Analyse structurale des interactions de la lamine régulant la stabilité du génome

par Agathe Marcelot

Projet de thèse en Biochimie et biologie structurale

Sous la direction de Sophie Zinn-Justin et de Sophie Zinn-Justin.

Thèses en préparation à l'Université Paris-Saclay (ComUE) , dans le cadre de École doctorale Innovation thérapeutique : du fondamental à l'appliqué (Châtenay-Malabry, Hauts-de-Seine ; 2015-....) , en partenariat avec Institut de Biologie Intégrative de la Cellule (laboratoire) et de Université Paris-Sud (établissement de préparation de la thèse) depuis le 01-10-2018 .


  • Résumé

    Le sujet proposé est un sujet de recherche en biologie fondamentale. Il porte sur la description des zones de contact entre l'enveloppe nucléaire, le nucléosquelette et la chromatine. En effet, des analyses par tomographie cryo-électronique ont très récemment montré que le nucléosquelette consistait en un réseau de filaments de lamines de 3-4 nm de diamètre (Mahamid et al., Science 2016; Turgay et al., Nature 2017). Ce nucléosquelette coopère avec les protéines de l'enveloppe nucléaire et la chromatine afin de protéger l'intégrité du génome. Des altérations dans les lamines sont associées à toute une variété de troubles dégénératifs, de syndromes de vieillissement prématuré et de cancers; plusieurs de ces troubles sont caractérisés par une instabilité du génome (Gay & Foiani, Int Rev Cell Mol Biol 2015). Dans mon équipe, nous avons récemment résolu la première structure 3D d'un domaine de lamine en complexe avec ses partenaires à la périphérie nucléaire (Samson et al., soumis). Nous avons montré que ce domaine de lamine forme un complexe ternaire avec le domaine LEM de la protéine émerine de la membrane nucléaire interne et la protéine BAF associée à la chromatine. De plus, nous avons établi in vitro et en cellules que certaines mutations des lamines et de BAF associées à des syndromes de vieillissement accéléré perturbent l'interaction entre les lamines et BAF. Nous voudrions aujourd'hui aller plus loin et analyser comment notre complexe ternaire interagit avec plusieurs protéines essentielles à l'assemblage de la chromatine (histones, chaperons d'histones, enzymes modifiant les histones), ainsi qu'avec des protéines liant la chromatine lors des processus de réparation des dommages de l'ADN (collab. avec Pascale Bertrand, IRCM/DRF).

  • Titre traduit

    Structural analysis of lamin interactions regulating genome stability


  • Résumé

    The spatial and temporal organization of the genome has emerged as an important level of regulation of nuclear functions (Gonzalo et al., Adv Exp Med Biol 2014). Structural proteins associated with the nuclear envelope play important roles in the organization of the genome. The nuclear lamina, a polymeric meshwork formed by lamins (A- and B-type) and lamin-associated proteins, is viewed as a scaffold for tethering chromatin and protein complexes regulating a variety of nuclear functions. It cooperates with nuclear envelope proteins and chromatin in order to protect the genome integrity. Alterations in lamins function dysregulate epigenetic modifications that change chromatin structure; they impact DNA transcription, replication, and repair (Camozzi et al., Nucleus 2014; Gonzalo & Eissenberg, Curr Opin Genet Dev 2016). Defective lamins are associated with a whole variety of degenerative disorders, premature aging syndromes, and cancer; several of these disorders are characterized by genome instability, which provides evidence for lamins operating as caretakers of the genome (Gay & Foiani, Int Rev Cell Mol Biol 2015; Irianto et al., Cell Mol Bioeng 2016). At the cellular level, lamins form filaments that are mainly present at the nuclear periphery but are also observed in the nucleoplasm (Gruenbaum & Foisner, Annu Rev Biochem 2015). Their complex molecular organization was recently described using cryo-electron tomography (Mahamid et al., Science 2016; Turgay et al., Nature 2017). Within the densely packed environment observed close to the nuclear envelope, only nuclear pores, lamin filaments and chromatin could be identified. Lack of high resolution 3D structures for proteins anchored at the inner nuclear membrane as well as complexes between these proteins, the lamina and chromatin precluded any further description of the nuclear periphery architecture and of the structural defects caused by mutations in lamin genes. We have recently solved the first 3D structure of a lamin domain in complex with its partners at the nuclear periphery (Samson et al., submitted). We have shown that the A-type lamin immunoglobulin-like domain forms a ternary complex with the LEM domain of the inner nuclear membrane protein emerin and the chromatin-associated protein BAF. Moreover, from our structural analysis, we have proposed that BAF is able to bind simultaneously to A-type lamins, emerin and DNA. Finally, we have demonstrated in vitro and in cells that A-type lamins and BAF mutations causing accelerated aging syndromes disrupt the interaction between A-type lamins and BAF. This suggests that the interactions between the lamina and chromatin-associated proteins have important functions in human cells. We now aim at understanding how this chromatin-associated complex interacts with the nuclear machineries responsible for the maintenance of genome integrity. We will focus on 2 related aspects: (i) Lamin and BAF defects identified in diseases cause nuclear morphological abnormalities and an altered pattern of heterochromatin distribution (Robin & Magdinier, Front Genet 2016; Loi et al., Oncotarget 2016). We will characterize interactions with modified histones (Histones H3 & H4; Monte de Oca et al., J Biol Chem 2005 and Nucleus 2011), histone chaperones (RBBP4; Pegorano et al., 2009) and enzymes modifying histones (Sirt6; Ghosh et al., Cell Rep. 2015) in collaboration with the chromatin expert F. Ochsenbein in our laboratory. (ii) A-type lamin deficient cells are highly sensitive to agents that cause replication stress (Singh et al., Mol Cell Biol. 2013). A-type lamins are required for restart of stalled replication forks, either because of their role in the retention of histones or histone chaperones, or because of their role in the regulation of DNA damage signaling and repair pathways. We will study the retention by lamins of the chromatin-binding protein 53BP1 (Gonzalez-Suarez & Gonzalo, Nucleus 2010), which is a key mediator in the cellular response to DNA damage, proposed to function at the interface of DNA replication, recombination, and repair (collaboration with P. Bertrand at IRCM/DRF). Here again, we will purify the protein complexes and solve their 3D structures using a combination of structural biology methods. This work will be performed in collaboration with the Synchrotron Soleil (Gif-sur-Yvette) for collecting SAXS and X-ray crystallography data, and in collaboration with E. Le Cam (IGR, Villejuif) for studying the assembly of complexes onto DNA by electron microscopy. We will also follow by NMR how the different partners are post-translationally modified using an approach now available in our team (Theillet et al., Nat Protoc 2013), in order to understand how these modifications regulate the formation of the complexes. Finally, we will analyze in vitro and in cells the impact of lamin A/C mutations causing accelerated aging syndromes on the formation of the complexes and the function of the associated proteins, in collaboration with the team of P. Bertrand (IRCM/DRF). This project should contribute to understand how A-type lamins interact with chromatin and participate to the cellular response to a replication stress.