Loss of transcriptional repression of three sterol-regulated genes in mutant hamster cells

• Published in: The Journal of biological chemistry 1989-09, Vol.264 (26), p.15634-15641
• Main Authors: Metherall, J E, Goldstein, J L, Luskey, K L, Brown, M S
• Format: Article
• Language: English
• Subjects: Amphotericin B - pharmacology; Animals; Biological and medical sciences; Cell Line; Cell Survival - drug effects; Cholesterol - biosynthesis; Cricetinae; Cricetulus; Drug Resistance; Enzyme Repression; Female; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation - drug effects; Genes; Hydroxycholesterols - pharmacology; Hydroxymethylglutaryl CoA Reductases - biosynthesis; Hydroxymethylglutaryl CoA Reductases - genetics; Hydroxymethylglutaryl-CoA Synthase - biosynthesis; Hydroxymethylglutaryl-CoA Synthase - genetics; Molecular and cellular biology; Molecular genetics; Mutation; Oleic Acid; Oleic Acids - metabolism; Ovary; Plasmids; Promoter Regions, Genetic; Receptors, LDL - biosynthesis; Receptors, LDL - genetics; Transcription, Genetic; Transcription. Transcription factor. Splicing. Rna processing; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)84879-2

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.