Download
1 / 2
20 likes | 131 Views
人類CYP21基因腎上腺專一性表現之調控元素的研究 Characterization of the regulatory element(s) controlling the adrenal-specific expression of human CYP21(P450c21) gene.
E N D
人類CYP21基因腎上腺專一性表現之調控元素的研究Characterization of the regulatory element(s) controlling the adrenal-specific expression of human CYP21(P450c21) gene. • 21氫氧化酵素 (steroid 21-hydroxylase) 是一種 cytochrome P450 酵素 ， 參與類固醇荷爾蒙的生合成 ， 而且只在腎上腺皮質表現 。 這個酵素的缺乏已知與腎上腺皮質增生症這種遺傳疾病的發生有關 。 負責表現人類 21 氫氧化酵素的基因有兩套 ， 一為活性基因 、 CYP21 ， 另一為 CYP21P 偽基因 。 這兩個基因與人類白血球抗原的第四個血清補體要素基因 、 C4A 及 C4B ， 在染色體上以全長約 75 kb ， 5''C4A-CYP21P-C4B-CYP213'' 的重覆方式排列 。近年來在此染色體區域內陸續發現的其它成對基因組：XA、XB 、XB-S、YA、YB、ZA 及 ZB 基因與 C4 及 CYP21 形成 5''C4A-ZA-CYP21P-YA-XA-C4B-ZB-CYP21-YB-XB-S-XB3''的排列 。 其中 ， 除了C4A，C4B，XB 之外 ， 其餘基因的轉錄產物都具有腎上腺專一性的表現 。 我們因此設計實驗以分析在這個基因組中是否存在一段類似 locus control region (LCR) 的基因元素 ， 可以調控這些基因在腎上腺具專一性的表現 。 我們以短暫轉染實驗 ， 利用 CAT assay 及引子延伸方法 ， 分析人類 CYP21P 基因上游 13.5 kb 以及 CYP21 基因上游 6.3 kb 的DNA 區域對於 CYP21 基因啟動子轉錄活性的影響 。 我們以老鼠腎上腺皮質腫瘤細胞 Y-1 ， 老鼠睪丸間隙細胞 MA10 ， 以及人類肝臟腫瘤細胞 Hep G-2 這三種細胞來分析基因在腎上腺皮質專一表現的性質。 實驗結果顯示在 CYP21 基因上游 -6328/-1668 bp 這整個 DNA 片段可於 Y-1 細胞中增強 CYP21 基本啟動子的轉錄活性約二倍 ， 但這種現象並未於 MA10 及 Hep G-2 細胞內被偵測到 。 而其它測試的 DNA 區域對 CYP21 基本啟動子的轉錄活性卻大都呈現抑制的效果 。 我們因此推測在這 -6328/-1668 區域內可能具有能夠調控 CYP21 基因具腎上腺專一性表現 ， 而類似 locus-control region 的 DNA 序列 。 一旦此區域的 DNA 以較小的片段存在時 ， 它對於 CYP21 基本啟動子的轉錄活性之增強作用卻消失了 ， 反而有抑制的效果 。 因此 ， 確認在此 4.7 kb 的DNA 區域內所存在的 LCR 之確切位置 ， 將是我們未來繼續研究的方向 。
Steroid 21 hydroxylase (P450c21) is specifically expressed in the adrenal cortex, and is required for the biosynthesis of steroid hormones. Enzymatic deficiency of 21-hydroxylase causes the congenital adrenal hyperplasia (CAH), an autosomal recessive disorder. There are two copies of human steroid 21-hydroxylase gene, the active CYP21 which encodes the enzyme, and the pseudogene, CYP21P.These two genes are localized on human chromosome 6p21.1, and duplicated in tandem with the genes for the fourth components of complement, C4A and C4B, andother newly characterized duplicated genes, organized as following: 5''C4A(ZA(CYP21P(YA(XA(C4B(ZB(CYP21(YB(XB-S3''. In this complex locus, most of the transcripts are adrenal-specific, including the CYP21, XA, YA, ZA,. XB-S, YB, ZB.To study the cis-elements controlling the adrenal-specific expression of these genes, we analyzed the effects of DNA sequences upstream from the CYP21P and CYP21 on the transcription activity of the basic promoter of the CYP21. We used mouse adrenocorticoid tumor Y-1 cell, testicular Leydig MA10 cell, and human liver Hep G-2 tumor cells to analyze the adrenal-specific expression. After transient transfection into cells, the transcription activity was analyzed using CAT and primer extension assays. The results showed that DNA sequences at the -6328/-1668 bp region upstream from the CYP21 enhanced the transcriptional activity of CYP21 to 2 fold in Y-1 cell, but not in MA10 or Hep G-2 cells. However, smaller DNA fragments covering the same region didn''t show enhanced effect but suppressed the transcription activity of the CYP21 in all three cell linestested. In addition, DNA regions within the 13.5 kb upstream from the CYP21P all suppressed the transcription activity of the CYP21 but to a different level. Therefore, we predict the regulatory element(s) controlling the adrenal-specific expression of the CYP21 may be located within the -6328/-1668 bp region.Other cis-elements at the C4/CYP21 gene locus which are not tested in this study may also be involved in the regulation of the tissue-specific expression of human steroid 21-hydroxylase. Further study will be performed to test this possibili
