Telomere protection by TPP1 is mediated by POT1a and POT1b

Abstract

Mammalian telomeres are protected by the shelterin complex, which contains single-stranded telomeric DNA binding proteins (POT1a and POT1b in rodents, POT1 in other mammals). Mouse POT1a prevents the activation of the ATR kinase and contributes to the repression of the nonhomologous end-joining pathway (NHEJ) at newly replicated telomeres. POT1b represses unscheduled resection of the 5'-ended telomeric DNA strand, resulting in long 3' overhangs in POT1b KO cells. Both POT1 proteins bind TPP1, forming heterodimers that bind to other proteins in shelterin. Short hairpin RNA (shRNA)-mediated depletion had previously demonstrated that TPP1 contributes to the normal function of POT1a and POT1b. However, these experiments did not establish whether TPP1 has additional functions in shelterin. Here we report on the phenotypes of the conditional deletion of TPP1 from mouse embryo fibroblasts. TPP1 deletion resulted in the release of POT1a and POT1b from chromatin and loss of these proteins from telomeres, indicating that TPP1 is required for the telomere association of POT1a and POT1b but not for their stability. The telomere dysfunction phenotypes associated with deletion of TPP1 were identical to those of POT1a/POT1b DKO cells. No additional telomere dysfunction phenotypes were observed, establishing that the main role of TPP1 is to allow POT1a and POT1b to protect chromosome ends.

FIG. 1.

Conditional deletion of TPP1 removes POT1a and -b from telomeres. (A) Schematic of the mouse TPP1/Acd locus on chromosome 8, the targeting vector, the conditional allele (TPP1^F), and the null allele (TPP1^Del). PCR primers (F1, F2, and R) and probes for analysis of genomic DNA are indicated. E, EcoRI. (B) PCR analysis of genomic DNA isolated from SV40-LT-immortalized TPP1^F/F MEFs at 96 h after Hit&Run-Cre infection. (C) Graph of the proliferation of the indicated MEFs with and without Cre infection. (D) Phase-contrast micrographs of TPP1^F/F MEFs at 10 days after Cre infection and the uninfected control. Cells were stained for SA-β-galactosidase. (E) ChIP with shelterin components in TPP1 KO cells. TPP1^F/F MEFs were fixed and analyzed at 96 h after Cre or mock infection. Crude sera were used for the IPs. Telomeric DNA was detected with a TTAGGG repeat probe. Sera used for IPs were the same as in panel F. PI, preimmune. (F) Quantification of the ChIP shown in panel E. Bars show average results of three or four independent experiments and standard deviations. The P value was derived from the Wilcoxon rank sum test. PI background ChIP values were subtracted from the other ChIP values. (G) TPP1^F/F cells were fractionated prior to or at 96 h after infection with Cre. Whole-cell lysate (WC), cytoplasmic proteins (CP), nucleoplasmic proteins (NP), and chromatin-bound proteins (CB) were analyzed by immunoblotting. α-Tubulin was used as a control for cytoplasmic proteins. Asterisk, nonspecific band.

FIG. 2.

ATR-dependent DNA damage signaling in TPP1 KO cells. (A) Induction of TIFs by deletion of TPP1. TPP1^F/F cells at 96 h after Cre (or mock) infection were analyzed using FISH for telomeres (FITC, green) and IF for 53BP1 (Alexa 555, red). (B) Quantification of the TIF response. Cells shown in panel A were scored for 10 or more telomeric 53BP1 foci. Bars show average results of three independent experiments and standard deviations. (C) Exogenous TPP1 expression represses TIF formations in TPP1 KO cells. The TIF response was quantified as for panels A and B. (D) γH2AX TIFs induced by TPP1 deletion. TPP1^F/F cells were analyzed using FISH for telomeres (FITC, green) and IF for γH2AX (RRX, red) at the same time point as in panel A after Cre infection. (E) Loss of TPP1 induces phosphorylation of Chk1 and Chk2. The indicated cells were harvested at 96 h after Cre infection and processed for immunoblotting for the indicated proteins. Asterisk, nonspecific band. (F) Immunoblots verifying shRNA-mediated knockdown of ATM and ATR. Cells infected with the indicated shRNAs were collected and analyzed at 96 h after Hit&Run-Cre infection. (G) ATR-dependent TIF formation upon TPP1 deletion. Cells infected with the indicated shRNAs were analyzed using FISH for telomeres (FITC, green) and IF for 53BP1 (Alexa 555, red) at the same time point as in panel F after Cre infection. (H) Quantification of the effect of ATM and ATR knockdown on the TIF response upon TPP1 deletion. Cells were treated as for panel G and scored for 10 or more telomeric 53BP1 foci. Bars show average results of two independent experiments and standard errors of the means.

FIG. 3.

TPP1 deficiency induces excess single-stranded telomeric DNA. (A) In-gel overhang assay with the indicated MEFs at the indicated time points after infection with Cre (or mock treatment). Molecular sizes are indicated in kilobases. (B) Quantification of the overhang signals of the gel shown in panel A. The single-stranded telomeric DNA signal was normalized to the double-stranded telomeric DNA signal.

FIG. 4.

Sister telomere fusions and infrequent chromosome-type fusions in TPP1 null cells. (A) Metaphase from TPP1^F/F cells at 144 h after Cre infection. DNA was stained with DAPI (false colored in red). Telomere DNA was detected by FISH (FITC, green). (B) Examples of chromosome-type fusions and sister telomere fusions in TPP1 KO cells. Experimental conditions are as for panel A. (C) Quantification of the types of telomere fusions in TPP1 null cells analyzed as for panel A. Telomere sister fusions were not scored for the short-arm telomeres because their proximity can lead to spurious coincidence of the telomeric signals.

FIG. 5.

The deletion of TPP1 induces endoreduplication. (A) DNA profiles of TPP^F/F cells before and after Cre. Cells were harvested and fixed at 5 days after Cre infection. DNA content was analyzed by FACS (PI staining). Sub-G₁ cells are not shown. (B) Telomere FISH on a metaphase spread derived from TPP1 KO cells showing diplochromosomes.