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...

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...

Mice were subjected to different dietary manipulations to selectively alter expression of hepatic sterol regulatory elementbinding protein 1 (SREBP-1) or SREBP-2. mRNA levels for key target genes were measured and compared with the direct binding of SREBP-1 and -2 to the associated promoters using isoform specific antibodies in chromatin immunoprecipitation studies. A diet supplemented with Zetia (ezetimibe) and lovastatin increased and decreased nuclear SREBP-2 and SREBP-1, respectively, whereas a fasting/refeeding protocol dramatically altered SREBP-1 but had modest effects on SREBP-2 levels. Binding of both SREBP-1 and -2 increased on promoters for 3-hydroxy-3-methylglutaryl-CoA reductase, fatty-acid synthase, and squalene synthase in livers of Zetia/lovastatin-treated mice despite the decline in total SREBP-1 protein. In contrast, only SREBP-2 binding was increased for the low density lipoprotein receptor promoter. Decreased SREBP-1 binding during fasting and a dramatic increase upon refeeding indicates that the lipogenic "overshoot" for fatty-acid synthase gene expression known to occur during high carbohydrate refeeding can be attributed to a similar overshoot in SREBP-1 binding. SREBP co-regulatory protein recruitment was also increased/decreased in parallel with associated changes in SREBP binding, and there were clear distinctions for different promoters in response to the dietary manipulations. Taken together, these studies reveal that there are alternative molecular mechanisms for activating SREBP target genes in response to the different dietary challenges of Zetia/lovastatin versus fasting/refeeding. This underscores the mechanistic flexibility that has evolved at the individual gene/promoter level to maintain metabolic homeostasis in response to shifting nutritional states and environmental fluctuations. 3-Hydroxy-3-methylglutaryl (HMG)3 -CoA reductase catalyzes a critical early reaction in the biosynthetic pathway for isoprenoids and cholesterol and is subject to multivalent regulation to ensure optimal pathway activity (1). The molecular events targeted for regulation include primary transcriptional as well as translational and post-translational mechanisms (2). The principal transcriptional regulators for HMG-CoA reductase gene expression are the basic helix-loop-helix (bHLH) leucine zipper sterol regulatory element-binding proteins (SREBPs), which have unique features that distinguish them from other bHLH leucine zipper transcription factors. The first of these is the presence of two closely spaced membrane-spanning helices that bisect the coding sequence and target the SREBPs to the endoplasmic reticulum membrane. These are followed by a carboxyl-terminal domain that interacts with regulatory proteins and controls their trafficking, proteolytic activation, and membrane release (3, 4). SREBPs also have a signature tyrosine residue in the basic DNA-binding domain that is not present in any other bHLH proteins; this one amino acid is key for allowing specific recognition of both the canonical inv...

We have evaluated the mechanism for sterol-regulated gene expression by the sterol regulatory element binding proteins (SREBPs) in intact cells. We show that activation of SREBPs by sterol depletion results in the increased binding of Sp1 to a site adjacent to SREBP in the promoter for the low density lipoprotein (LDL) receptor gene in vivo. Similarly, sterol depletion resulted in the increased recruitment of two distinct SREBP coregulatory factors, NF-Y and CREB, to the promoter for hydroxymethyl glutaryl CoA reductase, another key gene of intracellular cholesterol homeostasis. Furthermore, increased acetylation of histone H3 but not H4 was also detected in chromatin from both promoters on SREBP activation. Thus, SREBP activation results in the similar selective recruitment of different coregulatory generic transcription factors to two separate cholesterol-regulated promoters. These studies demonstrate the utility of the chromatin immunoprecipitation technique for analyzing the differential action of low-abundance transcription factors in fundamental regulatory events in intact cells. Our results also provide key in vivo support for the mechanism proposed from cell-free experiments, where SREBP increased the binding of Sp1 to the LDL receptor promoter. Finally, our findings also indicate that subtle differences in the pattern of core histone acetylation play a role in selective gene activation. F eedback regulation of mammalian cholesterol homeostasis is mediated by a positive regulatory mechanism through the sterol regulatory element binding proteins (SREBPs) (1). The SREBPs also regulate key genes of fatty acid metabolism so they control flux into the two major lipid classes in mammalian cells (1, 2). When sterol levels fall in cultured cells, the SREBPs are released from membranes of the endoplasmic reticulum and nuclear envelope through the action of two sequential proteolytic activities (3). The resulting soluble mature transcription factor enters the nucleus, where it activates a set of target genes that are involved in lipid accumulation.In all promoters for SREBP target genes that have been carefully studied thus far, SREBP-dependent regulation requires an additional generic coregulatory DNA binding factor(s) for efficient expression (4). The identity of the coregulatory factor and the position of its binding site relative to the binding site(s) for the SREBPs differs from promoter to promoter. The common use of SREBP provides a mechanism for coordinate regulation. However, the unique coregulatory factors and subtle differences in promoter architecture provide the opportunity for more subtle promoter-specific regulatory effects that integrate other cellular signaling pathways with simple nutrient sensing to provide optimal control of cellular lipid levels.To understand the mechanism for coordinate and genespecific activation by the SREBPs, we have been investigating how they function to activate transcription synergistically with distinct coregulatory factors in different promoters. The gene that encode...

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, a key regulatory enzyme in the pathway for endogenous cholesterol synthesis, is a target for negative feedback regulation by cholesterol. When cellular sterol levels are low, the sterol regulatory element-binding proteins (SREBPs) are released from the endoplasmic reticulum membrane, allowing them to translocate to the nucleus and activate SREBP target genes. However, in all SREBP-regulated promoters studied to date, additional co-regulatory transcription factors are required for sterol-regulated activation of transcription. We have previously shown that, in addition to SREBPs, NF-Y/CBF is required for sterol-regulated transcription of HMG-CoA synthase. This heterotrimeric transcription factor has recently been shown to function as a co-regulator in several other SREBP-regulated promoters, as well. In addition to cis-acting sites for both SREBP and NF-Y/CBF, the sterol regulatory region of the synthase promoter also contains a consensus cAMP response element (CRE), an element that binds members of the CREB/ATF family of transcription factors. Here, we show that this consensus CRE is essential for sterolregulated transcription of the synthase promoter. Using in vitro binding assays, we also demonstrate that CREB binds to this CRE, and mutations within the CRE that result in a loss of CREB binding also result in a loss of sterol-regulated transcription. We further show that efficient activation of the synthase promoter in Drosophila SL2 cells requires the simultaneous expression of all three factors: SREBPs, NF-Y/CBF, and CREB. To date this is the first promoter shown to require CREB for efficient sterol-regulated transcription, and to require two different co-regulatory factors in addition to SREBPs for maximal activation.3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) 1 synthase is a key rate-limiting enzyme of cholesterol biosynthesis, converting acetoacetyl-CoA and acetyl-CoA into HMG-CoA. In order to achieve cellular cholesterol homeostasis, this and other genes of cholesterol metabolism are regulated by classical feedback repression: they are up-regulated when sterol levels fall and down-regulated when sterol levels rise (1-5). This control is exerted primarily at the transcriptional level through the action of the sterol regulatory element-binding proteins (SREBPs), a unique subfamily of basic helix-loop-helix zipper (bHLHZip) proteins. SREBPs are expressed as 125-kDa precursor proteins that are anchored to the endoplasmic reticulum and nuclear membranes. When cellular sterol levels fall, the precursor is released from the membrane by a two-step proteolytic mechanism, allowing the mature, transcriptionally active SREBPs to translocate to the nucleus (6). Once inside the nucleus, SREBPs activate cholesterogenic target genes through binding to sterol regulatory elements (SREs) present in their promoters. SREBPs also activate key genes of fatty acid metabolism (7-9) thus, they are central transcription factors of mammalian lipid metabolism. SREBPs alone...