In our present study, we demonstrate that mutation of this HSS-SRE-1 element significantly reduced, but did not abolish, the response of HSS promoter to change in sterol concentration. Mutation scanning indicates that two additional DNA promoter sequences are involved in sterol-mediated regulation. The first sequence contains an inverted SRE-3 element (Inv-SRE-3) and the second contains an inverted Y-box (Inv-Y-box) sequence. A single mutation in any of these sequences reduced, but did not completely remove, the response to sterols. Combination mutation studies showed that the HSS promoter activity was abolished only when all three elements were mutated simultaneously. Co-expression of SRE-1-or SRE-2-binding proteins (SREBP-1 or SREBP-2) with HSS promoter-luciferase reporter resulted in a dramatic increase of HSS promoter activity. Gel mobility shift studies indicate differential binding of the SREBPs to regulatory sequences in the HSS promoter. These results indicate that the transcription of the HSS gene is regulated by multiple regulatory elements in the promoter.Squalene synthase (farnesyl diphosphate-farnesyl diphosphate farnesyltransferase, EC 2.5.1.21) catalyzes the reductive head-to-head condensation of two molecules of farnesyl diphosphate (FPP) 1 to form squalene, the first specific intermediate in the cholesterol biosynthesis pathway. The expression of squalene synthase, as that of several other key enzymes in the pathway, such as 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, HMG-CoA synthase, and FPP synthase, is highly regulated by the cholesterol homeostasis in cells (1-4). It has previously been demonstrated that the expression of squalene synthase is regulated transcriptionally (5), but the mechanism for this regulation is unknown. Low density lipoprotein receptor (LDLR) and HMG-CoA synthase are regulated by interaction between recently described transcriptional factors, sterol regulatory element-binding-proteins (SREBPs). These interact with sequences called sterol regulatory elements (SRE-1 and SRE-2) that exist in the promoters of the two genes (6 -8). Two functionally related SREBPs, SREBP-1 and SREBP-2, have been purified from human cells and hamster cells, and the mechanism by which they regulate the expression of LDLR and HMG-CoA synthase has been studied extensively (9 -11). Human SREBP-1 and SREBP-2 are 47% identical. At the NH 2 -terminal region of each protein, there is a basic-helix-loop-helix leucine zipper (bHLH-Zip) structure that serves as a transcriptionally active domain. Next to the bHLH-Zip domain there are two membrane attachment domains. Nascent SREBP-1 and SREBP-2 are localized in the ER by these domains, and they are inactive in stimulating transcription. At lower concentrations of sterol in cells, an ER-associated, sterol-sensitive protease is activated and proteolytically activates the SREBPs by a cleavage at a site between the leucine zipper and the membrane attachment domains to release the bHLH-Zip domain (12). The active bHLH-Zip segment of SREBP-1 was...

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Transcription of the human squalene synthase (HSS) gene is regulated by variations in the level of cellular cholesterol. Three regulatory elements in the HSS promoter region are known to be involved in the regulation: 1) a modified sterol regulatory element (SRE) 1 (HSS-SRE-1), 2) an inverted SRE-3 (Inv-SRE-3), 3) an inverted Y box (Inv-Y-Box).We report here the regulatory role of distinct cis-elements in the HSS promoter by using mutants of an HSSluciferase promoter reporter. The activity of a wild-type promoter reporter transiently transfected into HepG-2 cells is increased by sterol depletion of the cells or by coexpression of mature forms of the SRE-binding proteins (SREBP) 1a and SREBP-2. Differential activation by SREBP-1a and SREBP-2 of the reporter gene mutated at various regions of the promoter is observed. Mutation of either the HSS-SRE-1 or the Inv-SRE-3 sequence diminished the activation by SREBP-1a and by sterol depletion but did not affect the activation by SREBP-2. Simultaneous mutations of both of these sequences almost completely abolished activation of the promoter by SREBP-1a or by sterol depletion, but activation by SREBP-2 was retained at 70%. Mutation of the Inv-Y-Box sequence element decreased the activity of the promoter by 50% or more, and if mutated together with both SREs, the activation was almost completely abolished. Mutation of any single GC box of the two located at ؊40 to ؊57 did not affect activity, whereas simultaneous mutation of the two decreased activation by SREBP-2 by 60%, by lipid depletion by 20%, and had no effect on the activation by SREBP-1a. A Y box motif at ؊159 to ؊166 and an SRE-like sequence element (SRE-1(8/10)) at position ؊101 to ؊108 are also involved in the sterol regulation. These results indicate that the complex sterol-mediated transcriptional regulation of the HSS gene is due to the presence of multiple copies of diverse cis elements in the HSS promoter. The differential activation of the HSS promoter may point to specific role of the SREBPs in cholesterogenesis.Squalene synthase (farnesyl-diphosphate:farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21), which catalyzes the formation of squalene by condensation of two molecules of farnesyl diphosphate, is the first committed enzyme in cholesterol biosynthesis. Its expression, like that of several other key enzymes in the pathway such as 3-hydroxy-3-methylglutarylcoenzyme A reductase, 3-hydroxy-3-methylglutaryl-coenzyme A synthase, and farnesyl diphosphate synthase, is highly regulated by cellular cholesterol homeostasis (1-4). The 5Ј-flanking regions of the genes encoding these enzymes contain either a 10-bp 1 sequence designated sterol regulatory element (SRE)-1 or a related sequence (5-8). Other sterol-sensitive genes such as the low density lipoprotein receptor, fatty acid synthase, and acetyl-coenzyme A carboxylase (ACC) also contain SRE elements in the promoter region (9 -13).Two structurally related protein transcription factors designated sterol regulatory element binding protein (SREBP) 1 and 2...

To determine the cis-and trans-regulatory elements which control the expression of the apolipoprotein (apo) A-I gene, several DNA-protein binding assays, namely, gel mobility shift, exonuclease 111 protection, and exonuclease 111 footprinting assays, were employed to identify these elements. It is demonstrated that nuclear proteins of Hep G2 cells bind to five regions of DNA sequences between 252 and 149 base pairs upstream from the transcription initiation site of the rat apo A-I gene. Using South-Western blot analysis, it is determined that DNA-binding protein has a molecular mass of approximately 90 kDa. It is also shown that the DNA-binding protein was present in Hep G2 cells and rat livers but absent in rabbit livers. The results suggest that the lack of expression of the apo A-I gene in rabbit livers is due to the absence of this DNA-binding protein.Apolipoprotein (apo) A-I is an important plasma protein.It is a major constituent of plasma high-density lipoproteins and a cofactor of plasma lecithin -cholesterol acyltransferase [l] which catalyzes the formation of most of the cholesteryl esters in plasma. It is thought that apo A-I, together with lecithin -cholesterol acyltransferase, facilitates the removal of cholesterol from peripheral tissues and transports it to the liver for catabolism [2]. Patients with a defective apo A-I gene show low levels of plasma high-density lipoprotein and apo A-I and accelerated atherosclerosis [3]. Several epidemiological studies have established that plasma levels of high-density lipoprotein and apo A-I are negatively correlated with the incidence of coronary heart diseases [4 -61. Hence it is beneficial to raise plasma levels of both high-density lipoproteinThe expression of apo A-I can be induced at transcriptional levels by several pharmacological agents: phenobarbital, estrogen, insulin and dexamethasone [7 -91. Usually, the expression of eukaryotic genes is regulated by the 5'-flanking promoter and enhancer elements of the genes. These elements interact with and bind to specific trans-acting nuclear proteins [lo -121. Thus, the identification of the trans-acting elements which bind to and interact with the enhancer elements of the apo A-S gene becomes crucial for designing therapeutic agents which intervene in the expression of this gene. The nucleotide sequences of the rat and human apo A-I genes have been reported [13, 141. Recently, we have identified a 45-base-pair nucleotide element, located between 235 and 190 nucleotides upstream from the transcription initiation site of the rat apo A-I gene, which is essential for the expression of this gene in Hep G2 cells and that this element exerts its function with an enhancerlike activity [l 51. In the current research we studied the binding of nuclear proteins from Hep G2 cells and rat livers to the enhancer element of the rat apo A-I gene; our data suggest and apo A-S. that the lack of expression of the apo A-I gene in rabbit livers is due to decreased levels of this binding protein in rabbit livers. MATERIALS AND ...