The p53-induced gene wig-1 : regulation of expression and role in embryonic development

Abstract

The p53 tumor suppressor is a transcription factor that responds to various forms of cellular stress. Activated p53 induces growth arrest or apoptosis through transcriptional regulation of a wide range of target genes. More than 50% of human tumors carry mutations in the p53 gene, most of which reside within the DNAbinding core domain of p53. Most mutant p53 proteins are unable to bind to p53 motifs in DNA and activate transcription of target genes, and are therefore unable to initiate cell cycle arrest or apoptosis in response to cellular stress.

The aims of this thesis were to characterize the regulation and function of the p53-induced gene wig-1 (wild type p53-induced gene-1). Wig-1 encodes a protein containing three Cys2His2 zinc fingers and a potential bipartite nuclear localization signal, NLS. Wig-1 is upregulated in response to activation of wild type p53 and this lead us to investigate whether wig-1 is a direct transcriptional target of p53. The promoter region of wig-1 was cloned and partially characterized. Three putative p53-binding motifs were found in the mouse wig-1 promoter region. Two of the sites were found to form DNA/p 53-complexes in electrophoretic mobility shift assay (EMSA) and were able to drive p53-dependent transcription in a cellular reporter assay. This demonstrates that wig-1 is a bona fide p53-target gene.

When analysing the zinc finger structure of wig-1 we observed that it shared some structural features with two other proteins, JAZ and dsRBP-Zfa, that have been found to bind dsRNA. Using EMSA we demonstrated that a recombinant GST-wig-1 fusion protein preferentially binds dsRNA over ssRNA or dsDNA. The first zinc finger in wig- I was found to be essential for the binding to dsRNA. Wig-1 represents the first confirmed p53-induced gene that encodes a dsRNA-binding protein. This suggests that dsRNA-binding plays a role in the p53dependent stress response.

To investigate whether the p53 paralogs, p73 and p63, could also regulate wig-1, we generated cell lines that express temperature sensitive mutant p73 or p63 that obtain wild type properties at the permissive temperature. Using these cells we could see that both p73 and p63 upregulate wig-1 on mRNA and protein level after temperature shift. Moreover, ectopically expressed wig-1 stabilized p53 protein levels and inhibition of endogenous wig-1 by siRNA reduced p53 protein in wild type p53-expressing MCF7 and HCT116 cells. In contrast, overexpression of wig-1 reduced p73 protein half-life and inhibition of endogenous wig-1 resulted in an increase in p73á and p73â protein levels. Our results demonstrate that wig-1 is transcriptionally induced by all members of the p53 family, and that wig-1 in turn acts as a positive regulator of p53 but antagonizes p73. Thus, wig-1 can be viewed as a rheostat that regulates the relative activity of the p53 and p73 pathways.

In order to study the role of wig-1 in embryonic development and its possible role in tumorigenesis, we generated mice carrying inactivated wig-1-alleles. We found that complete lack of wig-1 is associated with embryonic lethality. The wig-1 null mouse embryos died before E 10.5 during gestation and showed severely deficient development of the brain. These results demonstrate that wig-1 is essential for normal embryonic development.