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, The SREBP proteins are special members of the basichelix-loop-helix-zipper (bHLHZip) family of DNA binding proteins since they bind the classic palindromic Ebox 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.TBalanced cholesterol metabolism in mammalian cells is maintained through the feedback regulation of key proteins involved in its cellular uptake and biosynthesis. A major control point is at the level of transcription for genes that encode important proteins of both processes (1, 2). DNA sequences required for cholesterol regulation of important enzymes of cholesterol biosynthesis and the low density lipoprotein (LDL) 1 receptor, the unique protein required for cholesterol uptake, have been identified by DNA transfection studies in cultured cells (1, 2). The original studies identified a sequence similarity in the target site for cholesterol regulation (called the SRE-1) in the promoter for the LDL receptor and HMG-CoA synthase (3)(4)(5). Further studies demonstrated that the SRE-1 of the LDL receptor is a 10-base pair site that binds a specific subfamily of basic-helix-loop-helix-zipper (bHLHZip) DNA binding proteins called the sterol regulatory element binding proteins or .When mammalian cells are starved for cholesterol the SREBP proteins are cleaved from the membrane of the endoplasmic reticulum and nuclear envelope by proteolysis and the soluble amino-terminal fragment containing the DNA binding and transcriptional activation functions is translocated to the nucleus where it activates expression of appropriate target genes. The first evidence for this maturation process came from classic pulse-chase and gel electrophoresis studies on cells that were cultured in the presence and absence of sterols (9). SREBP-1 and -2 proteins were regulated in an identical fashion in tissue culture cells (9). However, a recent study in animals has suggested that the two SREBPs are regulated independently by cholesterol deprivation in the liver (10). Nonetheless, the two proteins have a similar domain structure, bind to the same DNA target sites in vitro, and appear to activate the same target genes when overexpressed in transfection studies (8,11,12).A fundamental role for SREBP processing in...

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 12-0-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 rRNA 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.It is now widely accepted that tumor-promoting phorbol esters, which mimic the action of diacylglycerol, can activate a calcium-and phospholipid-dependent protein kinase, protein kinase C (reviewed in references 4, 17, and 20). We have previously observed that protein synthesis can be stimulated within minutes in nondividing Drosophila cells by reagents that act through a pathway involving calcium and the activation of protein kinase C (32). We found that phorbol esters stimulated protein synthesis and that this stimulation was dependent on the presence of calcium, suggesting a common signaling pathway. Since in most systems, the cellular processes of protein and rRNA synthesis are closely coupled (reviewed in reference 21), we have investigated whether in nondividing cells, activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA) can bring about changes in rRNA synthesis parallel to its affects on protein synthesis.Since TPA is known to affect growth-related events in dividing cells, we also chose to investigate the effects of this reagent on rRNA synthesis in dividing Drosophila cultured cells which were either growing slowly, i.e., under serumreduced conditions, or growing rapidly, in the presence of a high concentration of serum.There are several levels at which stimulation or inhibition of RNA synthesis could potentially be controlled. We have investigated the changes in rDNA transcription which take place in Drosophila cells treated with TPA and serum by using nuclear run-on assays to examine the number of active polymerase I (Pol I) complexes present on the 18S and 28S rRNA genes. MATERIALS AND METHODS Drosophila culture and male accessory gland isolation.Wild-type Drosophila melanogaster (Oregon-R) was grown at 25°C on a standard cornmeal-Karo-yeast agar medium supplemented with live yeast cells. After eclosion, the flies were separated according to sex. Accessory glands from 10 virgin males (7 to 10 days old) were ...

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.

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The expression of genes that code for the large ribosomal RNAs (rRNAs) and tRNAs can be regulated by calcium, serum, insulin and a tumor-promoting phorbol ester, TPA. These effectors can rapidly alter rRNA and tRNA synthesis in dividing and nondividing Drosophila cells. In an in vitro assay system of the nondividing cells of the male accessory glands, calcium, insulin and TPA were shown to increase both rRNA and tRNA synthesis. Exposure of actively dividing Drosophila culture cells to differing serum concentrations or TPA also altered rRNA and tRNA synthesis. Nuclear run-on assays demonstrate that the exposure of these cells to increased serum concentrations coordinately alters RNA polymerase I loading on both 18S and 28S rDNA. These data indicate that calcium, growth factors and a tumor-promoter each can signal changes in ribosomal and tRNA gene expression.