 alleles were detected with anti-Myc antibodies and Alexa 647 secondary antibodies and 53BP1 with anti-53BP1 antibodies and Alexa 555 secondary antibodies. DNA was stained with DAPI. Images were false colored for presentation purposes. (C) Quantification of q arm fragile telomeres detected by FISH in cells expressing DA or WT Myc-FokI-ERT2-TRF1 after shRNA knockdown of Luc or POLD3 (96 h). (D) Western blot to monitor POLD3 knockdown with shRNA (96 h) in cells expressing DA or WT Myc-FokI-ERT2-TRF1. γ-Tubulin serves as the loading control. (E) Quantification of the TIF response in cells expressing DA or WT Myc-FokI-ERT2-TRF1 after shRNA knockdown of POLD3. Cells were induced with 4-OHT for 24 h before fixation. Data are means ± SD of three independent experiments of >50 nuclei each. (F) Quantification of q arm fragile telomere frequency in cells expressing DA or WT Myc-FokI-ERT2-TRF1 by FISH and CO-FISH. Cells were treated with 4-OHT for 24 h before harvesting for metaphase analysis. (G) Quantification of q arm fragile telomeres detected by CO-FISH in cells expressing DA or WT Myc-FokI-ERT2-TRF1 after shRNA knockdown of Luc or POLD3 (96 h). For all fragile telomere analyses, data are means ± SD of three independent experiments of ∼2000 telomeres analyzed per experiment. All P-values in this figure were derived from two-tailed unpaired t-test. (***) P ≤ 0.001, (**) P ≤ 0.01, (*) P ≤ 0.05, (n.s.) P > 0.05.

Article Snippet: The following antibodies were used for IF: Myc (Cell Signaling 9B11), 53BP1 (Abcam ab175933), goat antirabbit A555 (Thermo Fisher A32732), and goat antimouse A647 (Thermo Fisher A21237).

Techniques: Western Blot, Expressing, Infection, Plasmid Preparation, Immunofluorescence, Staining, shRNA, Derivative Assay, Two Tailed Test

Journal: Genes & Development

Article Title: TRF1 uses a noncanonical function of TFIIH to promote telomere replication

doi: 10.1101/gad.349975.122

Figure Lengend Snippet: Helix2 of the TRF1 TRFH domain contributes to the suppression of telomere fragility. ( A ) Immunoblot analysis of TRF1 in Trf1 F/F MEFs ± Cre (96 h). γ-Tubulin served as the loading control. ( B ) Telomere FISH on metaphase spreads of Trf1 F/F MEFs ± Cre (96 h) with Cy3-[CCCTAA] 3 probes (green) and DAPI staining (red). (Asterisks) Fragile telomeres, (open circles) sister telomere associations. ( C ) Schematic representation of wild-type TRF1, TRF2, and the swapping mutants (domains are not to scale), with the Helix2 sequences in the TRF1 and TRF2 TRFH domains aligned in the middle , and the structure of the human TRF1 TRFH domain dimer at the bottom . The hTIN2 peptide is in red, Helix2 is in yellow, and E86 (E83 in mice) is in green. ( D ) Immunoblot to monitor the expression of the indicated TRF1 constructs in Trf1 F/F MEFs ± Cre (96 h). γ-Tubulin served as the loading control. ( E ) IF-FISH analysis of Trf1 F/F MEFs + Cre (96 h) complemented with the indicated versions of TRF1 or with the vector. Telomeres were detected by FISH with Alexa 647-[TTAGGG] 3 probes. The TRF1 proteins were detected with anti-FLAG antibodies and Alexa 488 secondary antibodies and 53BP1 with anti-53BP1 antibodies and Alexa 555 secondary antibodies. DNA was stained with DAPI (blue). Images were false-colored for presentation purposes. ( F ) Quantification of fragile telomeres detected by FISH in Trf1 F/F MEFs ± Cre (96 h) complemented with the corresponding constructs. Only long (q) arm telomeres were scored to avoid the confounding juxtaposition of telomeric signals at the centromeric end of the acrocentric mouse chromosomes. ( G ) Quantification of 53BP1 TIFs in Trf1 F/F MEFs ± Cre (96 h) complemented with the indicated versions of TRF1. Data are means ± SD of three independent experiments of >50 nuclei each. ( H ) Quantification of long arm STAs detected by FISH in Trf1 F/F MEFs ± Cre (96 h) complemented with the indicated version of TRF1. Data are means ± SD from three experiments, with ∼2000 telomeres analyzed per experiment. All P -values were derived from two-tailed unpaired t -test. (****) P ≤ 0.0001, (**) P ≤ 0.01, (*) P ≤ 0.05, (n.s.) P > 0.05.

Article Snippet: The following antibodies were used for IF: Myc 9B11 (Cell Signaling Technologies 2276), FLAG M2 (Sigma F1804), 53BP1 (Abcam ab175933), goat antirabbit A555 (Thermo Fisher A32732), goat antimouse A647 (Thermo Fisher A21237), and goat antimouse A488 (Thermo Fisher A32723).

Techniques: Western Blot, Staining, Expressing, Construct, Plasmid Preparation, Derivative Assay, Two Tailed Test

Journal: Genes & Development

Article Title: TRF1 uses a noncanonical function of TFIIH to promote telomere replication

doi: 10.1101/gad.349975.122

Figure Lengend Snippet: Epistasis and interaction of TFIIH and TRF1. ( A ) Immunoblot to monitor XPB, XPD, and TRF1 in Trf1 F/F MEFs ± Cre (96 h) with CRISPR/Cas9 targeting of Luc , Xpb , or Xpd (96 h) as indicated. γ-Tubulin served as the loading control. ( B ) Quantification of long arm fragile telomeres detected by FISH in Trf1 F/F MEFs ± Cre (96 h) with CRISPR/Cas9 targeting of Luc , Xpb , or Xpd (96 h) as in A . ( C ) Anti-Myc co-IPs of Myc-tagged mouse TRF1 and FLAG-tagged mouse TFIIH subunits from cotransfected 293FT cells. Immunoblots were probed with anti-Myc and anti-FLAG antibodies. Four percent of the lysate was run as the input. ( D ) Anti-Myc co-IPs of Myc-tagged mouse TRF1, TRF1-E83K/LW-TI, and TRF2 and FLAG-tagged mouse XPB, GTF2H3, and GTF2H4 from cotransfected 293FT cells. IP samples were treated with 25 U of benzonase where indicated. Immunoblots were probed with anti-Myc and anti-FLAG antibodies. Four percent of the lysate was run as the input. ( E ) Immunoblot to monitor XPB and TRF1 in Trf1 F/F MEFs with overexpression of wild-type TRF1 and TRF1-E83K/LW-TI and with CRISPR/Cas9 targeting of Luc or Xpb (96 h). γ-Tubulin served as the loading control. ( F ) Quantification of long arm fragile telomeres detected by FISH in Trf1 F/F MEFs as in E . All metaphase analyses are means ± SD of three experiments of ∼2000 telomeres analyzed. P -values were derived from two-tailed unpaired t -test. (****) P ≤ 0.0001, (***) P ≤ 0.001, (**) P ≤ 0.01, (n.s.) P > 0.05. ( G ) Model of how different motifs in TRF1 function to suppress telomere fragility, replication defects, ATR signaling, and STAs.

Article Snippet: The following antibodies were used for IF: Myc 9B11 (Cell Signaling Technologies 2276), FLAG M2 (Sigma F1804), 53BP1 (Abcam ab175933), goat antirabbit A555 (Thermo Fisher A32732), goat antimouse A647 (Thermo Fisher A21237), and goat antimouse A488 (Thermo Fisher A32723).

Techniques: Western Blot, CRISPR, Over Expression, Derivative Assay, Two Tailed Test