Identification of Regulatory Element(s) Controlling the Adrenal-Specific Expression of Genes within Human C4/CYP21 Gene Locus

• Main Authors: Wei-Chih Hsiao, 蕭偉志
• Other Authors: Shwu-Fen Chang
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/32170503317386721354

碩士 === 台北醫學院 === 細胞及分子生物研究所 === 88 === Abstract: Steroid c21-hydroxylase regulates the adrenal steroid hormone synthesis. Enzymatic deficiency of this enzyme disrupts the homeostasis of steroid hormone, resulting in a genetic disease, the congenital adrenal hyperplasia (CAH). Gene encodes this enzyme is CYP21, along with the pseudogene CYP21P, and other duplicated genes together to form the C4/CYP21 locus on human chromosome 6. Many genes in this locus show specific expression pattern in adrenal gland, thus a possible locus control region (LCR) was proposed to control the tissue-specific expression of these genes. In order to identify the possible LCR within this locus, we have analyzed the influence of DNA fragments upstream from CYP21P and CYP21 genes on the transcription activity of CYP21 basal promoter. Mouse adrenal carcinoma Y1, testicular Leydig MA10 and human hepatocarcinoma HepG2 cell lines were used in the in-vitro assay. Results showed that DNA fragments (-7519/-6315, -4602/-3442 and -3268/-2583 bp) upstream from CYP21 have enhancer activity on CYP21 basal promoter in Y1 cell, but not in non-adrenal MA10 and HepG2 cells. These DNA fragments were proposed that they might contribute the adrenal-specific expression activity of genes within this locus. In addition, our previous data showed that a 1.3 kb DNA fragment (-10981/-9654 bp) upstream from CYP21P gene has steroidogenic enhancer activity on the CYP21 basal promoter in steroidogenic Y1 and MA10 cells, but not in non-steroidogenic HepG2 cells in an orientation-dependent manner. Results showed that the enhancer activity of the 1.3kb fragment is supplied by the central 475 bp (-10427/-9952 bp) fragment. The activity of this 475 bp fragment is stronger then that of 1.3 kb fragment in an orientation-independent manner indicating that flanking sequence involves in regulating the steroidogenic enhancer activity. Electrophoresis mobility shift assay results showed that the -10157/-10073 bp fragment would interact with specific protein which from Y1 or MA10 cells but from HepG2 cells. We proposed that this special DNA-protein interaction maybe involve in controlling the steroidogenic enhancer activity of the 475 bp fragment. All these results indicate that there may be special regulatory element(s) consisting of multiple short sequences together in the tested regions involve in controlling tissue-specific expression.