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-hydroxy-3-methylglutaryl coenzyme A reductase was shown to be distinct from the classic sterol regulatory element (SRE-1) of the low density lipoprotein receptor gene (Osborne, T. F. (1991) J. Biol. Chem. 266, 13947-13951). However, oxysterol-resistant cells that continuously overproduce one of the sterol regulatory element binding proteins in the nucleus result in high unregulated expression of both genes (Yang, J., Brown, 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 activation of the reductase gene. In the present studies we demonstrate that SREBP-1 binds to two adjacent sites within the previously identified sterol regulatory element of the reductase gene even though there is only limited homology with the SRE-1 of the receptor. We also show that SREBP-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 activation by SREBP-1. Although specific, the magnitude of the activation observed is considerably lower than for the low density lipoprotein (LDL) receptor analyzed in parallel, suggesting there is an additional protein required for activation of the reductase promoter that is limiting in the transient assay. SREBP also binds to two additional sites in the reductase promoter which probably play an auxiliary role in expression. 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 special members of the basic-helix-loop-helix-zipper (bHLHZip) family of DNA binding proteins since they bind the classic palindromic E-box site as well as the direct repeat SRE-1 element. The SREBP binding sites in both the reductase and those recently identified in other sterol regulated promoters appear to contain a half-site with considerable divergence in the flanking residues. Here we also show that a 22-amino acid domain located immediately adjacent to the basic domain of the bHLHZip region is required for SREBP to efficiently recognize divergent sites in the reductase and 3-hydroxy-3-methylglutaryl-CoA synthase promoters but, interestingly, this domain is not required for efficient binding to the LDL direct repeat SRE-1 or to a palindromic high-affinity E-box element.