Irreversible inhibition of TRF2TRFH recruiting functions by a covalent cyclic peptide induces telomeric replication stress in cancer cells.

The TRF2 shelterin component is an essential regulator of telomere homeostasis and genomic stability. Mutations in the TRF2TRFH domain physically impair t-loop formation and prevent the recruitment of several factors that promote efficient telomere replication, causing telomeric DNA damage. Here, we design, synthesize, and biologically test covalent cyclic peptides that irreversibly target the TRF2TRFH domain. We identify APOD53 as our most promising compound, as it consistently induces a telomeric DNA damage response in cancer cell lines. APOD53 forms a covalent adduct with a reactive cysteine residue present in the TRF2TRFH domain and induces phenotypes consistent with TRF2TRFH domain mutants. These include induction of a telomeric DNA damage response, increased telomeric replication stress, and impaired recruitment of RTEL1 and SLX4 to telomeres. We demonstrate that APOD53 impairs cancer cell growth and find that co-treatment with APOD53 can exacerbate telomere replication stress caused by the G4 stabilizer RHPS4 and low dose aphidicolin (APH).

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.

Increase in lamin B1 promotes telomere instability by disrupting the shelterin complex in human cells.

Telomere maintenance is essential to preserve genomic stability and involves telomere-specific proteins, DNA replication and repair proteins. Lamins are key components of the nuclear envelope and play numerous roles, including maintenance of the nuclear integrity, regulation of transcription, and DNA replication. Elevated levels of lamin B1, one of the major lamins, have been observed in some human pathologies and several cancers. Yet, the effect of lamin B1 dysregulation on telomere maintenance remains unknown. Here, we unveil that lamin B1 overexpression drives telomere instability through the disruption of the shelterin complex. Indeed, lamin B1 dysregulation leads to an increase in telomere dysfunction-induced foci, telomeric fusions and telomere losses in human cells. Telomere aberrations were preceded by mislocalizations of TRF2 and its binding partner RAP1. Interestingly, we identified new interactions between lamin B1 and these shelterin proteins, which are strongly enhanced at the nuclear periphery upon lamin B1 overexpression. Importantly, chromosomal fusions induced by lamin B1 in excess were rescued by TRF2 overexpression. These data indicated that lamin B1 overexpression triggers telomere instability through a mislocalization of TRF2. Altogether our results point to lamin B1 as a new interacting partner of TRF2, that is involved in telomere stability.

The Long Linker Region of Telomere-Binding Protein TRF2 Is Responsible for Interactions with Lamins.

Telomere-binding factor 2 (TRF2) is part of the shelterin protein complex found at chromosome ends. Lamin A/C interacts with TRF2 and influences telomere position. TRF2 has an intrinsically disordered region between the ordered dimerization and DNA-binding domains. This domain is referred to as the long linker region of TRF2, or udTRF2. We suggest that udTRF2 might be involved in the interaction between TRF2 and lamins. The recombinant protein corresponding to the udTRF2 region along with polyclonal antibodies against this region were used in co-immunoprecipitation with purified lamina and nuclear extracts. Co-immunoprecipitation followed by Western blots and mass spectrometry indicated that udTRF2 interacts with lamins, preferably lamins A/C. The interaction did not involve any lamin-associated proteins, was not dependent on the post-translation modification of lamins, nor did it require their higher-order assembly. Besides lamins, a number of other udTRF2-interacting proteins were identified by mass spectrometry, including several heterogeneous nuclear ribonucleoproteins (hnRNP A2/B1, hnRNPA1, hnRNP A3, hnRNP K, hnRNP L, hnRNP M), splicing factors (SFPQ, NONO, SRSF1, and others), helicases (DDX5, DHX9, and Eif4a3l1), topoisomerase I, and heat shock protein 71, amongst others. Some of the identified interactors are known to be involved in telomere biology; the roles of the others remain to be investigated. Thus, the long linker region of TRF2 (udTRF2) is a regulatory domain responsible for the association between TRF2 and lamins and is involved in interactions with other proteins.

Transcriptional regulation of telomeric repeat-containing RNA by acridine derivatives.

Telomere is a specialized DNA-protein complex that plays an important role in maintaining chromosomal integrity. Shelterin is a protein complex formed by six different proteins, with telomeric repeat factors 1 (TRF1) and 2 (TRF2) binding to double-strand telomeric DNA. Telomeric DNA consists of complementary G-rich and C-rich repeats, which could form G-quadruplex and intercalated motif (i-motif), respectively, during cell cycle. Its G-rich transcription product, telomeric repeat-containing RNA (TERRA), is essential for telomere stability and heterochromatin formation. After extensive screening, we found that acridine derivative 2c and acridine dimer DI26 could selectively interact with TRF1 and telomeric i-motif, respectively. Compound 2c blocked the binding of TRF1 with telomeric duplex DNA, resulting in up-regulation of TERRA. Accumulated TERRA could bind with TRF1 at its allosteric site and further destabilize its binding with telomeric DNA. In contrast, DI26 could destabilize telomeric i-motif, resulting in down-regulation of TERRA. Both compounds exhibited anti-tumour activity for A549 cells, but induced different DNA damage pathways. Compound 2c significantly suppressed tumour growth in A549 xenograft mouse model. The function of telomeric i-motif structure was first studied with a selective binding ligand, which could play an important role in regulating TERRA transcription. Our results showed that appropriate level of TERRA transcript could be important for stability of telomere, and acridine derivatives could be further developed as anti-cancer agents targeting telomere. This research increased understanding for biological roles of telomeric i-motif, TRF1 and TERRA, as potential anti-cancer drug targets.

Non-duplex G-Quadruplex DNA Structure: A Developing Story from Predicted Sequences to DNA Structure-Dependent Epigenetics and Beyond.

The story of the non-duplex DNA form known as the G-quadruplex (G4) has traversed a winding path. From initial skepticism followed by debate to a surge in interest, the G4 story intertwines many threads. Starting with computational predictions of a gene regulatory role, which now include epigenetic functions, our group was involved in many of these advances along with many other laboratories. Following a brief background, set in the latter half of the last century when the concept of the G4 as a structure took ground, here we account the developments. This is through a lens that though focused on our groups' research presents work from many other groups that played significant roles. Together these provide a broad perspective to the G4 story. Initially we were intrigued on seeing potential G4 (pG4)-forming sequences, then known to be found primarily at the telomeres and immunoglobin switch regions, occurring throughout the genome and being particularly prevalent in promoters of bacteria. We further observed that pG4s were not only prevalent but also conserved through evolution in promoters of human, chimpanzee, mouse and rat genomes. This was between 2005 and 2007. Encouraged by these partly and partly in response to the view held by many that genome-wide presence of G4s were genomic "accidents", the focus shifted to seeking experimental evidence.In the next year, 2008, two independent findings showed promise. First, on treating human cancer cells with G4-binding ligands, we observed widespread change in gene expression. Second, our search for the missing G4-specific transcription factor, without which, importantly, G4s in promoters posed only half the story, yielded results. We determined how NM23-H2 (also known as NME2 or NDPK-B) interacts with G4s and how interaction of NM23-H2 with a G4 in the promoter of the oncogene c-myc was important for regulation of c-myc transcription. NM23-H2, and subsequently many other similar factors discovered by multiple groups, is possibly giving shape to what might be the "G4-transcriptome". Later, a close look at NM23-H2-G4 interaction in regulation of the human reverse transcriptase gene (hTERT) revealed the role of G4s in local epigenetic modifications. Meanwhile work from others showed how G4s impact histone modifications following replication. Together these show the intrinsic role of DNA sequence, through formation of DNA structure, in epigenetics.More recent work, however, was waiting to reveal aspects that tend to bring forth a completely new understanding of G4s. We observed that the telomere-repeat-binding-factor-2 (TRF2), known canonically to be telomere-associated, binds extensively outside telomeres throughout the genome. Moreover, a large fraction of the non-telomeric TRF2 sites comprise G4s. Second, the extent of non-telomeric TRF2 binding at promoters was dependent on telomere length. Thereby TRF2-induced epigenetic gene regulation was telomere-dependent. Together these implicate underlying connections that show signs of addressing an intriguing unanswered question that takes us back to the beginning: Why are G4s prevalent in two distinct regions, the telomeres and gene promoters?

Microcephalin 1/BRIT1-TRF2 interaction promotes telomere replication and repair, linking telomere dysfunction to primary microcephaly.

Telomeres protect chromosome ends from inappropriately activating the DNA damage and repair responses. Primary microcephaly is a key clinical feature of several human telomere disorder syndromes, but how microcephaly is linked to dysfunctional telomeres is not known. Here, we show that the microcephalin 1/BRCT-repeats inhibitor of hTERT (MCPH1/BRIT1) protein, mutated in primary microcephaly, specifically interacts with the TRFH domain of the telomere binding protein TRF2. The crystal structure of the MCPH1-TRF2 complex reveals that this interaction is mediated by the MCPH1 330YRLSP334 motif. TRF2-dependent recruitment of MCPH1 promotes localization of DNA damage factors and homology directed repair of dysfunctional telomeres lacking POT1-TPP1. Additionally, MCPH1 is involved in the replication stress response, promoting telomere replication fork progression and restart of stalled telomere replication forks. Our work uncovers a previously unrecognized role for MCPH1 in promoting telomere replication, providing evidence that telomere replication defects may contribute to the onset of microcephaly.

CDK phosphorylation of TRF2 controls t-loop dynamics during the cell cycle.

The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3' single-stranded telomere end can assemble into a lasso-like t-loop configuration1,2, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks2. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3' end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.

Functional Comparison of Short and Long Isoforms of the TRF2 Protein in Drosophila melanogaster.

TRF2 protein (TBP-related factor 2) can substitute for TBP forming alternative transcription initiation complexes on TATA-less promoters, including the promoters of histone H1 and piRNA clusters required for transposon repression. The Drosophilatrf2 gene codes for two isoforms: a "short" and a "long" one, in which the same short TRF2 sequence is preceded by a long N-terminal domain. Here, we demonstrated that the long TFR2 isoform has a greater functional activity than the short isoform by expressing each of them at a reduced rate under the endogenous promoters. Expression of the long isoform alone affects neither the flies' viability nor the sex ratio. Expression of the short isoform alone leads to the phenotype described for the trf2 gene insufficiency and derepression of transposable elements, that is, decreased viability, disturbance of homologous chromosome pairing and segregation, and apparent female-biased sex ratio.

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 30 years, 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.

Telomere Loop Dynamics in Chromosome End Protection.

Telomeres regulate DNA damage response (DDR) and DNA repair activity at chromosome ends. How telomere macromolecular structure contributes to ATM regulation and its potential dissociation from control over non-homologous end joining (NHEJ)-dependent telomere fusion is of central importance to telomere-dependent cell aging and tumor suppression. Using super-resolution microscopy, we identify that ATM activation at mammalian telomeres with reduced TRF2 or at human telomeres during mitotic arrest occurs specifically with a structural change from telomere loops (t-loops) to linearized telomeres. Additionally, we find the TRFH domain of TRF2 regulates t-loop formation while suppressing ATM activity. Notably, we demonstrate that ATM activation and telomere linearity occur separately from telomere fusion via NHEJ and that linear DDR-positive telomeres can remain resistant to fusion, even during an extended G1 arrest, when NHEJ is most active. Collectively, these results suggest t-loops act as conformational switches that specifically regulate ATM activation independent of telomere mechanisms to inhibit NHEJ.

Extratelomeric Binding of the Telomere Binding Protein TRF2 at the PCGF3 Promoter Is G-Quadruplex Motif-Dependent.

Telomere repeat binding factor 2 (TRF2) is critical for the protection of chromosome ends. Mounting evidence suggests that TRF2 associates with extratelomeric sites and TRF2 functions may not be limited to telomeres. Here, we show that the PCGF3 promoter harbors a sequence capable of forming the DNA secondary structure G-quadruplex motif, which is required for binding of TRF2 at the PCGF3 promoter. We demonstrate that promoter binding by TRF2 mediates PCGF3 promoter activity, and both the N-terminal and C-terminal domains of TRF2 are necessary for promoter activity. Altogether, this shows for the first time that a telomere binding factor may regulate a component of the polycomb group of proteins.

The structurally similar TRFH domain of TRF1 and TRF2 dimers shows distinct behaviour towards TIN2.

The telomere repeat binding-factor 1 and 2 (TRF1 and TRF2) proteins of the shelterin complex bind to duplex telomeric DNA as homodimers, and the homodimerization is mediated by their TRFH (TRF-homology) domains. We performed molecular dynamic (MD) simulations of the dimer forms of TRF1TRFH and TRF2TRFH in the presence/absence of the TIN2TBM (TIN2, TRF-interacting nuclear protein 2, TBM, TRF-binding motif) peptide. The MD results suggest that TIN2TBM is necessary to ensure the stability of TRF1TRFH homodimer but not the TRF2TRFH homodimer. In TRF1-TIN2-TRF2 complex, the peptide enhances the protein-protein interactions to yield a stable heterodimer. Both monomers in TRF1TRFH homodimer interact almost equally with the peptide, whereas in TRF2TRFH homodimer, monomer TRF2TRFH(M1) exhibits more dominant interactions than the TRF2TRFH(M2). The common residues of TRF1/2TRFH(M1) that form interactions with TIN2TBM in all peptide-bound systems originate from the H3 (helix) and L3 (loop) regions. Additionally, in the homodimer systems, residues of TRF1/2TRFH(M2) also interact with the peptide. The residue pair E71-K213 is responsible for different conformations of TRF1TRFH homodimers; specifically, this residue pair enhances the protein-peptide/protein interactions in peptide-bound/unbound systems, respectively. TRF1TRFH and TRF2TRFH proteins have a conserved but different interface responsible for the protein-protein/peptide interactions that exist in the corresponding dimers.

TRF1 participates in chromosome end protection by averting TRF2-dependent telomeric R loops.

The shelterin protein TRF2 assembles protective T loops at chromosome ends by stimulating intramolecular invasion of the telomeric G-rich single-stranded DNA (ssDNA) overhang into the duplex telomeric array. The other shelterin factor, TRF1, is thought to mainly facilitate telomeric dsDNA replication without directly participating in end protection. Here we show that in vitro human TRF2 stimulates invasion of G-rich TERRA-like RNA into telomeric dsDNA, leading to formation of telomeric RNA-DNA hybrids (telR loops). The N-terminal basic domain of TRF2 binds to TERRA-like RNA and enables TRF2 to promote efficient RNA invasion. TRF1, through its N-terminal acidic domain, counteracts TRF2-mediated RNA invasion but not ssDNA invasion. In vivo, when TRF1 is depleted or replaced with a variant lacking the acidic domain, TRF2 induces formation of telR loops, which in turn cause telomere loss. Hence, uncontrolled TRF2 threatens telomere integrity, and TRF1 directly supports end protection by suppressing harmful telR loops.

Structural and functional analyses of the mammalian TIN2-TPP1-TRF2 telomeric complex.

Telomeres are nucleoprotein complexes that play essential roles in protecting chromosome ends. Mammalian telomeres consist of repetitive DNA sequences bound by the shelterin complex. In this complex, the POT1-TPP1 heterodimer binds to single-stranded telomeric DNAs, while TRF1 and TRF2-RAP1 interact with double-stranded telomeric DNAs. TIN2, the linchpin of this complex, simultaneously interacts with TRF1, TRF2, and TPP1 to mediate the stable assembly of the shelterin complex. However, the molecular mechanism by which TIN2 interacts with these proteins to orchestrate telomere protection remains poorly understood. Here, we report the crystal structure of the N-terminal domain of TIN2 in complex with TIN2-binding motifs from TPP1 and TRF2, revealing how TIN2 interacts cooperatively with TPP1 and TRF2. Unexpectedly, TIN2 contains a telomeric repeat factor homology (TRFH)-like domain that functions as a protein-protein interaction platform. Structure-based mutagenesis analyses suggest that TIN2 plays an important role in maintaining the stable shelterin complex required for proper telomere end protection.

Basic domain of telomere guardian TRF2 reduces D-loop unwinding whereas Rap1 restores it.

Telomeric repeat binding factor 2 (TRF2) folds human telomeres into loops to prevent unwanted DNA repair and chromosome end-joining. The N-terminal basic domain of TRF2 (B-domain) protects the telomeric displacement loop (D-loop) from cleavage by endonucleases. Repressor activator protein 1 (Rap1) binds TRF2 and improves telomeric DNA recognition. We found that the B-domain of TRF2 stabilized the D-loop and thus reduced unwinding by BLM and RPA, whereas the formation of the Rap1-TRF2 complex restored DNA unwinding. To understand how the B-domain of TRF2 affects DNA binding and D-loop processing, we analyzed DNA binding of full-length TRF2 and a truncated TRF2 construct lacking the B-domain. We quantified how the B-domain improves TRF2's interaction with DNA via enhanced long-range electrostatic interactions. We developed a structural envelope model of the B-domain bound on DNA. The model revealed that the B-domain is flexible in solution but becomes rigid upon binding to telomeric DNA. We proposed a mechanism for how the B-domain stabilizes the D-loop.

TRF2 binds branched DNA to safeguard telomere integrity.

Although t-loops protect telomeres, they are at risk of cleavage by Holliday junction (HJ) resolvases if branch migration converts the three-way t-loop junction into four-way HJs. T-loop cleavage is repressed by the TRF2 basic domain, which binds three- and four-way junctions and protects HJs in vitro. By replacing the basic domain with bacterial-protein domains binding three- and four-way junctions, we demonstrated the in vivo relevance of branched-DNA binding. Branched-DNA binding also repressed PARP1, presumably by masking the PARP1 site in the t-loop junction. Although PARP1 recruits HJ resolvases and promotes t-loop cleavage, PARP1 activation alone did not result in t-loop cleavage, thus suggesting that the basic domain also prevents formation of HJs. Concordantly, removal of HJs by BLM helicase mitigated t-loop cleavage in response to loss of the basic domain. We propose that TRF2 masks and stabilizes the t-loop three-way junction, thereby protecting telomeres from detrimental deletions and PARP1 activation.

Disruption of direct 3D telomere-TRF2 interaction through two molecularly disparate mechanisms is a hallmark of primary Hodgkin and Reed-Sternberg cells.

In classical Hodgkin's lymphoma (cHL), specific changes in the 3D telomere organization cause progression from mononuclear Hodgkin cells (H) to multinucleated Reed-Sternberg cells (RS). In a post-germinal center B-cell in vitro model, permanent latent membrane protein 1 (LMP1) expression, as observed in Epstein-Barr virus (EBV)-associated cHL, results in multinuclearity and complex chromosomal aberrations through downregulation of key element of the shelterin complex, the telomere repeat binding factor 2 (TRF2). Thus, we hypothesized that the three-dimensional (3D) telomere-TRF2 interaction was progressively disturbed during transition from H to RS cells. To this end, we developed and applied for the first time a combined quantitative 3D TRF2-telomere immune fluorescent in situ hybridization (3D TRF2/Telo-Q-FISH) technique to monolayers of primary H and RS cells, and adjacent benign internal control lymphocytes of lymph node biopsy suspensions from diagnostic lymph node biopsies of 14 patients with cHL. We show that H and RS cells are characterized by two distinct patterns of disruption of 3D telomere-TRF2 interaction. Disruption pattern A is defined by massive attrition of telomere signals and a considerable increase of TRF2 signals not associated with telomeres. This pattern is restricted to EBV-negative cHL. Disruption pattern B is defined by telomere de-protection due to an impressive loss of TRF2 signals, physically linked to telomeres. This pattern is typical of, but is not restricted to, LMP1+EBV-associated cHL. In the disruption pattern B group, so-called 'ghost' end-stage RS cells, void of both TRF2 and telomere signals, were identified, whether or not associated with EBV. Our findings demonstrate that two molecularly disparate mechanisms converge on the level of 3D telomere-TRF2 interaction in the formation of RS cells.

NBS1 Phosphorylation Status Dictates Repair Choice of Dysfunctional Telomeres.

Telomeres employ TRF2 to protect chromosome ends from activating the DNA damage sensor MRE11-RAD50-NBS1 (MRN), thereby repressing ATM-dependent DNA damage checkpoint responses. How TRF2 prevents MRN activation at dysfunctional telomeres is unclear. Here, we show that the phosphorylation status of NBS1 determines the repair pathway choice of dysfunctional telomeres. The crystal structure of the TRF2-NBS1 complex at 3.0 Å resolution shows that the NBS1 429YQLSP433 motif interacts specifically with the TRF2TRFH domain. Phosphorylation of NBS1 serine 432 by CDK2 in S/G2 dissociates NBS1 from TRF2, promoting TRF2-Apollo/SNM1B complex formation and the protection of leading-strand telomeres. Classical-NHEJ-mediated repair of telomeres lacking TRF2 requires phosphorylated NBS1S432 to activate ATM, while interaction of de-phosphorylated NBS1S432 with TRF2 promotes alternative-NHEJ repair of telomeres lacking POT1-TPP1. Our work advances understanding of how the TRF2TRFH domain orchestrates telomere end protection and reveals how the phosphorylation status of the NBS1S432 dictates repair pathway choice of dysfunctional telomeres.

SIRT6 interacts with TRF2 and promotes its degradation in response to DNA damage.

Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.