Peshavaria M, Cissell M A, Henderson E, Petersen H V, Stein R
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville, Tennessee 37232, USA.
Mol Endocrinol. 2000 Dec;14(12):1907-17. doi: 10.1210/mend.14.12.0563.
PDX-1 is a homeodomain transcription factor whose targeted disruption results in a failure of the pancreas to develop. Mutations in the human pdx-1 gene are linked to an early onset form of non-insulin-dependent diabetes mellitus. PDX-1 binds to and transactivates the promoters of several physiologically relevant genes within the beta-cell, including insulin, glucose transporter 2, glucokinase, and islet amyloid polypeptide. This study focuses on the mechanisms by which PDX-1 activates insulin gene transcription. To evaluate the role of PDX-1 in transcription of the insulin gene, a chloramphenicol acetyltransferase reporter construct was designed with a single yeast GAL4-DNA binding site in place of the A3/PDX-1 binding element in the rat insulin II enhancer. In the presence of GAL4:PDX chimeras containing N-terminal transactivation domain sequences, this GAL4-substituted insulin construct was active in PDX-1-expressing beta-cells and not non-beta-cells. PDX-1 activation was mediated through three highly conserved segments of the transactivation domain. In addition, when cotransfected together with the GAL4-substituted insulin enhancer reporter gene in glucose-responsive MIN-6 beta-cells, glucose-induced activation is observed with GAL4:PDX-1 but not with fusions of the heterologous activation domains from herpes virus VP16 or adenovirus-5 E1A proteins. Using A3 element-substituted GAL4 insulin enhancer reporter constructs containing mutations in two additional key control elements, E1 and C1, we also show that full activation requires cooperative interactions between other enhancer-bound factors, particularly the E1 element activators. In contrast, the activity of the VP16 activation factor was not dependent on the activators of either the E1 or C1 sites. These results suggest that the PDX-1 transactivation domain is specifically required for appropriate regulation of insulin enhancer function in beta-cells.
PDX-1是一种同源结构域转录因子，其靶向破坏会导致胰腺发育失败。人类pdx-1基因的突变与非胰岛素依赖型糖尿病的早发形式有关。PDX-1与β细胞内几个生理相关基因的启动子结合并使其反式激活，这些基因包括胰岛素、葡萄糖转运蛋白2、葡萄糖激酶和胰岛淀粉样多肽。本研究聚焦于PDX-1激活胰岛素基因转录的机制。为了评估PDX-1在胰岛素基因转录中的作用，设计了一种氯霉素乙酰转移酶报告基因构建体，其中用单个酵母GAL4-DNA结合位点取代了大鼠胰岛素II增强子中的A3/PDX-1结合元件。在含有N端反式激活结构域序列的GAL4:PDX嵌合体存在的情况下，这种GAL4取代的胰岛素构建体在表达PDX-1的β细胞中具有活性，而在非β细胞中无活性。PDX-1的激活是通过反式激活结构域的三个高度保守区段介导的。此外，当与GAL4取代的胰岛素增强子报告基因一起共转染到葡萄糖反应性MIN-6β细胞中时，观察到GAL4:PDX-1可诱导葡萄糖激活，而来自疱疹病毒VP16或腺病毒5 E1A蛋白的异源激活结构域的融合体则不能。使用在另外两个关键控制元件E1和C1中含有突变的A3元件取代的GAL4胰岛素增强子报告基因构建体，我们还表明，完全激活需要其他增强子结合因子之间的协同相互作用，特别是E1元件激活剂。相比之下，VP16激活因子的活性不依赖于E1或C1位点的激活剂。这些结果表明，PDX-1反式激活结构域是β细胞中胰岛素增强子功能适当调节所特需的。
