No Overt Nucleosome Eviction at Deprotected Telomeres

Through their unique structure and composition, specialized nucleoprotein complexes known as telomeres protect the ends of linear chromosomes. Vertebrate telomeres can form so-called t-loops through strand invasion of their long 3′ overhang into the double-stranded region of telomeric TTAGGG repeats (17, 32). Beyond this ultrastructure, the packaging of telomeric DNA in nucleosomal chromatin lends an additional layer of complexity to vertebrate telomeres. Whereas Saccharomyces cerevisiae and Tetrahymena spp. have short, nonnucleosomal telomeres (7, 16, 43), the results of electron microscopy and micrococcal nuclease (MNase) digestion studies have demonstrated the presence of nucleosomes at telomeres in a number of vertebrates, including chickens, rodents, and humans (26, 28, 32, 38). Telomeric chromatin resembles bulk chromatin in its composition, consisting of the canonical core histone components, but the nucleosomal repeat length is short, and the nucleosomal core particle shows hypersensitivity to MNase (26, 28, 38). The results of in vitro studies have shown that nucleosomes assembled on TTAGGG repeats exhibit higher mobility than on other sequences (33). In addition, telomeric chromatin is enriched for heterochromatic marks, such as trimethylation of H3K9 and H4K20, and the results of studies in mice have shown that the loss of such marks correlates with abnormally elongated telomeres (3, 14, 15). Short human telomeres show evidence of an unusual chromatin structure, as deduced from the more diffuse nature of the MNase digestion patterns (38), but the molecular basis of this change is not known. Shelterin, a six-protein complex unique to chromosome ends, binds TTAGGG repeats within their nucleosomal context and protects telomeres from being recognized as DNA double-strand breaks (DSBs) or other sites of DNA damage (reviewed in reference 12). Telomere dysfunction caused by the inhibition of shelterin or replicative attrition can lead to the activation of the ATM or ATR kinase signaling cascade, resulting in cell cycle arrest and inappropriate repair of telomeres by nonhomologous end joining (NHEJ) or homologous recombination (HR). The DNA damage response at dysfunctional telomeres also results in the presence of DNA damage factors (e.g., 53BP1 and γ-H2AX) at chromosome ends, forming so-called telomere dysfunction-induced foci (TIFs) (37). The shelterin protein TRF2 binds double-stranded telomeric repeats through its C-terminal Myb/SANT DNA binding domain (5, 6). Its inhibition by either the introduction of a dominant-negative allele or conditional deletion in mouse embryonic fibroblasts (MEFs) activates the ATM kinase at all stages of the cell cycle and results in end-to-end telomere fusions mediated by Ku70- and DNA ligase IV-dependent NHEJ (8, 9, 36, 42). HR at telomeres gives rise to telomere-sister chromatid exchanges (T-SCEs), which are repressed by both TRF2 and Ku70 (9). On the other hand, the ATR kinase is repressed by POT1, which binds single-stranded TTAGGG repeats (2) and is recruited to telomeres through its interaction with another shelterin component, TPP1 (19, 20, 25, 45). In mice, which contain two POT1 genes (19), deletion of both POT1a and POT1b or POT1a alone leads to the activation of the ATR kinase (25). Like TRF2, the two POT1 proteins also repress HR at telomeres when Ku70 is absent (W. Palm, D. Hockemeyer, and T. de Lange, unpublished data). However, POT1a and -b do not contribute to the repression of NHEJ at telomeres. POT1b has a specific role in limiting the resection of the C-rich telomeric strand, a process that gives rise to the 3′ overhang (19-21). As a result, POT1b-null MEFs have two- to threefold-more single-stranded telomeric DNA. The cellular machinery that responds to DNA damage executes its functions within the context of chromatin, and the results of several recent studies have suggested that chromatin remodeling and modifications contribute to the recognition and repair of DSBs. Chromatin decondensation at DSBs has been inferred to occur in living cells subjected to DNA-damaging agents, and such conformational changes have been proposed to activate the ATM kinase (1, 24). In addition, nucleosome loss has been observed at sites of induced DSBs in several model systems. In S. cerevisiae, the induction of a single DSB by HO endonuclease leads to histone displacement that depends on the DNA damage sensor MRX and the chromatin remodeling complex INO80 (39, 41). In human cells, break sites introduced by the I-PpoI nuclease exhibit nucleosome disruption that requires NBS1 and ATM (4). Furthermore, several steps in NHEJ or HR are stimulated by one or more of the three major ATP-dependent chromatin remodeling complexes, INO80, Swi/Snf, and RSC (10, 31, 34, 35, 40, 41). Given that telomere dysfunction activates the canonical response to DSBs, exploring the role of chromatin remodeling at damaged telomeres may lend insight into the mechanisms by which shelterin prevents telomeres from being recognized as DNA damage. For this study, we investigated the nucleosomal organization of telomeres following their deprotection to determine whether the telomeric DNA damage signaling and repair processes involve chromatin remodeling.