Homepage   general_banner
imprimer
Print
     Nuclear Cell Biology - URA 2582 CNRS


  Director : Bachellier-Bassi Sophie* ; Nehrbass Ulf***par interim jusqu'au 31/12/05, puis interim par Vincent GALY. (bachelli@pasteur.fr ; nehrbass@pasteur.fr)


  abstract

 

We are currently studying nuclear structure function relationships, namely the regulatory function of spatial positioning. Using epifluorescence and spinning disk confocal microscopy, we are developing tools allowing us to follow spatial positioning of a discrete locus or larger DNA regions under active or inactive conditions. We also have started to study nuclear architecture-linked gene regulation in differenciation and developpment processes in the model organism Caenorhabditis elegans.



  report

cale

The laboratory has been engaged in nuclear structure function analysis, using the following approaches :

We have analyzed how nuclear processes are influenced by nuclear architecture on the example of transcriptional regulation

We have analyzed how viral particles make use of intranuclear structures to integrate in specific sites

We analyze the effect role of nuclear architecture during differenciation and developpment.

1. The structure/function analysis of transcriptional regulation. (S. Bachellier-Bassi, A. Berger, G. Cabal, B. David-Watine, F. Donnadieu, F. Feuerbach, O. Gadal, M. Mhlanga, A. Romano)

1.1 Contribution of transcription and/or export machineries to the spatial positioning of a transcribed locus.

Knowing that nuclear architecture is relevant for transcriptional silencing, we have started to analyze the significance of spatial positioning on transcriptional activation. We are currently trying to better understand the involvement of the transcription machinery, namely of the stress-related SAGA complex, as well as Sac3-Thp1-Cdc31, in GAL genes relocation when transcriptionaly activatede. Upon galactose induction, the Gal locus is confined in an area of the nucleus close to the enveloppe during the 15 minutes observations. This suggests the presence of factor(s) located at the nuclear periphery with the ability to tether this locus in this nuclear sub-domain. Nuclear pores components are good candidates for that function, given their location, their role in mRNA export and their in anchoring of telomeric chromatin. We have started studying mutants deficient for Nup1, which interacts with the Sac3-Thp1-Cdc31 complex.

1.2 Contribution of mRNAs to the spatial positioning of a transcribed locus.

We are also studying spatial positioning of a budding yeast heat shock gene. Comparison of the tagged gene distribution within the nuclear volume after a heat shock clearly shows a peripheral location of this locus upon temperature increase. This phenomenon did not occur for a gene which is not induced during heat shock.

In order to better understand the mechanisms responsible for this relocation, we are studying strains mutated in the promoter area, either abolishing transcription or the heat shock-induced response. We are also studying the effect of mutants of nuclear pore complex components on the heat shock protein locus positioning during stress response.

1.3 The rDNA as an element of nuclear spatial organization.

Ribosome biogenesis occurs within a specific domain of the nucleus : the nucleolus. In the budding yeast S. cerevisiae, the nucleolus displays an assymetric distribution flanksin contact with the nuclear enveloppe and occupies about a third of the nuclear volume. Interestingly, we have shown that peripheral proteins Mlp1 and Mlp2 are also assymetric since they exclusively associate with nuclear pores in the nucleoplasmic area, and are excluded from the nucleolar area. In order to better understand the role of the nucleolus in spatial architecture, we have studied a HMG-box protein, Hmo1, which concentrates in the DNA region encoding large ribosomal RNAs, the rDNA, localized in the nucleolus. We have shown that this protein interacts both with the rDNA and the genes encoding ribosomal proteins, which led us to suggest a nucleolar-dependant nuclear architecture.

tRNA-encoding genes have been shown by D. Engelke group to be transcribed in foci localized near the nucleolus. Our data suggest that the ribosomal-encoding genes share the same localization. To study the nucleolar localization of all the components of the translation machinery, we have developped - in collaboration with Christophe Zimmer (AIQ) - an algorithm dedicated to image analysis, which allows the study of gene positionning as compared to the nucleolus in three dimensions. We are testing our model by localizing the rDNA and the genes encoding the other components of the translation apparatus.

2. HIV/INI1 interaction and its role in subnuclear targeting (A. Boese, P. Sommer)

We have investigated the role of hSNF5/Ini1, a component of all mammalian SWI/SNF chromatin remodelling complexes, in the process of HIV integration site selection. To this end we developed a reporter system to monitor HIV transcriptional activity and RNAi technology to deplete hSNF5/Ini1in target cells. We have shown that integration in the absence of hSNF5/Ini1is as efficient as in the wild-type situation, but the integrated proviruses differ significantly in terms of transcriptional potential. Integration in the absence of hSNF5/Ini1results in a significant increase of HIV expression in the first few days after infection followed by a decline to normal levels at later phases. Moreover, the HIV promoter of proviruses established in the absence of hSNF5/Ini1demonstrates a significantly higher level of H3-K9 dimethylation, an epigenetic signature for transcriptional repression. The results show that the HIV promoter is variegated when integration occurs in the absence of hSNF5/Ini1, which is in striking contrast to the wild-type situation, and this phenomenon correlates with a distinct epigenetic regulation. This is the first experimental evidence that target site preferences of HIV are linked to the chromatin context and that a cellular binding partner of integrase, namely hSNF5/Ini1, confers the specificity in the selection process.

3. Structural and functional nuclear organization during C. elegans development (V. Galy)

We are using C. elegans as a model organism to study the mechanisms of nuclear enveloppe formation, as well as the role of the enveloppe in the nucleus architecture, and in the regulation and imprinting of genes during development. The C. elegans protein orthologous to mammal Tpr and yeast Mlp proteins is one of the candidate proteins which could be involved in nuclear organization. We have identified this protein, Ce-TPR, and started its functional analysis ; it is localized at the nuclear enveloppe, but also in a lesser amount in the nucleoplasm, as shown for the human or drosophila othologous proteins. Moreover, as shown in Drosophila, Ce-TPR seems to be localized at the mitotic spindle at metaphase and at the beginning of anaphase. We started characterizing the phenotype linked to Ce-TPR RNAi depletion-induced Ce-TPR inactivation : a drastic decrease of Ce-TPR in embryos causes either development arrets at larval stage, or morphological problems in the offspring of treated worms. In parallel, we have optimized the conditions of image acquisition in phase contrast and fluorescence microscopy during the first seven hours of development of C. elegans, to later be able to better characterize development abnormalities.

Keywords: nuclear architecture, gene regulation



  publications

puce Publications 2005 of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Dulieu, Isabelle, idulieu@pasteur.fr Bachellier-Bassi, Sophie, IP, (CR, bachelli@pasteur.fr)

David-Watine, Brigitte, INSERM, (CR, bdwati@pasteur.fr)

Feuerbach-Fournier, Frank, IP, (CR, ffeuerba@pasteur.fr)

Gadal, Olivier, CNRS, (CR, ogadal@pasteur.fr)

Galy, Vincent, CNRS (CR1, vgaly@pasteur.fr)

Berger, Axel, PhD Student

Boese, Annette, Postdoc

Cabal, Ghislain, PhD Student

Mhlanga, Musa, Postdoc

Romano, Alper, Postdoc

Sommer, Peter, Postdoc

Donnadieu, Françoise, (Technician, fdonna@pasteur.fr)

Doualot, Harry, (hdoualot@pasteur.fr)


Activity Reports 2005 - Institut Pasteur
filet

Page Top research Institut Pasteur homepage

If you have problems with this Web page, please write to rescom@pasteur.fr