|PDF Version||Developmental Genetics and Biochemistry|
|Director : ROUGEON François (firstname.lastname@example.org)|
The main focus of our laboratory is to understand at the molecular level how the mechanisms which contribute to genome evolution and stability have been functionaly integrated at the somatic level to generate antibody diversity. We are studying the relationship between double strand DNA break repair and V(D)J recombination on one hand, and recombination repair and hypermutation on the other hand. We have also set up research program aiming at the analysis of genetic and cellular events involved in the generation of single domain antibody repertoire in camelides. Finally the research program on rodent submaxillary gland peptidic mediators is now developed within the laboratory directed by Catherine Rougeot in the Department of Structural Biology and Chemistry.
1. Developmental regulation of immunoglobulin gene rearrangement (Michele Goodhardt)
Antigen receptor genes are assembled during lymphocyte development by a series of highly regulated site-specific recombination events known as V(D)J recombination. To study the regulation of immunoglobulin (Ig) k gene rearrangement, we have produced a series of transgenic mice lines containing unrearranged k genes. These studies have shown that both positive and negative-regulating elements are involved in control of rearrangement : (i) sequences associated with the intronic enhancer region which induce k gene rearrangement, (ii) a repressor element situated in the V-J intervening region which restricts k gene rearrangement to the late stages of B cell differentiation.
Several lines of evidence suggest that developmental- and lineage- specific changes in the accessibility of Ig genes to recombination factors underlie control of V(D)J recombination. In order to understand the structural basis of accessibility of Ig genes to V(D)J recombination factors, we have undertaken a detailed analysis of the chromatin structure of Ig heavy and k light chain V and J segments in RAG-deficient pro-B and pre-B cell precursors. The results of our study show that during B cell differentiation onset and inhibition of V(D)J recombination at the heavy and light chain loci are preceded by a reorganisation of Ig gene chromatin structure : nucleosomal organisation, histone acetylation, nuclease sensitivity and DNA methylation.
2. Regulation of terminal transferase expression during lymphoid development (Noëlle Doyen)
N regions diversity in antigen receptors is a developmentally regulated process in B and T lymphocytes which correlates with the differential expression of terminal deoxynucleotidyl transferase. In order to identify the different regulatory elements involved in the lymphoid specific expression we determined the onset of TdT gene activation during T cell differentiation and thymic ontogeny. We showed that TdT expression could be induced in immature thymocytes from fetal day 17 much earlier than previously reported and may depend on microenvironmental cues. Moreover, in order to identify the cis regulatory elements involved in TdT transcriptional activation, we assayed for Dnase I hypersensitive sites in a 35 kb region of the TdT locus. We found several hypersensitive sites upstream minimal promoter region and in the first intron.
The manipulation of repertoire diversity and its consequences in the immune response to external challenge might be evaluated using an experimental model of infection. We use Shigella Flexneri since immune responses to these pathogenes are extremely specific for the infecting serotype. Analysis of immune responses after infection with Shigella Flexneri were performed in two murine models with variable repertoire diversity : the TdT transgenic mice that we have developed and TdT Ko mice from (Gilfilan et al, 1993 Science 261 : 1176). Surprisingly, we didn't find any difference both in recovering of primary Shigella Flexneri infection and in protection against re-infection between the different strains of mice. However differences are observed in the specificity of their B cell responses which are investigated.
3. Structure and mechanisms of terminal deoxynucleotidyl transferase (TdT) (Catherine Papanicolaou)
We have shown that the two murine TdT isoforms share a common mechanism in vitro, although only the short isoform can add N regions at the B and T receptor V(D)J junctions, and that the long isoform has no intrinsic 3'-5' exonuclease activity. In collaboration with M. Delarue (Unité de Biochimie Structurale), we have resolved the structure of the catalytic core of the dominant isoform (TdTS) at 2.35 Angströms. Our data suggest that the inability of TdT to accommodate a single-stranded DNA template could be due to a sterical barrier formed by an eighteen amino acid loop which is absent in polymerase b, a replicative DNA polymerase related to TdT.
4. Chromosomic breaks repair and genomic stability (Miria Ricchetti)
The induction and repair of chromosomal double strand breaks (DSBs) is of fundamental importance for physiological processes such as the reshuffling of genetic material in meiosis and for immunological diversity. Spontaneous chromosome breaks do play a role in genome evolution, for examplewe have shown that repair of DSBs is the mechanism by which mitochondrial DNA integrate into the nucleuar genome (Ricchetti et al, 1999). We have then analysed this process in huhmans and found new markers to investigate the evolution of human populations (manuscript in preparation). DSBs can however be dangerousfor the integrity of the genome. When the repair process is deregulated, it can lead to cell death, genetic disorders, and a variety of different pathologies. We explore the repair potential of normal cells, using genetically modified mice, whose genome contains two I-SceI sites flanking a selection cassette (see collaboration). I-SceI is the target site of a rare-cutting endonuclease which, once expressed in the cells, induces a chromosome break. Since the chromosomal location of the break is known, we are able to analyse the precise nature of repair strategies adopted by the cell. We use this system in ES mouse cells, in cycling and post-mitotic cells, in embryonary cells as well as in cells originated from young and old animals. The goal is to understand how the repair of DSBs varies in different cell types during the life of the animal and how these variations are related to cell fate in a mammalian system. We use the I-SceI strategy also in human cell lines to analyse the role of error-prone DNA polymerases in DSB repair.
Keywords: deoxynucleotidyl terminal transferase, V(D)J recombination, chromatin structure, transcriptional regulation, DNA repair
|Publications of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|BOUTOUT Laurence, IP, email@example.com||DOYEN Noëlle*, IP, Researcher, E-mail : firstname.lastname@example.org
GOODHARDT Michèle*, CNRS, Researcher, E-mail : email@example.com
LAFAYE Pierre, IP, Researcher, E-mail : firstname.lastname@example.org (arrival 04/11/02)
LEDUC Mireille, University Paris XI, associate professor, E-mail : email@example.com
PAPANICOLAOU Catherine, CNRS, Researcher, E-mail : firstname.lastname@example.org
RICCHETTI Miria, IP, Researcher, E-mail : email@example.com
ROUGEON François, CNRS/IP, DR1 CNRS, Professor at Pasteur Institute, Head of Unit, E-mail : firstname.lastname@example.org
|BONNET Marie, DEA (arrival 01/03/03)
BOUBAKOUR Imenne, PhD student , E-mail : imenneb@ pasteur.fr (arrival 19/12/02)
CHERRIER Marie*, PhD student, E-mail : email@example.com
HUAULME Jean-François, PhD student, E-mail : firstname.lastname@example.org
JOVANIC Tihana,, PhD student , E-mail : email@example.com (arrival 29/10/02)
LE DEZ Gaëlle, CNRS, Technician , E-mail : firstname.lastname@example.org (arrival 04/11/02)
MAES Jérôme*, post-doctoral trainee, E-mail : email@example.com
|BONNEFOY Géraldine, IP, Technician, E-mail : firstname.lastname@example.org
CAVELIER Patricia, CNRS, Technician, E-mail : email@example.com
HERMITTE Véronique, IP, Technician, E-mail : firstname.lastname@example.org
KLIMCZAK Martine*, IP, Technician, E-mail : email@example.com