The Molecular Mechanism Of FATS-mediated Regulation Of P53Protein Expression

ObjectiveThe purpose of this study is to further understand the molecular mechanism underlying the regulation of p53expression by FATS, a new fragile-site gene, through molecular and cell biology approaches including investigating the protein interaction between FATS and p53, posttranslational modifications of p53protein in response to DNA damage, the impact of FATS on the transcriptional activity and protein stability of p53. This study will also test the hypothesis that FATS forms a positive feedback loop with p53.Methods1. Cells were transfected by a FATS-expressing vector. After24h, the protein lysate or total RNA were prepared. The expression of p53protein was detected by Western blot, and the expression of p53mRNA in cells was detected by RT-PCR.2. The transfected cells were treated with Cycloheximide (CHX), a inhibitor of protein synthesis, or Etoposide, a chemical compound inducing DNA damage. The expression levels of p53protein were detected by Western blot to test the effect of FATS on p53protein stability and modification including acetylation and phosphorylation.3. The protein interaction between FATS and p53in vivo was examined by Co-immunoprecipitation (IP) and Western blot assay.4. The vector GST-FATS(1-363) was trasnformed into E. coli BL21. After the induction of fusion protein with IPTG, the GST-tagged FATS(1-363) protein was purified for studying the protein interaction in vitro.5. The purified p53protein was obtained by in vitro translation.6. Purified p53protein and FATS (1-363) protein were subjected to GST pull-down assay to verify the direct interaction between N-terminal FATS protein and p53protein.7. Chromatin immunoprecipitation (ChIP) assay was performed to detect the binding of endogenous p53to the promoter of p21gene.8. The luciferase reporter pGL2-p21-luc was transfected alone, or cotransfected with p53or FATS expression vector into H1299(p53silent) cells. The luciferase activities were measured subsequently to determine the effect of FATS on p53transcription factivity.Result1. Compared with Control group, the expression level of p53protein in the cells was significantly increased after cells FATS transfection, while the expression of p53was significantly decreased after inhibition of FATS expression using FATS-shRNA.2. The express of FATS did not change the level of p53mRNA.3. After inhibition of protein synthesis by Cycloheximide (CHX), FATS expression significantly inhibited the degradation of p53protein.4. The results of coimmunoprecipitation and GST-pull-down showed that FATS protein interacted with p53protein in vivo and in vitro. The N-terminal (1-363) region of FATS protein directly bound to p53.5. After DNA damage, FATS expression further facilitated the accumulation of p53protein, and enhanced the acetylation and phosphorylation of p53protein.6. FATS expression increased the mRNA level of p21, and enhanced the binding of p53to p21promoter. The results of luciferase reporter assay indicated that FATS by itself did not function as a transcription factor and FATS-mediated regulation of p21transcription was p53-dependent.Conclusion1. FATS upregulates p53through increasing the stability of p53protein.2. FATS is a binding protein of p53, and p53directly binds to the N-terminal domain of FATS.3. FATS promotes p53activation after DNA damage, further enhancing the stability of p53protein and increasing the status of p53acetylation and phosphorylation.4. FATS increases the transcriptional activity of p53, and has strong impact on regulating p53-p21pathway.