TRF1 Binding Research Articles

The Altered Functions of Shelterin Components in ALT Cells

Telomeres are nucleoprotein complexes that cap the ends of eukaryotic linear chromosomes. Telomeric DNA is bound by shelterin protein complex to prevent telomeric chromosome ends from being recognized as damaged sites for abnormal repair. To overcome the end replication problem, cancer cells mostly preserve their telomeres by reactivating telomerase, but a minority (10–15%) of cancer cells use a homologous recombination-based pathway called alternative lengthening of telomeres (ALT). Recent studies have found that shelterin components play an important role in the ALT mechanism. The binding of TRF1, TRF2, and RAP1 to telomeres attenuates ALT activation, while the maintenance of ALT telomere requires TRF1 and TRF2. POT1 and TPP1 can also influence the occurrence of ALT. The elucidation of how shelterin regulates the initiation of ALT remains elusive. This review presents a comprehensive overview of the current findings on the regulation of ALT by shelterin components, aiming to enhance the insight into the altered functions of shelterin components in ALT cells and to identify potential targets for the treatment of ALT tumor cells.

Structural basis of telomeric nucleosome recognition by shelterin factor TRF1.

Shelterin and nucleosomes are the key players that organize mammalian chromosome ends into the protective telomere caps. However, how they interact with each other at telomeres remains unknown. We report cryo-electron microscopy structures of a human telomeric nucleosome both unbound and bound to the shelterin factor TRF1. Our structures reveal that TRF1 binds unwrapped nucleosomal DNA ends by engaging both the nucleosomal DNA and the histone octamer. Unexpectedly, TRF1 binding shifts the register of the nucleosomal DNA by 1 bp. We discovered that phosphorylation of the TRF1 C terminus and a noncanomical DNA binding surface on TRF1 are critical for its association with telomeric nucleosomes. These insights into shelterin-chromatin interactions have crucial implications for understanding telomeric chromatin organization and other roles of shelterin at telomeres including replication and transcription.

Assembly path dependence of telomeric DNA compaction by TRF1, TIN2, and SA1

Telomeres, complexes of DNA and proteins, protect ends of linear chromosomes. In humans, the two shelterin proteins TRF1 and TIN2, along with cohesin subunit SA1, were proposed to mediate telomere cohesion. Although the ability of the TRF1-TIN2 and TRF1-SA1 systems to compact telomeric DNA by DNA-DNA bridging has been reported, the function of the full ternary TRF1-TIN2-SA1 system has not been explored in detail. Here, we quantify the compaction of nanochannel-stretched DNA by the ternary system, as well as its constituents, and obtain estimates of the relative impact of its constituents and their interactions. We find that TRF1, TIN2, and SA1 work synergistically to cause a compaction of the DNA substrate, and that maximal compaction occurs if all three proteins are present. By altering the sequence with which DNA substrates are exposed to proteins, we establish that compaction by TRF1 and TIN2 can proceed through binding of TRF1 to DNA, followed by compaction as TIN2 recognizes the previously bound TRF1. We further establish that SA1 alone can also lead to a compaction, and that compaction in a combined system of all three proteins can be understood as an additive effect of TRF1-TIN2 and SA1-based compaction. Atomic force microscopy of intermolecular aggregation confirms that a combination of TRF1, TIN2, and SA1 together drive strong intermolecular aggregation as it would be required during chromosome cohesion.

In Vitro Characterization of the Physical Interactions between the Long Noncoding RNA TERRA and the Telomeric Proteins TRF1 and TRF2.

RNA-protein interactions drive key cellular pathways such as protein translation, nuclear organization and genome stability maintenance. The human telomeric protein TRF2 binds to the long noncoding RNA TERRA through independent domains, including its N-terminal B domain. We previously demonstrated that TRF2 B domain binding to TERRA supports invasion of TERRA into telomeric double stranded DNA, leading to the formation of telomeric RNA:DNA hybrids. The other telomeric protein TRF1, which also binds to TERRA, suppresses this TRF2-associated activity by preventing TERRA-B domain interactions. Herein, we show that the binding of both TRF1 and TRF2 to TERRA depends on the ability of the latter to form G-quadruplex structures. Moreover, a cluster of arginines within the B domain is largely responsible for its binding to TERRA. On the other side, a patch of glutamates within the N-terminal A domain of TRF1 mainly accounts for the inhibition of TERRA-B domain complex formation. Finally, mouse TRF2 B domain binds to TERRA, similarly to its human counterpart, while mouse TRF1 A domain lacks the inhibitory activity. Our data shed further light on the complex crosstalk between telomeric proteins and RNAs and suggest a lack of functional conservation in mouse.

Vascular senescence in progeria: role of endothelial dysfunction.

AimsHutchinson–Gilford progeria syndrome (HGPS) is a pre-mature aging disorder caused by the mutation of the LMNA gene leading to an irreversibly farnesylated lamin A protein: progerin. The major causes of death in HGPS are coronary and arterial occlusive disease. In the murine model of HGPS, vascular smooth muscle cell (VSMC) loss is the primary vascular manifestation, which is different from the arterial occlusive disease seen in older patients.Methods and resultsTo identify the mechanisms of HGPS vascular disease in humans, we differentiated isogenic endothelial cells (ECs) and VSMCs from HGPS-induced pluripotent stem cells (iPSCs) and control-iPSCs. Both HGPS-ECs and HGPS-VSMCs manifested cellular hallmarks of aging, including dysmorphic nuclei, impaired proliferation, increased β-galactosidase staining, shortened telomeres, up-regulated secretion of inflammatory cytokines, increased DNA damage, loss of heterochromatin, and altered shelterin protein complex (SPC) expression. However, at similar days after differentiation, even with lower levels of progerin, HGPS-ECs manifested more severe signs of senescence, as indicated in part by a higher percentage of β-galactosidase positive cells, shorter telomere length, and more DNA damage signals. We observed increased γH2A.X binding to RAP1 and reduced TRF2 binding to lamin A in HGPS-ECs but not in HGPS-VSMCs. The expression of γH2A.X was greater in HGPS-ECs than in HGPS-VSMCs and is associated with greater telomere shortening, impaired SPC interactions, and loss of heterochromatin.ConclusionAlthough progerin expression has a deleterious effect on both ECs and VSMCs, the dysfunction is greater in HGPS-ECs compared with HGPS-VSMCs. This study suggests that an endothelial-targeted therapy may be useful for HGPS patients.

Shelterin Components Modulate Nucleic Acids Condensation and Phase Separation in the Context of Telomeric DNA

Telomeres are nucleoprotein complexes that protect the ends of chromosomes and are essential for chromosome stability in Eukaryotes. In cells, individual telomeres form distinct globules of finite size that appear to be smaller than expected for bare DNA. Moreover, telomeres can cluster together, form telomere-induced-foci or co-localize with promyelocytic leukemia (PML) nuclear bodies. The physical basis for collapse of individual telomeres and coalescence of multiple ones remains unclear, as does the relationship between these two phenomena. By combining single-molecule force spectroscopy measurements, optical microscopy, turbidity assays, and simulations, we show that the telomere scaffolding protein TRF2 can condense individual DNA chains and drives coalescence of multiple DNA molecules, leading to phase separation and the formation of liquid-like droplets. Addition of the TRF2 binding protein hRap1 modulates phase boundaries and tunes the specificity of solution demixing while simultaneously altering the degree of DNA compaction. Our results suggest that the condensation of single telomeres and formation of biomolecular condensates containing multiple telomeres are two different outcomes driven by the same set of molecular interactions. Moreover, binding partners, such as other telomere components, can alter those interactions to promote single-chain DNA compaction over multiple-chain phase separation.

AKT-dependent signaling of extracellular cues through telomeres impact on tumorigenesis.

The telomere-bound shelterin complex is essential for chromosome-end protection and genomic stability. Little is known on the regulation of shelterin components by extracellular signals including developmental and environmental cues. Here, we show that human TRF1 is subjected to AKT-dependent regulation. To study the importance of this modification in vivo, we generate knock-in human cell lines carrying non-phosphorylatable mutants of the AKT-dependent TRF1 phosphorylation sites by CRISPR-Cas9. We find that TRF1 mutant cells show decreased TRF1 binding to telomeres and increased global and telomeric DNA damage. Human cells carrying non-phosphorylatable mutant TRF1 alleles show accelerated telomere shortening, demonstrating that AKT-dependent TRF1 phosphorylation regulates telomere maintenance in vivo. TRF1 mutant cells show an impaired response to proliferative extracellular signals as well as a decreased tumorigenesis potential. These findings indicate that telomere protection and telomere length can be regulated by extracellular signals upstream of PI3K/AKT activation, such as growth factors, nutrients or immune regulators, and this has an impact on tumorigenesis potential.

Human Telomerase (hTERT) is Directly Regulated by the Non-Telomeric Telomere-Binding Factor TRF2

Human telomerase reverse transcriptase hTERT remains suppressed in most normal adult somatic cells. Resulting erosion of telomeres leads eventually to replicative senescence. Reactivation of hTERT maintains telomeres and triggers progression of >90% human cancers. However any direct causal link between telomeres and telomerase regulation remains unclear. Here we show that the telomere-repeat-binding-factor TRF2 binds to the hTERT promoter and recruits the polycomb-repressor EZH2/PRC2 complex. This is causal for H3K27 trimethylation at the hTERT promoter and represses hTERT in cancer as well as normal cells. TRF2 interacts with hTERT promoter G-quadruplexes. Two highly recurrent hTERT promoter mutations found in many cancers, including ~83% glioblastoma-multiforme, that are known to destabilize hTERT promoter G-quadruplexes, obliterated TRF2 binding in patient-derived primary glioblastoma-multiforme cells. Ligand-induced G-quadruplex stabilization restored TRF2 binding, H3K27-trimethylation and hTERT re-suppression. These results uncover a previously unknown mechanism of hTERT regulation through a telomeric factor, implicating telomere-telomerase molecular links that might be important in neoplastic transformation, ageing and regenerative therapy.

Role for the shelterin protein TRF2 in human herpesvirus 6A/B chromosomal integration.

Human herpesviruses 6A and 6B (HHV-6A/B) are unique among human herpesviruses in their ability to integrate their genome into host chromosomes. Viral integration occurs at the ends of chromosomes within the host telomeres. The ends of the HHV-6A/B genomes contain telomeric repeats that facilitate the integration process. Here, we report that productive infections are associated with a massive increase in telomeric sequences of viral origin. The majority of the viral telomeric signals can be detected within viral replication compartments (VRC) that contain the viral DNA processivity factor p41 and the viral immediate-early 2 (IE2) protein. Components of the shelterin protein complex present at telomeres, including TRF1 and TRF2 are also recruited to VRC during infection. Biochemical, immunofluorescence coupled with in situ hybridization and chromatin immunoprecipitation demonstrated the binding of TRF2 to the HHV-6A/B telomeric repeats. In addition, approximately 60% of the viral IE2 protein localize at cellular telomeres during infection. Transient knockdown of TRF2 resulted in greatly reduced (13%) localization of IE2 at cellular telomeres (p<0.0001). Lastly, TRF2 knockdown reduced HHV-6A/B integration frequency (p<0.05), while no effect was observed on the infection efficiency. Overall, our study identified that HHV-6A/B IE2 localizes to telomeres during infection and highlight the role of TRF2 in HHV-6A/B infection and chromosomal integration.

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

Amyloid β and Tumorigenesis: Amyloid β-Induced Telomere Dysfunction in Tumor Cells

TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment

The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients.

Analysis of DNA-Protein Complexes by Atomic Force Microscopy Imaging: The Case of TRF2-Telomeric DNA Wrapping.

Atomic force microscopy (AFM) is a non-optical microscopy that enables the acquisition at the nanoscale level of a 3D topographical image of the sample. For 30years, AFM has been a valuable tool in life sciences to study biological samples in the field of tissue, cellular and molecular imaging, of mechanical properties and of force spectroscopy. Since the early beginnings of the technique, AFM has been extensively exploited as an imaging tool for structural studies of nucleic acids and nucleoprotein complexes. The morphometric analysis performed on the images can unveil specific structural and functional aspects of the sample, such as the multimerization state of proteins bound to DNA, or DNA conformational changes led by the DNA-binding proteins. Herein, a method for analyzing a complex formed by a telomeric DNA sequence wrapped around the TRF2 binding protein is presented. The described procedure could be applied to the study of any type of DNA-protein complex.

The Effect of Ethanol on Telomere Dynamics and Regulation in Human Cells.

Telomeres (TLs) protect chromosome ends from chromosomal fusion and degradation, thus conferring genomic stability, and play crucial roles in cellular aging and disease. Recent studies have found a correlation between environmental, physiological and even mental stresses on TL dynamics in humans. However, the causal relationship between stress and TL length and the molecular mechanisms underlying that relationship are far from being understood. This study describes the effect of moderate concentrations of ethanol, equivalent to social drinking, on human TL dynamics and partially elucidates the mechanism mediating this effect. The exposure of Immortalized human foreskin fibroblast, primary human foreskin fibroblast and human hepatocellular carcinoma cells to 25 mM ethanol for one week moderately shortened telomeres in all cells. Similar TL shortening was obtained following cells’ exposure to 25 µM acetaldehyde (AcH) and to a much lower extent after exposure to 4-methylpyrazolean, an inhibitor of alcoholdehydrogenase, suggesting that AcH plays a key role in ethanol-dependent telomere shortening. Telomerase activity was not involved in this effect. TRF2 and several TRF2 binding proteins increased their binding to TLs after ethanol treatment, implying their involvement in this effect. The methylation status of several sub-telomeric regions increased in response to EtOH exposure. Gene expression profiling showed distinct patterns in cells treated with EtOH and in cells recovered from EtOH. In addition to cellular ageing, the described telomere shortening may contribute to the carcinogenic potential of acute alcohol consumption; both are associated with the shortening of TLs and provide new insights regarding the moderate consumption of alcohol referred to as “social drinking.”

Modulation of telomere protection by the PI3K/AKT pathway

Telomeres and the insulin/PI3K pathway are considered hallmarks of aging and cancer. Here, we describe a role for PI3K/AKT in the regulation of TRF1, an essential component of the shelterin complex. PI3K and AKT chemical inhibitors reduce TRF1 telomeric foci and lead to increased telomeric DNA damage and fragility. We identify the PI3Kα isoform as responsible for this TRF1 inhibition. TRF1 is phosphorylated at different residues by AKT and these modifications regulate TRF1 protein stability and TRF1 binding to telomeric DNA in vitro and are important for in vivo TRF1 telomere location and cell viability. Patient-derived breast cancer PDX mouse models that effectively respond to a PI3Kα specific inhibitor, BYL719, show decreased TRF1 levels and increased DNA damage. These findings functionally connect two of the major pathways for cancer and aging, telomeres and the PI3K pathway, and pinpoint PI3K and AKT as novel targets for chemical modulation of telomere protection.

How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1.

Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation.

TRF1 phosphorylation on T271 modulates telomerase-dependent telomere length maintenance as well as the formation of ALT-associated PML bodies

TRF1, a component of the shelterin complex, plays a key role in both telomerase-dependent telomere maintenance and alternative lengthening of telomeres, the latter also known as ALT. Characteristics of ALT cells include C-circles and ALT-associated PML bodies, referred to as APBs. The function of TRF1 is tightly regulated by post-translational modification including phosphorylation, however TRF1 phosphorylation sites have yet to be fully characterized. Here we report a novel TRF1 phosphorylation site threonine 271. We show that a nonphosphorylatable mutation of T271A impairs TRF1 binding to telomeric DNA in vivo and renders TRF1 defective in inhibiting telomerase-dependent telomere elongation. On the other hand, TRF1 carrying a phosphomimic mutation of T271D is competent in not only binding to telomeric DNA but also inhibiting telomerase-mediated telomere lengthening. These results suggest that TRF1 phosphorylation on T271 negatively regulates telomerase-mediated telomere maintenance. We find that in telomerase-negative ALT cells, TRF1 carrying either a T271A or T271D mutation is able to promote C-circle production but fails to support APB formation. These results suggest that TRF1 phosphorylation on T271 is necessary for APB formation but dispensable for C-circle production. These results further imply that APB formation can be mechanistically separated from C-circle production.

Molecular Architecture of Full-length TRF1 Favors Its Interaction with DNA

Telomeres are specific DNA-protein structures found at both ends of eukaryotic chromosomes that protect the genome from degradation and from being recognized as double-stranded breaks. In vertebrates, telomeres are composed of tandem repeats of the TTAGGG sequence that are bound by a six-subunit complex called shelterin. Molecular mechanisms of telomere functions remain unknown in large part due to lack of structural data on shelterins, shelterin complex, and its interaction with the telomeric DNA repeats. TRF1 is one of the best studied shelterin components; however, the molecular architecture of the full-length protein remains unknown. We have used single-particle electron microscopy to elucidate the structure of TRF1 and its interaction with telomeric DNA sequence. Our results demonstrate that full-length TRF1 presents a molecular architecture that assists its interaction with telometic DNA and at the same time makes TRFH domains accessible to other TRF1 binding partners. Furthermore, our studies suggest hypothetical models on how other proteins as TIN2 and tankyrase contribute to regulate TRF1 function.

Sequence-Specific Binding and Diffusion of TRF1 on Telomeric DNA Studied by Molecular Dynamics

Telomeres are nucleoprotein complexes that cap the ends of linear chromosomes. Their protective and structural function depends on several protein factors that form the so-called shelterin complex, anchored to telomeric DNA via three different proteins: double-strand binding TRF1 and TRF2, and single-strand binding POT1. Homologous DNA-binding domains of TRF1 and TRF2 share the homeodomain fold commonly found among DNA-interacting factors, and both recognize the repeating telomeric motif (5'-TTAGGG-3') in a structurally similar fashion. Therefore, while both domains exhibit a high degree of similarity to other DNA-binding folds, they differ from them in the availability of target binding sites, which in the case of telomere-binding proteins are arranged in long (several kbp) tandem arrays. In such a system, diffusion between neighboring binding sites becomes a relevant aspect of protein dynamics at telomeres, facilitating e.g. the assembly of shelterin units on telomeric DNA.In this work, we present a comprehensive study of the sequence specific binding of TRF1 to telomeric DNA and the diffusion of TRF1 along the telomeric sequence. We try to identify the factors that facilitate the formation of sequence-specific protein-DNA complex, and explore the free energy landscape of TRF1 on telomeric DNA using both simulations of spontaneous complex formation and umbrella sampling along a helical path on DNA. The obtained results agree well with experimental data on the kinetics of diffusion on telomeric DNA, allowing us to extract the large-scale coarse grained picture of protein-DNA interaction in the telomeric region.

High Mobility Group A2 protects cancer cells against telomere dysfunction

The non-histone chromatin binding protein High Mobility Group AT-hook protein 2 (HMGA2) plays important roles in the repair and protection of genomic DNA in embryonic stem cells and cancer cells. Here we show that HMGA2 localizes to mammalian telomeres and enhances telomere stability in cancer cells. We present a novel interaction of HMGA2 with the key shelterin protein TRF2. We found that the linker (L1) region of HMGA2 contributes to this interaction but the ATI-L1-ATII molecular region of HMGA2 is required for strong interaction with TRF2. This interaction was independent of HMGA2 DNA-binding and did not require the TRF2 interacting partner RAP1 but involved the homodimerization and hinge regions of TRF2. HMGA2 retained TRF2 at telomeres and reduced telomere-dysfunction despite induced telomere stress. Silencing of HMGA2 resulted in (i) reduced binding of TRF2 to telomere DNA as observed by ChIP, (ii) increased telomere instability and (iii) the formation of telomere dysfunction-induced foci (TIF). This resulted in increased telomere aggregation, anaphase bridges and micronuclei. HMGA2 prevented ATM-dependent pTRF2T188 phosphorylation and attenuated signaling via the telomere specific ATM-CHK2-CDC25C DNA damage signaling axis. In summary, our data demonstrate a unique and novel role of HMGA2 in telomere protection and promoting telomere stability in cancer cells. This identifies HMGA2 as a new therapeutic target for the destabilization of telomeres in HMGA2+ cancer cells.

TRF1 and TRF2 binding to telomeres is modulated by nucleosomal organization.

The ends of eukaryotic chromosomes need to be protected from the activation of a DNA damage response that leads the cell to replicative senescence or apoptosis. In mammals, protection is accomplished by a six-factor complex named shelterin, which organizes the terminal TTAGGG repeats in a still ill-defined structure, the telomere. The stable interaction of shelterin with telomeres mainly depends on the binding of two of its components, TRF1 and TRF2, to double-stranded telomeric repeats. Tethering of TRF proteins to telomeres occurs in a chromatin environment characterized by a very compact nucleosomal organization. In this work we show that binding of TRF1 and TRF2 to telomeric sequences is modulated by the histone octamer. By means of in vitro models, we found that TRF2 binding is strongly hampered by the presence of telomeric nucleosomes, whereas TRF1 binds efficiently to telomeric DNA in a nucleosomal context and is able to remodel telomeric nucleosomal arrays. Our results indicate that the different behavior of TRF proteins partly depends on the interaction with histone tails of their divergent N-terminal domains. We propose that the interplay between the histone octamer and TRF proteins plays a role in the steps leading to telomere deprotection.