TRF1 negotiates TTAGGG repeat-associated replication problems by recruiting the BLM helicase and the TPP1/POT1 repressor of ATR signaling

The semiconservative replication of telomeres is facilitated by the shelterin component TRF1. Without TRF1, replication forks stall in the telomeric repeats, leading to ATR kinase signaling upon S-phase progression, fragile metaphase telomeres that resemble the common fragile sites (CFSs), and the association of sister telomeres. In contrast, TRF1 does not contribute significantly to the end protection functions of shelterin. We addressed the mechanism of TRF1 action using mouse conditional knockouts of BLM, TRF1, TPP1, and Rap1 in combination with expression of TRF1 and TIN2 mutants. The data establish that TRF1 binds BLM to facilitate lagging but not leading strand telomeric DNA synthesis. As the template for lagging strand telomeric DNA synthesis is the TTAGGG repeat strand, TRF1-bound BLM is likely required to remove secondary structures formed by these sequences. In addition, the data establish that TRF1 deploys TIN2 and the TPP1/POT1 heterodimers in shelterin to prevent ATR during telomere replication and repress the accompanying sister telomere associations. Thus, TRF1 uses two distinct mechanisms to promote replication of telomeric DNA and circumvent the consequences of replication stress. These data are relevant to the expression of CFSs and provide insights into TIN2, which is compromised in dyskeratosis congenita (DC) and related disorders.     

A human interstitial telomere associates in vivo with specific TRF2 and TIN2 proteins

Mammalian telomeres are composed of long arrays of TTAGGG repeats that form a nucleoprotein complex which protects the chromosome ends. Human telomere function is known to require two TTAGGG repeat factors, TRF1 and TRF2, and several interacting proteins, but the mechanism by which the DNA/protein complex prevents end to end fusion in vivo has not been elucidated. In order to better understand the role of specific telomere-associated proteins in the organisation of chromosome ends, we have studied a patient with a rare chromosome rearrangement that has given rise to an interstitial telomere. Using specific antibodies and immuno-FISH on unfixed metaphase chromosomes, we show that the proteins TRF2 and TIN2 (TIN2 interacts with TRF1) co-localise with the interstitial TTAGGG repeats. Our results demonstrate, for the first time in humans, that TRF2 and TIN2 proteins associate with interstitial duplex TTAGGG repeats, in vivo. They confirm that double stranded-telomeric repeats, even when complexed with specific proteins, are not sufficient to create a functional telomere. Finally, they suggest a possible role for proteins in stabilising interstitial TTAGGG repeats.     

Post-translational modifications of TRF1 and TRF2 and their roles in telomere maintenance

Telomeres, heterochromatic structures, found at the ends of linear eukaryotic chromosomes, function to protect natural chromosome ends from nucleolytic attack. Human telomeric DNA is bound by a telomere-specific six-subunit protein complex, termed shelterin/telosome. The shelterin subunits TRF1 and TRF2 bind in a sequence-specific manner to double-stranded telomeric DNA, providing a vital platform for recruitment of additional shelterin proteins as well as non-shelterin factors crucial for the maintenance of telomere length and structure. Both TRF1 and TRF2 are engaged in multiple roles at telomeres including telomere protection, telomere replication, sister telomere resolution and telomere length maintenance. Regulation of TRF1 and TRF2 in these various processes is controlled by post-translational modifications, at times in a cell-cycle-dependent manner, affecting key functions such as DNA binding, dimerization, localization, degradation and interactions with other proteins. Here we review the post-translational modifications of TRF1 and TRF2 and discuss the mechanisms by which these modifications contribute to the function of these two proteins.     

A role for heterochromatin protein 1γ at human telomeres

Human telomere function is mediated by shelterin, a six-subunit complex that is required for telomere replication, protection, and cohesion. TIN2, the central component of shelterin, has binding sites to three subunits: TRF1, TRF2, and TPP1. Here we identify a fourth partner, heterochromatin protein 1γ (HP1γ), that binds to a conserved canonical HP1-binding motif, PXVXL, in the C-terminal domain of TIN2. We show that HP1γ localizes to telomeres in S phase, where it is required to establish/maintain cohesion. We further demonstrate that the HP1-binding site in TIN2 is required for sister telomere cohesion and can impact telomere length maintenance by telomerase. Remarkably, the PTVML HP1-binding site is embedded in the recently identified cluster of mutations in TIN2 that gives rise to dyskeratosis congenita (DC), an inherited bone marrow failure syndrome caused by defects in telomere maintenance. We show that DC-associated mutations in TIN2 abrogate binding to HP1γ and that DC patient cells are defective in sister telomere cohesion. Our data indicate a novel requirement for HP1γ in the establishment/maintenance of cohesion at human telomeres and, furthermore, may provide insight into the mechanism of pathogenesis in TIN2-mediated DC.     

端粒及端粒酶活性的调控机制

广义的端粒由帽子、双链的串联重复序列的DNA核心部分及亚端粒构成,其结合蛋白是一个复合体,由TRF1、TRF2、TIN2、Pot1、TPP1、RAP1 6个亚单位组成;另外,还结合组蛋白的特定成分H3K9三甲基聚合体和H4K20三甲基聚合体.端粒酶主要由hTerc、hTert、dyskerin构成.端粒的功能主要受端粒酶的活性调控;而端粒酶活性主要受hTert及hTerc的转录水平和转录后的剪切、hTert的翻译等因素的调控.端粒与端粒酶结构和功能的异常与细胞衰老及肿瘤的发生、发展关系密切.     

DNA damage responses in neural cells: Focus on the telomere

Postmitotic neurons must survive for the entire life of the organism and be able to respond adaptively to adverse conditions of oxidative and genotoxic stress. Unrepaired DNA damage can trigger apoptosis of neurons which is typically mediated by the ataxia telangiectasia mutated (ATM)-p53 pathway. As in all mammalian cells, telomeres in neurons consist of TTAGGG DNA repeats and several associated proteins that form a nucleoprotein complex that prevents chromosome ends from being recognized as double strand breaks. Proteins that stabilize telomeres include TRF1 and TRF2, and proteins known to play important roles in DNA damage responses and DNA repair including ATM, Werner and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). We have been performing studies of developing and adult neurons aimed at understanding the effects of global and telomere-directed DNA damage responses in neuronal plasticity and survival in the contexts of aging and neurodegenerative disorders. Deficits in specific DNA repair proteins, including DNA-PKcs and uracil DNA glycosylase (UDG), render neurons vulnerable to adverse conditions of relevance to the pathogenesis of neurodegenerative disorders such as Alzheimer's disease and stroke. Similarly, early postmitotic neurons with reduced telomerase activity exhibit accentuated responses to DNA damage and are prone to apoptosis demonstrating a pivotal role for telomere maintenance in both mitotic cells and postmitotic neurons. Our recent findings suggest key roles for TRF2 in regulating the differentiation and survival of neurons. TRF2 affects cell survival and differentiation by modulating DNA damage pathways, and gene expression. A better understanding of the molecular mechanisms by which neurons respond to global and telomere-specific DNA damage may reveal novel strategies for prevention and treatment of neurodegenerative disorders. Indeed, work in this and other laboratories has shown that dietary folic acid can protect neurons against Alzheimer's disease by keeping homocysteine levels low and thereby minimizing the misincorporation of uracil into DNA in neurons.     

Inside the mammalian telomere interactome: regulation and regulatory activities of telomeres

Work in model organisms, such as mouse, yeast, Tetrahymena, ciliates, and plants, has led to a deeper understanding of telomere biology. Telomeres together with telomere-binding proteins have evolved to protect chromosomal ends and maintain chromosomal length and integrity. Over the last two decades, biochemical, molecular, cellular, and genetic studies have greatly enhanced our knowledge of the unique function and structure of telomeres and telomere-associated factors. In this review, we focus on the important advances, in terms of our knowledge and the methods used, in understanding mammalian telomere regulation by telomeric proteins. Recently, the 6 telomeric proteins (TRF1, TRF2, POT1, TIN2, RAP1, and TPP1) were found to form a high-order complex. This complex and its associated partners provide the basis for constructing an interaction map of telomere regulators in mammalian cells, which we named the Telomere Interactome. The Telomere Interactome incorporates the various telomere signaling pathways and represents the molecular machinery that regulates mammalian telomeres. The establishment of the Telomere Interactome will also enable the integration of the intricate circuitries that regulate telomeres with other cellular interactomes in vertebrates.     

Cell cycle control of telomere protection and NHEJ revealed by a ts mutation in the DNA-binding domain of TRF2

TRF2 is a component of shelterin, the telomere-specific protein complex that prevents DNA damage signaling and inappropriate repair at the natural ends of mammalian chromosomes. We describe a temperature-sensitive (ts) mutation in the Myb/SANT DNA-binding domain of TRF2 that allows controlled and reversible telomere deprotection. At 32 degrees C, TRF2ts was functional and rescued the lethality of TRF2 deletion from conditional TRF2(F/-) mouse embryonic fibroblasts (MEFs). When shifted to the nonpermissive temperature (37 degrees C), TRF2ts cells showed extensive telomere damage resulting in activation of the ATM kinase and nonhomologous end-joining (NHEJ) of chromosome ends. The inactivation of TRF2ts at 37 degrees C was rapid and reversible, permitting induction of short periods (3-6 h) of telomere dysfunction in the G0, G1, and S/G2 phases of the cell cycle. The results indicate that both the induction of telomere dysfunction and the re-establishment of the protected state can take place throughout interphase. In contrast, the processing of dysfunctional telomeres by NHEJ occurred primarily in G1, being repressed in S/G2 in a cyclin-dependent kinase (CDK)-dependent manner.     

Dynamic relocation of telomere complexes in mouse meiotic chromosomes

Telomeric DNA repeats as well as different specific proteins such as TRF1 and Rap1 associate in functional telomere complexes found at chromosome ends. Using spreading techniques, the presence of TRF1 and Rap1 has been reported at mammalian meiotic telomeres during prophase I. In the present study, we have analysed, by fluorescence in-situ hybridization and immunofluorescence, the appearance and location of telomere complexes during both male mouse meiotic divisions. Additionally, we have studied their relationship with different centromere/kinetochore proteins and the synaptonemal complex protein SCP3. Our results show that telomere complexes are not located at condensed meiotic chromosome tips. Therefore, a change in chromosome structure may occur from pachytene up to metaphase I involving the dynamic relocation of telomere complexes in condensed chromosomes. Moreover, we have found that proximal telomere complexes are relocated internally to kinetochores from metaphase I up to anaphase II. We discuss the functional significance of the location of telomere complexes into internal domains of condensed meiotic chromosomes.     

Telomere shortening and cell cycle arrest in Trypanosoma brucei expressing human telomeric repeat factor TRF1

Trypanosoma brucei has telomeres composed of 15 kb tracts of TTAGGG repeats that end in 3' overhangs and form t-loops. This structure is also present in mammalian cells and is thought to reflect the presence of telomere-binding proteins. The human TTAGGG repeat-binding factor TRF1 binds to telomeres and regulates their length. We attempted to interfere with the normal function of trypanosome telomeres by expressing human TRF1 in T. brucei. TRF1 localized to telomeres in cultured procyclic (midgut-stage) trypanosomes with great fidelity, but not in bloodstream-stage trypanosomes. Procyclic trypanosomes expressing high levels of TRF1 for extended periods of time exhibited shortening and increased size heterogeneity of their telomeres and the cell cycle was arrested in G1-S. These effects were not detected in cells expressing a TRF1 mutant incapable of binding to TTAGGG repeats. We argue that TRF1 displaces putative endogenous trypanosome telomere-binding proteins, not yet identified, and affects telomeres in ways that reflect its role as a negative regulator of telomere length in human cells.     

The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione S-transferase pull-down assay indicated that the C-terminal region of POT1a/b binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2 dose-dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.     

Functional interaction between telomere protein TPP1 and telomerase

Human chromosome end-capping and telomerase regulation require POT1 (Protection of Telomeres 1) and TPP1 proteins, which bind to the 3′ ssDNA extension of human telomeres. POT1–TPP1 binding to telomeric DNA activates telomerase repeat addition processivity. We now provide evidence that this POT1–TPP1 activation requires specific interactions with telomerase, rather than it being a DNA substrate-specific effect. First, telomerase from the fish medaka, which extends the same telomeric DNA primer as human telomerase, was not activated by human POT1–TPP1. Second, mutation of a conserved glycine, Gly100 in the TEN (telomerase essential N-terminal) domain of TERT, abolished the enhancement of telomerase processivity by POT1–TPP1, in contrast to other single amino acid mutations. Chimeric human–fish telomerases that contained the human TEN domain were active but not stimulated by POT1–TPP1, showing that additional determinants of processivity lie outside the TEN domain. Finally, primers bound to mouse POT1A and human TPP1 were activated for extension by human telomerase, whereas mPOT1A–mTPP1 was most active with mouse telomerase, indicating that these mammalian telomerases have specificity for their respective TPP1 proteins. We suggest that a sequence-specific interaction between TPP1 in the TPP1–POT1–telomeric DNA complex and the G100 region of the TEN domain of TERT is necessary for high-processivity telomerase action.     

Essential roles of Xenopus TRF2 in telomere end protection and replication

TRF1 and TRF2 are double-stranded (ds) telomere DNA-binding proteins and the core members of shelterin, a complex that provides the structural and functional basis of telomere functions. We have reported that unlike mammalian TRF1 that constitutively binds to chromatin, Xenopus TRF1 (xTRF1) associates with mitotic chromatin but dissociates from interphase chromatin reconstituted in Xenopus egg extracts. This finding raised the possibility that xTRF1 and Xenopus TRF2 (xTRF2) contribute to telomere functions in a manner different from mammalian TRF1 and TRF2. Here, we focused on the role of xTRF2. We prepared chromatin reconstituted in egg extracts immunodepleted for xTRF2. Compared to mock-depleted nuclei, DNA damage response at telomeres was activated, and bulk DNAs were poorly replicated in xTRF2-depleted nuclei. The replication defect was rescued by inactivating ATR through the addition of anti-ATR neutralizing antibody, suggesting that ATR plays a role in the defect. Interestingly, the bulk DNA replication defect, but not the DNA damage response at telomeres, was rescued by supplementing the xTRF2-depleted extracts with recombinant xTRF2 (rTRF2). We propose that xTRF2 is required for both efficient replication of bulk DNA and protection from the activation of the DNA damage checkpoints pathway, and that those two functions are mechanistically separable.     

Telomere‐associated proteins: cross‐talk between telomere maintenance and telomere‐lengthening mechanisms

Telomeres, the ends of eukaryotic chromosomes, have been the subject of intense investigation over the last decade. As telomere dysfunction has been associated with ageing and developing cancer, understanding the exact mechanisms regulating telomere structure and function is essential for the prevention and treatment of human cancers and age‐related diseases. The mechanisms by which cells maintain telomere lengthening involve either telomerase or the alternative lengthening of the telomere pathway, although specific mechanisms of the latter and the relationship between the two are as yet unknown. Many cellular factors directly (TRF1/TRF2) and indirectly (shelterin‐complex, PinX, Apollo and tankyrase) interact with telomeres, and their interplay influences telomere structure and function. One challenge comes from the observation that many DNA damage response proteins are stably associated with telomeres and contribute to several other aspects of telomere function. This review focuses on the different components involved in telomere maintenance and their role in telomere length homeostasis. Special attention is paid to understanding how these telomere‐associated factors, and mainly those involved in double‐strand break repair, perform their activities at the telomere ends. Copyright © 2008 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

POT1-interacting protein PIP1: a telomere length regulator that recruits POT1 to the TIN2/TRF1 complex

Human telomere length is controlled by a negative feedback loop based on the binding of TRF1 to double-stranded telomeric DNA. The TRF1 complex recruits POT1, a single-stranded telomeric DNA-binding protein necessary for cis-inhibition of telomerase. By mass spectrometry, we have identified a new telomeric protein, which we have named POT1-interacting protein 1 (PIP1). PIP1 bound both POT1 and the TRF1-interacting factor TIN2 and could tether POT1 to the TRF1 complex. Reduction of PIP1 or POT1 levels with shRNAs led to telomere elongation, indicating that PIP1 contributes to telomere length control through recruitment of POT1.     

Tpz1 controls a telomerase-nonextendible telomeric state and coordinates switching to an extendible state via Ccq1

Telomeres are nucleoprotein complexes comprising telomeric DNA repeats bound by the multiprotein shelterin complex. A dynamic binary switch between telomerase-extendible and telomerase-nonextendible telomeric states determines telomere length homeostasis. However, the molecular nature of the nonextendible state is largely unknown. Here, we show that, in fission yeast, Tpz1 (the ortholog of human TPP1)-mediated complete linkage within the shelterin complex, bridging telomeric dsDNA to ssDNA, controls the telomerase-nonextendible state. Disruption of this linkage leads to unregulated telomere elongation while still retaining the shelterin components on telomeres. Therefore, the linkage within the shelterin components, rather than the individual shelterin components per se, defines the telomerase-nonextendible state. Furthermore, epistasis analyses reveal that Tpz1 also participates in the activation of telomeres to the extendible state via its interaction with Ccq1. Our results suggest critical regulatory roles of Tpz1 in the telomere binary switch.     

Breaks at telomeres and TRF2-independent end fusions in Fanconi anemia

Fanconi anemia (FA) is a rare genetic disease characterized by chromosome instability, progressive pancytopenia and cancer susceptibility. Telomeres are intimately related to chromosome stability and play an important role in organismal viability at the hematological level. Since previous works suggested an accelerated shortening of telomeres in FA, we have studied several markers of telomere integrity and function in FA patients and age-matched controls to get insights into the mechanisms and consequences of telomere erosion in FA. A higher frequency of extra-chromosomic TTAGGG signals and of chromosome ends with undetectable TTAGGG repeats was observed in FA cells by fluorescence in situ hybridization (FISH), suggesting intensive breakage at telomeric sequences. This was proven by measuring the frequency of excess of telomeric signals per cell, which was 2.8-fold higher in FA. Consistent with previous reports, quantitative FISH analysis showed an accelerated telomere shortening of 0.68 kb in FA, which occurred concurrently in both chromosome arms in a similar magnitude. Our data therefore suggest that the telomere erosion in FA is caused by a higher rate of breakage at TTAGGG sequences in vivo in differentiated cells, in addition to mere replicative shortening during lymphocyte proliferation. Consistent with impaired telomeres in FA patients, we observed a >10-fold increase in chromosome end fusions in FA compared to normal controls. This observation was independent of TRF2, a telomere binding factor that protects human telomeres from end fusions, since immunohistochemistry studies in FA cell lines and corrected counterparts by retrovirus-mediated transfer of FANCA and FANCD2 cDNA showed that a functional FA pathway is not required for telomere binding of TRF2.     

The proper connection between shelterin components is required for telomeric heterochromatin assembly

Telomeric regions contain prominent sites of heterochromatin, which is associated with unique histone modification profiles such as the methylation of histone H3 at Lys9 (H3K9me). In fission yeast, the conserved telomeric shelterin complex recruits the histone H3K9 methyltransferase complex CLRC to establish subtelomeric heterochromatin. Although many shelterin mutations affect subtelomeric heterochromatin assembly, the mechanism remains elusive due to the diverse functions of shelterin. Through affinity purification, we found that shelterin directly associates with CLRC through the Ccq1 subunit. Surprisingly, mutations that disrupt interactions between shelterin subunits compromise subtelomeric heterochromatin without affecting CLRC interaction with shelterin component Pot1, located at chromosome ends. We further discovered that telomeric repeats are refractory to heterochromatin spreading and that artificial restoration of shelterin connections or increased heterochromatin spreading rescued heterochromatin defects in these shelterin mutants. Thus, subtelomeric heterochromatin assembly requires both the recruitment of CLRC by shelterin to chromosome ends and the proper connection of shelterin components, which allows CLRC to skip telomeric repeats to internal regions.