Role of TRF2 and its partners in the formation and the processing of the t-loop of human telomeres – TELOLOOP

Our project combines techniques from structural, molecular, cellular biology and biochemistry. In cells, we are studying the formation of T-loops, their removal and the proteins that participate in these processes. For the study of molecular mechanisms, we are building molecular models and analyze the former reactions in vitro. Finally, 3D structures are studied by different biophysical techniques.

The studies we performed during the last 18 months have been especially devoted to the establishment of techniques and to the production of tools for future studies. We have also established a few important points:
- Data that we have published in an international journal (Nucleic Acids Research) reveal the important role played by the first domain of TRF2. Indeed, depending on its nature and the modifications it can sustain, this domain can profoundly alter TRF2 functions.
- We have unveiled new functional domains in the SLX4 protein, a protein involved in T-loop removal which could play an important role in telomere biology.
- We have obtained the low resolution 3D image of TRF2 and of its complex with its partner RAP1.

During the next period, stress will be put on four main topics:
- The biochemical tools that we have produced will be put to use to elucidate the mechanisms controlling T-loop formation. We hope to determine which domain(s) of the TRF2 protein are involved and which TRF2 partner(s) participate.
- A TRF2 mutant that we recently produced will be used to determine the role of T-loop in telomere protection.
- Using several mutant of SLX4, we will study the role of this protein in T-loop removal.
- We will endeavor to obtain high resolution (atomic resolution) and low resolution 3D structures of TRF2, RAP1 and complexes of these proteins with DNA.

We have published an article in an international journal:
Poulet A, Pisano S, Faivre-Moskalenko C, Pei B, Tauran Y, Haftek-Terreau Z, Brunet F, Le Bihan YV, Ledu MH, Montel F, Hugo N, Amiard S, Argoul F, Chaboud A, Gilson E, Giraud-Panis MJ. The N-terminal domains of TRF1 and TRF2 regulate their ability to condense telomeric DNA. Nucleic Acids Research, 2012, 40, 2566-2576.

Although the importance of telomeres in genome stability and cell viability has been discovered long ago, the underlying mechanisms have only recently begun to be elucidated. Today a wealth of data attests of the involvement of these chromosomic elements in cancer and ageing. Indeed, telomeres shorten at each DNA replication which ultimately leads to cell senescence. This considered as an anti-tumor barrier and is thought to participate in the ageing process. In fact, telomeric modifications and alterations are hallmarks of ageing and cancers. One of the main concepts that arose from recent studies is that the size of the telomere per se cannot be considered as the only parameter that defines a functional telomere state. Indeed, other parameters are equally important such as appropriate folding and appropriate dynamics of the telomeric complexes. In human cells, one of these complexes is formed through the association of six major telomeric proteins TRF1, TRF2, RAP1, TIN2, TPP1 and POT1 and is called shelterin complex or telosome. Other sub-complexes containing less than the six members of the shelterin/telosome have also been described and the proteins that compose it exhibit separate functions on telomeres. TRF1 and its complexes act as regulators of telomeres size, POT1 participates in telomeres length regulation and protection. TRF2 and its partners are in charge of the protection of telomeric ends against illicit recognition by checkpoint proteins and recombinases. At least some of these functions are thought to originate from the ability of TRF2 to fold DNA into a lasso-like structure called the T-loop in vitro, a structure that can be observed on purified telomeres from cells or produced in vitro in the presence of TRF2 and telomeric DNA. Formation of T-loops has been proposed to rely on invasion of the telomeric 3’ single strand that ends the telomeric tract into the telomeric double helix in a cis-oriented reaction that would create, at the base of the loop, a D-loop (a bubble-like structure) and a Holliday junction (HJ, a four-stranded DNA structure known as an intermediate in Homologous Recombination). In two recent publications (Amiard et al., Nature Struct. & Mol. Biol. 2007, Poulet et al., EMBO J 2009), Partner 1 has unveiled new striking intrinsic properties of TRF2 related to T-loop formation and stability : i) its ability to stimulate telomeric invasion by modifying DNA topology locally, a property that has far-reaching implications in terms of telomere recombination, replication and transcription; ii) its capacity to bind and protect HJs from cleavage by resolving enzymes, which can explain the anti-recombinogenic properties of TRF2. These properties could also be at the heart of the folding of telomeres in the form of the T-loop. This is this latter topic that we propose to investigate in the TELOLOOP project. Surprisingly, although the T-loop is often presented as a key structure to protect chromosome ends, no functional evidence exists that sustains this assertion. Our strategy to address this question of the role of the T-loop in telomere protection is to understand how TRF2 and its partners contribute to T-loop homeostasis and to use this information to study the biological effects of loss-of-function mutations of TRF2 and of its partners that specifically impairs T-loop formation. Therefore, the major objective of TELOLOOP is to unravel the biological role of TRF2 and its partners in the formation and processing of the T-loop by combining biological, biochemical, biophysical and structural approaches. This project is organized in 4 inter-connected tasks and involves 4 partners with complementary expertises in cell biology, biochemistry, single-molecule technology and structural biology. Important knowledge about telomeric complexes is expected to be brought to light and technological breakthroughs are anticipated through the development of new techniques and the use of newly designed technologies.