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  Director : ROUGEON François (frougeon@pasteur.fr)


  abstract

 

Work in our laboratory is centered around two main themes : (i) molecular mechanisms underlying the clonal expression of immunoglobulin genes in B lymphocytes ; (ii) transcriptional and post-transcriptional regulation of the expression of renin and other rodent submaxillary gland-specific polypeptides.



  report

cale

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 and consequences of junctional diversity on the immune response (Noëlle Doyen)

Terminal transferase (TdT) gene is expressed exclusively at early stages of both B and T lymphocyte development : TdT is differentially expressed during development and is not found until the first week after birth in mice. The amount of TdT expressed in vivo correlates with the degree of N region diversity in the antigen receptors. Transactivating factors have been shown to contribute to the lymphoid specific expression of TdT but they do not entirely account for TdT expression patterns. We established that changes in the methylation status of the TdT gene were involved in its regulation. We identify the different regulatory elements involved in T and B specific expression. We search whether changes in chromatin structure could be implicated in the differential TdT expression observed in fetal and adult life.

Differentially regulated TdT expression during development is responsible for a limited antigen receptor repertoire in embryos, and a more complex one in adults. Questions concerning the functional significance of N-region diversity in the adult repertoire and the impact of a more limited repertoire at the fetal stage on the immune response remain to be answered. We are analyzing immune responses after infection with pathogens (as shigella flexneri) 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 : 1175). The manipulation of repertoire diversity and its consequences on the immune response to external challenge might be evaluated using experimental models of infection.

3. Analysis in vitro of proteins involved in V(D)J recombination (Catherine Papanicolaou)

Our novel method of production of heterologous proteins in E. coli (DB/MD-99/112) allowed us to produce the two murine TdT isoforms, a truncated version of TdT (TdTdelta130) which remains active, the full length RAG1 and RAG2 proteins and the HMG1 and HMG2 proteins : (i) we have compared the two murine TdTs and shown that, for the long isoform, a twenty amino acid insertion in a highly conserved region modifies the thermosensitivity but not the catalytic activity ; (ii) we have shown that TdT, unlike any other polymerases, does not discriminate at the incorporation step between ribo and deoxyribonucleotides but that it does not accommodate easily nucleic acids terminated with ribonucleotides ; (iii) in collaboration with Marc Delarue (Unité d'Immunologie Structurale), we have obtained crystals of TdTdelta130 and the structure has been resolved. Alone and as binary complexes with either a single-stranded DNA or a nucleotide, TdT resembles DNA polymerase beta. This reinforces the hypothesis of a common evolutionnary origin. TdT adopts in these conditions a closed conformation such as the conformation adopted by pol beta when it interacts with its two substrates simultaneously. The presence of an additional lariat-like loop in TdT could account for the nucleic acid specificity differences (single versus double-stranded) between the two polymerases ; (iv) we have expanded, in light of the tridimensional data, our analysis of mutated TdT enzymes in order to study the mechanisms involved in the selection of single stranded DNA and nucleotide substrates ; (v) we are continuing the design of a V(D)J acellular system with full length proteins.

4. Molecular and functional characterization of a new peptide mediator : sialorphin (Catherine Rougeot)

In a genomic approach, a tissue- and sex-specific cDNA from male rat submandibular gland (SMG) has been characterized in the laboratory. We have demonstrated that this androgen-regulated gene encodes a new hormonal precursor, named SMR1 (SubMandibular Rat1 protein).

In an integrative post-genomic approach we have established the molecular and functional bases providing the existence of a novel exocrine and endocrine peptide-mediator, whose expression is under activational androgenic regulation and secretion is evoked under adrenergic-mediated response to environmental stress, in rats. This signaling messenger is the peptide generated from the final maturation step of the SMR1 prohormone : the SMR1-Pentapeptide, which is named today sialorphin. Sialorphin is involved in the tonic and dynamic coordination of components of the adaptive behavioral response of the organism to an aggression. Thus, on one hand, this specific signal of exocrine and endocrine communication is mobilized in vivo under neuroendocrine control and, more particularly upon urgent situations. And, on the other hand, we have showed that the circulating sialorphin is a physiological antagonist of a membrane metalloectopeptidase activity, which catalyzes the inactivation of hormones and neuropeptides controlling the cardiovascular and inflammatory responses, the nociceptive response and the phosphate and sodium homeostatic exchanges.

5. Evolution and regulation of expression of the gene encoding SMR1-pentapeptide (I. Rosinski-Chupin)

SMR1 gene expression is induced by androgens in the submandibular gland (SMG) of rats. Using an in situ hybridization approach and an analysis at the single cell level, we have shown that SMR1 expression is mosaic in conditions of low androgen concentrations in the SMG acinar cells. The increase in androgen concentrations during male sexual maturation leads to an increase in the frequency of transcriptionally active cells (binary response) and in SMR1 mRNA content per cell (graded response). This led us to propose a model for SMR1 regulation, where, in conditions of low androgen concentrations, transcription is repressed at the cell level, probably through a chromatin-mediated mechanism.

The gene encoding SMR1 belongs to a multigene family whose organization was characterized in the rat, the mouse and in human. A diversification of the coding sequences is associated with an increase in the size of this family, in the rodents.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

BOUTOUT Laurence, IP, lboutout@pasteur.fr

ROSINSKI-CHUPIN Isabelle, IP, Researcher, E-mail : ichupin@pasteur.fr (departure 31/12/01)

DOYEN Noëlle, IP, Researcher, E-mail : ndoyen@pasteur.fr

GOODHARDT Michèle, CNRS, Researcher, E-mail : migood@pasteur.fr

LEFEVERE-LAOIDE Brid, IP, Researcher (departure august 2001)

PAPANICOLAOU Catherine, CNRS, Researcher, E-mail : papanico@pasteur.fr

RICCHETTI Miria, IP, Researcher, E-mail : mricch@pasteur.fr (arrival 01/10/01)

ROUGEON François, CNRS/IP, DR1 CNRS, Professor at Pasteur Institute, Head of Unit, E-mail : frougeon@pasteur.fr

ROUGEOT Catherine, IP, Researcher, E-mail : crougeot@pasteur.fr

BOULE Jean-Baptiste, PhD student, E-mail : jbboule@pasteur.fr

CHERRIER Marie, PhD student, E-mail : cherrier@pasteur.fr

COQUILLEAU Isabelle, PhD student, E-mail : icoq@pasteur.fr (Thesis presented 09/07/01 - departure 31/07/01)

HUAULME Jean-François, PhD student, E-mail : jhuaulme@pasteur.fr

MAES Jérôme, PhD student, E-mail : jmaes@pasteur.fr (Thesis presented 18/12/01)

BONNEFOY Géraldine, IP, Technician (arrival 27/11/01)

CAVELIER Patricia, CNRS, Technician, E-mail : cavelier@pasteur.fr

HERMITTE Véronique, IP, Technician, E-mail : hermitte@pasteur.fr (arrival 02/10/01)

KLIMCZAK Martine, IP, Technician, E-mail : mfanton@pasteur.fr

RIGAULT Anne-Gaëlle, IP, Technician, E-mail : arigault@pasteur.fr (departure 03/10/01)


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