Research in the Unit is centred around four projects :

1) Inactivation of the mouse X chromosome

2) Genetic analysis of type 1 diabetes in the mouse as a model for traits under multifactorial and polygenic control

3) The role of the X-linked gene Nap1l2 in the control of neuronal cell division and neuronal stem cell proliferation

4) Mouse genome analysis undertaken in collaboration with Genoscope (Centre National de Sequençage and the Centre National de Genotypage, Evry)

One of the major research interests of the Mouse Molecular Genetics Unit is X chromosome inactivation.This is a complex biological process which depends on the presence of two copies of the X-inactivation centre (Xic) and which results in the inactivation of one of the two X chromosomes present in the female cell. The process involves both the sensing and counting of the number of X chromosomes in the cell in relation to the autosomal complement. The X chromosome chosen to remain active is thought to be protected by an as yet hypothetical blocking factor. Several genetic elements including the genetically identified locus Xce (X-controlling element) seem to be able to affect the choice of which X chromosome will be chosen to be inactivated. Xce is distinct from Xist (X-inactive specific transcript) which codes for a large non-coding RNA which plays a major role in the initiation of the inactivation process.

The candidate region for the Xce locus was recently refined to a region of less than 50kb and targeted deletion approaches to the candidate region initiated. Both in vitro and in vivo studies of the effect of the mutation have been initiated and will hopefully confirm the molecular characterisation of the Xce locus first identified some forty years ago.

Other experiments undertaken in the laboratory are aimed at the functional identification and analysis of the various components of the Xic by targeted mutagenesis using the cre-lox system. This approach has allowed us to define a region lying 3' to Xist which both controls Xist expression and the counting process, possibly functioning as a binding site for the hypothesised 'blocking' factor. The analysis of the original deletion is currently being refined by a so-called add-back strategy.This has allowed at least two subregions to be defined one of which is implicated in the counting process. The other which controls both the expression of Xist and Tsix antisense appears to influence, amongst other things the retention of Xist RNA at its chromosomal site of transcription.

The importance of including a genomic approach in such projects is demonstrated by the new genes within the Xic which have identified from the recently completed annotation of the sequence of the mouse Xic and the results of our comparative sequence analysis of the mouse, human and bovine Xic regions. Conserved regions which are not coding sequences are candidates for regulatory roles in the function of the Xic region . This analysis is providing the basis for all our ongoing functional studies of the Xic.

A better understanding of some of the processes involved in the initiation of X-inactivation is likely to come from combining such genetic and cell biology approaches with an analysis of chromatin structure. Several different approaches to such an analysis including ChIP (Chromatin Immunoprecipitation) have been established in the laboratory. ChIP studies have recently proved of great importance in characterising the role played by histone H3 methylation in the earliest stages of X-inactivation. A region 5' to Xist was defined which may act as a nucleation centre allowing the inactivation to spread from the Xic over the whole X chromosome. Approaches aimed at obtaining purified chromatin from the Xic region for biochemical analysis are also underway.

Genetics factors involved in the X-inactivation process lying outside of the Xic and in all probability elsewhere than on the X chromosome, are being looked for by a complementary approach based on a comparative transcriptome analysis of 6.5 dpc female and male embryos using SAGE (Serial Analysis of Gene Expression) analysis. Currently 50000 transcript tags from each are being exploited.

A second interest of the Unit concerns the study of mouse phenotypes under multifactorial and polygenic control. We have taken as our prototype type 1 diabetes or insulino dependent diabetes for which the NOD mouse represents an interesting model. Our studies are aimed at defining the genetic factors (Idd) implicated in this pathology which is known to depend on a complex interaction between environmental and genetic factors. Our studies are concentrated on the characterisation of Idd loci controlling diabetes susceptibility/resistance locating to the distal part of mouse chromosome 6. The past year has seen major advances in the definition and localisation of three loci Idd6, Idd19 and Idd20 lying in the distal part of mouse chromosome 6.

The continued refining of the candidate regions for these loci by establishment of congenic mouse strains has allowed candidate gene approaches to be undertaken to defining and characterising the genes responsible for these traits and in particular for Idd6. The immunological characterisation of these congenic strains has allowed definition of the probable site of action/tissue involved in resistance to diabetes onset conferred by the Idd6 locus.

One means of reducing the genetics complexity underlying type 1 diabetes is to systematically study sub-phenotypes associated with the disease. The identification of the genes coding for target autoantigens identified by T-cell clones isolated from diabetic or pre-diabetic animals appears a particularly promising approach and has allowed us to identify a region on mouse chromosome 17 concerned in the regulation of the expression of several such antigens. The molecular analysis of the region(s) is underway.

Promising results have also been obtained for the X-linked , Nap1l2 gene. Mutations in this gene are associated with embryonic lethality, spina bifida and exencephaly linked to a massive overproliferation of neuronal cells. Current experiments which are aimed at understanding the role and mechanism of action of this gene in tissue specific cell cycle regulation and to better defining the population of neuronal cells, including neuronal stem cells which are the site of its action.

Collaborations:

Dr Bruce Cattanach

Medical Research Council, Genetics Division, Harwell, GB- Analysis of the Xce locus

Laboratoire de Biometrie et Biologie Evolutive (UMR CNRS 5558), Université Claude Bernard - Lyon 1, F- Xic sequencing project : sequence annotation

CEA Grenoble, Laboratoire de Chimie des Protéines, Grenoble, F- Biochemical identification and mass spectroscopy analysis of Xic binding proteins

Institut Curie, F- Methyl histones H3 in X-inactivation , Trophoblast cell line characterisation, X-inactivation and nuclear architecture

UNC-Chapel Hill, Department of Genetics, The University of North Carolina, Chapel Hill, USA- Polycomb proteins and X-inactivation

Centre National de Génotypage, Evry, F- Studies on Xce sequence polymorphism

Department of Molecular, Cellular and Developmental Biology, Yale Univ., New Haven, USA- Construction of Xic microarrays

Chromatin and Gene Expression Group, Anatomy Department, University of Birmingham Medical School, GB- Analysis of histone modification and the Xic

Max Planck Institut für Molekulaire Genetik, Berlin, D- Methylation analysis of the Xic region

Medical University of South Carolina, Hollings Cancer Center, Charleston,USA- Nap1l2 and mutational characterisation

Institute of Reproductive and Developmental Medicine, Imperial College, London, GB- Spina bifida and the role of Nap1l2 in man

Hopital St Vincent de Paul, INSERM U 342, Pathologie Métabolique et Hormonale, Paris, F- Immunological characterisation of type 1 diabetes

Equipe ATIPE Genetique et Pathologie du Developpement, CNRS-FRE2134, Institut de Transgenose, Orleans, F- ENU mutagenesis programmes

Centre National de Génotypage, Evry, F- Microarray provision and mouse mutagenesis programme

Centre National de Sequencage, Genoscope, Evry, F- Mouse transcript mapping and cDNA sequencing

Unité de Neurobiologie Moléculaire, Institut Pasteur- Nap1l2 and neurulation

Service d'Informatique Scientifique, Institut Pasteur- Informatics and sequence annotation

BOURDET Agnès Fondation pour la Recherche Médicale (fellowship)

GRIMM Christina European Community (fellowship)

MISE Natan Fondation pour la Recherche Médicale (fellowship)

MOREY Céline Ministère de la Recherche (grant)

PRISSETTE Marine Association pour la Recherche contre le Cancer (fellowship)

ROGNER Ute Centre National de Génotypage (salary)

DEMOND Anne, ademond@pasteur.fr or labavner@pasteur.fr

ROUGEULLE Claire - CNRS - Senior Staff Scientist, rougeull@pasteur.fr

ROGNER Ute - Postdoctoral fellow, urogner@pasteur.fr

WISZNIOWSKI Karine IP