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TZAP: A telomere-associated protein involved in telomere length control

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Science  10 Feb 2017:
Vol. 355, Issue 6325, pp. 638-641
DOI: 10.1126/science.aah6752

A protein to trim too-long telomeres

Telomeres cap the ends of linear eukaryotic chromosomes. They consist of multiple copies of short DNA repeats. The length of telomeres is important to genome stability; if they become too short, individuals can become prone to cancer and premature aging. Li et al. discovered a protein, TZAP (telomeric zinc finger–associated protein), which instead prevents telomeres from becoming too long (see the Perspective by Lossaint and Lingner). TZAP binds directly to the telomeric DNA repeats, competing with the shelterin complex. It stimulates telomere trimming, preventing aberrantly long telomeres.

Science, this issue p. 638; see also p. 578

Abstract

Telomeres are found at the end of chromosomes and are important for chromosome stability. Here we describe a specific telomere-associated protein: TZAP (telomeric zinc finger–associated protein). TZAP binds preferentially to long telomeres that have a low concentration of shelterin complex, competing with the telomeric-repeat binding factors TRF1 and TRF2. When localized at telomeres, TZAP triggers a process known as telomere trimming, which results in the rapid deletion of telomeric repeats. On the basis of these results, we propose a model for telomere length regulation in mammalian cells: The reduced concentration of the shelterin complex at long telomeres results in TZAP binding and initiation of telomere trimming. Binding of TZAP to long telomeres represents the switch that triggers telomere trimming, setting the upper limit of telomere length.

Telomere length homeostasis is essential for proper cellular function (13). The telomere proteome consists of ~200 proteins that have been associated with different aspects of telomere biology, including telomere protection, telomeric DNA synthesis, and telomere elongation (48). Here we describe the characterization of the Kruppel-like zinc finger protein ZBTB48 as a telomere-associated factor involved in telomere length regulation. On the basis of the telomere-specific localization of ZBTB48 (Fig. 1, A to C, and fig. S1, A to C), we renamed this factor as telomeric zinc finger–associated protein (TZAP). TZAP bound to telomeres in telomerase-positive cells as well as telomerase-negative cells (Fig. 1, A and B, and fig. S1, A to C). Using a rabbit polyclonal antibody raised against human TZAP, we confirmed that endogenous TZAP resides at telomeres (Fig. 1B). The specificity of this antibody was verified using TZAP−/− U2OS clones generated by CRISPR-Cas9 genome editing (Fig. 1B and fig. S1, D to F). Using chromatin immunoprecipitation (ChIP) followed by next-generation sequencing (ChIP-seq), we confirmed that the level of TZAP association with telomeres in U2OS cells is comparable to that of TRF1 (Fig. 1C). Notably, TZAP was not significantly enriched at any genetic locus other than telomeres in U2OS cells. To date, the only proteins known to exclusively and specifically associate with TTAGGG are the components of the shelterin complex. Our data indicate that TZAP binds to telomeres independently of the shelterin complex, as shown by the fact that it does not interact with members of this complex (Fig. 1D and fig. S1, G and H) and by its localization to telomeres in shelterin-free mouse embryonic fibroblasts (MEFs) (9) (Fig. 1E and fig. S1I).

Fig. 1 TZAP, a telomere-associated protein.

(A) Fluorescence in situ hybridization–immunofluorescence (FISH-IF) images showing localization of MYC-TZAP (green) at telomeres (red) in mouse embryonic fibroblasts (MEFs). (B) IF images showing endogenous TZAP at telomeres in U2OS cells. TZAP knockout clones (KO#E3 and KO#E5) and overexpressing cells are used to confirm staining specificity. In (A) and (B), the bottom panels are zoomed-in views of the areas outlined by dashed rectangles. (C) (Left) Chromatin immunoprecipitation (ChIP) shows association of FLAG-TRF1 or FLAG-TZAP to telomeric DNA in U2OS cells. WB, Western blot. (Right) Bars represent the fraction of raw sequences containing four repeats of the indicated sequences from the total reads obtained by ChIP-seq analysis. (D) FLAG immunoprecipitation was performed from lysates of human embryonic kidney 293T cells expressing the indicated FLAG-tagged and MYC-tagged constructs. (E) MYC-TZAP localizes at telomeres in TRF1F/FTRF2F/F Rs26CRE-ER MEFs, either untreated (-OHT) or 4 days after tamoxifen (+OHT) treatment.

To test whether TZAP binds directly to TTAGGG repeats, we used a domain-swap approach aimed at assessing whether the zinc finger domains of TZAP could replace the DNA binding domain of the telomeric protein TRF2. We generated TRF2F/F, Rs26CRE-ER MEFs expressing the following MYC-tagged constructs: full-length TRF2 (TRF2), a TRF2 allele lacking its TTAGGG binding domain (TRF2ΔMyb), and a chimeric protein containing TRF2ΔMyb fused to the 11 zinc fingers of TZAP (TRF2Znc1-11) (Fig. 2A). As expected, in TRF2-null conditions [4-hydroxytamoxifen (+OHT)], full-length TRF2 localized at telomeres, whereas the TRF2ΔMyb allele did not (Fig. 2B). The TRF2Znc1-11 chimera allele localized at telomeres (Fig. 2B) and functionally complemented the loss of TRF2 in suppressing chromosome end-to-end fusions (Fig. 2, C and D). Next, using an array of truncation mutants, we found that the three terminal zinc finger domains of TZAP (Znf9-11) were both required and sufficient for telomeric localization (Fig. 2E and figs. S2, E to I, and S3, A to D). Finally, using purified recombinant TZAPznf9-11 harboring a Hi6xs-MBP tag (Fig. 2F and fig. S3E), we carried out an electrophoretic mobility shift assay to show that TZAPznf9-11 exhibited binding affinity specific to dsTTAGGG repeats in vitro (Fig. 2F and fig. S3F). Thus, TZAP is a telomere-associated protein that directly binds dsTTAGGG repeats via zinc finger domains.

Fig. 2 The zinc finger domains of TZAP directly bind telomeric repeats.

(A) Schematic representation of the constructs used to show that the zinc finger domains of TZAP are sufficient for telomeric localization. (B) FISH-IF images for telomeric localization of the indicated constructs in TRF2-null MEFs. (C) Metaphase spreads derived from TRF2-null MEFs expressing the indicated constructs were stained for telomeric DNA (green) and 4′,6-diamidino-2-phenylindole (DAPI) (red). (D) Genomic DNA isolated from TRF2-proficient (-OHT) or TRF2-null (+OHT) MEFs expressing the indicated constructs was digested with MboI, resolved on a pulsed field gel, and probed using a radioactive telomeric probe. EV, empty vector. (E) The terminal zinc finger domains (9, 10, and 11) of TZAP are required for telomeric localization, as shown by FLAG-ChIP in U2OS cells expressing the indicated FLAG-tagged constructs. (F) A recombinant HisMBP-tagged TZAPZnf9-11 allele was expressed in bacteria, purified, and used in gel shift assays. (Left) HisMBP-TZAPZnf9-11 binds telomeric repeats and is supershifted by a His-tag antibody. (Right) Competition assay using increasing amounts (0-, 1-, 10-, 100-, and 1000-fold molar excess) of cold nontelomeric or telomeric competitor double-stranded DNA (dsDNA).

We next explored whether TZAP and TRFs compete for localization to telomeres. To test this hypothesis, we generated a U2OS cell line with doxycycline-inducible expression of FLAG-TZAP (Flp-IN T-REX FLAG-TZAP) (Fig. 3A). Overexpression of TRF2 in these cells caused a large reduction in the localization of TZAP to telomeres (Fig. 3, A to C). In contrast, induction of TZAP did not affect the localization of TRF2 to telomeres (fig. S4A). We confirmed these data in MEFs and found that TRF2 overexpression displaced TZAP (fig. S4, D to F) as well as the TRF2Znc1-11 chimera (Fig. 3, D and E, and fig. S4B) but not the shelterin component TRF1. Quantification of the protein levels of shelterin show that the abundance of this complex does not change in relation to telomere length (10). As a result, cells with long telomeres have a lower density of the shelterin complex compared with cells that have shorter telomeres, a difference that is not detected by ChIP (11). We therefore tested whether TZAP preferentially binds long telomeres with low shelterin density. To test this hypothesis, we transduced two subclones of HeLa cells that differ in telomere length: (i) HeLa VST (very short telomeres), with an average telomere length of ~5 kb, and (ii) HeLa 1.2.11, with longer telomeres (~20 kb). Although TZAP readily localized to telomeres in HeLa 1.2.11 cells, no distinguishable TZAP localization was detected in HeLa VST cells (Fig. 3, F and G). In agreement with this finding, an inverse correlation between telomere length and TZAP localization at telomeres was observed in a wide array of cell lines, independently of their transformation status, mode of telomere length regulation, and species of origin (fig. S5, A and B).

Fig. 3 TZAP binding to telomeres is inhibited by elevated TRF2 levels.

(A) IF images for doxycycline-inducible expression of FLAG-TZAP at telomeres in FLP-IN T-Rex U2OS cells with and without MYC-TRF2 overexpression. TRF1, red; anti-FLAG IF, green; and anti-MYC IF, blue. (B) Quantification of the data shown in (A), indicating the percentage of cells with doxycycline-inducible FLAG-TZAP localized at >90% of telomeres. (C) ChIP for telomeric DNA associated with doxycycline-inducible expression of FLAG-TZAP in FLP-IN T-Rex U2OS cells with and without MYC-TRF2 overexpression. Immunoblot of FLAG-tagged and MYC-tagged proteins from cells used for ChIP. (D) IF images for MYC-TRF2 or MYC-TRF2Znf1-11 ectopically expressed in TRF2F/F Rs26CRE-ER MEFs at day 4 after tamoxifen treatment (+OHT) with or without overexpression of FLAG-TRF2. Anti-TRF1, red; anti-MYC, green; and anti-FLAG, blue. (E) Quantification of data shown in (D), indicating the percentage of cells with MYC localized at >90% of telomeres. (F) Localization of FLAG-TRF1 or FLAG-TZAP in HeLa cells with very short telomeres (HeLa VST) or with long telomeres (HeLa 1.2.11). Anti-TRF2, red; anti-FLAG, green; and DAPI, blue. (G) Quantification of the data shown in (F), indicating the percentage of cells with FLAG localized at >90% of telomeres.

On the basis of its localization at long telomeres, we reasoned that TZAP might play a role in telomere length regulation. Overexpression of TZAP in U2OS cells confirmed this hypothesis, showing a progressive reduction in telomere length (Fig. 4A) that culminated in the accumulation of telomere-free chromosome ends (Fig. 4, D and E). Because U2OS cells lack telomerase, these data suggest that TZAP promotes rapid deletion of telomeres, a mechanism of telomere length regulation identified in yeast (12) and conserved in plants (13) and mammals (1416). Telomere trimming involves the deletion of the secondary telomeric structures known as T-loops (17), leading to telomere shortening and accumulation of extra chromosomal telomeric DNA (ECT-DNA) (1416). TZAP overexpression resulted in the accumulation of ECT-DNA, as shown by single-stranded C-circle assay (C-circle), telomeric circle (T-circle) assay, and two-dimensional gels (1820) (Fig. 4, B and C, and fig. S6, A, D, E, G, and H). Moreover, TZAP overexpression induced the formation of APBs [alternative lengthening of telomeres (ALT)–associated promyelocytic leukemia nuclear bodies], a phenotype associated with telomere trimming (14) (fig. S6, I to K). Conversely, decreasing TZAP levels resulted in a reduction of ECT-DNA in U2OS and GM847 cells (fig. S6, B to H). Thus, alteration in the levels of TZAP at telomeres controls the level of telomere trimming, leading to substantial changes in telomere length and the presence of ECT-DNA.

Fig. 4 TZAP contributes to telomere length homeostasis promoting telomere trimming.

(A) Telomere restriction fragment analysis of U2OS cells ectopically expressing the indicated constructs at the indicated time points after infection (days). (B) C-circle analysis to detect extra chromosomal telomeric DNA (ECT-DNA) in U2OS cells expressing the indicated constructs 6 days after infection. (C) Two-dimensional electrophoresis to detect ECT-DNA in U2OS cells expressing the indicated constructs 6 days after infection. Gels were denatured and probed with a telomeric probe; arrows indicate circular telomeric species. (D) Metaphase spreads of U2OS cells infected with the indicated constructs, analyzed by FISH. Telomeric DNA probe, green; DAPI, red. (E) Quantification of chromosome ends lacking detectable telomeric signal, indicative of telomere shortening. Bars average quantification from three independent experiments (error bars indicate SD). (F) Q-FISH analysis of mouse embryonic stem (mES) cells of the indicated genotypes, either with or without lentiviral infection of TZAP. WT, wild type.

Telomere trimming plays an important role in the control of telomere length in embryonic stem (ES) (15, 16) cells. To define whether the loss of TZAP would affect telomere length homeostasis in these cells, we generated multiple clones of mouse ES (mES) cells depleted of TZAP by CRISPR-Cas9 gene editing (fig. S7, A and B). These TZAP−/− cells showed significant telomere elongation, as measured by telomere restriction fragment analysis and quantitative fluorescence in situ hybridization (Q-FISH) (Fig. 4). Complementation of three independent TZAP−/− mES clones with exogenous TZAP restored telomere length back to the lower threshold (Fig. 4F and fig. S7H). When measuring C-circle abundance, we found that four out of five independent TZAP−/− clonal cell lines show reduced levels of ECT-DNA (fig. S7, D and E).

In conclusion, we propose a model for the function of TZAP at telomeres in which TZAP is preferentially recruited to long telomeres containing reduced concentration of the shelterin complex (fig. S8). In these settings, the binding of TZAP to telomeres initiates telomere trimming, a process that prevents the accumulation of aberrantly long telomeres. In agreement with our data, Tzap was recently identified as a down-regulated gene in mouse ES cells characterized by extremely long telomeres (21). Additional factors are likely to modulate the function of TZAP at telomeres, as suggested by the fact that TZAP-mediated telomere trimming is exacerbated in ALT-positive cells. The discovery of mechanisms that control telomere elongation explains how mammalian cells bypass the end-replication problem. This study provides insight into the mechanisms that control the upper limit of telomere length, a key determinant of life span and cancer susceptibility across mammalian species (22).

Supplementary Materials

www.sciencemag.org/content/355/6325/638/suppl/DC1

Materials and Methods

Figs. S1 to S8

References (2330)

References and Notes

  1. Acknowledgments: We thank A. Sfeir for performing experiment involving shelterin-free cells and for comments on the manuscript. We thank P. Wright and members of his lab for critical support in the purification of TZAP. This work was supported by a grant from the American Cancer Society (RSG-14-186-01) (E.L.D.), the Swedish Research Council International postdoc grant (D0730801) (J.M.F.), and NIH grants (R01GM087476 and R01CA174942) (J.K.).
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