鑑定 p53 在 p27 promoter 上的調控位置

Abstract

Angiogenesis is a process of the new outgrowth of capillaries from the pre-existing blood vessels. It has been indicated that female sex hormones could regulate the process of angiogenesis. The presence of progesterone receptor in human vascular endothelial cells led us to propose that progesterone might regulate the endothelial behavior. Previously, our lab has demonstrated that progesterone (at a concentration of 500nM) inhibited DNA synthesis of cultured human umbilical vein endothelial cells (HUVEC) through a p53-dependent pathway. Progesterone increased the level of p53 protein, which in turn increased the levels of p21 and p27 protein, subsequently inhibited the activity of cyclin-CDK2 complex, and finally arrested the cell cycle at the G0/G1 phase and decreased the DNA synthesis. Previous studies have demonstrated that the p53 protein directly activates p21 expression though binding to the p21 promoter at 75 bp and 2.4 Kb site upstream the start codon of p21 gene. The p53-regulated p27 expression has not been studied before. Accordingly the aim of this thesis research is to investigate further the precise binding domains of p53 protein on the p27 promoter. In order to analyze the p53 binding domains on the p27 promoter, two different p27 promoter fragments were subcloned into the pGL3-luciferase reporter vector. Luciferase assay showed that the potential p53 binding region spans on site 258 to site 310 upstream the start codon of the p27 gene. Within this range there are three likely binding fragments including p27PF-Δ1, p27PF-Δ2, and p27PF-Δ3, and between each fragment is separated by less than 13 base pairs. We individually deleted p27PF-Δ1, p27PF-Δ2, and p27PF-Δ3 fragment, and then subcloned these mutated clones into luciferase reporter vector. Promoter activity assay showed that these mutated clones of the p27 promoter did not response to progesterone treatment, suggesting that all these three fragments are essential for p53 protein to regulate the p27 promoter activity. In addition, chromatin-immunoprecipitation (ChIP) analysis indicated that progesterone receptor could bind onto the p27 promoter which lacks a canonical progesterone responsive element (PRE) after progesterone administration. Moreover, immunoprecipitation (IP) analysis demonstrated an association of progesterone receptor and p53 protein in nucleus after progesterone treatment. This result suggested that progesterone receptor might be involved in regulating p27 gene expression. Taken together, the findings of this thesis study suggested that p53 protein induced p27 gene expression through binding onto the p27 promoter at site 258 to site 310 upstream the start codon of the p27 gene. Our results also suggest that progesterone receptor might also participate in mediating the p27 promoter activity. However, the involvement of progesterone receptor in regulating the p27 gene expression needs further investigation.