Molecular studies of Wig-1, a p53-induced zinc finger protein

Abstract

The fact that p53 is one of the most commonly mutated gene in human tumors indicates that it has a key role in tumor suppression. p53 accumulates after DNA-damage and other types of cellular stress, leading to the initiation of a p53-dependent biological response, including cell cycle arrest and apoptosis. The mechanisms by which p53 accomplishes its biological functions are still not completely understood. Several lines of evidence clearly support the notion that the ability of p53 to transcriptionally regulate target genes is important for its tumor suppressor activity. Hence, the identification of transcriptional targets of p53 is essential to gain insight into the molecular mechanisms underlying p53 mode of tumor suppression.

We have previously used the PCR-based differential display technique to identify mRNAs induced by wild type p53 expressed from the temperature-sensitive Val-135 mouse mutant p53 construct in J3D mouse T lymphoma cells. This system led us to the identification of a novel p53-induced mouse gene designated wig-1 (for wild type p53-induced gene), which encodes a Cys2His2-type zinc finger protein of unknown function.

In order to evaluate the role of wig-1 in the p53 response in human cells we have isolated and characterized the human ortholog of wig-1. Human wig-1 (hwig-1) is upregulated by wild type p53 using the temperature sensitive (ts) p53 system in BL41 cells and by DNA damage in cells carrying wild type p53. It localizes to chromosome 3q26.3-27, a region altered in a variety of human tumors. In addition, we found that hwig-1 inhibits tumor cell growth in a colony formation assay.

The fact that the wig-1 mRNA is upregulated within 2-4 hours in response to p53 activation suggested that wig-1 is a direct transcriptional target of p53. Indeed, we showed that two p53-binding motifs located at the promoter region could form DNA-protein complexes with p53 and drive p53-dependent transcription in a luciferase reporter assay. These results demonstrate that wig-1 is a bona fide p 53-target gene.

The observation that the distribution of zinc fingers in Wig-1 resembles that of the previously described double-stranded RNA-binding proteins, dsRBP-ZFa and JAZ, led us to analyze the nucleic acids binding properties of Wig-1. We found that both mouse and human Wig-1 bind dsRNA with higher affinity compared to dsDNA, ssRNA and DNA-RNA hybrids through its most N-terminal zinc finger domain in vitro. Immunoprecipitation experiments showed that Wig-1 binds dsRNA also in vivo. In addition, we demonstrated that Wig-1 binds dsRNA of a minimal length of 23-50 bp and 5'-overhangs. Interestingly, Wig-1 could also efficiently bind to a 21-nucleotides dsRNA probe resembling the structure of several noncoding RNAs including small interfering RNAs (siRNAs) and micro RNAs (miRNAs). Taken together, these results raise the possibility that p53 and Wig-1 play a role in the regulation of RNA interference and/or translational repression mediated by siRNAs and miRNAs.

Many nuclear proteins are regulated by acetylation. Here, we have studied whether Wig-1 is also posttranslationally modified by acetylation. We found that Wig-1 interacts with p300 and that it is a substrate for p300/CBP-mediated acetylation. Moreover, we found that binding of Wig-1 to dsRNA inhibits Wig-1 acetylation by p300 in vitro.

In summary, this thesis provides evidence that suggest the implication of dsRNA-binding in the p53-mediated biological response and opens a new avenue toward the comprehensive understanding of p53 function.