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

NMR studies of telomeric nucleoprotein complexes involving the Myb-like domain of the human telomeric protein TRF2

In order to study the binding of the Myb-like domain of the human telomeric protein TRF2 (Myb-TRF2) with different structural components of the t-loop model, we report NMR studies of the binding of Myb-TRF2 protein with two repeats human telomeric DNA under three conformations. Our results showed that Myb-TRF2 binds to the duplex and even to the quadruplex and the random coil G-rich strand. The solution structure of Myb-TRF2 reported here looks like Myb-TRF1 suggesting similar DNA binding mode. As a matter of fact, we have shown that its binding to the human telomeric duplex presents great similarities with this of Myb-TRF1

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

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

Essonne committee of the Ligue contre le cancer M18756 M22897 Foundation ARC pour la Recherche sur le Cancer SFI20121205503International audienceTelomere 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 shel-terin, 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

The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA

TRF1 and TRF2 are key proteins in human telomeres, which, despite their similarities, have different behaviors upon DNA binding. Previous work has shown that unlike TRF1, TRF2 condenses telomeric, thus creating consequential negative torsion on the adjacent DNA, a property that is thought to lead to the stimulation of single-strand invasion and was proposed to favor telomeric DNA looping. In this report, we show that these activities, originating from the central TRFH domain of TRF2, are also displayed by the TRFH domain of TRF1 but are repressed in the full-length protein by the presence of an acidic domain at the N-terminus. Strikingly, a similar repression is observed on TRF2 through the binding of a TERRA-like RNA molecule to the N-terminus of TRF2. Phylogenetic and biochemical studies suggest that the N-terminal domains of TRF proteins originate from a gradual extension of the coding sequences of a duplicated ancestral gene with a consequential progressive alteration of the biochemical properties of these proteins. Overall, these data suggest that the N-termini of TRF1 and TRF2 have evolved to finely regulate their ability to condense DNA

Dynamique et régulation des assemblages nucléoprotéiques des télomères humains (fonctions de la protéine TRF2)

La protéine TRF2 est une protéine clé dans la dynamique des télomères, ces structures nucléoprotéiques présentes à l extrémité des chromosomes linéaires et responsables de leur protection. Bien que la manière dont les télomères s organisent pour protéger l ADN soit encore méconnue, il a été montré récemment que TRF2 est à l origine de la formation d une structure en boucle, ou boucle télomérique qui empêcherait les extrémités télomériques d être reconnus comme des coupures double brin. Un modèle propose que TRF2 permette la formation de cette boucle en induisant l invasion du simple brin télomérique terminal à l intérieur de la séquence double brin après repliement du télomère sur lui même. Les études réalisées lors de cette thèse montrent que TRF2 est en mesure de stimuler l invasion télomérique de manière indirecte facilitant l ouverture de la double hélice grâce à des modifications d ordre topologique de l ADN cible. Par ailleurs, les travaux réalisés mettent également en évidence un second mode de fixation à l ADN de TRF2, par l intermédiaire de son domaine N-terminal qui possède une affinité remarquable pour la structure des jonctions de Holliday. La dernière partie de cette thèse met en évidence l activité 5 exonucléase d une nouvelle protéine télomérique, la protéine Apollon, qui serait impliquée dans la protection des télomères. Tous ces résultats participent à une meilleure compréhension du fonctionnement de TRF2 sur les télomères et en particulier de son rôle dans la formation de la boucle télomérique.ORLEANS-BU Sciences (452342104) / SudocSudocFranceF

Multifunctionality of the Telomere-Capping Shelterin Complex Explained by Variations in Its Protein Composition

International audienceProtecting telomere from the DNA damage response is essential to avoid the entry into cellular senescence and organismal aging. The progressive telomere DNA shortening in dividing somatic cells, programmed during development, leads to critically short telomeres that trigger replicative senescence and thereby contribute to aging. In several organisms, including mammals, telomeres are protected by a protein complex named Shelterin that counteract at various levels the DNA damage response at chromosome ends through the specific function of each of its subunits. The changes in Shelterin structure and function during development and aging is thus an intense area of research. Here, we review our knowledge on the existence of several Shelterin subcomplexes and the functional independence between them. This leads us to discuss the possibility that the multifunctionality of the Shelterin complex is determined by the formation of different subcomplexes whose composition may change during aging

Erratum to: “Interstrand cross-links of cisplatin induce striking distortions in DNA”

International audienceErratum to: ‘‘Interstrand cross-links of cisplatin induce striking distortions in DNA’’ [J. Inorg. Biochem. 77 (1999) 23–29

Interstrand cross-links of cisplatin induce striking distortions in DNA

International audienceIn the reaction between cellular DNA and cisplatin, different bifunctional adducts are formed including intrastrand and interstrand cross-links. The respective role of these lesions in the cytotoxicity of the drug is not yet elucidated. This paper deals with the current knowledge on cisplatin interstrand cross-links and presents results on the formation, stability and structure of these adducts. A key step in the studies of these lesions is the recent determination of solution and crystallographic structures of double-stranded oligonucleotides containing a unique interstrand cross-link. The DNA distortions induced by this adduct exhibit unprecedented features such as the location of the platinum residue in the minor groove, the extrusion of the cytosines of the cross-linked d(GpC).d(GpC) site, the bending of the helix axis towards the minor groove and a large DNA unwinding. In addition to a detailed determination of the distortions, the high resolution of the crystal structure allowed us to locate the water molecules surrounding the adduct. The possible implications of this structure for the chemical properties and the cellular processing of cisplatin interstrand cross-links are discussed

Drosophila DSP1 and rat HMGB1 have equivalent DNA binding properties and share a similar secondary fold.

International audienceThe protein DSP1 belongs to the group of HMG-box proteins, which share the common structural feature of the HMG-box. This approximately 80 amino acid long motif binds DNA via the minor groove. DSP1 was discovered as a transcriptional co-repressor of Dorsal in Drosophila melanogaster and then was shown to participate to the remodeling of chromatin. By means of sequence alignment and gene organization, DSP1 was classified as the fly homologue of the vertebrate proteins HMGB1/2. DSP1 contains two HMG boxes flanked by two glutamine-rich domains at the N-terminus. In addition, the HMG domain of DSP1 displays two differences in its primary sequence as compared to the vertebrate HMGB1: a shorter acidic tail and a linker between the two boxes longer by 6 amino acids. By comparing several functional parameters of DSP1 with those of HMGB1, the present study establishes the functional equivalence of both proteins in terms of DNA recognition. The major structural difference between the two proteins, the glutamine-rich N-terminal tail of DSP1, which does not exist in HMGB1, did not interfere with any of the studied DNA-binding properties of the proteins