人端粒结合蛋白TRF1的克隆、表达和抗体制备

利用RT-PCR技术,从HeLa细胞的cDNA文库中扩增到人端粒结合蛋白1(hTRF1)基因编码区序列,克隆至pUCm-T载体,测序正确后,构建带His₆-tag原核表达载体pET-28c-TRF1,经IPTG诱导表达的His₆-TRF1融合蛋白分子量约为65kD,Western-blot证实表达产物可特异地与TRF1抗体sc-6165结合。用Ni^2＋NTA胶亲和层析纯化可得到电泳均一的融合蛋白,免疫新西兰纯种大白兔,获得特异性好的多克隆抗体,该抗体可用于免疫荧光染色和Western-blot方法检测哺乳动物细胞内源性的TRF1分子。     

The human telomeric protein TRF1 specifically recognizes nucleosomal binding sites and alters nucleosome structure

Telomeres are dynamic nucleoprotein structures that cap the ends of eukaryotic chromosomes. In humans, the long (TTAGGG)(n) double-stranded telomeric DNA repeats are bound specifically by the two related proteins TRF1 and TRF2, and are organized in nucleosomes. Whereas the role of TRF1 and TRF2 in telomeric function has been studied extensively, little is known about the involvement of telomeric nucleosomes in telomere structures or how chromatin formation may affect binding of the TRFs. Here, we address the question of whether TRF1 is able to bind to telomeric binding sites in a nucleosomal context. We show that TRF1 is able to specifically recognize telomeric binding sites located within nucleosomes, forming a ternary complex. The formation of this complex is strongly dependent on the orientation of binding sites on the nucleosome surface, rather than on the location of the binding sites with respect to the nucleosome dyad. Strikingly, TRF1 binding causes alterations in nucleosome structure without dissociation of histone subunits. These results indicate that nucleosomes contribute to the establishment of a telomeric capping complex, whose structure and dynamics can be modulated by the binding of telomeric factors.     

Zinc binding catalytic domain of human tankyrase 1

Tankyrases are recently discovered proteins implicated in many important functions in the cell including telomere homeostasis and mitosis. Tankyrase modulates the activity of target proteins through poly(ADP-ribosyl)ation, and here we report the structure of the catalytic poly(ADP-ribose) polymerase (PARP) domain of human tankyrase 1. This is the first structure of a PARP domain from the tankyrase subfamily. The present structure reveals that tankyrases contain a short zinc-binding motif, which has not been predicted. Tankyrase activity contributes to telomere elongation observed in various cancer cells and tankyrase inhibition has been suggested as a potential route for cancer therapy. In comparison with other PARPs, significant structural differences are observed in the regions lining the substrate-binding site of tankyrase 1. These findings will be of great value to facilitate structure-based design of selective PARP inhibitors, in general, and tankyrase inhibitors, in particular.     

Lack of age-associated telomere shortening in long- and short-lived species of sea urchins

The red sea urchin, Strongylocentrotus franciscanus, can live in excess of 100 years while the sea urchin Lytechinus variegatus has an estimated lifespan of only 3-4 years. In an effort to understand the molecular mechanism underlying the difference in their longevity we characterized telomere biology in these species of sea urchins. Telomerase activity was found throughout early stages of development in L. variegatus and is maintained in adult tissues of L. variegatus and S. franciscanus. Terminal restriction fragment analysis indicated a lack of age-associated telomere shortening. These data suggest that long- and short-lived sea urchins do not utilize telomerase repression as a mechanism to suppress neoplastic transformation.     

Telomere elongation by a mutant tankyrase 1 without TRF1 poly(ADP-ribosyl)ation

Telomeres are the capping structures of the eukaryotic chromosome ends. Tankyrase 1 is a poly(ADP-ribose) polymerase that elongates telomeres in a telomerase-dependent manner. This function of tankyrase 1 is mediated by down-regulation of TRF1, a negative regulator of telomere access to telomerase. Namely, tankyrase 1 poly(ADP-ribosyl)ates (PARsylates) TRF1, which in turn dissociates TRF1 from telomeres. The resulting telomeres become better substrates for telomerase-mediated DNA extension. Tankyrase 1 has five independent TRF1 binding sites, ARC (ANK repeat cluster) I to V. Among them, the most C-terminal ARC V is required for TRF1 PARsylation and its release from telomeres. By contrast, functional significance of other four ARCs remains elusive. In this study, we generated a mutant tankyrase 1 that had inactive ARC IV and lacked ARC V but elongated telomeres without TRF1 PARsylation. Consistent with the failure in PARsylation, this mutant only marginally released TRF1 from telomeres. Still, it decreased telomere binding of POT1, a downstream effector of TRF1-mediated telomere length control, and elongated the telomeric 3'-overhang as the wild-type tankyrase 1 did. Thus even without TRF1 PARsylation, this mutant tankyrase 1 seemed to loosen the closed structure of the telomeric heterochromatin. These findings suggest a new role for multiple ARCs in telomere extension by tankyrase 1.     

端粒蛋白TRF1与肌动蛋白结合蛋白PFN2相互作用的鉴定

目的:鉴定端粒蛋白TRF1和肌动蛋白结合蛋白PFN2是否存在相互作用,并且两者的相互作用是否与端粒在细胞核周的锚定有关。方法:将TRF1构建到myc标签载体,PFN2构建到GST标签载体,采用GST-pull down技术,验证两者是否存在相互作用;同时将TRF1构建到EGFP标签的绿色荧光载体,PFN2构建到RED标签的红色荧光载体,两者共转入细胞,利用荧光显微镜观察两者在细胞中的共定位情况。结果:GST-pull down证明TRF1与PFN2存在直接相互作用,两者在细胞中可以共定位。结论:TRF1与PFN2存在相互作用,且这种相互作用发生在细胞核周。     

Topoisomerase IIIalpha is required for normal proliferation and telomere stability in alternative lengthening of telomeres

Topoisomerase (Topo) IIIalpha associates with BLM helicase, which is proposed to be important in the alternative lengthening of telomeres (ALT) pathway that allows telomere recombination in the absence of telomerase. Here, we show that human Topo IIIalpha colocalizes with telomeric proteins at ALT-associated promyelocytic bodies from ALT cells. In these cells, Topo IIIalpha immunoprecipitated with telomere binding protein (TRF) 2 and BLM and was shown to be associated with telomeric DNA by chromatin immunoprecipitation, suggesting that these proteins form a complex at telomere sequences. Topo IIIalpha depletion by small interfering RNA reduced ALT cell survival, but did not affect telomerase-positive cell lines. Moreover, repression of Topo IIIalpha expression in ALT cells reduced the levels of TRF2 and BLM proteins, provoked a strong increase in the formation of anaphase bridges, induced the degradation of the G-overhang signal, and resulted in the appearance of DNA damage at telomeres. In contrast, telomere maintenance and TRF2 levels were unaffected in telomerase-positive cells. We conclude that Topo IIIalpha is an important telomere-associated factor, essential for telomere maintenance and chromosome stability in ALT cells, and speculate on its potential mechanistic function.     

Conservation of telomere protein complexes: shuffling through evolution

The rapid evolution of telomere proteins has hindered identification of orthologs from diverse species and created the impression that certain groups of eukaryotes have largely non-overlapping sets of telomere proteins. However, the recent identification of additional telomere proteins from various model organisms has dispelled this notion by expanding our understanding of the composition, architecture and range of telomere protein complexes present in individual species. It is now apparent that versions of the budding yeast CST complex and mammalian shelterin are present in multiple phyla. While the precise subunit composition and architecture of these complexes vary between species, the general function is often conserved. Despite the overall conservation of telomere protein complexes, there is still considerable species-specific variation, with some organisms having lost a particular subunit or even an entire complex. In some cases, complex components appear to have migrated between the telomere and the telomerase RNP. Finally, gene duplication has created telomere protein paralogs with novel functions. While one paralog may be part of a conserved telomere protein complex and have the expected function, the other paralog may serve in a completely different aspect of telomere biology.     

Role of Pin2/TRF1 in telomere maintenance and cell cycle control

Telomeres are specialized structures found at the extreme ends of chromosomes, which have many functions, including preserving genomic stability, maintaining cell proliferative capacity, and blocking the activation of DNA-damage cell cycle checkpoints. Deregulation of telomere length has been implicated in cancer and ageing. Telomere maintenance is tightly regulated by telomerase and many other telomere-associated proteins and is also closely linked to cell cycle control, especially mitotic regulation. However, little is known about the identity and function of the signaling molecules connecting telomere maintenance and cell cycle control. Pin2/TRF1 was originally identified as a protein bound to telomeric DNA (TRF1) and as a protein involved in mitotic regulation (Pin2). Pin2/TRF1 negatively regulates telomere length and importantly, its function is tightly regulated during the cell cycle, acting as an important regulator of mitosis. Recent identification of many Pin2/TRF1 upstream regulators and downstream targets has provided important clues to understanding the dual roles of Pin2/TRF1 in telomere maintenance and cell cycle control. These results have led us to propose that Pin2/TRF1 functions as a key molecule in connecting telomere maintenance and cell cycle control.     

The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats

The study of the proteins that bind to telomeric DNA in mammals has provided a deep understanding of the mechanisms involved in chromosome-end protection. However, very little is known on the binding of these proteins to nontelomeric DNA sequences. The TTAGGG DNA repeat proteins 1 and 2 (TRF1 and TRF2) bind to mammalian telomeres as part of the shelterin complex and are essential for maintaining chromosome end stability. In this study, we combined chromatin immunoprecipitation with high-throughput sequencing to map at high sensitivity and resolution the human chromosomal sites to which TRF1 and TRF2 bind. While most of the identified sequences correspond to telomeric regions, we showed that these two proteins also bind to extratelomeric sites. The vast majority of these extratelomeric sites contains interstitial telomeric sequences (or ITSs). However, we also identified non-ITS sites, which correspond to centromeric and pericentromeric satellite DNA. Interestingly, the TRF-binding sites are often located in the proximity of genes or within introns. We propose that TRF1 and TRF2 couple the functional state of telomeres to the long-range organization of chromosomes and gene regulation networks by binding to extratelomeric sequences.     

BRCA1 and CtIP promote alternative non-homologous end-joining at uncapped telomeres

Loss of telomere protection occurs during physiological cell senescence and ageing, due to attrition of telomeric repeats and insufficient retention of the telomere-binding factor TRF2. Subsequently formed telomere fusions trigger rampant genomic instability leading to cell death or tumorigenesis. Mechanistically, telomere fusions require either the classical non-homologous end-joining (C-NHEJ) pathway dependent on Ku70/80 and LIG4, or the alternative non-homologous end-joining (A-NHEJ), which relies on PARP1 and LIG3. Here, we show that the tumour suppressor BRCA1, together with its interacting partner CtIP, both acting in end resection, also promotes end-joining of uncapped telomeres. BRCA1 and CtIP do not function in the ATM-dependent telomere damage signalling, nor in telomere overhang removal, which are critical for telomere fusions by C-NHEJ. Instead, BRCA1 and CtIP act in the same pathway as LIG3 to promote joining of de-protected telomeres by A-NHEJ. Our work therefore ascribes novel roles for BRCA1 and CtIP in end-processing and fusion reactions at uncapped telomeres, underlining the complexity of DNA repair pathways that act at chromosome ends lacking protective structures. Moreover, A-NHEJ provides a mechanism of previously unanticipated significance in telomere dysfunction-induced genome instability.     

Cell cycle-dependent 3D distribution of telomeres and telomere repeat-binding factor 2 (TRF2) in HaCaT and HaCaT-myc cells

Telomeres are specialized structures at the ends of the chromosomes that, with the help of proteins--such as the telomere repeat-binding factor TRF2 -, form protective caps which are essential for chromosomal integrity. Investigating the structure and three-dimensional (3D) distribution of the telomeres and TRF2 in the nucleus, we now show that the telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells, while they extend more widely during mitosis. TRF2 is present at the telomeres at all cell cycle phases. During mitosis additional TRF2 protein concentrates all around the chromosomes. This change in staining pattern correlates with a significant increase in TRF2 protein at the S/G2 transition as seen in Western blots of synchronized cells and is paralleled by a cell cycle-dependent regulation of TRF2 mRNA, arguing for a specific role of TRF2 during mitosis. The distinct territorial localization of telomeres is abrogated in a HaCaT variant that constitutively expresses c-Myc--a protein known to contribute to genomic instability. These cells are characterized by overlapping telomere territories, telomeric aggregates (TAs), that are accompanied by an overall irregular telomere distribution and a reduced level in TRF2 protein. These TAs which are readily detectable in interphase nuclei, are similarly present in mitotic cells, including cells in telophase. Thus, we propose that TAs, which subsequently also cluster their respective chromosomes, contribute to genomic instability by forcing an abnormal chromosome segregation during mitosis.     

Control of telomere length by a trimming mechanism that involves generation of t-circles

Telomere lengths are maintained in many cancer cells by the ribonucleoprotein enzyme telomerase but can be further elongated by increasing telomerase activity through the overexpression of telomerase components. We report here that increased telomerase activity results in increased telomere length that eventually reaches a plateau, accompanied by the generation of telomere length heterogeneity and the accumulation of extrachromosomal telomeric repeat DNA, principally in the form of telomeric circles (t-circles). Telomeric DNA was observed in promyelocytic leukemia bodies, but no intertelomeric copying or telomere exchange events were identified, and there was no increase in telomere dysfunction-induced foci. These data indicate that human cells possess a mechanism to negatively regulate telomere length by trimming telomeric DNA from the chromosome ends, most likely by t-loop resolution to form t-circles. Additionally, these results indicate that some phenotypic characteristics attributed to alternative lengthening of telomeres (ALT) result from increased mean telomere length, rather than from the ALT mechanism itself.     

NBS1 deficiency promotes genome instability by affecting DNA damage signaling pathway and impairing telomere integrity

Studies revealed that Nijmegen Breakage Syndrome protein 1 (NBS1) plays an important role in maintaining genome stability, but the underlying mechanism is controversial and elusive. Our results using clinical samples showed that NBS1 was involved in ataxia-telangiectasia mutated (ATM)-dependent pathway. NBS1 deficiency severely affected the phosphorylation of ATM as well as its downstream targets. BrdU proliferation assay revealed a delay of NBS cells in inhibiting DNA synthesis after Doxorubicin (Dox) treatment. In addition, under higher concentrations of Dox, NBS cells exhibited a much lower level of apoptosis compared to their normal counterparts, indicating a resistance to Dox treatment. Accelerated telomere shortening was also observed in NBS fibroblasts, consistent with an early onset of cellular replicative senescence in vitro. This abnormality may be due to the shelterin protein telomeric binding factor 2 (TRF2) which was found to be upregulated in NBS fibroblasts. The dysregulation of telomere shortening rate and of TRF2 expression level leads to telomere fusions and cellular aneuploidy in NBS cells. Collectively, our results suggest a possible mechanism that NBS1 deficiency simultaneously affects ATM-dependent DNA damage signaling and TRF2-regulated telomere maintenance, which synergistically lead to genomic abnormalities.     

端粒结合蛋白TRF2的研究进展

端粒DNA结合蛋白TRF2(TTAGGG repeat binding factor-2)以二聚体形式通过Myb结构域与端粒重复序列TTAGGG结合,并与TRF1、TIN2、Rap1、TINT1及POT1蛋白组成Shelterin蛋白复合物,协同在端粒动态平衡维持过程中起关键作用,进而影响整个基因组的稳定性。此外,TRF2在细胞DNA损伤应答过程中可能发挥重要作用。本文将对TRF2结构和功能研究的最新进展进行综述。