Analysis of human Dynamin IV (Dymple) gene promoter

• Main Authors: Wei-Dih Sy, 思瑋玓
• Other Authors: Hong, Yi-Ren
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/95094024753912977066

碩士 === 國立中山大學 === 生物科學系研究所 === 91 === We first identified the transcriptional regulatory element of the human dynamin IV gene (Hdyn IV; dymple). The Hdyn IV belongs to a large GTPase family. This protein has a N-terminal highly conserved tripartite GTP-binding domain, coiled-coil (CC) region, but it lacks the pleckstrin homology (PH) domain and a modestly conserved C-terminal proline rich domain (PRD). Hdyn IV gene is enriched in subcellular membrane fractions of cytoplasmic vesicles and endoplasmic reticulum, and the function of Hdyn IV gene is considered to be associated with the functions of mitochondria. The Hdyn IV is expressed as four alternative splicing variants in all eukaryotic organisms. Our question concerning why expressions of four alternative splicing variants in brain tumor tissues? To elucidate the regulatory mechanism and the transcription factors involved, we firstly determined the transcriptional start site by 5’ RACE. We next cloned the 5’-flanking region of the Hdyn IV gene and determined the nucleotide sequence of 999 bases upstream from the transcription start site. The promoter has several potential binding sites for AP2, Sp1 binding protein, but it lacks TATA and CAAT boxes. Transfection studies using a series of Hdyn IV promoter luciferase constructs in HeLa cell demonstrate that the 5’flanking region has a promoter activity. Functional promoter element of the Hdyn IV gene was located within the –140~ +29 region. Deletion analyses demonstrated that the minimal promoter activity for the transcriptional element of Hdyn IV was detected in the sequence between nucleotides –110 and –100. Electorphoretic mobility shift assay demonstrated that a putative transcriptional factor bound to the –119 to –90 region. Site-directed mutagenesis analysis of this region revealed that nucleotides at positions –108 to –100 were essential for transactivation mediated by this element. To summary, the data indicated that the ’’CTCCCAGCA’’ (-108~ -100) sequence is capable of regulating Hdyn IV gene expression. However, the protein involved in the binding of this novel sequence requires further study.