Study The Molecular Mechanism Of Thap11: The Regulation Of Gene Transcription

THAP11 belongs to the newly identified THAP family which was characterized by the presence of an evolutionarily conserved motif. Many findings suggest that THAP proteins may function as sequence-specific DNA binding factors with roles in cell proliferation, apoptosis, chromatin modification and transcriptional regulation. Our previous results have demonstrated that THAP11 represses the expression of c-Myc by binding to the promoter sites of c-Myc then inhibits the cell growth. In addition, THAP11 can regulate self-renewal and the cell proliferation of the embryonic stem cells. But recently the mechanism remained to study. We discovered the double transcriptional activity of THAP11 and confirmed the functional domain to explore the reporter gene assay and serial deletion mutant, the repressive domain of THAP11 was located at the N terminal 82 amino acids, in addition to a repressive domain, the transactivation domain at C terminal 185 amino acids and the transcriptional repression activity was not related with the chromatin modification factors. Further our labs performed a yeast two-hybrid screening of a human liver cDNA library, C-terminal binding protein 1(CtBP1) was identified as an interacting substrate for THAP11. We confirmed the interaction between THAP11 and CtBP1 and managed to find the interaction region of THAP11 with CtBP1 through the coimmunoprecipitation.As to CtBP1 that serve as a transcriptional corepressor and recruited by other proteins which regulated the transcriptional repression activities, it may be a platform for regulating the transcriptional repression activities of THAP11 through THAP11 interaction with CtBP1.To further identify the molecular mechanism of THAP11 that regulates the expression of target gene, we discovered the potential binding site of THAP11 in the Oct4 promoter by study the human genome. We constructed the human Oct4 promoter and discovered that the Oct4 promoter activity was up-regulation by THAP11 and it depended on the C terminal of THAP11 but the activity was not related with the DNA binding capability.THAP11 can bind to the forecasted binding site of Oct4 promoter region from the EMSA,but it may be not directly bind to the binding site of Oct4 promoter on the analysis of the transcriptional activity of deletion mutants of the hOct4 promoter upstream region, which suggested that THAP11 regulated the Oct4 gene expression through other mechanism. In vivo the expression of endogenous Oct4 was up-regulation when THAP11 was overexpression in PA-1 cells through Real-time PCR, which suggested Oct4 was the regulational target gene of THAP11. To further study in ES cells, we constructed the lentiviral vector containing human Thanatos-assotiated protein 11 gene and established an expression system, and then produced the THAP11 lentivrirus.