研究人類C4/CYP21基因座中CpG島內核酸甲基化與基因腎上腺專一表現的關係

The Relationship of DNA Methylation within C4/CYP21 Locus and Adrenal-Specific Gene Expression

張恬慈(Tien-Tzu Chang)

指導教授 ： 張 淑 芬

臺北醫學大學/醫學院/細胞及分子生物研究所/碩士(2003年)

摘要

Steroid 21-hydroxylase 在體內參與調節鹽類皮質素和糖類皮質素這兩種類固醇賀爾蒙的生合成。造成先天性腎上腺增生症主要就是Steroid 21-hydroxylase 這個蛋白質的活性發生問題。人體中負責製造此酵素的是CYP21 基因， CYP21 有一個相似度很高的偽基因稱做 CYP21P。這兩個基因位在 C4/CYP21 基因座中，在這個基因座內還存在著其他成對的基因，而且這些基因大部分都專一的表現在腎上腺中。核酸的甲基化被認為和基因在特定的組織表現有關，所以我們認為核酸的甲基化可能是調控 CYP21 基因腎上腺專一表現的機制之ㄧ。所以本論文藉由重亞硫酸鈉修飾的方法 ( sodium bisulfite modification ) ，比較， 在 C4/CYP21 基因座內的 CpG 島在腎上腺皮質細胞 H295和人類肝臟細胞 HepG2 是否 cytosine 甲基化的形式不同。雖然結果顯示在CYP21P 基因上游 -13873/-13813 bp、-13316/-13230 bp、 -12223 /-12138 bp、 -11461 bp /-11350bp、 -8450/-8332 bp和 -3574/-3473 bp這些區域中的 CpG 序列都被甲基化而且在HepG2 和H295這兩種細胞中沒有不同，但是這些區域的序列與活性基因 CYP21的相似度高達98％。所以無法推斷在這些區域中的序列是否會影響 CYP21基因腎上腺專一的表現。只有在-12561/-12516bp、 -11075 bp /-11015 bp和 -4176/-4113 bp在這些區域中 CpG 序列都被甲基化而且在HepG2 和H295這兩種細胞中結果相同，所以推論這些區域的序列不會影響 CYP21基因腎上腺專一的表現。

關鍵字

CYP21

並列摘要

Steroid 21-hydroxylase mediates biosynthesis of glucocorticoids and mineralcorticoids. Enzymatic deficiency of steroid 21-hydroxylase is associated with congenital adrenal hyperplasia. There are two copies of human steroid 21 hydroxylase genes, the active CYP21 gene and pseudogene CYP21P and these two genes exhibit approximately 98% homology. These duplicated genes are located within the C4/CYP21 locus. Now, more and more pairs of duplicated genes which specifically express in the adrenal gland have been found within this locus. DNA methylation is thought to be one of the facts that can regulate tissue specific expression. We therefore used sodium bisulfite modification to analyze the methylation status of predicted CpG islands in the C4/CYP21 locus and compared the methylation status of these CpG islands of genome from adrenal H295 and non-adrenal HepG2 cells. The results showd that the CpG sequence within -13873/-13813 bp、 -13316/-13230 bp、 -12223 /-12138 bp、 -11461 bp /-11350bp、 -8450/-8332 bp and -3574/-3473 bp upstream of CYP21P gene are all methylated and there were no differences between genomes of H295 and HepG2 cells. In addition, within -12561/-12516bp、 -11075 bp /-11015 bp and -4176/-4113 bp upstream of CYP21P gene are all methylated and there were no difference between H295 and HepG2 cells so the sequence in these regions may not influence the adrenal-specific expression of CYP21 gene.

並列關鍵字

CYP21

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