Shelterin Protein TRF2 Research Articles

PML induces compaction, TRF2 depletion and DNA damage signaling at telomeres and promotes their alternative lengthening.

The alternative lengthening of telomeres (ALT) mechanism allows cancer cells to escape senescence and apoptosis in the absence of active telomerase. A characteristic feature of this pathway is the assembly of ALT-associated promyelocytic leukemia (PML) nuclear bodies (APBs) at telomeres. Here, we dissected the role of APBs in a human ALT cell line by performing an RNA interference screen using an automated 3D fluorescence microscopy platform and advanced 3D image analysis. We identified 29 proteins that affected APB formation, which included proteins involved in telomere and chromatin organization, protein sumoylation and DNA repair. By integrating and extending these findings, we found that APB formation induced clustering of telomere repeats, telomere compaction and concomitant depletion of the shelterin protein TRF2 (also known as TERF2). These APB-dependent changes correlated with the induction of a DNA damage response at telomeres in APBs as evident by a strong enrichment of the phosphorylated form of the ataxia telangiectasia mutated (ATM) kinase. Accordingly, we propose that APBs promote telomere maintenance by inducing a DNA damage response in ALT-positive tumor cells through changing the telomeric chromatin state to trigger ATM phosphorylation.

Shading the TRF2 recruiting function: a new horizon in drug development.

The shelterin protein TRF2 has come to the limelight for its role in telomere maintenance and tumorigenesis. Herein, the application of rational design and synthesis allowed identifying the first TRF2TRFH binder able to elicit a marked DNA damage response in cancer cells. This work paves the way for the unprecedented employment of a chemical tool to finely tune specific mechanisms underlying telomere maintenance.

Novel Function of the Fanconi Anemia Group J or RECQ1 Helicase to Disrupt Protein-DNA Complexes in a Replication Protein A-stimulated Manner

Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.

Genomic Instability: The Driving Force behind Refractory/Relapsing Hodgkin’s Lymphoma

In classical Hodgkin’s lymphoma (HL) the malignant mononuclear Hodgkin (H) and multinuclear, diagnostic Reed-Sternberg (RS) cells are rare and generally make up <3% of the total cellular mass of the affected lymph nodes. During recent years, the introduction of laser micro-dissection techniques at the single cell level has substantially improved our understanding of the molecular pathogenesis of HL. Gene expression profiling, comparative genomic hybridization analysis, micro-RNA expression profiling and viral oncogene sequencing have deepened our knowledge of numerous facets of H- and RS-cell gene expression deregulation. The question remains whether disturbed signaling pathways and deregulated transcription factors are at the origin of refractory/relapsing Hodgkin’s lymphoma or whether these hallmarks are at least partially related to another major factor. We recently showed that the 3D nuclear organization of telomeres and chromosomes marked the transition from H- to RS-cells in HL cell lines. This transition is associated with progression of telomere dysfunction, shelterin disruption and progression of complex chromosomal rearrangements. We reported analogous findings in refractory/relapsing HL and identified the shelterin proteins TRF1, TRF2 and POT1 as targets of the LMP1 oncogene in post-germinal center B-cells. Here we summarize our findings, including data not previously published, and propose a model in which progressive disruption of nuclear integrity, a form of genomic instability, is the key-player in refractory/relapsing HL. Therapeutic approaches should take these findings into account.

Abstract 743: Pu-27 G-quadruplex induces uncompensated DNA damage response by destabilizing shelterin complex at telomere and non-telomeric region leading to cell death.

Abstract Throughout the genome as well as telomere region, there is presence of DNA sequences which forms quadruplex. Here we show Pu-27 triggers the damage of cells by interfering telomeric ‘shelterin’ protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks (DSB). We found that Pu-27 frequently prompted chromosomal aberrations including abnormal metaphases (45%), chromatid breaks (40%) chromosomal break (27%) by karyotyping of U937 metaphase cells and was associated with induction of DNA damage response sensor γ-H2AX. Analysis of DNA damage response regulators by RT-PCR array of Pu-27 treated U937 cells revealed downregulation of telomeric ‘shelterin’ proteins (TRF2, TRF1, POT1 and TIN2), upstream kinase ATM, DNA damage response mediators (RAD17, RAD50 and 53BP1), and cell cycle arrest molecules (CHK1 and CHK2). Interestingly, there were no changes or low expression of DNA repair molecules (H2AX and BRCA1) and telomere maintenance gene TERT respectively. αB-U937 cell, where basic-domain of shelterin protein TRF2 was deleted became relatively resistance to Pu-27 and showed no changes of constitutively active form of γ-H2AX. RT-PCR array of Pu-27 treated αB-U937 cell appeared to be no changes of telomeric ‘shelterin’ proteins (TRF2, TRF1, POT1 and TIN2), DNA damage response mediators (RAD17, RAD50 and 53BP1); down regulation of upstream kinase ATM; up regulation of cell cycle arrest molecules (SMC1, CHK1 and CHK2), DNA repair molecules (H2AX and BRCA1), and telomere maintenance gene TERT. Shelterin protein, TRF1 co-associated with γH2AX by inducing TIFs (Telomere-dysfunction Induced Foci) when U937 cell was treated with Pu-27. Sk-Lu-1, an Alt (Alternating Lengthening to Telomere) cell which does not use TERT for their growth and propagation is relatively resistance to Pu-27. ATM preserves the telomeric homeostasis by maintaining the integrity of ‘shelterin’ protein. To substantiate the specificity of Pu-27 to shelterin protein complex, we found ATM-deficient cell was more sensitive to Pu-27 than ATM-proficient cell. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, these results suggest that Pu-27-treated sensitivity at least in part due to compromising telomeric shelterin complex and perhaps stabilizing telomeric quadruplex and not by directly inhibiting telomerase enzyme. U937 cell with chromosomal aberration and unstable telomere progress through cell cycle without arrest and repair leading to cell death whereas αB-U937 cells is continuously repaired by arresting and induction of repair machinery. Continuous exposure of Pu-27 results in refractory to repair of DNA damage leading to uncompensated genomic stability leading to cell death. Citation Format: Md Ashraful Islam, Shelia D. Thomas, Kara J. Sedoris, Donald M. Miller. Pu-27 G-quadruplex induces uncompensated DNA damage response by destabilizing shelterin complex at telomere and non-telomeric region leading to cell death. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 743. doi:10.1158/1538-7445.AM2013-743 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

TRF1 interacts with XRCC4 and XLF at telomeres

At telomeres, the shelterin complex is entrusted with a protective role against DNA repair mechanisms that could mistakenly recognize the natural ends of chromosomes as the product of a double strand break, and create telomeric end‐to‐end fusions that can drive genome instability and tumorigenesis. Paradoxically, telomeres also harbor several members of the DNA repair machinery whose actions they are trying to block, like members of the NHEJ pathway. How these NHEJ proteins are prevented from engaging in NHEJ at telomeres is not fully understood. Using protein fragment complementation assay, we have detected novel interactions between shelterin proteins TRF1 and TRF2 with XRCC4 and XLF, two NHEJ proteins that interact and are involved in later steps during DSB repair. Furthermore, cells expressing interacting TRF1 and XRCC4 proteins displayed clear punctate staining that co‐localized with other telomeric proteins, indicating that XRCC4 and TRF1 can interact at telomeres. Mutations that disrupt XRCC4‐XLF interaction or known phosphorylation sites for ATM and DNA‐PKcs kinases did not impair XRCC4‐TRF1 interaction nor did they impair XRCC4 localization at telomeres. Overall our results suggest that NHEJ proteins XRCC4 and XLF can be recruited to telomeres through interactions with shelterin proteins TRF1 and TRF2 in ways that require neither XRCC4‐XLF interaction nor ATM or DNA‐PKcs phosphorylation.

Role of 53BP1 oligomerization in regulating double-strand break repair

Tumor suppressor p53-binding protein 1 (53BP1) regulates the repair of dysfunctional telomeres lacking the shelterin protein TRF2 by promoting their mobility, their nonhomologous end-joining (NHEJ), and, as we show here, by blocking 5' resection by CtIP. We report that these functions of 53BP1 required its N-terminal ATM/ATR target sites and its association with H4K20diMe, but not the BRCT domain, the GAR domain, or the binding of 53BP1 to dynein. A mutant lacking the oligomerization domain (53BP1(oligo)) was only modestly impaired in promoting NHEJ of dysfunctional telomeres and showed no defect with regard to the repression of CtIP. This 53BP1(oligo) allele was previously found to be unable to support class switch recombination or to promote radial chromosome formation in PARP1 inhibitor-treated Brca1-deficient cells. The data therefore support two conclusions. First, the requirements for 53BP1 in mediating NHEJ at dysfunctional telomeres and in class switch recombination are not identical. Second, 53BP1-dependent repression of CtIP at double-strand breaks (DSBs) is unlikely to be sufficient for the generation of radial chromosomes in PARP1 inhibitor-treated Brca1-deficient cells.

The nuclease hSNM1B/Apollo is linked to the Fanconi anemia pathway via its interaction with FANCP/SLX4

The recessive genetic disorder Fanconi anemia (FA) is clinically characterized by congenital defects, bone marrow failure and an increased incidence of cancer. Cells derived from FA patients exhibit hypersensitivity to DNA interstrand crosslink (ICL)-inducing agents. We have earlier reported a similar cellular phenotype for human cells depleted of hSNM1B/Apollo (siRNA). In fact, hSNM1B/Apollo has a dual role in the DNA damage response and in generation and maintenance of telomeres, the latter function involving interaction with the shelterin protein TRF2. Here we find that ectopically expressed hSNM1B/Apollo co-immunoprecipitates with SLX4, a protein recently identified as a new FA protein, FANCP, and known to interact with several structure-specific nucleases. As shown by immunofluorescence analysis, FANCP/SLX4 depletion (siRNA) resulted in a significant reduction of hSNM1B/Apollo nuclear foci, supporting the functional relevance of this new protein interaction. Interestingly, as an additional consequence of FANCP/SLX4 depletion, we found a reduction of cellular TRF2, in line with its telomere-related function. Finally, analysis of human cells following double knockdown of hSNM1B/Apollo and FANCP/SLX4 indicated that they function epistatically. These findings further substantiate the role of hSNM1B/Apollo in a downstream step of the FA pathway during the repair of DNA ICLs.

Abstract 2123: ATM mediated homeostasis of ‘shelterin’ at telomere is compromised by G-quadruplex leading to DNA damage

Abstract Quadruplex-forming DNA sequences are present throughout the genome, including in the telomeres. We have recently shown that the c-myc promoter quadruplex-forming sequence, Pu-27 inhibits growth of malignant cells. Telomere integrity is maintained by the ‘shelterin’ protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks. ATM has been shown to play a role in the maintenance of telomere integrity. U937, histiocytic lymphoma cells were sensitive to Pu-27 as evident by MTT and colony formation assay and frequently showed chromosomal aberrations including abnormal metaphases (45%), chromatid breaks (40%) chromosomal breaks (27%). RT-PCR array of Pu-27 treated U937 cells revealed downregulation of ‘shelterin’ proteins TRF2, TRF1, POT1 and TIN2; induction of DNA damage response sensor γ-H2AX; downregulation of upstream kinase ATM and other downstream DNA damage mediator RAD17, RAD50, BRCA1, 53BP1, CHK1, CHK2. To understand the specific role of ‘shelterin’ protein complex, we stably transfected U937 cells with a vector expressing a dominant negative mutant of a shelterin protein TRF2 (dn-U937). These dn-UP37 cells became relatively resistant when treated with Pu-27 and showed no changes of basal level expression of ‘shelterin’ protein complex but induction of γ-H2AX and DNA repair inducing molecules BRCA1 and CHK1. This suggests that intact TRF2 is required for Pu-27 mediated U937 cellular sensitivity and it may acted by altering the telomere-‘shelterin’ complex. This idea was further substantiated by the fact that ALT (Alternative Lengthening of Telomere) cells were also relatively resistant to Pu27. To elucidate the specific role of ATM, we have shown by MTT and colony formation assay that ATM-deficient cells are more sensitive to growth inhibition by Pu-27 whereas ATM-proficient cells were relatively resistant to Pu-27. Co-immunostaining with γ-H2AX and TRF1 revealed that the sensitivity to Pu-27 in ATM deficient cells was associated with phosphorylation of H2AX, an indicator of DNA double strand break and induction of TIFs (Telomere-dysfunction Induced Foci). There was induction of γ-H2AX and no alteration of basal level of ‘shelterin’ protein complex in Pu-27 treated ATM deficient cells (as determined by RT-PCR array). A549 cells, which were sensitive to Pu-27, also showed abundant presence of TIF compared to untreated cells. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, these data suggest that downregulation of ATM destabilizes telomere-shelterin homeostasis at telomere ends in response to Pu-27 of U937 cells. Continuous exposure of G-quadruplex results in refractory DNA damage repair leading to uncompensated genomic stability and cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2123. doi:1538-7445.AM2012-2123

RECQL4, the Protein Mutated in Rothmund-Thomson Syndrome, Functions in Telomere Maintenance

Telomeres are structures at the ends of chromosomes and are composed of long tracks of short tandem repeat DNA sequences bound by a unique set of proteins (shelterin). Telomeric DNA is believed to form G-quadruplex and D-loop structures, which presents a challenge to the DNA replication and repair machinery. Although the RecQ helicases WRN and BLM are implicated in the resolution of telomeric secondary structures, very little is known about RECQL4, the RecQ helicase mutated in Rothmund-Thomson syndrome (RTS). Here, we report that RTS patient cells have elevated levels of fragile telomeric ends and that RECQL4-depleted human cells accumulate fragile sites, sister chromosome exchanges, and double strand breaks at telomeric sites. Further, RECQL4 localizes to telomeres and associates with shelterin proteins TRF1 and TRF2. Using recombinant proteins we showed that RECQL4 resolves telomeric D-loop structures with the help of shelterin proteins TRF1, TRF2, and POT1. We also found a novel functional synergistic interaction of this protein with WRN during D-loop unwinding. These data implicate RECQL4 in telomere maintenance.

Abstract 3886: The G-quadruplex induced DNA damage at telomeres is regulated by ATM

Abstract Quadruplex-forming DNA sequences are present throughout the genome, including in the telomeres. Our group has shown that quadruplex-forming oligonucleotides selectively inhibit proliferation of transformed cells. One of these oligonucleotides, AS1411, is currently in a Phase IIb clinical trial. We have recently shown that the c-myc promoter quadruplex-forming sequence, Pu-27, also inhibits growth of malignant cells. We have hypothesized that one potential mechanism of this effect is competition for telomere-binding proteins. If this is correct, we would expect this effect to be mediated by the DNA repair response. Telomere integrity is maintained by the shelterin protein complex which is required to prevent chromosomal ends from being recognized as DNA double strand breaks. Like many other DNA damage response proteins, ATM has been shown to play a role in the maintenance of telomere integrity. We have shown that ATM deficient cells are more sensitive to growth inhibition by Pu-27 whereas ATM proficient cells were relatively resistant to Pu-27. Co-immunostaining with γ-H2AX and TRF1 revealed that the sensitivity to Pu-27 in ATM deficient cells was associated with phosphorylation of H2AX, an indicator of DNA double strand break and induction of TIFs (Telomere-dysfunction Induced Foci). There was induction of γ-H2AX and downregulation of shelterin proteins TRF2 and TRF1 in Pu-27 treated ATM deficient cells (as determined by western blotting). U937 leukemia cells, which were sensitive to Pu-27, became relatively resistant when they were stably transfected with a vector expressing a dominant negative mutant of the shelterin protein TRF2 (dn-TRF2). This suggests that Pu-27 may act by altering the telomere-shelterin complex. This idea was further substantiated by the fact that ALT (Alternative Lengthening of Telomere) cells were also relatively resistant to Pu27. A549 cells, which were sensitive to Pu-27, also showed abundant presence of TIF compared to untreated cells. Lastly, we showed that both p53 wild type and deficient colorectal cells (HCT116 cell line) were relatively resistant to Pu-27. Taken together, this data suggests that ATM plays a pivotal role in Pu-27 induced DNA damage at telomere ends, which occurs in response to a Pu-27 induced alteration in telomere-shelterin homeostasis. It is likely that this response is induced in a p53 independent manner. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3886. doi:10.1158/1538-7445.AM2011-3886

The Epstein–Barr virus nuclear antigen-1 promotes telomere dysfunction via induction of oxidative stress

The Epstein–Barr virus (EBV) nuclear antigen (EBNA)-1 promotes the accumulation of chromosomal aberrations in malignant B cells by inducing oxidative stress. Here we report that this phenotype is associated with telomere dysfunction. Stable or conditional expression of EBNA1 induced telomere abnormalities including loss or gain of telomere signals, telomere fusion and heterogeneous length of telomeres. This was accompanied by the accumulation of extrachromosomal telomeres, telomere dysfunction-induced foci (TIFs) containing phosphorylated histone H2AX and the DNA damage response protein 53BP1, telomere-associated promyelocytic leukemia nuclear bodies (APBs), telomeric-sister chromatid exchanges and displacement of the shelterin protein TRF2. The induction of TIFs and APBs was inhibited by treatment with scavengers of reactive oxygen species (ROS) that also promoted the relocalization of TRF2 at telomeres. These findings highlight a novel mechanism by which EBNA1 may promote malignant transformation and tumor progression.

Abstract 3207: Telomerase inhibition affects fludarabine sensitivity in quiescent primary chronic lymphocytic leukemia lymphocytes

Abstract B- Cell chronic leukemia (CLL) is the most common leukemia in adults. It is clinically very heterogeneous and has a highly variable clinical course. Telomere length and telomerase activity have been shown to be excellent prognostic factors in CLL. Moreover, telomere maintenance is deficient in CLL lymphocytes. These studies have prompted the assessment of the telomerase inhibitor GRN163L in a phase I-II clinical trial in CLL and lymphomas. Here we report that telomerase activity is not required for the survival of quiescent primary CLL lymphocytes. In contrast telomerase activity seems to be required for the survival of quiescent primary CLL lymphocytes after treatment with fludarabine (FLU) in vitro. Furthermore, the effect of fludarabine on telomerase activity was associated with the basal expression of the telomere shelterin protein TRF2. In summary, our results suggest that GRN163L in combination with FLU may be useful to decrease the tumor burden in CLL. To our knowledge this is the first report assessing the effect of telomerase inhibition in combination with a chemotherapeutic agent in non-stimulated primary CLL lymphocytes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3207.