A DIRECT ROLE FOR STEROL REGULATORY ELEMENT-BINDING PROTEIN IN ACTIVATION OF 3-HYDROXY-3-METHYLGLUTARYL COENZYME-A REDUCTASE GENE

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.