Induced chromosome deletions cause hypersociability and other features of Williams-Beuren syndrome in mice

The neurodevelopmental disorder Williams-Beuren syndrome is caused by spontaneous similar to 1.5 Mb deletions comprising 25 genes on human chromosome 7q11.23. To functionally dissect the deletion and identify dosage-sensitive genes, we created two half-deletions of the conserved syntenic region on mouse chromosome 5G2. Proximal deletion (PD) mice lack Gtf2i to Limk1, distal deletion (DD) mice lack Limk1 to Fkbp6, and the double heterozygotes (D/P) model the complete human deletion. Gene transcript levels in brain are generally consistent with gene dosage. Increased sociability and acoustic startle response are associated with PD, and cognitive defects with DD. Both PD and D/P males are growth-retarded, while skulls are shortened and brains are smaller in DD and D/P. Lateral ventricle (LV) volumes are reduced, and neuronal cell density in the somatosensory cortex is increased, in PD and D/P. Motor skills are most impaired in D/P. Together, these partial deletion mice replicate crucial aspects of the human disorder and serve to identify genes and gene networks contributing to the neural substrates of complex behaviours and behavioural disorders

Isolation and characterization of genes involved in glucocorticoid-induced thymocyte apoptosis

Apoptosis is characterized by a series of well defined morphological and biochemical features that allow cells to initiate self-destruction in response to a variety of stimuli. CD4⁺CD8⁺ is a sub-population of immature thymocytes that are especially prone to the action of apoptosis-inducing agents and are sensitive to glucocorticoid-induced apoptosis, an event that plays a critical role in eliciting the antigen-specific thymocyte repertoire. Glucocorticoids induce apoptosis through activation of the GR, a ligand-induced transcription factor that transduces the hormonal signals into the regulated expression of target genes. While much is known about the structure and function of GR, key steroid-regulated genes believed to be required for thymocyte apoptosis have not been found. Based on the transcriptional-regulation of apoptosis by ecdysone-mediated induction of reaper gene expression in Drosophila, and p53-mediated transcriptional-activation of Bax gene expression in mammalian cells, our laboratory has set out to identify GR-regulated genes in a glucocorticoid-sensitive mouse thymocyte cell line called WEHI 7.2. Identification of such genes involved in this process, and elucidation of the underlying mechanisms by which they mediate apoptosis, have prime importance because malfunctioning of this system has been shown to cause severe human diseases and developmental abnormalities. I have isolated and characterized the full-length nucleotide sequence of mGIG1, a novel gene product that had previously been shown to be up-regulated in thymocytes undergoing glucocorticoid-induced apoptosis. The human homolog of the mouse GIG1 was also identified and mapped to chromosome 8, band q21. A peptide segment of GIG1 protein was expressed in bacteria and used to raise antibodies in rabbits in order to dissect its role in thymocyte apoptosis. Results of DNA sequence analyses suggest that mGIG1 may encode a DNA-binding protein that could function as a transcription factor in thymocytes. In a second project, I established a transient expression cloning system in mouse thymoma cells and isolated a cDNA sequence called mArg-2 on the basis of a functional cloning strategy. However, determination of the possible involvement of both GIG1 and Arg-2 in thymocyte apoptosis will require further experimentation. Finally, spatial and temporal coordination of abrupt changes in mismatch repair (MMR) gene expression with occurrence of apoptosis in mouse thymocytes prompted me to study apoptosis in MMR-deficient mice. My findings indicate that glucocorticoid and anti-Fas antibody-induced cell death machinery is intact and functional in MMR⁻/⁻ mice

Characterization of Apt- cell lines exhibiting crossresistance to glucocorticoid- and Fas-mediated apoptosis

Apoptosis induction by staurosporine, ceramide, and Fas stimulation was investigated in the mouse thymoma cell line W7.2 and a panel of dexamethasone (dex)-resistant W7.2 mutant cell lines, Apt3.8, Apt4.8 and Apt5.8, and a Bcl-2 transfectedW7.2 cell line (Wbcl2).WhileW7.2 cellswere found to be sensitive to these apoptosis inducers, the Apt- mutants andWbcl2 cells were shownto be resistant tosome or all of the treatments.Specifically, all threeApt-mutantsandWbcl2cells were found to be resistant to ceramide and Fas-mediated apoptosis, whereas, Apt4.8 and Apt5.8 were sensitive to staurosporine-induced apoptosis under conditions in which Apt3.8 and Wbcl2 cells were resistant. Measurements of caspase activity and cytochrome c release in cytosolic extracts of dex and staurosporine-treated cells indicated that the recessive Apt- mutations effect steps upstream of mitochondrial dysfunction. Steady-state RNA levels of apoptosis-associated gene transcripts showed that the observed differential resistance of the Apt- cell lines could not be explained by altered expression of numerous Bcl-2 or Fas related genes. Transient transfection of human Fas gene coding sequences into the Apt- mutants and Wbcl2 cells did not induce apoptosis, even though these same cell lines were sensitive to ectopic expression of the FADD and caspase 8 genes. Taken together, these data provide genetic evidence for the existence of shared components in the dex- and Fasmediated apoptotic pathways in W7.2 cells