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(Received for publication, August 19, 1996, and in revised form, February 7, 1997)
From the 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
Volume 272, Number 15,
Issue of April 11, 1997
pp. 10295-10302
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
,
,
Department of Biochemistry, F. Edward
Hébert School of Medicine, Uniformed Services University of the
Health Sciences, Bethesda, Maryland 20814, * Department of Molecular
Biology and Biochemistry, University of California, Irvine, California
92717, and the § Dana-Farber Cancer Institute and Department
of Cell Biology, Harvard Medical School,
Boston, Massachusetts 02115
-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.
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