Regulation of the low density lipoprotein (LDL) receptor promoter by cholesterol requires a well defined sterol regulatory site and an adjacent binding site for the universal transcription factor Sp1. These elements are located in repeats 2 and 3 of the wild type promoter, respectively. The experiments reported here demonstrate that Sp1 participates in sterol regulation of the LDL receptor in an orientation-specific fashion. We present data which suggest that sterol regulatory element-binding protein (SREBP) increases the binding of Sp1 to the adjacent repeat 3 sequence. We also demonstrate that SREBP and Sp1 synergistically activate expression from the LDL receptor promoter inside the cell by cotransfecting expression vectors encoding each protein into Drosophila tissue culture cells that are devoid of endogenous Sp1. In addition, other transcription factor sites were unable to substitute for Sp1 in sterol regulation when placed next to the SREBP-binding site. These studies together with recent data from others provide the basis of a working model for sterol regulation of the LDL receptor promoter. The presence of Sp1 sites in several other regulated promoters suggests that this universal transcription factor has been recruited to participate in many regulatory responses possibly by a similar mechanism.

The gene encoding fatty acid synthase, the essential multi-functional enzyme of fatty acid biosynthesis, is shown to be regulated by cellular sterol levels similar to genes that encode important proteins of cholesterol metabolism. We show that expression of the endogenous FAS gene is repressed when regulatory sterols are included in the culture medium of HepG2 cells and that the FAS promoter is subject to similar regulation when fused to the luciferase reporter gene. Mutational studies demonstrate that sterol regulation is mediated by binding sites for the sterol regulatory element-binding protein (SREBP) and transcription factor Sp1, making it mechanistically similar to sterol regulation of the low density lipoprotein receptor gene. It is also demonstrated that SREBP and Sp1 synergistically activate the FAS promoter in Drosophila tissue culture cells, which lack endogenous Sp1. These experiments provide key molecular evidence that directly links the metabolism of fatty acids and cholesterol together.Coordinate regulation of fatty acid and cholesterol accumulation is essential for balanced membrane biosynthesis and turnover to accommodate metabolic fluctuations that occur during normal cellular growth. Also, these two important lipids are simultaneously required in the liver for regular-ordered assembly of very low density lipoprotein particles, which deliver their lipid load of cholesterol and fatty acids from the liver to other sites in the body to maintain lipid homeostasis (1, 2).Co-regulation of genes that encode important enzymes of both fatty acid and cholesterol metabolism would be an ideal way to coordinate lipid regulation, and recent work has identified a family of transcriptional regulatory proteins that could link the two pathways together (3-5). Sterol regulatory element-binding proteins 1 and 2 (SREBP 1 and 2) 1 are highly related proteins that bind to the same cis-acting elements in the LDL receptor and HMG-CoA synthase promoters and activate expression only when cellular sterol stores are depleted (3, 5, 6). The cDNAs for both were cloned based on amino acid sequence information obtained from the purified proteins (3, 5).Independently, the rat equivalent of SREBP-1 was cloned from an adipocyte cDNA expression library (4). The rat mRNA was expressed at very high levels in brown fat and was also abundant in white fat and liver. The rat SREBP-1 mRNA was also induced during adipocyte differentiation in cell culture, so it was named the adipocyte determination-and differentiationdependent factor 1 (ADD1). These observations suggested that SREBP-1/ADD1 is a regulator of genes that are important for lipid accumulation in the adipocyte.The above studies indicate that the activity of SREBP-1/ ADD1 may provide a direct link between the regulation of cholesterol and fatty acid metabolism. In the present paper we demonstrate that expression of the mRNA for fatty acid synthase (FAS), an essential enzyme of fatty acid biosynthesis, is regulated by sterols in a manner similar to genes that encode important pro...

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...

Receptor tyrosine kinase erbB2, which is activated by neuregulin, is expressed in Schwann and muscle cells in the developing neuromuscular junction (NMJ). In vitro studies have shown that neuregulin promotes the survival and migration of Schwann cells and stimulates acetylcholine receptor gene transcription in cultured muscle cells. These findings suggest an important role for erbB2 in the development of the NMJ. Here we examine erbB2-deficient mice to determine whether erbB2 is required for NMJ development in vivo. Our analysis shows that there are pre-and postsynaptic defects of developing NMJ in erbB2-deficient embryos. The presynaptic defects include defasciculation and degeneration of the motor nerves, and an absence of Schwann cells. The postsynaptic defect features an impairment of junctional folds at the neuromuscular synapse in the mutants. These results demonstrate that erbB2 is essential for in vivo development of the NMJ.

Transcription from the housekeeping promoter for the acetyl coenzyme A carboxylase (ACC) gene, which encodes the rate-controlling enzyme of fatty acid biosynthesis, is shown to be regulated by cellular sterol levels through novel binding sites for the sterol-sensitive sterol regulatory element binding protein (SREBP)-1 transcription factor. The position ofthe SREBP sites relative to those for the ubiquitous auxiliary transcription factor Spl is reminiscent of that previously described for the sterol-regulated low density lipoprotein receptor promoter. The experiments provide molecular evidence that the metabolism of fatty acids and cholesterol, two different classes of essential cellular lipids, are coordinately regulated by cellular lipid levels.Fatty acids and cholesterol are both major constituents of animal cell membranes and their cellular levels must rise and fall together to provide a balanced supply for membrane biosynthesis and turnover during normal cellular growth. In addition, the liver plays a special role in lipid homeostasis since cholesterol and fatty acids are packaged into very low density lipoprotein (VLDL) particles that deliver their lipid to other sites in the body to maintain fat homeostasis (1). It is likely that there is a feedback mechanism to modulate the accumulation of both cholesterol and fatty acids for optimal cell growth and VLDL assembly.Coordinate regulation could be accomplished at least in part through the AMP-dependent protein kinase, which phosphorylates and inactivates both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase and acetyl coenzyme A carboxylase (ACC), the rate-controlling enzymes of cholesterol and fatty acid biosynthesis, respectively (2). While it is clear that a high ratio of AMP to ATP stimulates the kinase, other metabolic factors that modulate its activity are not well understood. Therefore, a role for the kinase in coordinating regulation in response to cellular lipid levels is unclear.Transcriptional control of genes that encode important enzymes of both cholesterol and fatty acid metabolism would be an alternative level for coordinate regulation to occur, and recent work has identified a family of activator proteins that could link the two pathways together. These sterol regulatory element binding proteins (SREBPs) activate transcription of the low density lipoprotein (LDL) receptor and HMG CoA synthase genes, which are essential for cholesterol uptake and biosynthesis, respectively (3, 4). cDNAs for SREBP-1 and -2 were cloned with specific oligonucleotide probes predicted by the amino acid sequence of the purified polypeptides (3, 4). Both SREBP proteins bind the same cis-acting elements in the LDL receptor and HMG CoA synthase promoters and activate expression only when cellular sterol levels fall below that required for optimal cell function (5).Independently, SREBP-1 was cloned from an adipocyte cDNA expression library with a DNA recognition site probe containing a special "E-box" sequence (6). Its mRNA wasThe publication costs of t...