X chromosome inactivation
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 non-coding RNA and 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 based on the analysis of additional critical recombinant animals and the characterisation of novel polymorphic markers including SNPs (Single Nucleotide Polymorphisms). The candidate region is currently under study using a genetic deletion approach which should allow molecular characterisation of this locus which was first identified some thirty 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 analysis has defined at least two subregions 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.
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 over the past eighteen months.
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 comparative sequence analysis of the mouse, human and bovine Xic regions. Conserved regions which are not coding are candidates for regulatory roles in the function of the Xic region.
Genetics factors involved in the X-inactivation process lying outside of the Xic are being looked for by a complementary approach based on a comparative transcriptome analysis of female and male cells of the early embryo using SAGE (Serial Analysis of Gene Expression) analysis.
Genome Research and Mouse Disease Models
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 mouse breeding has allowed candidate gene approaches to be undertaken to defining and characterising the genes responsible for these traits.
One means of reducing the genetics complexity underlying type 1 diabetes is to 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 of the mouse genome 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 another X-linked gene, Nap1l2. Mutations in this gene are associated with embryonic lethality, spina bifida and exencephaly linked to a massive over proliferation 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 have suggested that the NAP1L2 protein cycles between the cytoplasm and the nucleus and may be involved in histone protein transfer to the nucleus.