Interactional Investigation Between Sp1 And P300 By The Mammalian Two-hybrid Assays

"Two-hybrid"or"interaction trap"is one of the tools for the research of protein-protein interactions in vivo by measuring the expression of the reporter gene activated by fused-proteins, based on reconstructing functional transcription factors. This technique enables not only the identification of interacting partners but also the characterization of known interaction couples. Mammalian Two-hybrid is the developed two-hybrid used in the higher eukaryotic cell lines.p16INK4a functions as a suppressor of cell cycle,and its binding to CDK4/CDK6 can prevent CDK4/CDK6 from binding to cyclinD,which leads to G1 arrest. Consequently, the mutants of p16 are found common in many kinds of tumors. There are several GC boxes, which is the classical Sp1-binding sites in the promoter of p16INK4a gene. It has been reported that Sp1 is a strong activator of this promoter. Meanwhile, p16INK4a is also regulated by histone acetylation. This study demonstrated that acyltransferase p300 can activate p16INK4a on promoter activity, mRNA level and protein expression with Sp1 synergistically. Then, we investigated the precise interactions between Sp1 and p300. Experimental data presented in this thesis show that the N-terminal region of Sp1 and the Q domain of p300 play important roles in the interaction between Sp1 and p300.