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  Director : Ulf Nehrbass (nehrbass@pasteur.fr)


  abstract

 

My laboratory studies nuclear structure function relationships. The underlying idea of our work is that the structural organization of the nucleus is relevant for the correct execution of nuclear function. In particular, transcriptional regulation has been proposed to depend upon the topological and structural organization of the nucleus. Recent studies have shown that transcriptionally repressed genes can relocate to heterochromatin rich nuclear sub-domains. However, it has not been possible to demonstrate that the subnuclear position of a gene directly contributes to the establishment of its transcriptional state.



  report

cale

Molecular Analysis of Nuclear Structure Function Relations

We have been able, for the first time, to determine the structural organization of transcriptionally repressive sub-compartments. Using the eukaryotic model system yeast we could demonstrate that the repressive sub-domain is formed with the help of Mlp-filaments, which link telomeric chromatin to the nuclear envelope by physically interacting with the telomere binding protein Yku70p. Mlp-proteins, in turn are docked to the nuclear envelope via the nuclear pore complex component Nup60p. Starting from these structural constituents we then analyzed how the integrity of perinuclear heterochromatin sub-domains affects the transcriptional state of reporter genes at various positions within the nucleus. We found that expression of HM reporter constructs with intact or truncated silencer elements can be regulated by altering the intranuclear positioning relative to intact perinuclear subdomains. Deletion of NUP60 or any of the other structural constituents, NUP145C-ter, MLP1/2, YKU leads to release of perinuclear Sir-silencing factors and equalizes the transcriptional potential of given intranuclear positions. We can directly show, moreover, that epigenetic variegation in the expression of a subtelomeric reporter gene depends on reversible positioning within telomeric heterochromatin domains. Together, these data strongly suggest that yeast cells encode regulatory information in the spatial coordination of silencing factors within the nucleus. Our findings thus support a model in which sub-compartments of immobilized factors allow chromatin to determine transcriptional states through relative positioning.

To independently confirm these results, and to find further constituents of silent domains we employed retro-transposable elements as probes. Ty5 retro-transposons specifically integrate into silent domains, a process that is mediated by the Ty5-integrase. First, we could demonstrate that deletion of either MLP or YKU abolishes targeting of Ty5 into silent domains, while leading to a striking increase in overall integration. Thus, employing an independent system, we were able to confirm the role of Mlp-proteins and YKu as structural constituents of silent domains. To identify the actual host factors mediating the targeting to silent domains we employed consecutive yeast double hybrid screen using Ty5 integrase as bait. We found SRL2, a suppressor of RAD53 lethality, to specifically interact with Ty5-intergase. Deletion of SRL2 significantly reduced overall integration. In a consecutive screen using SRL2 as bait RIS1 was identified as a specific double hybrid interactor. RIS1 has previously been shown to play a role in silencing, and to directly bind to silencing factor Sir4p, an intrinsic constituent of silent domains. Thus, using Ty5 as a molecular probe, we have identified the mechanism underlying the targeting of Ty5 to silent domains, and have at the same time identified a number of novel silent domain players. The functional connection of SRL2 with RAD53 suggests that silent domains may have additional roles in DNA repair and/or replication.

Following up on this assumption we could indeed show that disruption of silent domains leads to impairment of DNA DSB-repair. The defect associated with deletion of MLP1 and MLP2 is not epistatic with RAD52, suggesting that the NHEJ pathway of DSB-repair was affected. To directly analyze the role of silent domains in DNA-DSB repair we developed a system to visualize double strand breaks in living cells by integrating a tetO repeat cassette next to an inducible HO-cleavage site. Preliminary data suggest that upon constitutive cleavage and in absence of DNA repair, the broken double strand breaks get relocated to silent domains. This could imply that broken ends, upon sustained damage, can get hetero-chromatinized ("telomerized?") and stabilized in peripheral silent domains.

Together, these results lead us to suggest that Mlp- proteins form a cross-connecting, lamina like network that is docked into position with the help of nuclear pore complexes. At the nuclear periphery it serves as an operational platform, which allows a variety of different processes to transiently dock and to thus gain a topological quality. Mlps are thus a common topological determinant, which allow a variety of nuclear metabolic processes to encode regulatory information in nuclear architecture.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
   

Ulf Nehrbass, Chef de laboratoire

Brigitte David-Watine , CR1 INSERM

Frank Feuerbach, Postdoctorant

Annette Boese, Postdoctorant

Vincent Galy, Etudiant en thèse

Chiara Conti, Etudiante en thèse

Gaelle Bourout, Etudiant de DEA

Sylvia Muenter, Etudiant en thèse

Olivier Gadal, Postdoctorant

Nadia Froger, Technicienne IP


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