Characterization ofp53 target genes identified by differential display: TIS11D and CYFIP2

The tumor suppressor, p53, is a ubiquitously expressed 53 kDa transcription factor that regulates cellular genotoxic stress responses to prevent incorporation of DNA damage into the genome. Loss of p53 function through mutation, allelic insufficiency, loss of heterozygosity, and disruption of upstream signaling to or downstream effector pathways from p53 frequently contribute to carcinogenesis. While p53 target genes that regulate the cell cycle---p21, 14-3-3sigma, and GADD45---have been identified and characterized, the gene network responsible for p53-dependent apoptosis remains unclear.; To identify novel p53-regulated genes, our lab has conducted a comprehensive Fluorescent Differential Display (FDD) screening of the tetracycline (TET)-regulated p53 cell lines, DLD1-p53 and H1299-p53. In addition to the detection of known targets such as p21 and MDM2, this screen identified TIS11D and CYFIP2 as two uncharacterized candidate p53-inducible genes. Induction of TIS11D mRNA was confirmed by Northern blot in response to p53 expression in DLD1-p53 cells. Inducible expression of a GFP-TIS11D fusion protein resulted in inhibition of cell proliferation and the induction of apoptosis. In a similar manner, induction of CYFIP2 mRNA in response to p53 expression was confirmed by Northern blot and quantitative real-time PCR in DLD1-p53 and H1299-p53 cells. The CYFIP2 promoter was detected to contain a p53-responsive element that conferred p53 binding as well as transcriptional activation of a heterologous reporter, respectively using chromatin immunoprecipitation (ChIP) and dual luciferase reporter assays. Inducible expression of a GFP-CYFIP2 fusion protein was sufficient for caspase activation and cellular apoptosis, reminiscent of p53 activation. In addition, the sensitivity of CYFIP2 protein subcellular localization to Leptomycin-B, a CAM-1/Exportin inhibitor, suggests that the biological functions of CYFIP2 may extend from the cytoplasmic compartment into the nucleus of the cell.; In summary, the mRNA-binding protein, TIS11D, and the adaptor protein, CYFIP2, may play an important role(s) in the cell as parts of the network of mediators of p53-dependent tumor suppression.