Fox H B, Gutman P D, Dave H P, Cao S X, Mittelman M, Berg P E, Schechter A N
Laboratory of Chemical Biology, National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD 20892.
Blood. 1989 Dec;74(8):2749-54.
We studied the effects of a known retroviral trans-activating factor, HTLV-I tax1, on transcription of human globin genes. Transfection of HeLa cells by the cloned tax1 gene stimulated activity of both the beta- and epsilon-globin promoters approximately 20-fold, as measured by chloramphenicol acetyl transferase (CAT) assays. Studies of promoter 5'-deletion mutants revealed that the trans-activation response required only 185 base pairs (bp) of beta-globin 5'-flanking sequence or 177 bp of epsilon-globin 5' flanking sequence. These promoter regions contain either two (for beta) or three (for epsilon) copies of the pentanucleotide sequence CTGAC, which is characteristic of previously described tax1-responsive promoters. We also stably transfected tax1 into the erythroid cell line K562. Transfectants expressing tax1 showed increased transcription of epsilon-, gamma-, zeta-, and alpha-globins. This indicates that tax1 can stimulate transcription of globin genes in their native chromosomal location. This was confirmed by measurements of increases in intracellular hemoglobin as determined by an increased percentage of cells staining with benzidine and by spectrophotometric measurements of hemoglobin. The observed trans-activation of globin genes by tax1 may provide insight into normal regulation of globin genes by clarifying cis regulatory sequences. Furthermore, it suggests that the trans-acting effects of tax1 on heterologous genes are more widespread than was previously appreciated.
我们研究了一种已知的逆转录病毒反式激活因子HTLV-I tax1对人珠蛋白基因转录的影响。通过氯霉素乙酰转移酶（CAT）分析测定，用克隆的tax1基因转染HeLa细胞可使β-珠蛋白和ε-珠蛋白启动子的活性增强约20倍。对启动子5'-缺失突变体的研究表明，反式激活反应仅需要β-珠蛋白5'-侧翼序列的185个碱基对（bp）或ε-珠蛋白5'-侧翼序列的177个bp。这些启动子区域含有五核苷酸序列CTGAC的两个（对于β-珠蛋白）或三个（对于ε-珠蛋白）拷贝，这是先前描述的tax1反应性启动子的特征。我们还将tax1稳定转染到红系细胞系K562中。表达tax1的转染子显示ε-、γ-、ζ-和α-珠蛋白的转录增加。这表明tax1可以在其天然染色体位置刺激珠蛋白基因的转录。通过联苯胺染色细胞百分比增加以及血红蛋白的分光光度测量所确定的细胞内血红蛋白增加的测量结果证实了这一点。tax1对珠蛋白基因的反式激活作用可能通过阐明顺式调控序列为珠蛋白基因的正常调控提供见解。此外，这表明tax1对异源基因的反式作用比以前认识到的更为广泛。
