Yin M J, Paulssen E J, Seeler J S, Gaynor R B
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8594, USA.
J Virol. 1995 Jun;69(6):3420-32. doi: 10.1128/JVI.69.6.3420-3432.1995.
Gene expression from the human T-cell leukemia virus type I (HTLV-I) long terminal repeat (LTR) is mediated by three cis-acting regulatory elements known as 21-bp repeats and the transactivator protein Tax. The 21-bp repeats can be subdivided into three motifs known as A, B, and C, each of which is important for maximal gene expression in response to Tax. The B motif contains nucleotide sequences known as a cyclic AMP response element (CRE) or tax-response element which binds members of the ATF/CREB family of transcription factors. Though mutations of this element in the HTLV-I LTR eliminate tax activation, Tax will not activate most other promoters containing these CRE sites. In this study, we investigated the mechanism by which Tax activates gene expression in conjunction with members of the ATF/CREB family. We found that Tax enhanced the binding of one member of the ATF/CREB family, CREB 1, to each of the three HTLV-I LTR 21-bp repeats but not another member designated CRE-BP1 or CREB2. Tax enhanced the binding of CREB1 to nonpalindromic CRE binding sites such as those found in the HTLV-I LTR, but Tax did not enhance the binding of CREB1 to palindromic CRE binding sites such as found in the somatostatin promoter. This finding may help explain the failure of Tax to activate promoters containing consensus CRE sites. These studies were extended by use of the mammalian two-hybrid system. Tax was demonstrated to interact directly with CREB1 but not with other bZIP proteins, including CREB2 and Jun. Site-directed mutagenesis of both Tax and CREB1 demonstrated that the amino terminus of Tax and both the basic and the leucine zipper regions of CREB1 were required for direct interactions between these proteins both in vivo and in vitro. This interaction occurred in vivo and thus did not require the presence of the HTLV-I 21-bp repeats, as previously suggested. These results define the domains required for interaction between Tax and CREB that are likely critical for the activation of HTLV-I gene expression.
人T细胞白血病病毒I型（HTLV-I）长末端重复序列（LTR）的基因表达由三种顺式作用调节元件介导，即21碱基重复序列和反式激活蛋白Tax。21碱基重复序列可细分为A、B、C三种基序，每个基序对于响应Tax实现最大程度的基因表达都很重要。B基序包含被称为环磷酸腺苷反应元件（CRE）或Tax反应元件的核苷酸序列，它能结合ATF/CREB转录因子家族的成员。尽管HTLV-I LTR中该元件的突变消除了Tax激活作用，但Tax不会激活大多数含有这些CRE位点的其他启动子。在本研究中，我们探究了Tax与ATF/CREB家族成员共同激活基因表达的机制。我们发现Tax增强了ATF/CREB家族的一个成员CREB 1与HTLV-I LTR的三个21碱基重复序列中每一个的结合，但没有增强与另一个名为CRE-BP1或CREB2的成员的结合。Tax增强了CREB1与非回文CRE结合位点（如在HTLV-I LTR中发现的那些位点）的结合，但Tax没有增强CREB1与回文CRE结合位点（如在生长抑素启动子中发现的那些位点）的结合。这一发现可能有助于解释Tax无法激活含有共有CRE位点的启动子的原因。这些研究通过使用哺乳动物双杂交系统得以扩展。结果表明Tax直接与CREB1相互作用，而不与包括CREB2和Jun在内的其他bZIP蛋白相互作用。对Tax和CREB1进行定点诱变表明，Tax的氨基末端以及CREB1的碱性区域和亮氨酸拉链区域对于这些蛋白质在体内和体外的直接相互作用都是必需的。这种相互作用发生在体内，因此并不像之前所认为的那样需要HTLV-I 21碱基重复序列的存在。这些结果确定了Tax和CREB之间相互作用所需的结构域，这些结构域可能对HTLV-I基因表达的激活至关重要。
