Einerhand A W, Kos W, Smart W C, Kal A J, Tabak H F, Cooper T G
Academic Medical Center, University of Amsterdam, The Netherlands.
Mol Cell Biol. 1995 Jun;15(6):3405-14. doi: 10.1128/MCB.15.6.3405.
Expression of the FOX3 gene, which encodes yeast peroxisomal 3-oxoacyl-coenzyme A thiolase, can be induced by oleate and repressed by glucose. Previously, we have shown that induction was mediated by an oleate response element. Just upstream of this element a negatively acting control region that mediated glucose repression was found. In order to study this negative control region, we carried out DNA-binding assays and analyzed phenotypes of mutations in this region and in the trans-acting factor CAR80, which is identical to UME6. DNA-binding assays showed that two multifunctional yeast proteins, ABF1 and RP-A, interacted with the negative control element independently of the transcriptional activity of the FOX3 gene. ABF1 and RP-A, the latter being identical to BUF, were able to bind to DNA independently of one another but also simultaneously. The phenotypes of mutations in either DNA-binding sites of ABF1, RP-A, or both, which affected the DNA binding of these factors in vitro, indicated that these sites and the proteins that interact with them participate in glucose repression. The involvement of the RP-A site in glucose repression was further supported by our observation that the CAR80 gene product, which is required for repression mediated by the RP-A site, was essential for maintenance of glucose repression. In addition to the RP-A site in the FOX3 promoter, similar sequences were observed in other genes involved in peroxisomal function. RP-A proved to bind to all of these sequences, albeit with various affinities. From these results it is concluded that the ABF1 and RP-A sites are being required in concert to mediate glucose repression of the FOX3 gene. In addition, coordinated regulation of expression of genes involved in peroxisomal function in response to glucose is mediated by proteins associated with the RP-A site, probably RP-A and CAR80.
编码酵母过氧化物酶体3-氧代酰基辅酶A硫解酶的FOX3基因的表达可被油酸诱导，并被葡萄糖抑制。此前，我们已经表明诱导是由油酸反应元件介导的。就在这个元件的上游，发现了一个介导葡萄糖抑制的负性作用控制区。为了研究这个负性控制区，我们进行了DNA结合试验，并分析了该区域以及与UME6相同的反式作用因子CAR80中的突变表型。DNA结合试验表明，两种多功能酵母蛋白ABF1和RP-A与负性控制元件相互作用，且与FOX3基因的转录活性无关。ABF1和RP-A（后者与BUF相同）能够彼此独立但同时结合到DNA上。ABF1、RP-A或两者的DNA结合位点发生突变的表型，这些突变在体外影响了这些因子的DNA结合，表明这些位点以及与它们相互作用的蛋白质参与了葡萄糖抑制。我们观察到，RP-A位点介导的抑制所必需的CAR80基因产物对于维持葡萄糖抑制至关重要，这进一步支持了RP-A位点参与葡萄糖抑制的观点。除了FOX3启动子中的RP-A位点外，在其他参与过氧化物酶体功能的基因中也观察到了类似序列。事实证明，RP-A能结合所有这些序列，尽管亲和力各不相同。从这些结果可以得出结论，ABF1和RP-A位点共同作用以介导FOX3基因的葡萄糖抑制。此外，参与过氧化物酶体功能的基因表达对葡萄糖的协调调节是由与RP-A位点相关的蛋白质介导的，可能是RP-A和CAR80。
