Dynamics of satellite binding protein CENP-B and telomere binding protein TRF2/MTBP in the nuclei of mouse spermatogenic line

The location of centromeric protein CENP-B and telomeric protein TRF2/MTBP in the mouse spermatogenic line has been studied using indirect immunofluorescent and immunoelectron microscopy. CENP-B localized to the heterochromatic parts of the nuclei at meiotic stages. A clearly distinct chromocenter forms in the nucleus at stages 3-4 of spermatid maturation; CENP-B localizes in it and in the area adjacent to the future acrosome. CENP-B localization in the subacrosomal area and in the chromocenters' periphery demonstrates that centromeres are organized in two groups in mouse spermatozoa, unlike human centromeres. TRF2/MTBP concentrates around the forming chromocenter at spermiogenesis early stages. The TRF2/MTBP main signal migrates into the area of acrosomal membrane at the course of spermatozoon maturation. TRF2/MTBP never localizes inside the synaptonemal complex but can be found in the areas where the synaptonemal complex attaches to the nuclear envelope. At the pachytene and diplotene stages when chromosomes separate from the nuclear envelope, some amount of the protein remains bound to the nuclear membrane while the other part reveals itself in chromosomes. TRF2/MTBP accumulates in the future acrosome from the very beginning of its formation. In the mature spermatozoon TRF2/MTBP decorates the acrosomal membrane as well as spreads in condensed chromatin.     

The telobox, a Myb-related telomeric DNA binding motif found in proteins from yeast, plants and human.

The yeast TTAGGG binding factor 1 (Tbf1) was identified and cloned through its ability to interact with vertebrate telomeric repeats in vitro. We show here that a sequence of 60 amino acids located in its C-terminus is critical for DNA binding. This sequence exhibits homologies with Myb repeats and is conserved among five proteins from plants, two of which are known to bind telomeric-related sequences, and two proteins from human, including the telomeric repeat binding factor (TRF) and the predicted C-terminal polypeptide, called orf2, from a yet unknown protein. We demonstrate that the 111 C-terminal residues of TRF and the 64 orf2 residues are able to bind the human telomeric repeats specifically. We propose to call the particular Myb-related motif found in these proteins the 'telobox'. Antibodies directed against the Tbf1 telobox detect two proteins in nuclear and mitotic chromosome extracts from human cell lines. Moreover, both proteins bind specifically to telomeric repeats in vitro. TRF is likely to correspond to one of them. Based on their high affinity for the telomeric repeat, we predict that TRF and orf2 play an important role at human telomeres.     

Rap1-independent telomere attachment and bouquet formation in mammalian meiosis

Attachment of telomeres to the nuclear envelope (NE) and their clustering in a chromosomal bouquet during meiotic prophase I is an evolutionary conserved event that promotes chromosome pairing and recombination. In fission yeast, bouquet formation fails when the telomeric protein Rap1 is absent or when the telomeric protein Taz1 fails to recruit Rap1 to telomeres. The mammalian Rap1 orthologue is a component of the shelterin complex and localises to telomeres through an interaction with a Taz1-like telomeric DNA binding factor, TRF2. Here, we investigated the role of mammalian Rap1 in meiotic telomere attachment and clustering by analysing spermatogenesis in Rap1-deficient mice. The results establish that the meiotic three-dimensional nuclear architecture and recombination are not affected by the absence of Rap1. Furthermore, Rap1-deficient meiotic telomeres assemble the SUN1 nuclear membrane protein, attach to the NE, and undergo bouquet formation indistinguishable from the wild-type setting. Thus, the role of Rap1 in meiosis is not conserved between fission yeast and mammals, suggesting that mammals have alternative modes for connecting telomeres to SUN proteins on the meiotic nuclear envelope.     

Sequence-specific binding of Taz1p dimers to fission yeast telomeric DNA

The fission yeast (Schizosaccharomyces pombe) taz1 gene encodes a telomere-associated protein. It contains a single copy of a Myb-like motif termed the telobox that is also found in the human telomere binding proteins TRF1 and TRF2, and Tbf1p, a protein that binds to sequences found within the sub-telomeric regions of budding yeast (Saccharomyces cerevisiae) chromosomes. Taz1p was synthesised in vitro and shown to bind to a fission yeast telomeric DNA fragment in a sequence specific manner that required the telobox motif. Like the mammalian TRF proteins, Taz1p bound to DNA as a preformed homodimer. The isolated Myb-like domain was also capable of sequence specific DNA binding, although with less specificity than the full-length dimer. Surprisingly, a protein extract produced from a taz1–fission yeast strain still contained the major telomere binding activity (complex I) we have characterised previously, suggesting that there could be other abundant telomere binding proteins in fission yeast. One candidate, SpX, was also synthesised in vitro, but despite the presence of two telobox domains, no sequence specific binding to telomeric DNA was detected.     

Mammalian telomeres end in a large duplex loop.

Mammalian telomeres contain a duplex array of telomeric repeats bound to the telomeric repeat-binding factors TRF1 and TRF2. Inhibition of TRF2 results in immediate deprotection of chromosome ends, manifested by loss of the telomeric 3' overhang, activation of p53, and end-to-end chromosome fusions. Electron microscopy reported here demonstrated that TRF2 can remodel linear telomeric DNA into large duplex loops (t loops) in vitro. Electron microscopy analysis of psoralen cross-linked telomeric DNA purified from human and mouse cells revealed abundant large t loops with a size distribution consistent with their telomeric origin. Binding of TRF1 and single strand binding protein suggested that t loops are formed by invasion of the 3' telomeric overhang into the duplex telomeric repeat array. T loops may provide a general mechanism for the protection and replication of telomeres.     

Identification of human Rap1: implications for telomere evolution

It has been puzzling that mammalian telomeric proteins, including TRF1, TRF2, tankyrase, and TIN2 have no recognized orthologs in budding yeast. Here, we describe a human protein, hRap1, that is an ortholog of the yeast telomeric protein, scRap1p. hRap1 has three conserved sequence motifs in common with scRap1, is located at telomeres, and affects telomere length. However, while scRap1 binds telomeric DNA directly, hRap1 is recruited to telomeres by TRF2. Extending the comparison of telomeric proteins to fission yeast, we identify S. pombe Taz1 as a TRF ortholog, indicating that TRFs are conserved at eukaryotic telomeres. The data suggest that ancestral telomeres, like those of vertebrates, contained a TRF-like protein as well as Rap1. We propose that budding yeast preserved Rap1 at telomeres but lost the TRF component, possibly concomitant with a change in the telomeric repeat sequence.     

Homologous recombination generates T-loop-sized deletions at human telomeres

The t-loop structure of mammalian telomeres is thought to repress nonhomologous end joining (NHEJ) at natural chromosome ends. Telomere NHEJ occurs upon loss of TRF2, a telomeric protein implicated in t-loop formation. Here we describe a mutant allele of TRF2, TRF2DeltaB, that suppressed NHEJ but induced catastrophic deletions of telomeric DNA. The deletion events were stochastic and occurred rapidly, generating dramatically shortened telomeres that were accompanied by a DNA damage response and induction of senescence. TRF2DeltaB-induced deletions depended on XRCC3, a protein implicated in Holliday junction resolution, and created t-loop-sized telomeric circles. These telomeric circles were also detected in unperturbed cells and suggested that t-loop deletion by homologous recombination (HR) might contribute to telomere attrition. Human ALT cells had abundant telomeric circles, pointing to frequent t-loop HR events that could promote rolling circle replication of telomeres in the absence of telomerase. These findings show that t-loop deletion by HR influences the integrity and dynamics of mammalian telomeres.     

POT1 and TRF2 cooperate to maintain telomeric integrity

Mammalian telomeric DNA contains duplex TTAGGG repeats and single-stranded overhangs. POT1 (protection of telomeres 1) is a telomere-specific single-stranded DNA-binding protein, highly conserved in eukaryotes. The biological function of human POT1 is not well understood. In the present study, we demonstrate that POT1 plays a key role in telomeric end protection. The reduction of POT1 by RNA interference led to the loss of telomeric single-stranded overhangs and induced apoptosis, chromosomal instability, and senescence in cells. POT1 and TRF2 interacted with each other to form a complex with telomeric DNA. A dominant negative TRF2, TRF2(DeltaBDeltaM), bound to POT1 and prevented it from binding to telomeres. POT1 overexpression protected against TRF2(DeltaBDeltaM)-induced loss of telomeric single-stranded overhangs, chromosomal instability, and senescence. These results demonstrate that POT1 and TRF2 share in part in the same pathway for telomere capping and suggest that POT1 binds to the telomeric single-stranded DNA in the D-loop and cooperates with TRF2 in t-loop maintenance.     

Distinct functions of POT1 at telomeres

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.     

ERCC1/XPF removes the 3' overhang from uncapped telomeres and represses formation of telomeric DNA-containing double minute chromosomes

Human telomeres are protected by TRF2. Inhibition of this telomeric protein results in partial loss of the telomeric 3' overhang and chromosome end fusions formed through nonhomologous end-joining (NHEJ). Here we report that ERCC1/XPF-deficient cells retained the telomeric overhang after TRF2 inhibition, identifying this nucleotide excision repair endonuclease as the culprit in overhang removal. Furthermore, these cells did not accumulate telomere fusions, suggesting that overhang processing is a prerequisite for NHEJ of telomeres. ERCC1/XPF was also identified as a component of the telomeric TRF2 complex. ERCC1/XPF-deficient mouse cells had a novel telomere phenotype, characterized by Telomeric DNA-containing Double Minute chromosomes (TDMs). We speculate that TDMs are formed through the recombination of telomeres with interstitial telomere-related sequences and that ERCC1/XPF functions to repress this process. Collectively, these data reveal an unanticipated involvement of the ERCC1/XPF NER endonuclease in the regulation of telomere integrity and establish that TRF2 prevents NHEJ at telomeres through protection of the telomeric overhang from ERCC1/XPF.     

PTOP interacts with POT1 and regulates its localization to telomeres

Telomere maintenance has been implicated in cancer and ageing, and requires cooperation between a multitude of telomeric factors, including telomerase, TRF1, TRF2, RAP1, TIN2, Tankyrase, PINX1 and POT1 (refs 1-12). POT1 belongs to a family of oligonucleotide-binding (OB)-fold-containing proteins that include Oxytricha nova TEBP, Cdc13, and spPot1, which specifically recognize telomeric single-stranded DNA (ssDNA). In human cells, the loading of POT1 to telomeric ssDNA controls telomerase-mediated telomere elongation. Surprisingly, a human POT1 mutant lacking an OB fold is still recruited to telomeres. However, the exact mechanism by which this recruitment occurs remains unclear. Here we identify a novel telomere protein, PTOP, which interacts with both POT1 and TIN2. PTOP binds to the carboxyl terminus of POT1 and recruits it to telomeres. Inhibition of PTOP by RNA interference (RNAi) or disruption of the PTOP-POT1 interaction hindered the localization of POT1 to telomeres. Furthermore, expression of the respective interaction domains on PTOP and POT1 alone extended telomere length in human cells. Therefore, PTOP heterodimerizes with POT1 and regulates POT1 telomeric recruitment and telomere length.     

Localization of proteins, specifically binding highly-repetitive sequences of DNA in human spermatozoids

Immunoblot revealed in spermatozoa alpha-satellite (sat) DNA-specific centromere protein B (CENP-B) and p70 (Enukashvily et al., 2000), a membrane telomere binding protein (MTBP/TRF2) (Podgornaya et al., 2000), and Alu-binding protein p68 (Lukyanov et al., 2000). The localization of some of these proteins in spermatozoa was defined using indirect immunofluorescence. Spermatozoa were fixed in methanol/acetic acid 3:1, or prior to fixation were treated with 5 mM heparin and 10 mM DTT. The heparin/DTT treatment causes the nuclear membrane destruction and a partial chromatin decondensation. In non-treated spermatozoa fluorescent signals from all ABs are registered near the membrane, with MTBP/TRF2 being localized closer to the acrosome than sat-DNA-specific proteins. In the treated spermatozoa MTBP/TRF2 was partially lost, whereas part of CENP-B and sat-p70 remained in contact with membrane. Another part of sat-binding proteins reveals a dot-like staining pattern, with dots confined to the DAPI-stained chromatin area, inside a nuclei. This is in partial agreement with the pattern of telomere and CEN position revealed by FISH. Commonly MTBP has a near membrane localization, being lost when the nuclear membrane is destroyed. Centromere-binding proteins are arranged in the order from the nuclear membrane towards the nuclear center, with CENP-B being situated more peripherally but not in the middle of the nucleus. This discrepancy may be explained by the fact, that some proteins are not associated with the appropriate sequences in a spermatozoon. Possibly, such a distribution of proteins may reflect their role in unpacking the paternal genetic material in a zygote.     

MRE11-RAD50-NBS1 and ATM function as co-mediators of TRF1 in telomere length control

Human telomeres are associated with ATM and the protein complex consisting of MRE11, RAD50 and NBS1 (MRN), which are central to maintaining genomic stability. Here we show that when targeted to telomeres, wild-type RAD50 downregulates telomeric association of TRF1, a negative regulator of telomere maintenance. TRF1 binding to telomeres is upregulated in cells deficient in NBS1 or under ATM inhibition. The TRF1 association with telomeres induced by ATM inhibition is abrogated in cells lacking MRE11 or NBS1, suggesting that MRN and ATM function in the same pathway controlling TRF1 binding to telomeres. The ability of TRF1 to interact with telomeric DNA in vitro is impaired by ATM-mediated phosphorylation. We propose that MRN is required for TRF1 phosphorylation by ATM and that such phosphorylation results in the release of TRF1 from telomeres, promoting telomerase access to the ends of telomeres.     

How the human telomeric proteins TRF1 and TRF2 recognize telomeric DNA: a view from high-resolution crystal structures

Human telomeres consist of tandem arrays of TTAGGG sequence repeats that are specifically bound by two proteins, TRF1 and TRF2. They bind to DNA as preformed homodimers and have the same architecture in which the DNA-binding domains (Dbds) form independent structural units. Despite these similarities, TRF1 and TRF2 have different functions at telomeres. The X-ray crystal structures of both TRF1- and TRF2-Dbds in complex with telomeric DNA (2.0 and 1.8 angstroms resolution, respectively) show that they recognize the same TAGGGTT binding site by means of homeodomains, as does the yeast telomeric protein Rap1p. Two of the three G-C base pairs that characterize telomeric repeats are recognized specifically and an unusually large number of water molecules mediate protein-DNA interactions. The binding of the TRF2-Dbd to the DNA double helix shows no distortions that would account for the promotion of t-loops in which TRF2 has been implicated.     

TRF2-Tethered TIN2 Can Mediate Telomere Protection by TPP1/POT1

The shelterin protein TIN2 is required for the telomeric accumulation of TPP1/POT1 heterodimers and for the protection of telomeres by the POT1 proteins (POT1a and POT1b in the mouse). TIN2 also binds to TRF1 and TRF2, improving the telomeric localization of TRF2 and its function. Here, we ask whether TIN2 needs to interact with both TRF1 and TRF2 to mediate the telomere protection afforded by TRF2 and POT1a/b. Using a TIN2 allele deficient in TRF1 binding (TIN2-L247E), we demonstrate that TRF1 is required for optimal recruitment of TIN2 to telomeres and document phenotypes associated with the TIN2-L247E allele that are explained by insufficient TIN2 loading onto telomeres. To bypass the requirement for TRF1-dependent recruitment, we fused TIN2-L247E to the TRF2-interacting (RCT) domain of Rap1. The RCT-TIN2-L247E fusion showed improved telomeric localization and was fully functional in terms of chromosome end protection by TRF2, TPP1/POT1a, and TPP1/POT1b. These data indicate that when sufficient TIN2 is loaded onto telomeres, its interaction with TRF1 is not required to mediate the function of TRF2 and the TPP1/POT1 heterodimers. We therefore conclude that shelterin can protect chromosome ends as a TRF2-tethered TIN2/TPP1/POT1 complex that lacks a physical connection to TRF1.     

Dynamics of protein binding to telomeres in living cells: implications for telomere structure and function

Telomeric proteins have an essential role in the regulation of the length of the telomeric DNA tract and in protection against end-to-end chromosome fusion. Telomere organization and how individual proteins are involved in different telomere functions in living cells is largely unknown. By using green fluorescent protein tagging and photobleaching, we investigated in vivo interactions of human telomeric DNA-binding proteins with telomeric DNA. Our results show that telomeric proteins interact with telomeres in a complex dynamic fashion: TRF2, which has a dual role in chromosome end protection and telomere length homeostasis, resides at telomeres in two distinct pools. One fraction ( approximately 73%) has binding dynamics similar to TRF1 (residence time of approximately 44 s). Interestingly, the other fraction of TRF2 binds with similar dynamics as the putative end-protecting factor hPOT1 (residence time of approximately 11 min). Our data support a dynamic model of telomeres in which chromosome end-protection and telomere length homeostasis are governed by differential binding of telomeric proteins to telomeric DNA.