Shim Y H, Bonner J J, Blumenthal T
Department of Biology, Indiana University, Bloomington 47405, USA.
J Mol Biol. 1995 Nov 10;253(5):665-76. doi: 10.1006/jmbi.1995.0581.
The GATA motif (WGATAR) is found in the promoter regions of numerous Caenorhabditis elegans genes, including two intestine-specific genes, vit-2 and ges-1, in which it has been shown to be required for promoter function. The protein ELT-1, encoded by a single-copy gene homologous to the GATA family of vertebrate transcription factors, is potentially capable of interacting with this element. In order to determine whether ELT-1 is a transcriptional activator that recognizes this sequence, we have expressed it under the control of the GAL1 promoter in yeast. lacZ driven by the CYC1 promoter lacking an upstream activation sequence (UAS) but containing GATA sequences was used as a reporter. beta-Galactosidase was expressed upon induction only when GATA sequences were present, and expression was increased dramatically by additional binding sites. Deletion analysis demonstrated that the C terminus, containing only one of the two zinc fingers, is sufficient for activation. In addition, the DNA-binding domain and two transactivation regions were identified by fusing these isolated domains to previously defined domains of heterologous transcription factors. While most single base alterations in the GATA core sequence eliminated activity, an A to C change in position four, creating a GATC core, was found to increase activity significantly. The deleted ELT-1 protein containing only the C-terminal Zn finger was sufficient for activation in response to GATA, but both fingers were required for activation at GATC. A variety of sites with non-optimal sequences surrounding the GATA core also were found to be excluded better by the protein containing both Zn fingers. Furthermore, a fusion protein containing the entire ELT-1 DNA binding domain fused to the VP16 activation domain was found to have an even greater preference for the GATC core, as well as the optimal flanking bases. We conclude that, although ELT-1 having only its C-terminal finger is capable of activation in response to the WGATAR site, the presence of the upstream finger supplies additional base specificity.
GATA基序（WGATAR）存在于许多秀丽隐杆线虫基因的启动子区域，包括两个肠道特异性基因vit-2和ges-1，研究表明它是启动子功能所必需的。由与脊椎动物转录因子GATA家族同源的单拷贝基因编码的ELT-1蛋白，可能能够与该元件相互作用。为了确定ELT-1是否是识别该序列的转录激活因子，我们在酵母中GAL1启动子的控制下表达了它。由缺乏上游激活序列（UAS）但含有GATA序列的CYC1启动子驱动的lacZ用作报告基因。仅当存在GATA序列时，诱导后才表达β-半乳糖苷酶，并且通过额外的结合位点表达显著增加。缺失分析表明，仅包含两个锌指之一的C末端足以激活。此外，通过将这些分离的结构域与异源转录因子的先前定义的结构域融合，鉴定出DNA结合结构域和两个反式激活区域。虽然GATA核心序列中的大多数单碱基改变消除了活性，但发现第4位的A到C变化产生了GATC核心，显著增加了活性。仅包含C末端锌指的缺失ELT-1蛋白足以响应GATA进行激活，但两个锌指对于GATC处的激活都是必需的。还发现，GATA核心周围具有非最佳序列的各种位点被含有两个锌指的蛋白质更好地排除。此外，发现含有与VP16激活结构域融合的整个ELT-1 DNA结合结构域的融合蛋白对GATC核心以及最佳侧翼碱基具有更大的偏好。我们得出结论，尽管仅具有C末端锌指的ELT-1能够响应WGATAR位点进行激活，但上游锌指的存在提供了额外的碱基特异性。
