Béraud C, Lombard-Platet G, Michal Y, Jalinot P
UMR 49 CNRS-ENS, Laboratoire de Biologie Moléculaire et Cellulaire, Lyon, France.
EMBO J. 1991 Dec;10(12):3795-803. doi: 10.1002/j.1460-2075.1991.tb04949.x.
Transcription driven by the HTLV-I promoter is strongly activated by the viral transactivator protein Tax1. This effect is mediated via a 21 bp sequence which is imperfectly repeated three times in the viral promoter. We showed previously that a single 21 bp copy exhibits a strong Tax1-inducible enhancer activity and is able to bind different cellular proteins, namely ATF, HEB1 and HEB2. We have further investigated the molecular mechanism involved in the Tax1 induction of the 21 bp motif's enhancer activity by analysing Tax1 interaction with this DNA sequence. For this purpose a HeLa cell line constitutively expressing a functional Tax1 protein was established and nuclear extracts of these cells were used to perform a DNA affinity precipitation assay. This experimental approach allowed us to show that Tax1 specifically binds to the 21 bp motif. The same sequence elements of the 21 bp motif are required both for Tax1 binding and for Tax1-induced enhancer activity. Chromatographic fractionation of the HeLa tax nuclear extract showed that the binding is indirect and is mediated by the cellular factor HEB 1.
由人嗜T细胞病毒I型（HTLV-I）启动子驱动的转录被病毒反式激活蛋白Tax1强烈激活。这种效应是通过一个21 bp的序列介导的，该序列在病毒启动子中不完全重复三次。我们之前表明，单个21 bp拷贝表现出强烈的Tax1诱导的增强子活性，并且能够结合不同的细胞蛋白，即活化转录因子（ATF）、HEB1和HEB2。我们通过分析Tax1与该DNA序列的相互作用，进一步研究了Tax1诱导21 bp基序增强子活性所涉及的分子机制。为此，建立了一个组成型表达功能性Tax1蛋白的HeLa细胞系，并使用这些细胞的核提取物进行DNA亲和沉淀试验。这种实验方法使我们能够证明Tax1特异性结合21 bp基序。Tax1结合和Tax1诱导的增强子活性都需要21 bp基序的相同序列元件。对HeLa tax核提取物进行色谱分级分离表明，这种结合是间接的，并且是由细胞因子HEB 1介导的。
