Identification Of Promoter Of Human Restin And Its Regulation By P53

restin was a novel gene isolated by our lab from the differentiated cancer cell (HL60) induced by all-trans retinoic acid (ATRA) in 1999, and its molecular functions were not well understood. Chomez and colleague also found this gene by homologous sequence scanning in the database of Genebank in 2001 and named it as melanoma associated antigen HI(MAGE-H1).Restin was a 219 amino acid residue alkaline protein, and contained a bipartite nuclear targeting sequence and a MHD (MAGE homology domain, MHD) located in the N-terminal and C-terminal region respectively. Restin is localized on the X chromatosome in Xp11.22 site. The phylogenetic analysis of MAGE family proteins and Restin shows that the relationship of Restin is closer to type II MAGE. The tissue expression analysis of Restin shows that Restin is highlyexpressed in normal terminal differentied cells, prostate, testis, nerves and fetal tissues, and low or less in the white blood cells or tumor tissues.The tumor cells transfected with restin are arrested at G1 phase and the cell proliferation is inhibited. It implies that restin might be correlated to cell cycle regulation. It is not well known about the regulation of restin's transcription.The regulation of gene expression has important biological value for the cell in the course of growth, differentiation, development, and adapting to enviroment. In eukaryonic cell, the temporal and spatial specificity of gene expression is controlled by specific factors. The whole expression of gene can be divided into several steps: gene activation, transcription, processing after transcription, translation, and posttranslational process. There are many checkpoints for each steps of gene expression. The most efficient regulation of gene occurs at transcriptional level by regulating the interaction between transcriptional factors and cis-acting recognizing element.The regulation of the target gene expression at transcriptional level was affected by the interaction between the relevant transcriptional factors and their recognizing cis-acting sequence in the promoterregion. Promoter, in a broad sense, consists of transcriptional start site, binding site of RNA polymerase (promoter in a narrow sense) and upstream cis-acting sequence. The most efficient regulation of gene occurs at transcriptional level by regulating the interaction between transcriptional factors and their upstream recognizing sequences. Thus, to investigate the regulation in promoter region of a target gene will be helpful to predicate the principle of molecule regulation, biological function of molecules and their significances in pathogenesis of some diseases.In our study, we first constructed the luciferases report system of restin gene, and detected the relative luciferase to define the location of promoter. By use of dot blot, RT-PCR assays, we further observed the relationship between P53 and restin gene expression at transcriptional level. The main results are as follows:1. Cloning of the promoter of restin and constructing of promoter luciferase report vector.We made the luciferase report system by cloning the upstream sequence of restin gene (2000bp from ATG), into the pG5-luc vector containing the downstream luciferase gene, then, we could detect theluciferase activity if the promoter was functional.We transfected luciferase report system into the Hela cell line, added with ATRA, then detected the rise of luciferase reading. The results demonstrated that the functional promoter of the restin has been cloned in the report vector.2. Functional location and characterization of restin promoter sequence .We cloned three truncations of Rpl 5' end (Rp2-luc, Rp3-luc, Rp4-luc) according to the promoter prediction result and transcription factor binding sequence. Then we transfected luciferase report system into cell line, the luciferase activity of three delection vector was increased after the use of ATRA. So we proposed the upstream region (-945bp from ATG) is the functional region of restin promoter.3. P53 upregulating the transcription of restin by detecting the relationship of restin gene expression with P53, transient co-transfection of p53 and luciferase report system.We transfected P53 into A549 cell line which has a low Restin level, and 48h later, results of RT-PCR and dot blot, showed that P53 upregulated the transcription of restin.We co-transfected luciferase report plasmid (Rpl-luc) with P53...