1. Control of V(D)J recombination : chromatin remodeling and allelic exclusion at the immunoglobulin loci (Michele Goodhardt)
Our previous studies have shown that during B cell development, initiation and inhibition of V(D)J recombination is associated with a reorganization of Ig gene chromatin structure and that both positive and negative-regulatory elements are involved in control of rearrangement. Our objective in the next few years is to understand how chromatin remodeling at the Ig loci is regulated and to determine whether establishment of allelic exclusion requires allele specific modifications prior to V(D)J recombination.
2. Study of differential TdT expression during development and its consequences for the immune response (Noëlle Doyen)
Terminal transferase (TdT) is expressed only in T and B lymphocyte precursors and the amount expressed in vivo correlates with the degree of N region diversity in the antigen receptors. N nucleotides are rare in V(D)J junction from fetal and newborn mice but constitute a major component of Ig and TCR diversity in adults. This phenomenon is due to the differential expression of TdT during development since TdT is not found until the first week after birth in mice. For these reasons we will focus our work on two themes : the first is to identify the factors responsible for differential TdT expression and the second is to study the consequences of TdT expression for the immune response.
3. Analysis in vitro of proteins involved in V(D)J recombination (Catherine Papanicolaou)
We have taken advantage of our novel method of production of heterologous proteins in E. coli (DB/MD-99/112). We have produced 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) in collaboration with Marc Delarue (Unité d'Immunologie Structurale), we have obtained crystals of TdTdelta130 and a set of diffraction data at 2.3 angströms; (iii) we have studied TdT discrimination between ribo and deoxyribonucleotides; (iv) we have identified by mutagenesis an aspartate residue essential for TdT catalytic activity and expanded 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. Characterization of a novel peptide modulator (SMR1-Pentapeptide) (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 established that the new androgen-regulated gene encodes a precursor protein named SMR1 protein which has the structural and functional characteristics of an hormone precursor. Indeed, as it is generally observed for major functional peptide hormones of the endocrine system, the final mature SMR1-Pentapeptide in vivo is selectively matured after processing at pairs of basic amino acid residues. It is synthesized predominantly in response to androgen steroids in a tissue (SMG, prostate) -, age - and sex - related manner and locally as well as systemically released in response to environmental stress.
Data gathered over the past decade in the laboratory by an integrative post-genomic approach provide convincing evidence that SMR1-Pentapeptide is a novel signaling peptide. It is adapted to the sex - and species - specific environmental, behavioral and physiological characteristics. It is tonically and dynamically mobilized upon urgent situations, in the way to coordinate both local and systemic nociceptive, inflammatory, pressor and/or mineral homeostatic responses.
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 and in SMR1 mRNA content per cell. 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. We are currently trying to characterize the changes in chromatin structure which are associated with the activation of SMR1 transcription in response to androgens.
The gene encoding SMR1 belongs to a multigene family which was characterized in the rat, the mouse and, more recently, in human. Our aim is also to precise the genetic events which have led to the diversification, in terms of coding and regulatory sequences, of this gene family during evolution.
6. Mechanistic aspects of somatic hypermutation in the variable regions of human immunoglobulin genes (Brid Laoide)
Somatic hypermutation (SHM) occurs during a brief period of B cell development, in the specialised environment of the germinal centers of secondary lymphoid tissues. SHM is accurately targeted and tightly regulated and introduces mutations into rearranged immunoglobulin genes at an estimated rate of 1 mutation/kilobase/generation. Our objective is to identify the novel mutator function essential for this process. This research will be carried out in the context of the Unité's interest in the evolutionary origins of the mechanistic aspects of sequence diversification in jawed vertebrates.