TRF2 controls telomeric nucleosome organization in a cell cycle phase-dependent manner

Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres.By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping.Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization.     

Distinct functions of POT1 at telomeres

The mammalian protein POT1 binds to telomeric single-stranded DNA (ssDNA), protecting chromosome ends from being detected as sites of DNA damage. POT1 is composed of an N-terminal ssDNA-binding domain and a C-terminal protein interaction domain. With regard to the latter, POT1 heterodimerizes with the protein TPP1 to foster binding to telomeric ssDNA in vitro and binds the telomeric double-stranded-DNA-binding protein TRF2. We sought to determine which of these functions-ssDNA, TPP1, or TRF2 binding-was required to protect chromosome ends from being detected as DNA damage. Using separation-of-function POT1 mutants deficient in one of these three activities, we found that binding to TRF2 is dispensable for protecting telomeres but fosters robust loading of POT1 onto telomeric chromatin. Furthermore, we found that the telomeric ssDNA-binding activity and binding to TPP1 are required in cis for POT1 to protect telomeres. Mechanistically, binding of POT1 to telomeric ssDNA and association with TPP1 inhibit the localization of RPA, which can function as a DNA damage sensor, to telomeres.     

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.     

TRF2-Tethered TIN2 Can Mediate Telomere Protection by TPP1/POT1

The shelterin protein TIN2 is required for the telomeric accumulation of TPP1/POT1 heterodimers and for the protection of telomeres by the POT1 proteins (POT1a and POT1b in the mouse). TIN2 also binds to TRF1 and TRF2, improving the telomeric localization of TRF2 and its function. Here, we ask whether TIN2 needs to interact with both TRF1 and TRF2 to mediate the telomere protection afforded by TRF2 and POT1a/b. Using a TIN2 allele deficient in TRF1 binding (TIN2-L247E), we demonstrate that TRF1 is required for optimal recruitment of TIN2 to telomeres and document phenotypes associated with the TIN2-L247E allele that are explained by insufficient TIN2 loading onto telomeres. To bypass the requirement for TRF1-dependent recruitment, we fused TIN2-L247E to the TRF2-interacting (RCT) domain of Rap1. The RCT-TIN2-L247E fusion showed improved telomeric localization and was fully functional in terms of chromosome end protection by TRF2, TPP1/POT1a, and TPP1/POT1b. These data indicate that when sufficient TIN2 is loaded onto telomeres, its interaction with TRF1 is not required to mediate the function of TRF2 and the TPP1/POT1 heterodimers. We therefore conclude that shelterin can protect chromosome ends as a TRF2-tethered TIN2/TPP1/POT1 complex that lacks a physical connection to TRF1.     

Apollo, an Artemis-related nuclease, interacts with TRF2 and protects human telomeres in S phase

Human chromosome ends are protected by shelterin, an abundant six-subunit protein complex that binds specifically to the telomeric-repeat sequences, regulates telomere length, and ensures that chromosome ends do not elicit a DNA-damage response (reviewed in). Using mass spectrometry of proteins associated with the shelterin component Rap1, we identified an SMN1/PSO2 nuclease family member that is closely related to Artemis. We refer to this protein as Apollo and report that Apollo has the ability to localize to telomeres through an interaction with the shelterin component TRF2. Although its low abundance at telomeres indicates that Apollo is not a core component of shelterin, Apollo knockdown with RNAi resulted in senescence and the activation of a DNA-damage signal at telomeres as evidenced by telomere-dysfunction-induced foci (TIFs). The TIFs occurred primarily in S phase, suggesting that Apollo contributes to a processing step associated with the replication of chromosome ends. Furthermore, some of the metaphase chromosomes showed two telomeric signals at single-chromatid ends, suggesting an aberrant telomere structure. We propose that the Artemis-like nuclease Apollo is a shelterin accessory factor required for the protection of telomeres during or after their replication.     

Distinct roles of TRF1 in the regulation of telomere structure and lengthening

The telomere is a functional chromatin structure that consists of G-rich repetitive sequences and various associated proteins. Telomeres protect chromosomal ends from degradation, provide escape from the DNA damage response, and regulate telomere lengthening by telomerase. Multiple proteins that localize at telomeres form a complex called shelterin/telosome. One component, TRF1, is a double-stranded telomeric DNA binding protein. Inactivation of TRF1 disrupts telomeric localization of other shelterin components and induces chromosomal instability. Here, we examined how the telomeric localization of shelterin components is crucial for TRF1-mediated telomere-associated functions. We found that many of the mTRF1 deficient phenotypes, including chromosomal instability, growth defects, and dysfunctional telomere damage response, were suppressed by the telomere localization of shelterin components in the absence of functional mTRF1. However, abnormal telomere signals and telomere elongation phenotypes were either not rescued or only partially rescued, respectively. These data suggest that TRF1 regulates telomere length and function by at least two mechanisms; in one TRF1 acts through the recruiting/tethering of other shelterin components to telomeres, and in the other TRF1 seems to play a more direct role.     

In Vivo Stoichiometry of Shelterin Components

Human telomeres bind shelterin, the six-subunit protein complex that protects chromosome ends from the DNA damage response and regulates telomere length maintenance by telomerase. We used quantitative immunoblotting to determine the abundance and stoichiometry of the shelterin proteins in the chromatin-bound protein fraction of human cells. The abundance of shelterin components was similar in primary and transformed cells and was not correlated with telomere length. The duplex telomeric DNA binding factors in shelterin, TRF1 and TRF2, were sufficiently abundant to cover all telomeric DNA in cells with short telomeres. The TPP1·POT1 heterodimer was present 50–100 copies/telomere, which is in excess of its single-stranded telomeric DNA binding sites, indicating that some of the TPP1·POT1 in shelterin is not associated with the single-stranded telomeric DNA. TRF2 and Rap1 were present at 1:1 stoichiometry as were TPP1 and POT1. The abundance of TIN2 was sufficient to allow each TRF1 and TRF2 to bind to TIN2. Remarkably, TPP1 and POT1 were ∼10-fold less abundant than their TIN2 partner in shelterin, raising the question of what limits the accumulation of TPP1·POT1 at telomeres. Finally, we report that a 10-fold reduction in TRF2 affects the regulation of telomere length but not the protection of telomeres in tumor cell lines.     

Give me a break: How telomeres suppress the DNA damage response

Linear organization of the genome requires mechanisms to protect and replicate chromosome ends. To this end eukaryotic cells evolved telomeres, specialized nucleoproteic complexes, and telomerase, the enzyme that maintains the telomeric DNA. Telomeres allow cells to distinguish chromosome ends from sites of DNA damage. In mammalian cells this is accomplished by a protein complex, termed shelterin, that binds to telomeric DNA and is able to shield chromosome ends from the DNA damage machinery. In recent years, we have seen major advances in our understanding of how this protein complex works due to the generation of mouse models carrying mutations of individual shelterin components. This review will focus on our current understanding of how the shelterin complex is able to suppress the DNA damage response pathways, and on the cellular and organismal outcomes of telomere dysfunction.     

Comparison between TRF2 and TRF1 of their telomeric DNA-bound structures and DNA-binding activities

Mammalian telomeres consist of long tandem arrays of double-stranded telomeric TTAGGG repeats packaged by the telomeric DNA-binding proteins TRF1 and TRF2. Both contain a similar C-terminal Myb domain that mediates sequence-specific binding to telomeric DNA. In a DNA complex of TRF1, only the single Myb-like domain consisting of three helices can bind specifically to double-stranded telomeric DNA. TRF2 also binds to double-stranded telomeric DNA. Although the DNA binding mode of TRF2 is likely identical to that of TRF1, TRF2 plays an important role in the t-loop formation that protects the ends of telomeres. Here, to clarify the details of the double-stranded telomeric DNA-binding modes of TRF1 and TRF2, we determined the solution structure of the DNA-binding domain of human TRF2 bound to telomeric DNA; it consists of three helices, and like TRF1, the third helix recognizes TAGGG sequence in the major groove of DNA with the N-terminal arm locating in the minor groove. However, small but significant differences are observed; in contrast to the minor groove recognition of TRF1, in which an arginine residue recognizes the TT sequence, a lysine residue of TRF2 interacts with the TT part. We examined the telomeric DNA-binding activities of both DNA-binding domains of TRF1 and TRF2 and found that TRF1 binds more strongly than TRF2. Based on the structural differences of both domains, we created several mutants of the DNA-binding domain of TRF2 with stronger binding activities compared to the wild-type TRF2.     

Requirement of DDX39 DEAD box RNA helicase for genome integrity and telomere protection

Human chromosome ends associate with shelterin, a six-protein complex that protects telomeric DNA from being recognized as sites of DNA damage. The shelterin subunit TRF2 has been implicated in the protection of chromosome ends by facilitating their organization into the protective capping structure and by associating with several accessory proteins involved in various DNA transactions. Here we describe the characterization of DDX39 DEAD-box RNA helicase as a novel TRF2-interacting protein. DDX39 directly interacts with the telomeric repeat binding factor homology domain of TRF2 via the FXLXP motif (where X is any amino acid). DDX39 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT but has no effect on telomerase activity. Whereas overexpression of DDX39 in telomerase-positive human cancer cells led to progressive telomere elongation, depletion of endogenous DDX39 by small hairpin RNA (shRNA) resulted in telomere shortening. Furthermore, depletion of DDX39 induced DNA-damage response foci at internal genome as well as telomeres as evidenced by telomere dysfunction-induced foci. Some of the metaphase chromosomes showed no telomeric signal at chromatid ends, suggesting an aberrant telomere structure. Our findings suggest that DDX39, in addition to its role in mRNA splicing and nuclear export, is required for global genome integrity as well as telomere protection and represents a new pathway for telomere maintenance by modulating telomere length homeostasis.     

Human Telomere Repeat Binding Factor TRF1 Replaces TRF2 Bound to Shelterin Core Hub TIN2 when TPP1 Is Absent

Human telomeric repeat binding factors TRF1 and TRF2 along with TIN2 form the core of the shelterin complex that protects chromosome ends against unwanted end-joining and DNA repair. We applied a single-molecule approach to assess TRF1–TIN2–TRF2 complex formation in solution at physiological conditions. Fluorescence cross-correlation spectroscopy was used to describe the complex assembly by analyzing how coincident fluctuations of differently labeled TRF1 and TRF2 correlate when they move together through the confocal volume of the microscope. We observed, at the single-molecule level, that TRF1 effectively substitutes TRF2 on TIN2. We assessed also the effect of another telomeric factor TPP1 that recruits telomerase to telomeres. We found that TPP1 upon binding to TIN2 induces changes that expand TIN2 binding capacity, such that TIN2 can accommodate both TRF1 and TRF2 simultaneously. We suggest a molecular model that explains why TPP1 is essential for the stable formation of TRF1–TIN2–TRF2 core complex.     

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.     

A higher-order entity formed by the flexible assembly of RAP1 with TRF2

Telomere integrity is essential to maintain genome stability, and telomeric dysfunctions are associated with cancer and aging pathologies. In human, the shelterin complex binds TTAGGG DNA repeats and provides capping to chromosome ends. Within shelterin, RAP1 is recruited through its interaction with TRF2, and TRF2 is required for telomere protection through a network of nucleic acid and protein interactions. RAP1 is one of the most conserved shelterin proteins although one unresolved question is how its interaction may influence TRF2 properties and regulate its capacity to bind multiple proteins. Through a combination of biochemical, biophysical and structural approaches, we unveiled a unique mode of assembly between RAP1 and TRF2. The complete interaction scheme between the full-length proteins involves a complex biphasic interaction of RAP1 that directly affects the binding properties of the assembly. These results reveal how a non-DNA binding protein can influence the properties of a DNA-binding partner by mutual conformational adjustments.     

TPP1 as a versatile player at the ends of chromosomes

Telomeres, the ends of linear eukaryotic chromosomes, are tandem DNA repeats and capped by various telomeric proteins. These nucleoprotein complexes protect telomeres from DNA damage response （DDR）, recombination, and end-to-end fusions, ensuring genome stability. The human telosome/shelterin complex is one of the best-studied telomere-associated protein complexes, made up of six core telomeric proteins TRF1, TRF2, TIN2, RAPI, POT1, and TPPI. TPP1, also known as adrenocortical dysplasia protein homolog （ACD）, is a putative mammalian homolog of TEBP-β and belongs to the oligonucleotide binding （OB）-fold-containing protein family. Three functional domains have been identified within TPP1, the N-terminal OB fold, the POT1 binding recruitment domain （RD）, and the carboxyl-terminal TIN2-interacting domain （TID）. TPP1 can interact with both POT1 and TIN2 to maintain telomere structure, and mediate telomerase recruitment for telomere elongation. These features have indicated TPP1 play an essential role in telomere maintenance. Here, we will review important findings that highlight the functional significance of TPP1, with a focus on its interaction with other telosome components and the telomerase. We will also discuss potential implications in disease therapies.     

PinX1 localizes to telomeres and stabilizes TRF1 at mitosis

Human telomeres are DNA-protein complexes that cap and protect the ends of chromosomes. The protein PinX1 associates with telomeres through an interaction with the resident double-stranded telomere-binding protein TRF1. PinX1 also binds to and inhibits telomerase, the enzyme responsible for complete replication of telomeric DNA. We now report that endogenous PinX1 associates with telomeres primarily at mitosis. Moreover, knockdown of PinX1 caused delayed mitotic entry and reduced the accumulation of TRF1 on telomeres during this stage of the cell cycle. Taking these findings together, we suggest that one function of PinX1 is to stabilize TRF1 during mitosis, perhaps to promote transition into M phase of the cell cycle.     

Telomeric 3' overhangs derive from resection by Exo1 and Apollo and fill-in by POT1b-associated CST

A 3' overhang is critical for the protection and maintenance of mammalian telomeres, but its synthesis must be regulated to avoid excessive resection of the 5' end, which could cause telomere shortening. How this balance is achieved in mammals has not been resolved. Here, we determine the mechanism for 3' overhang synthesis in mouse cells by evaluating changes in telomeric overhangs throughout the cell cycle and at leading- and lagging-end telomeres. Apollo, a nuclease bound to the shelterin subunit TRF2, initiates formation of the 3' overhang at leading-, but not lagging-end telomeres. Hyperresection by Apollo is blocked at both ends by the shelterin protein POT1b. Exo1 extensively resects both telomere ends, generating transient long 3' overhangs in S/G2. CST/AAF, a DNA polα.primase accessory factor, binds POT1b and shortens the extended overhangs produced by Exo1, likely through fill-in synthesis. 3' overhang formation is thus a multistep, shelterin-controlled process, ensuring functional telomeric overhangs at chromosome ends.