Sm. Vallett et al., A DIRECT ROLE FOR STEROL REGULATORY ELEMENT-BINDING PROTEIN IN ACTIVATION OF 3-HYDROXY-3-METHYLGLUTARYL COENZYME-A REDUCTASE GENE, The Journal of biological chemistry, 271(21), 1996, pp. 12247-12253
In earlier studies the DNA site required for sterol regulation of 3-hy
droxy-3-methylglutaryl coenzyme A reductase was shown to be distinct f
rom the classic sterol regulatory element (SRE-1) of the low density l
ipoprotein receptor gene (Osborne, T. F. (1991) J. Biol. Chem. 266, 13
947-13951), However, oxysterol-resistant cells that continuously overp
roduce one of the sterol regulatory element binding proteins in the nu
cleus result in high unregulated expression of both genes (Yang, J., B
rown, M. S., Ho, Y. K., and Goldstein, J. L. (1995) J. Biol, Chem. 270
, 12152-12161) suggesting a direct role for the SREBPs in the activati
on of the reductase gene, In the present studies we demonstrate that S
REBP-1 binds to two adjacent sites within the previously identified st
erol regulatory element of the reductase gene even though there is onl
y limited homology with the SRE-1 of the receptor. We also show that S
REBP-1 specifically activates the reductase promoter in transient DNA
transfection studies in HepG2 cells and that mutations which eliminate
sterol regulation and SREBP-1 binding also abolish transient activati
on by SREBP-1, Although specific, the magnitude of the activation obse
rved is considerably lower than for the low density lipoprotein (LDL)
receptor analyzed in parallel, suggesting there is an additional prote
in required for activation of the reductase promoter that is limiting
in the transient assay, SREBP also binds to two additional sites in th
e reductase promoter which probably play an auxiliary role in expressi
on. When the DNA sequence within the sites are aligned with each other
and with the LDL receptor SRE-1, a consensus half-site is revealed 5'
-PyCAPy-3'. The LDL receptor element contains two half-sites oriented
as a direct repeat spaced by one nucleotide.The SREBP proteins are spe
cial members of the basic-helix-loop-helix-zipper (bHLHZip) family of
DNA binding proteins since they bind the classic palindromic E-box sit
e as well as the direct repeat SRE-1 element. The SREBP binding sites
in both the reductase and those recently identified in other sterol re
gulated promoters appear to contain a half-site with considerable dive
rgence in the flanking residues, Here we also show that a 22-amino aci
d domain located immediately adjacent to the basic domain of the bHLHZ
ip region is required for SREBP to efficiently recognize divergent sit
es in the reductase and 3-hydroxy-3-methylglutaryl-CoA synthase promot
ers but, interestingly, this domain is not required for efficient bind
ing to the LDL direct repeat SRE-1 or to a palindromic high-affinity E
-box element.