Identification, cloning and characterization of the p53 induced gene human Wig-1

Abstract

The tumor suppressor p53 is a transcription factor that responds to various kinds of cellular stress. In respons to the sensed stress it is able to induces cell cycle arrest and apoptosis. In order to achieve these effects it transactivates target genes. Thus knowledge about p53 target genes and their function is vital for the understandnig of this important tumor suppressor.

We previously identified a novel p53-induced mouse gene, wig-1, that encodes a 290 amino acid zinc finger protein. Here we have identified and characterized the human homolog of mouse wig-1. The human wig-1 protein is 87% identical to the mouse protein and contains three zinc finger domains and a putative nuclear localization signal. Human wig-1 mRNA and protein is induced following activation of wild type p53. Northern blotting detected low levels of wig-1 mRNA in normal human tissues. Fluorescence in situ hybridization mapped human wig-1 to chromosome 3q26.3-27. FLAG-tagged human wig-1 localizes to the nucleus. Ectopic overexpression of human wig-1 inhibits tumor cell growth in a colony formation assay. These results suggest that human wig-1 has a role in the p53dependent growth regulatory pathway.

To further characterize the effect of Wig-1 on cell growth and survival, we generated Saos-2 osteosarcoma cells expressing tetracycline-inducible Flag-tagged human Wig-1. We found that induction of Wig-1 expression by addition of doxycycline caused a significant inhibition of growth but no changes in cell cycle distribution could be detected, nor did we see an increased fraction of cells with sub-G1 DNA content indicative of apoptosis. Colony formation assays in Saos-2 cells with wild type and zinc finger mutants of Wig-1 indicated that the first zinc finger domain is important for Wig-1mediated growth suppression. Wig-1 also inhibited colony formation in U20S osteosarcoma cells that carry wild type p53. Colony formation assays in HCT1 16 colon carcinoma cells revealed that the growth inhibitory effect of Wig-1 is more pronounced in the presence of wild type p53.

Furthermore, we show that human wig-1 has a functional p53 binding motif within its first intron, confirming its status as bona fide p53 target gene. Using a polyclonal Wig-1 antiserum, we show that the basal levels of the two Wig-1 protein species vary in human tumor cell lines. We also demonstrate that the two forms are generated by translation initiation from different AUG initiation codons. Wig-1 was induced by gamma irradiation and 5fluorouracil (5-FU) in a p53-dependent manner, whereas UV light only caused a modest induction of Wig-1 expression. Hypoxia did not induce any increased expression of Wig-1 despite accumulation of p53. Thus, Wig-1 is differentially regulated by p53 in response to stress stimuli.

In search of wig-1 orthologs we demonstrate its presence in Gallus gallus (chicken), Silurana tropicalis (frog) and Fugu rubripes (fish) by assembly of EST sequence data from a range of data bases into putative cDNAs. The zinc finger regions of Wig-1 are almost completely conserved from fish to man. A short upstream open reading frame is conserved in mammals and birds but not in amphibians. Sequence information from clones of Macaca mulatta (Rhesus macaque), Sus scrofa (pig), Danio rerio (Zebra fish) and Tetraodon nigroviridis (fresh water puffer fish) indicates that Wig-1 orthologs exist also in these species. No genes with striking similarities to wig-1 were found in invertebrates, Our results suggest that Wig-1 and particularly the zinc finger domains has an important biological function in the p53 pathway in vertebrates.