Bantignies F, Rousset R, Desbois C, Jalinot P
Laboratoire de Biologie Moléculaire et Cellulaire, UMR49 CNRS/ENSL, France.
Mol Cell Biol. 1996 May;16(5):2174-82. doi: 10.1128/MCB.16.5.2174.
To achieve a better understanding of the mechanism of transactivation by Tax of human T-cell leukemia virus type 1 Tax-responsive element 1 (TRE-1), we developed a genetic approach with Saccharomyces cerevisiae. We constructed a yeast reporter strain containing the lacZ gene under the control of the CYC1 promoter associated with three copies of TRE-1. Expression of either the cyclic AMP response element-binding protein (CREB) or CREB fused to the GAL4 activation domain (GAD) in this strain did not modify the expression of the reporter gene. Tax alone was also inactive. However, expression of the reporter gene was induced by coexpression of Tax and CREB. This effect was stronger with the GAD-CREB fusion protein. Analysis of different CREB mutants with this genetic system indicated that the C-terminal 92 amino acid residues, which include the basic domain and the leucine zipper, are necessary and sufficient to mediate transactivation by Tax. To identify cellular proteins binding to TRE-1 in a Tax-dependent manner, this strain was also used to screen a library of human cDNAs fused to GAD. Of five positive clones isolated from 0.75 x 10(6) yeast colonies, four were members of the CREB/activating transcription factor (ATF) family: CREB, two isoforms of the cyclic AMP-responsive element modulator (CREM), and ATF-1. Interestingly, these three proteins can be phosphorylated by protein kinase A and thus form a particular subgroup within the CREB/ATF family. Expression of ATF-2 in S. cerevisiae did not activate TRE-1 in the presence of Tax. This shows that in a eukaryotic nucleus, Tax specifically interacts with the basic domain-leucine zipper region of ATF-1, CREB, and CREM. The fifth clone identified in this screening corresponded to the Ku autoantigen p70 subunit. When fused to GAD, the C-terminal region of Ku was able to activate transcription via TRE-1 but this activation was not dependent on Tax.
为了更好地理解1型人类T细胞白血病病毒的Tax蛋白对Tax反应元件1（TRE-1）的反式激活机制，我们利用酿酒酵母开发了一种遗传学方法。我们构建了一个酵母报告菌株，该菌株含有在与三个拷贝的TRE-1相关的CYC1启动子控制下的lacZ基因。在该菌株中，单独表达环磷酸腺苷反应元件结合蛋白（CREB）或与GAL4激活域（GAD）融合的CREB均不会改变报告基因的表达。单独的Tax蛋白也无活性。然而，Tax蛋白与CREB的共表达可诱导报告基因的表达。GAD-CREB融合蛋白的这种作用更强。利用该遗传系统对不同的CREB突变体进行分析表明，包含碱性结构域和亮氨酸拉链的C末端92个氨基酸残基对于介导Tax蛋白的反式激活是必需且足够的。为了鉴定以Tax依赖方式与TRE-1结合的细胞蛋白，该菌株还用于筛选与GAD融合的人类cDNA文库。从7.5×10⁵个酵母菌落中分离出的五个阳性克隆中，有四个是CREB/激活转录因子（ATF）家族的成员：CREB、环磷酸腺苷反应元件调节剂（CREM）的两种同工型和ATF-1。有趣的是，这三种蛋白可被蛋白激酶A磷酸化，因此在CREB/ATF家族中形成一个特殊的亚组。在酿酒酵母中表达ATF-2在有Tax蛋白存在时不会激活TRE-1。这表明在真核细胞核中，Tax蛋白特异性地与ATF-1、CREB和CREM的碱性结构域-亮氨酸拉链区域相互作用。在该筛选中鉴定出的第五个克隆对应于Ku自身抗原p70亚基。当与GAD融合时，Ku的C末端区域能够通过TRE-1激活转录，但这种激活不依赖于Tax蛋白。
