Transcriptional Regulation Of FATS Gene And It's Inhibiting Effect On The Degradation Of P21

ObjectiveThrough Genome-wide dissection of mouse tumor genome using microarray-based comparative genomic hybridization (array-CGH), we have identified a previously uncharacterized gene and named it FATS (for Fragile-site Associated Tumor Suppressor). Furthermore, we identified a novel transcript variant of FATS (GenBank accession number:GQ499374) through screening a cDNA library from mouse testis. Our previous studies confirmed that FATS inhibits HDAC1 binding to p21, a key regulator of cell cycle,and enhances its acetylation and stability. The purpose of this study is to further elucidate the molecular mechanism underlying the transcriptional regulation of FATS gene and to investigate the protein-protein interaction involved in FATS-mediated stabilization of p21.Methods1. The results of bioinformatics analysis indicated that the promoter region of FATS gene contains potential p53-binding motifs. The DNA fragment of human FATS gene promoter (about 746Kb) was amplified by PCR, and a luciferase reporter driven by FATS gene promoter (746-luc) was subsequently generated. The reporter 746-luc was transfected alone or co-transfected with a p53-expressing vector into U87 (p53 wild-type) cells. The luciferase activity in trasfected cells were detected by a luciferase assay kit (Promega)2. U87 cells were transfected with or without p53, then chromatin immunoprecipitation (ChIP) was performed to detect the specific binding of p53 to the FATS promoter DNA.3. A point mutation in p53 responsive element (RE) was introduced though site-directed mutagenesis to generate M-746-luc.746-Luc or M-746-Luc was transfected alone or co-transfected with p53 into U87 cells, respectively. The luciferase activities in transfected cells were determined subsequently.4. As p53 is one known transcription factor in response to DNA damage, we further examined FATS expression in U87 cells with or without the treatment of etoposide, a chemical DNA-damage inducer, by RT-PCR and Western Blot.5. A GST-C8 expressing vector was trasnformed into E. coli BL21. After the induction of fusion protein with IPTG, the GST-C8 protein was purified.6. The Ac-p21 (139-164) and p21(139-164) peptide were synthezied, then GST pull-down assay was performed to evaluate the efffect of acetylation modification on their binding to proteasome C8 subunit.7. The effect of TSA, a deacetylase inhibitor, on the protein level of endogenous p21 in the presence of protein synthesis inhibitor cycloheximide (CHX) was determined by immunoblotting.Result1. The luciferase activity in cells co-trasnfected with a p53-expressing vector and 746-Luc was significantly higher than that in cells transfected with 746-Luc alone.2. The transcription factor p53 specifically bind to the promoter of FATS gene.3. The luciferase activity driven by M-746-luc in the presence of p53 was significantly decreased in comparison to that by 746-Luc under the same condition.4. The expression level of FATS was significantly induced in cells treated with epitoside.5. GST pull-down assay indicated that the purified GST-C8 protein strongly asso-ciated with p21 peptide. However, only tiny amount of Ac-p21 peptide was bound to GST-C8.6. In the presence of TSA, the protein level of endogenous p21 in cells was significantly increased within lh after CHX treatmnet.Conclusion1. FATS is a new trasncriptional target of p53. 2. Site-directed mutagenesis of p53-RE abolishes p53-mediated transcription activity of FATS promoter.3. The transcription of FATS is induced in cells after DNA damage.4. The acetylation of p21 protein enhances its stability.