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 plan 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.

Two separate sites contribute to AP-1 activation of the promoter for 3-hydroxy-3-methylglutaryl coenzyme A synthase.

Metabolic flux into the mevalonate pathway is regulated by end product repression and cell growth. In the experiments reported here the transcriptional promoter for an early enzyme of the pathway, 3-hydroxy-3-methylglutaryl coenzyme A synthase, is shown to be activated by the growth stimulatory agent tetraphorbol acetate (TPA). We show that TPA has a direct stimulatory action on the promoter and further that this is mediated by the AP-1 transcription factor. In addition, we show that there are two separate cis-acting sites that bind AP-1 and both are required for maximal stimulation. We further show that in AP-1-deficient cells ectopic expression of AP-1 stimulates synthetic promoters containing two copies of each synthase element upstream of a minimal promoter. The physiological rationale of having both end product repression and direct activation by growth stimulatory cues is discussed.

In vivo regulation of rRNA transcription occurs rapidly in nondividing and dividing Drosophila cells in response to a phorbol ester and serum.

The synthesis of ribosomes is an essential cellular process which requires the transcription of the rRNA genes by RNA polymerase I (Pol I). The regulation of rRNA synthesis is known to be coupled to growth regulation. In nongrowing, slowly growing, and rapidly growing Drosophila cells, exposure to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) increases the synthesis of precursor and mature rRNAs. Using nuclear run-on assays, we show that TPA enhances transcription of the rRNA genes. These results suggest that TPA regulates expression of RNA genes transcribed by Pol I, irrespective of the growth state of the cells. In slowly dividing Drosophila cells, increasing the serum concentration rapidly alters the accumulation of rRNA by enhancing rDNA transcription within 1 h. Thus, TPA and serum are each able to rapidly regulate rRNA gene expression in Drosophila cells. These results indicate that the RNA Pol I transcription system can be regulated by agents which have previously been shown to effect specific genes transcribed by the RNA Pol II system.