Abstract
The expression of human squalene synthase (HSS) gene is transcriptionally regulated in HepG-2 cells, up to 10-fold, by variations in cellular cholesterol homeostasis. An earlier deletion analysis of the 5'-flanking region of the HSS gene demonstrated that most of the HSS promoter activity is detected within a 69-base pair sequence located between nucleotides -131 and -200. ADD1/SREBP-1c, a rat homologue of sterol regulatory element-binding protein (SREBP)-1c binds to sterol regulatory element (SRE)-1-like sequence (HSS-SRE-1) present in this region (Guan, G., Jiang, G., Koch, R. L. and Shechter, I. (1995) J. Biol. Chem. 270, 21958-21965). 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.
Highlights
The expression of human squalene synthase (HSS) gene is transcriptionally regulated in HepG-2 cells, up to 10-fold, by variations in cellular cholesterol homeostasis
We have shown that ADD1/ sterol regulatory element-binding protein (SREBP)-1c binds to an HSS-sterol regulatory element (SRE)-1 element existing in this region [22]
To verify the regulatory effect of this interaction between ADD1/SREBP-1c and HSS-SRE-1 in cultured cells, the effect of ADD1/SREBP-1c on HSS promoter activity was tested by expressing different ADD1/SREBP-1c constructs together with pHSS1kb-Luc in HepG-2 cells grown in the presence of sterol
Summary
Vol 272, No 15, Issue of April 11, pp. 10295–10302, 1997 Printed in U.S.A. Multiple Sequence Elements are Involved in the Transcriptional Regulation of the Human Squalene Synthase Gene*. The active bHLH-Zip segment of SREBP-1 was shown to localize at the nucleus This form of SREBP binds to SRE-1 in promoters of LDLR and HMG-CoA synthase, which results in the transcriptional activation of the two genes [13, 14]. It was recently demonstrated that the expression of fatty acid synthase (16 –19), an essential enzyme in fatty acid biosynthesis, and acetyl-coenzyme A carboxylase [20] are transcriptionally activated by the binding of SREBP-1 to the sterol regulatory elements in their promoters This observation provides evidence that directly links the metabolism of fatty acids and cholesterol and the importance of the SREBP family of transcription factors in the regulation of lipid metabolism. The same sequence exists in the promoter of HSS as well [22]
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