Screening And Research On The Transcription Factors Regulating Of P53Protein

The development and progression of tumor involves activation and inactivation ofa variety of oncogenes and tumor suppressor genes. P53is an important tumorsuppressor which is associated with many cellular processes including, but not limitedto, regulation of the cell cycle, DNA repair, cell senescence and apoptosis. p53has longbeen recognized as a major tumor suppressor and the most commonly mutated genes inhuman cancer. The frequent inactivation of p53in tumors fostered the attractive notionthat restoring the function of p53in tumors will provide an effective tumor-specifictherapy, therefore, the strategies of restoring the function of wildtype of p53have thevital significance and some have reached to clinical trials, which highlight theimportance of the research on the regulation of p53on transcriptional level. Through thereview of the literature, we found that there are more than60,000PubMed-listedpublications, however, most of them are focused on the study of post-transcriptionallevel. Based on the above reasons, we cloned p53promoter and inserted it into aluciferase reporter gene vector. By screening multiple transcriptional factors that mayregulate p53transcription using luciferase reporter system, we obtained a noveltranscriptional factor regulating p53, temporarily named as TF1.Until now, little is known about the function of TF1. The previous research of TF1was mainly focused on the constitutive interaction with ERβ to regulate antioxidativeenzyme Quinone reductase (QR). We first demonstrated that TF1activated p53transcription in a dose-dependent manner. qRT-PCR and Western blot analysisconfirmed that overexpression of TF1increased both the transcriptional level andprotein level of p53, respectively. Immunofluorescence and subcellular fractionationassay revealed that TF1as a transcription factor mainly located in nucleus. Chromatinimmunoprecipitation (ChIP) further showed that TF1could be directly recruited to thep53promoter. Epigenetic studies have demonstrated that mono-, di-and tri-methylations of thefourth lysine (K) of histone H3in gene promoter region activate the gene transcription.Therefore, we studied the molecular mechanisms of transcriptional activation of p53gene by TF1through research on H3K4methylation. ChIP assays showed that TF1enhanced the recruitment of H3K4me, H3K4me2, H3K4me3to the p53promoter, andthe recruitment of endogenous methylation transferase SETD7to p53promoter.Overexpression of SETD7also enhanced the recruitment of TF1to the p53promoter.These results suggest that, TF1activates p53transcription by enhancing therecruitment of SETD7to the p53promoter and SETD7in turn facilitates therecruitment of TF1to the p53promoter.Finally, using hepatocellular carcinoma (HCC) cell line as a model, we studied thebiological function of TF1. After TF1knockdown cell lines were established, weexamined the expression of p53downstream target genes. The results of western blotand qRT-PCR both showed that TF1selectively increased the expression of p53downstream target genes, such as p21and BAX, but not GADD45and PIG3. Celltoxicity test indicated that TF1inhibited the proliferation of HCC cells, and suppressedthe formation and metastasis of tumor in BAL B/C mice.In summary, the transcription factor TF1as a novel transcription activator of p53,not only increases the level of p53protein, but also selectively changes downstreamtarget genes of p53. In addition, TF1inhibits the growth and progression of tumor cellsboth in vitro and in vivo. Therefore, further studies on the functions and mechanisms ofTF1are expected to provide clearer clues for the study of mechanism of how p53functions in tumors, which will provide new ideas and strategy for clinical treatment oftumors.