Activity of a C. elegans GATA transcription factor, ELT-1, expressed in yeast.
Activity of a C. elegans GATA transcription factor, ELT-1, expressed in yeast.
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酵母中表达的线虫 GATA 转录因子 ELT-1 的活性。
DOI：
10.1006/jmbi.1995.0581
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发表时间：
1995
期刊：
影响因子：
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通讯作者：
Blumenthal,T
中科院分区：
文献类型：
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作者：
Shim,YH;Bonner,JJ;Blumenthal,T
GATA基序(WGATAR)存在于许多秀丽线虫基因的启动子区域，其中包括两个肠道特异基因Vit-2和GES-1，已被证明是启动子功能所必需的。ELT-1蛋白由与脊椎动物转录因子GATA家族同源的单拷贝基因编码，具有与该元件相互作用的潜力。为了确定ELT-1是否是识别该序列的转录激活子，我们在GAL1启动子的控制下在酵母中表达了ELT-1，以没有上游激活序列(UAS)但含有GATA序列的CyC1启动子驱动的lacZ作为报告基因。|lcbeta|-Galactosidase只有在GATA序列存在的情况下才能在诱导时表达，并且通过额外的结合位点显著增加表达。缺失分析表明，C末端只包含两个锌指中的一个，足以激活。此外，通过将这些分离的结构域与预先定义的异源转录因子的结构域融合，鉴定了DNA结合区和两个反式激活区。虽然GATA核心序列中的大多数单碱基改变消除了活性，但第四位的A到C改变，创建了GATC核心，发现显著增加了活性。缺失的只含有C-末端锌指的ELT-1蛋白足以激活GATA，但在GATC激活两个手指是必需的。还发现，含有两个锌指的蛋白质更好地排除了围绕GATA核心具有非最佳序列的各种位点。此外，包含整个ELT-1DNA结合域和VP16激活域的融合蛋白被发现对GATC核心以及最佳侧翼碱基有更大的偏好。我们的结论是，虽然ELT-1只有其C-末端手指能够响应WGATAR位点而被激活，但上游手指的存在提供了额外的碱基特异性。
The GATA motif (WGATAR) is found in the promoter regions of numerousCaenorhabditis elegansgenes, including two intestine-specific genes,vit-2 andges-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 theGAL1promoter in yeast.lacZdriven by theCYC1promoter lacking an upstream activation sequence (UAS) but containing GATA sequences was used as a reporter. |lcbeta|-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.