Metherall J E, Goldstein J L, Luskey K L, Brown M S
Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1989 Sep 15;264(26):15634-41.
Two genes that encode enzymes in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and HMG-CoA synthase, and the gene encoding the low density lipoprotein (LDL) receptor are repressed when sterols accumulate in animal cells. Their 5'-flanking regions contain a common element, designated sterol regulatory element-1 (SRE-1). In the HMG-CoA synthase and LDL receptor promoters, the SRE-1 enhances transcription in the absence of sterols and is inactivated in the presence of sterols. In the HMG-CoA reductase promoter, the region containing the SRE-1 represses transcription when sterols are present. In the current studies, we show that the SRE-1 retains enhancer function but loses sterol sensitivity in mutant Chinese hamster ovary cells that are resistant to the repressor, 25-hydroxycholesterol. In the absence of sterols, the mutant cells produced high levels of all three sterol-regulated mRNAs, and there was no repression by 25-hydroxycholesterol. When transfected with plasmids containing each of the regulated promoters fused to a bacterial reporter gene, the mutant cells showed high levels of transcription in the absence of sterols and no significant repression by sterols. When the SRE-1 in the LDL receptor and HMG-CoA synthase promoters was mutated prior to transfection into the mutant cells, transcription was markedly reduced. Thus, the 25-hydroxycholesterol-resistant cells retain a protein that enhances transcription by binding to the SRE-1 in the absence of sterols, but they have lost the function of a protein that abolishes this enhancement in the presence of sterols. Mutation of a 30-base pair segment of the HMG-CoA reductase promoter that contains the SRE-1 did not reduce transcription in the mutant cells, indicating that this promoter is driven by elements other than the SRE-1. Nevertheless, this promoter failed to be repressed by sterols in the mutant cells. These data suggest that a common factor mediates the effects of sterols on the SRE-1 in all three promoters and that this factor has been functionally lost in the 25-hydroxycholesterol-resistant cells.
当动物细胞中固醇积累时，编码胆固醇生物合成中酶的两个基因，即3-羟基-3-甲基戊二酰辅酶A（HMG-CoA）还原酶和HMG-CoA合酶，以及编码低密度脂蛋白（LDL）受体的基因会受到抑制。它们的5'-侧翼区域含有一个共同元件，称为固醇调节元件-1（SRE-1）。在HMG-CoA合酶和LDL受体启动子中，SRE-1在无固醇时增强转录，在有固醇时失活。在HMG-CoA还原酶启动子中，当存在固醇时，含有SRE-1的区域会抑制转录。在当前研究中，我们发现SRE-1在对抑制剂25-羟基胆固醇具有抗性的突变中国仓鼠卵巢细胞中保留了增强子功能，但失去了固醇敏感性。在无固醇时，突变细胞产生高水平的所有三种固醇调节的mRNA，并且不受25-羟基胆固醇的抑制。当用含有与细菌报告基因融合的每个调节启动子的质粒转染时，突变细胞在无固醇时显示高水平的转录，并且不受固醇的显著抑制。当LDL受体和HMG-CoA合酶启动子中的SRE-1在转染到突变细胞之前发生突变时，转录明显减少。因此，对25-羟基胆固醇具有抗性的细胞保留了一种在无固醇时通过与SRE-1结合来增强转录的蛋白质，但它们失去了一种在有固醇时消除这种增强作用的蛋白质的功能。HMG-CoA还原酶启动子中包含SRE-1的一个30碱基对片段的突变并没有降低突变细胞中的转录，表明该启动子由SRE-1以外的元件驱动。然而，该启动子在突变细胞中不受固醇的抑制。这些数据表明，一个共同因子介导了固醇对所有三个启动子中SRE-1的作用，并且该因子在对25-羟基胆固醇具有抗性的细胞中已在功能上丧失。
