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     Nuclear Cell Biology - URA 2582 CNRS


  Director : Bachellier Sophie, Nehrbass Ulf (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 are also using retroelements to define specific functional sub-domains in the chromosomes.



  report

cale

The laboratory has been engaged in nuclear structure function analysis.

Two major approaches have been taken:

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

Both areas have seen significant advance during this year:

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

1.1 Perinuclear retention of pre-mRNA.

Building on previous work on the yeast structural proteins Mlp1 and Mlp2, we have shown that both proteins are distributed asymmetrically in the nuclear envelope, adjacent to chromatin. Using a genetic screen (in collaboration with the Unité de Génétique des Interactions Macromoléculaires), we could show that Mlp1 implements a quality control step prior to mRNA export, physically retaining faulty precursors. Therefore, pre-mRNA retention processes might take advantage of this unusual Mlp distribution.

1.2 A molecular model for a provocative hypothesis: "gene gating".

Knowing that nuclear architecture is relevant for transcriptional silencing, we have started to analyze the significance of spatial positioning on transcriptional activation. Building on work from last year suggesting that Cadmium-stress related genes require intact perinuclear architecture to properly activate transcription, we have been zooming in on one transcriptionally regulatory complex which is associated with stress gene transcription, the SAGA complex. In this project, in collaboration with the Unité d'Analyse d'Images Quantitative, we have been able for the first time to track a gene in 3D over time in either an active or inactive state. We can document the relocation of genes upon activation and can further show that this recruitment is mediated by components of the SAGA complex interacting with the mRNA export machinery at the NPC extensions. These results constitute the first direct molecular proof for the gene-gating hypothesis first formulated 20 years ago.

1.3 Can mRNA contribute to the spatial positioning of a transcribed locus ?

Other projects have started to elucidate the role of mRNA in mediating the commitment of a transcribed locus to the perinuclear platform. In this project the location of a tetO-labeled heat shock gene in yeast is established relative to GFP-tagged Mlp-proteins delineating the nuclear periphery. Intriguingly, upon heat shock Mlp1 change their perinuclear distribution, forming few foci mostly at the nuclear periphery. These Mlp1 foci occasionally colocalize with the heat shock locus. We are currently analyzing whether the RNA of the very highly transcribed heat shock locus can recruit Mlp1 towards the transcription site. Whilst we have shown before that Mlp proteins can specifically interact with RNAs, the current experiment goes further in that it suggests an additional role of the transcribed mRNA in the localization of the transcribed gene. This activity of RNA in committing a highly transcribed gene to a given spatial locus could be in synergy with promoter binding factors. In coming experiments the correlation between dynamic Mlp1 delocalization and high turnover transcription sites will be analyzed as a function of individual RNA binding proteins such as SUB2 and YRA1.

1.4 Using rDNA repeats as a model to study spatial positioning during gene regulation.

We have used fluorescent probes to label the repeated ribosomal DNA (rDNA) region. Using a confocal spinning disk set-up and a super-resolution detection algorithm, in collaboration with C. Zimmer (Unité d'Analyse d'Images Quantitative), we could show that histone deacetylase Sir2, a known regulator of rDNA structure, affects the labelled rDNA architecture. We are planning to extend our study to 3D plus time to analyse rDNA dynamics in vivo, and the effect of its extremely active transcription by switching it on and off. Under these conditions, we expect rDNA dynamics to be drastically affected, thus confirming the validity of this quantitative approach. Further, the modified rDNA will be evaluated in electron microscopy after cryo-fixation (EM) (in collaboration with P-E Gleizes). This study should provide more insight in structure function relationship between transcription and chromosome dynamics.

2. Viral elements in the structure function analysis of nuclei. (A. Boese, F. Feuerbach, P. Sommer)

2.1 Ty retrotransposons as tools to investigate modification in chromatin metabolism.

In the viral element related projects, we have used yeast retrotransposons as probes to explore the structural and functional organization of the nucleus. Deletion of genes encoding Mlp-proteins leads to an increase in recombination of cDNA copies of Ty5 with endogenous targets, involving these proteins in homologous recombination (HR) suppression. Analysis of retrotransposons' transposition in cells deficient for Mlp-proteins as well as the Rad52 protein, a component of the HR pathway, reveals the existence of two cell populations differing in their abilities to sustain Ty replication. We are currently analysing these two populations in order to understand their behaviours and exploring the relationships between Mlp-proteins and DNA repair/recombination pathways.

2.2 HIV/INI1 interaction and its role in subnuclear targeting.

The HIV project has analyzed whether HIV routes to and integrates into transcriptionally active sub-domains by the help of an interaction between HIV integrase and the hSNF5 (INI1) component of the SWI/SNF chromatin remodeling complex. Using an LTR-driven HIV reporter construct to infect cells in which INI1 has been reversibly knocked down using the RNA interference (RNAi) technology, we made the unexpected observation that the absence of INI1 at the time of viral infection leads to an increase in transcription rate of the reporter. By contrast, resurgence of INI1 levels after release of RNAi leads to repression of viral transcription. Intriguingly, a second RNAi depletion of INI1 only leads to a modest increase in expression of the already integrated viral reporter, indicating that the repressive release is not reversible. Further work has shown that this may be due to imprinting of a repressive chromatin code through H3 K9 methylation.

2.3 Global correlation between transcriptional potential and spatial positioning of integrated HIV viruses.

HIV further constitutes a very elegant system to correlate intra-nuclear positioning with transcriptional potential. We have generated clones of cells transduced with an HIV-based reporter construct, and determined the integration site via sequencing. Clones expressing varying amounts of LTR-driven proteins, which further vary in their ability to respond to tat activation, will be subjected to FISH analysis to determine HIV localization relative to the according chromosome territory. This work should help to elucidate whether a correlation exists between the expression levels of the viral reporters and their integration into specific functional sub-domains. It will further allow to study the effect of HIV integration on changes in spatial positioning of target sites, both in active and inactive conditions.

3. Further projects : Analyzing actin based locomotion associated with nuclei. (S. Münter)

A functionally compartmentalized nucleus should require some form of communication in-between compartments. In a system where spatial positioning encodes functional information, movement in-between these compartments could assume a regulatory role. Actin, which is associated with nuclei in its monomeric form, as well as ARP-proteins have been associated with nuclear function in a variety of contexts, such as chromatin remodeling and transcriptional activation. We therefore investigated whether actin treadmilling could constitute a possible nuclear locomotion principle. Using live cell confocal imaging combined with image processing, we observed micro-injected fluorescent actin monomers accumulate into a ring-structure around the nuclear envelope (NE) minutes after injection. A fluorescent analog of cytochalasin D (CD-BODIPY) also revealed this distinct ring of actin around NE-membranes. Both CD-BODIPY and fluorescent actin were detected deep inside NE-membrane invaginations. Further, in vitro, isolated NE-membranes nucleated actin polymerization, which was inhibited by the presence of antibodies directed against nucleoporins. Accordingly, we concluded that actin polymerization occurs specifically at the cytoplasmic face of NE-membranes, and depends upon steric availability of nucleoporins.

Keywords: nuclear architecture, gene regulation



  publications

puce Publications 2004 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, (CR1, bdwati@pasteur.fr)

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

Gadal, Olivier, CNRS, (CR2, ogadal@pasteur.fr)

Boese, Annette, Postdoc

Cabal, Ghislain, PhD Student

Mhlanga, Musa, Postdoc

Münter, Sylvia, Postdoc

Romano, Alper, Postdoc

Sommer, Peter, Postdoc

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

Doualot, Harry, (hdoualot@pasteur.fr)


Activity Reports 2004 - Institut Pasteur
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