YY1 is a positive regulator of transcription of the Col1a1 gene.

Both cell-specific and ubiquitous transcription factors in fibroblasts have been identified as critical for expression of the Col1a1 gene, which encodes the alpha1 chain of type I collagen. Here, we report that Yin Yang 1 (YY1) binds to the Col1a1 promoter immediately upstream of the TATA box, and we examine the functional implications of YY1 binding for regulation of Col1a1 gene expression in BALBc/3T3 fibroblasts. The Col1a1 promoter region spanning base pairs (bp) -56 to -9 bound purified recombinant YY1 and the corresponding binding activity in nuclear extracts was supershifted using a YY1-specific antibody. Mutation of the TATA box to TgTA enhanced YY1 complex formation. Mutation analysis revealed two YY1 core binding sites at -40/-37 bp (YY1A) and, on the reverse strand, at -32/-29 bp (YY1B) immediately adjacent to the TATA box. In transfections using Col1a1-luciferase constructs, mutation of YY1A decreased activity completely (wild-type p350 (p350wt), -222/+113 bp) or partially (p130wt, -84 bp/+13 bp), whereas mutation of YY1B blocked the expression of both promoter constructs. Cotransfection with pCMV-YY1 increased p350wt and p130wt activities by as much as 10-fold, whereas antisense YY1 decreased constitutive expression and blocked the increased activity due to pCMV-YY1 overexpression. The mTgTA constructs were devoid of activity, arguing for a requirement for cognate binding of the TATA box-binding protein (TBP). Electrophoretic mobility shift assays performed under conditions permitting TBP binding showed that recombinant TBP/TFIID and YY1 could bind to the -56/-9 bp fragment and that YY1B was the preferred site for YY1 binding. Our results indicate that YY1 binds to the Col1a1 proximal promoter and functions as a positive regulator of constitutive activity in fibroblasts. Although YY1 is not sufficient for transcriptional initiation, it is a required component of the transcription machinery in this promoter.

Transcriptional regulation of the murine acetyl-CoA synthetase 1 gene through multiple clustered binding sites for sterol regulatory element-binding proteins and a single neighboring site for Sp1.

Cytosolic acetyl-CoA synthetase (AceCS1) activates acetate to supply the cells with acetyl-CoA for lipid synthesis. The cDNA for the mammalian AceCS1 has been isolated recently, and the mRNA was shown to be negatively regulated by sterols in cultured cells. In the current study, we describe the molecular mechanisms directing the sterol-regulated expression of murine AceCS1 by cloning and functional studies of the 5'-flanking region of the AceCS1 gene. An AceCS1 promoter-reporter gene (approximately 2.1 kilobase pairs) was negatively regulated when sterols were added to the medium of cultured cells, and the promoter was markedly induced by co-transfection of a plasmid that expresses the transcriptionally active nuclear form of either sterol regulatory element-binding protein (SREBP)-1a or -2 in HepG2 cells. Sequence analysis suggested that the AceCS1 promoter contains an E-box, two putative CCAAT-boxes, eight sterol regulatory element (SRE) motifs, and six GC-boxes. Gel shift assays demonstrated that all eight SRE motifs bound purified SREBP-1a in vitro with similar affinity. Luciferase reporter gene assays revealed that sterol regulation was critically dependent on three closely spaced SRE motifs and an adjacent GC-box. However, mutation of two putative upstream CCAAT-boxes did not affect SREBP dependent activation. Electrophoretic mobility "supershift" analyses confirmed that both Sp1 and Sp3 bound to the critical GC-box. In addition, transfection studies in Drosophila SL2 cells demonstrated that SREBP synergistically activated the AceCS1 promoter along with Sp1 or Sp3 but not with nuclear factor-Y.

Conditional response of the human steroidogenic acute regulatory protein gene promoter to sterol regulatory element binding protein-1a.

The steroidogenic acute regulatory protein (StAR) gene controls the rate-limiting step in the biogenesis of steroid hormones, delivery of cholesterol to the cholesterol side-chain cleavage enzyme on the inner mitochondrial membrane. We determined whether the human StAR promoter is responsive to sterol regulatory element-binding proteins (SREBPs). Expression of SREBP-1a stimulated StAR promoter activity in the context of COS-1 cells and human granulosa-lutein cells. In contrast, expression of SREBP-2 produced only a modest stimulation of StAR promoter activity. One of the SREBP-1a response elements in the StAR promoter was mapped in deletion constructs and by site-directed mutagenesis between nucleotides -81 to -70 from the transcription start site. This motif bound recombinant SREBPs in electrophoretic mobility shift assays, but with lesser affinity than a low density lipoprotein receptor SREBP-binding site. An additional binding site for the transcriptional modulator, yin yang 1 (YY1), was observed within the SREBP-binding site (nucleotides -73 to -70). Mutation of the YY1-binding site increased the responsiveness of the StAR promoter to exogenous SREBP-1a, but did not alter the affinity for SREBP-1a binding in electrophoretic mobility gel shift assays. Manipulations that altered endogenous mature SREBP-1a levels (e.g. culture in lipoprotein-deficient medium and addition of 27-hydroxycholesterol) did not affect StAR promoter function, but influenced low density lipoprotein receptor promoter activity. We conclude that 1) the human StAR promoter is conditionally responsive to SREBP-1a such that promoter activity is up-regulated in the presence of high levels of SREBP-1a, but is unaffected when mature SREBP levels are suppressed; and 2) the human StAR promoter is selectively responsive to SREBP-1a.

Nutrient regulation of gene expression by the sterol regulatory element binding proteins: increased recruitment of gene-specific coregulatory factors and selective hyperacetylation of histone H3 in vivo.

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.

Different sterol regulatory element-binding protein-1 isoforms utilize distinct co-regulatory factors to activate the promoter for fatty acid synthase.

Sterol regulatory element-binding proteins (SREBPs) activate genes of cholesterol and fatty acid metabolism. In each case, a ubiquitous co-regulatory factor that binds to a neighboring recognition site is also required for efficient promoter activation. It is likely that gene- and pathway-specific regulation by the separate SREBP isoforms is dependent on subtle differences in how the individual proteins function with specific co-regulators to activate gene expression. In the studies reported here we extend these observations significantly by demonstrating that SREBPs are involved in both sterol regulation and carbohydrate activation of the FAS promoter. We also demonstrate that the previously implicated Sp1 site is largely dispensable for sterol regulation in established cultured cells, whereas a CCAAT-binding factor/nuclear factor Y is critically important. In contrast, carbohydrate activation of the FAS promoter in primary hepatocytes is dependent upon SREBP and both the Sp1 and CCAAT-binding factor/nuclear factor Y sites. Because 1c is the predominant SREBP isoform expressed in hepatocytes and 1a is more abundant in sterol depleted established cell lines, this suggests that the different SREBP isoforms utilize distinct co-regulatory factors to activate target gene expression.

Co-stimulation of promoter for low density lipoprotein receptor gene by sterol regulatory element-binding protein and Sp1 is specifically disrupted by the yin yang 1 protein.

Sterol regulation of gene expression in mammalian cells is mediated by an interaction between the cholesterol-sensitive sterol regulatory element-binding proteins (SREBPs) and promoter-specific but generic co-regulatory transcription factors such as Sp1 and NF-Y/CBF. Thus, sterol-regulated promoters that require different co-regulatory factors could be regulated independently through targeting the specific interaction between the SREBPs and the individual co-regulatory proteins. In the present studies we demonstrate that transiently expressed yin yang 1 protein (YY1) inhibits the SREBP-mediated activation of the low density lipoprotein (LDL) receptor in a sensitive and dose-dependent manner. The inhibition is independent of YY1 binding directly to the LDL receptor promoter, and we show that the same region of YY1 that interacts in solution with Sp1 also interacts with SREBP. Furthermore, other SREBP-regulated genes that are not co-regulated by Sp1 are either not affected at all or are not as sensitive to the repression. Thus, the specific interaction that occurs between SREBPs and Sp1 to stimulate the LDL receptor promoter is a specific target for inhibition by the YY1 protein, and we provide evidence that the mechanism can be at least partially explained by the ability of YY1 to inhibit the interaction between SREBP and Sp1 in solution in vitro. The LDL receptor is the key gene of cholesterol uptake, and the rate-controlling genes of cholesterol synthesis are stimulated by the concerted action of SREBPs along with coregulators that are distinct from Sp1. Therefore, repression of gene expression through specifically targeting the interaction between SREBP and Sp1 would provide a molecular mechanism to explain how cholesterol uptake can be regulated independently from cholesterol biosynthesis in mammalian cells.

A critical role for cAMP response element-binding protein (CREB) as a Co-activator in sterol-regulated transcription of 3-hydroxy-3-methylglutaryl coenzyme A synthase promoter.

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 sterol-regulated 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.

Oxysterol regulation of steroidogenic acute regulatory protein gene expression. Structural specificity and transcriptional and posttranscriptional actions.

Oxysterols exert a major influence over cellular cholesterol homeostasis. We examined the effects of oxysterols on the expression of steroidogenic acute regulatory protein (StAR), which increases the delivery of cholesterol to sterol-metabolizing P450s in the mitochondria. 22(R)-hydroxycholesterol (22(R)-OHC), 25-OHC, and 27-OHC each increased steroidogenic factor-1 (SF-1)-mediated StAR gene transactivation by approximately 2-fold in CV-1 cells. In contrast, cholesterol, progesterone, and the 27-OHC metabolites, 27-OHC-5beta-3-one and 7alpha,27-OHC, had no effect. Unlike our findings in CV-1 cells, SF-1-dependent StAR promoter activity was not augmented by 27-OHC in COS-1 cells, Y-1 cells, BeWo choriocarcinoma cells, Chinese hamster ovary (CHO) cells, and human granulosa cells. Studies examining the metabolism of 27-OHC indicated that CV-1 cells formed a single polar metabolite, 3beta-OH-5-cholestenoic acid from radiolabeled 27-OHC. However, this metabolite inhibited StAR promoter activity in CV-1, COS-1 and CHO cells. Because 7alpha,27-OHC was unable to increase SF-1-dependent StAR promoter activity, we examined 27-OHC 7alpha-hydroxylase in COS-1 and CHO cells. COS-1 cells contained high 7alpha-hydroxylase activity, whereas the enzyme was undetectable in CHO cells. The hypothesis that oxysterols act in CV-1 cells to increase StAR promoter activity by reducing nuclear levels of sterol regulatory element binding protein was tested. This notion was refuted when it was discovered that sterol regulatory element binding protein-1a is a potent activator of the StAR promoter in CV-1, COS-1, and human granulosa cells. Human granulosa and theca cells, which express endogenous SF-1, contained more than 5-fold more StAR protein following addition of 27-OHC, whereas StAR mRNA levels remained unchanged. We conclude that 1) there are cell-specific effects of oxysterols on SF-1-dependent transactivation; 2) the ability to increase transactivation is limited to certain oxysterols; 3) there are cell-specific pathways of oxysterol metabolism; and 4) oxysterols elevate StAR protein levels through posttranscriptional actions.

Molecular aspects in feedback regulation of gene expression by cholesterol in mammalian cells.

Intracellular cholesterol balance is maintained by a tight feedback mechanism that prevents the overaccumulation of cholesterol to cytotoxic levels. This is achieved through the coordinate regulation of genes of cholesterol uptake and biosynthesis by the sterol regulatory element binding proteins (SREBPs). The SREBPs are synthesized as membrane bound precursors that are released from their membrane tether when the cell needs new cholesterol. In the present article we present a model for how the cholesterol uptake pathway may be activated before the biosynthetic pathway to prevent wasting cellular energy and carbon on unneeded synthesis. Then we introduce a system for analyzing the differential localization and cellular trafficking of the different SREBP isoforms that can be performed over time in living cells.

Polyunsaturated fatty acids decrease expression of promoters with sterol regulatory elements by decreasing levels of mature sterol regulatory element-binding protein.

Membrane physiology, plasma lipid levels, and intracellular sterol homeostasis are regulated by both fatty acids and cholesterol. Sterols regulate gene expression of key enzymes of cholesterol and fatty acid metabolism through proteolysis of the sterol regulatory element-binding protein (SREBP), which binds to sterol regulatory elements (SRE) contained in promoters of these genes. We investigated the effect of fatty acids on SRE-dependent gene expression and SREBP. Consistent results were obtained in three different cell lines (HepG2, Chinese hamster ovary, and CV-1) transfected with SRE-containing promoters linked to the luciferase expression vector. We show that micromolar concentrations of oleate and other polyunsaturated fatty acids (C18:2-C22:6) dose-dependently (0.075-0.6 mmol) decreased transcription of SRE-regulated genes by 20-75%. Few or no effects were seen with saturated free fatty acids. Fatty acid effects on SRE-dependent gene expression were independent and additive to those of exogenous sterols. Oleate decreased levels of the mature sterol regulatory element-binding proteins SREBP-1 and -2 and HMG-CoA synthase mRNA. Oleate had no effect in sterol regulation defective Chinese hamster ovary cells or in cells transfected with mutant SRE-containing promoters. We hypothesize that unsaturated fatty acids increase intracellular regulatory pools of cholesterol and thus affect mature SREBP levels and expression of SRE-dependent genes.

Sterol regulatory element binding protein-1 activates the cholesteryl ester transfer protein gene in vivo but is not required for sterol up-regulation of gene expression.

The plasma cholesteryl ester transfer protein (CETP) plays a central role in high density lipoprotein metabolism and reverse cholesterol transport. Plasma CETP levels are increased in response to dietary or endogenous hypercholesterolemia as a result of increased gene transcription in liver and periphery. Deletional analysis in human CETP transgenic mice localized this response to a region of the proximal promoter which contains a tandem repeat of the sterol regulatory element (SRE) of the 3-hydroxy-3-methylglutaryl-CoA reductase gene. The purpose of the present study was to evaluate the role of the SRE-like element in CETP promoter activity. Gel shift assays using CETP promoter fragments containing these elements showed binding of the transcription factors, sterol regulatory element-binding protein-1 (SREBP-1) and Yin Yang-1 (YY-1). Point mutations in the SRE-like element, designated MUT1 and MUT2, resulted in decreased binding of SREBP-1 (MUT1) or SREBP-1 and YY-1 (MUT2). To determine the in vivo significance of this binding activity, CETP transgenic mice were prepared containing these promoter point mutations. MUT1 and MUT2 transgenic mice expressed CETP activity and mass in plasma. In response to high fat, high cholesterol diets, both MUT1-CETP and MUT2-CETP transgenic mice displayed induction of plasma CETP activity similar to that observed in natural flanking region (NFR) CETP transgenic mice. Moreover, in stably transfected adipocyte cell lines, MUT1 and MUT2 CETP promoter-reporter genes showed significant induction of reporter activity in response to sterols. To evaluate transactivation by SREBP-1, NFR- and MUT1-CETP transgenic mice were crossed with SREBP-1 transgenic mice. Induction of the SREBP transgene in the liver with a low carbohydrate diet resulted in a 3-fold increase in plasma CETP activity in NFR-CETP/SREBP transgenic mice, but there was no significant change in activity in MUT1-CETP/SREBP transgenic mice. Thus, SREBP-1 transactivates the NFR-CETP transgene in vivo, as a result of interaction with the CETP promoter SREs. However, this interaction is not required for positive sterol induction of CETP gene transcription. The results suggest independent regulation of the CETP gene by SREBP-1 and a distinct positive sterol response factor.

Specificity in cholesterol regulation of gene expression by coevolution of sterol regulatory DNA element and its binding protein.

When demand for cholesterol rises in mammalian cells, the sterol regulatory element (SRE) binding proteins (SREBPs) are released from their membrane anchor through proteolysis. Then, the N-terminal region enters the nucleus and activates genes of cholesterol uptake and biosynthesis. Basic helix-loop-helix (bHLH) proteins such as SREBPs bind to a palindromic DNA sequence called the E-box (5'-CANNTG-3'). However, SREBPs are special because they also bind direct repeat elements called SREs. Importantly, sterol regulation of all promoters studied thus far is mediated by SREBP binding only to SREs. To study the reason for this we converted the direct repeat SRE from the sterol-regulated low-density lipoprotein receptor promoter into an E-box. In this report we show that SREBPs are still able to bind and activate this promoter however, sterol regulation is lost. The results are consistent with the mutant promoter being a target for promiscuous activation by constitutively expressed E-box binding bHLH proteins that are not regulated by cholesterol. Kim and coworkers [Kim, J. B., Spotts, G. D., Halvorsen, Y.-D., Shih, H.-M., Ellenberger, T., Towle, H. C. & Spiegelman, B. M. (1995) Mol. Cell. Biol. 15, 2582-2588] demonstrated that the dual DNA binding specificity of SREBPs is caused by a specific tyrosine in the conserved basic region of the DNA binding domain that corresponds to an arginine in all other bHLH proteins that recognize only E-boxes. Taken together the data suggest an evolutionary mechanism where a DNA binding protein along with its recognition site have coevolved to ensure maximal specificity and sensitivity in a crucial nutritional regulatory response.

HLH106, a Drosophila sterol regulatory element-binding protein in a natural cholesterol auxotroph.

In mammalian cells, sterol regulatory element-binding proteins (SREBPs) coordinate metabolic flux through the cholesterol and fatty acid biosynthetic pathways in response to intracellular cholesterol levels. We describe experiments that evaluate the functional equivalence of mammalian SREBPs and the insect homologue of SREBP-1a, HLH106, in both mammalian and insect cell culture systems. HLH106 binds to both palindromic E-boxes and direct repeat sterol regulatory elements (SREs) efficiently, suggesting that it has a dual DNA binding specificity similar to the mammalian proteins. The amino-terminal "mature" protein activates transcription from mammalian SREs in both mammalian and Drosophila tissue culture cells. Additionally, HLH106 also requires a ubiquitous regulatory co-activator to efficiently activate transcription from mammalian SREs. These properties are shared with its mammalian counterparts. When expressed in mammalian cells, the carboxyl-terminal portion also localizes to perinuclear membranes similar to mammalian SREBPs. Furthermore, membrane-bound HLH106 is proteolytically processed in response to intracellular sterol levels in mammalian cells in an SREBP cleavage-activating protein-stimulated fashion. The presence of an SREBP homologue in Drosophila whose processing is regulated by intracellular sterol levels when expressed in mammalian cells suggests that related processing machinery exists in insect cells. This is notable, since insects are reportedly incapable of de novo sterol biosynthesis.

Related membrane domains in proteins of sterol sensing and cell signaling provide a glimpse of treasures still buried within the dynamic realm of intracellular metabolic regulation.

Recent discoveries in the regulation of cholesterol metabolism have documented a two step proteolytic pathway that regulates nuclear targeting of the sterol regulatory element binding proteins. Sterol regulatory element binding protein cleavage activating protein is a newly identified protein that modulates the proteolytic maturation of the sterol regulatory element binding proteins. It contains a domain that is quite similar in sequence to the membrane spanning region of the rate controlling enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A reductase. The membrane domain of the reductase is involved in its post-translational regulation by cholesterol. The molecular defect in the intracellular cholesterol storage disease, Niemann-Pick type C, has also recently been identified. Surprisingly, the affected gene encodes a protein with similarity to the membrane domains that are conserved in 3-hydroxy-3-methylglutaryl reductase and sterol regulatory element binding protein cleavage activating protein. Furthermore, the cell surface receptor for the sterol modified hedgehog morphogen, Patched, also contains a membrane domain with significant similarity to this putative sterol monitoring domain. These recent developments suggest a common mechanism for sensing intracellular sterol levels and cell signaling, which is based on the function of related membrane domains that are contained in key regulatory proteins.

Sterol regulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase gene through a direct interaction between sterol regulatory element binding protein and the trimeric CCAAT-binding factor/nuclear factor Y.

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. The promoter for HMG-CoA synthase contains two binding sites for the sterol regulatory element-binding proteins (SREBPs). When cellular sterol levels are low, the SREBPs are released from the endoplasmic reticulum membrane, allowing them to translocate to the nucleus and activate SREBP target genes. In all SREBP-regulated promoters studied to date, additional co-regulatory transcription factors are required. In the HMG-CoA synthase promoter there are several potential co-regulatory transcription factor binding sites, including an inverted CCAAT box. A similar element has been shown to function with SREBP to mediate sterol regulation of another gene involved in cholesterol metabolism, farnesyl diphosphate synthase. Here, we show that CCAAT binding factor/nuclear factor Y (CBF/NF-Y) binding to the CCAAT box is required for sterol-regulated transcription of HMG-CoA synthase. The SREBP sites and the inverted CCAAT box are normally separated by 17 base pairs, and we show that increasing this distance results in a decrease in the level of transcriptional regulation by sterols. Furthermore, we provide evidence that there is a direct interaction between CBF/NF-Y and the basic helix-loop-helix-zipper region of SREBP. Interestingly, this interaction does not occur efficiently with any of the isolated subunits and appears to require all three nonidentical CBF/NF-Y subunits in a preassembled complex. Since CBF/NF-Y only binds to DNA when all three subunits are in a complex, this would prevent SREBP from forming nonproductive associations with the individual subunits.

Promoter selective transcriptional synergy mediated by sterol regulatory element binding protein and Sp1: a critical role for the Btd domain of Sp1.

Cellular cholesterol and fatty acid levels are coordinately regulated by a family of transcriptional regulatory proteins designated sterol regulatory element binding proteins (SREBPs). SREBP-dependent transcriptional activation from all promoters examined thus far is dependent on the presence of an additional binding site for a ubiquitous coactivator. In the low-density lipoprotein (LDL) receptor, acetyl coenzyme A carboxylase (ACC), and fatty acid synthase (FAS) promoters, which are all regulated by SREBP, the coactivator is the transcription factor Sp1. In this report, we demonstrate that Sp3, another member of the Sp1 family, is capable of substituting for Sp1 in coactivating transcription from all three of these promoters. Results of an earlier study showed that efficient activation of transcription from the LDL receptor promoter required domain C of Sp1; however, this domain is not crucial for activation of the simian virus 40 promoter, where synergistic activation occurs through multiple Sp1 binding sites and does not require SREBP. Also in the present report, we further localize the critical determinant of the C domain required for activation of the LDL receptor to a small region that is highly conserved between Sp1 and Sp3. This crucial domain encompasses the buttonhead box, which is a 10-amino-acid stretch that is present in several Sp1 family members, including the Drosophila buttonhead gene product. Interestingly, neither the buttonhead box nor the entire C domain is required for the activation of the FAS and ACC promoters even though both SREBP and Sp1 are critical players. ACC and FAS each contain two critical SREBP sites, whereas there is only one in the LDL receptor promoter. This finding suggested that buttonhead-dependent activation by SREBP and Sp1 may be limited to promoters that naturally contain a single SREBP recognition site. Consistent with this model, a synthetic construct containing three tandem copies of the native LDL receptor SREBP site linked to a single Sp1 site was also significantly activated in a buttonhead-independent fashion. Taken together, these studies indicate that transcriptional activation through the concerted action of SREBP and Sp1 can occur by at least two different mechanisms, and promoters that are activated by each one can potentially be identified by the number of critical SREBP binding sites that they contain.

Sterol regulation of acetyl coenzyme A carboxylase promoter requires two interdependent binding sites for sterol regulatory element binding proteins.

The sterol regulatory element binding proteins (SREBPs) are central regulators of lipid homeostasis in mammalian cells. Their activity is controlled by a sterol-regulated two-step proteolytic process that releases the nuclear targeted amino-terminal domain from the membrane anchored carboxyl-terminal remnant. This ensures that transcriptional stimulation of the appropriate genes occurs only when increased intracellular sterol accumulation is required. Gene targets for SREBP encode key proteins of cholesterol metabolism as well as essential proteins of fatty acid biosynthesis, providing a mechanism for coordinate control of these two major lipid pathways when sterols and fatty acids need to accumulate together. However, the regulatory mechanism must provide a way to uncouple these two pathways to allow separate regulation when sterol or fat levels need to increase independently of each other. We compared the similarities and differences for how SREBP activates the promoter for the low density lipoprotein (LDL) receptor, which is the key protein involved in cholesterol uptake, relative to how it activates promoters for acetyl coenzyme A carboxylase (ACC) and fatty acid synthase (FAS), which are both key enzymes of fatty acid biosynthesis. In the current studies we show there are two distinct sites for SREBP binding that control activation of the ACC PII promoter whereas previous work has shown there is only a single SREBP site in the LDL receptor. Additionally, disruption of either ACC site results in a total loss in promoter function and a severe decrease in SREBP binding even to the neighboring unaltered site. Thus, the two sites are equally important and dependent on one another for optimal function. This is in contrast to the FAS promoter where SREBP binds to two adjacent sites independently and the one located closer to the binding site for the Sp1 co-regulator is more critical for sterol regulation and activation by SREBP over-expression.

Multiple sequence elements are involved in the transcriptional regulation of the human squalene synthase gene.

The expression of human squalene synthase (HSS) gene is transcriptionally regulated in HepG-2 cells, up to 10-fold, by variations in cellular cholesterol homeostasis. An earlier deletion analysis of the 5'-flanking region of the HSS gene demonstrated that most of the HSS promoter activity is detected within a 69-base pair sequence located between nucleotides -131 and -200. ADD1/SREBP-1c, a rat homologue of sterol regulatory element-binding protein (SREBP)-1c binds to sterol regulatory element (SRE)-1-like sequence (HSS-SRE-1) present in this region (Guan, G., Jiang, G., Koch, R. L. and Shechter, I. (1995) J. Biol. Chem. 270, 21958-21965). In our present study, we demonstrate that mutation of this HSS-SRE-1 element significantly reduced, but did not abolish, the response of HSS promoter to change in sterol concentration. Mutation scanning indicates that two additional DNA promoter sequences are involved in sterol-mediated regulation. The first sequence contains an inverted SRE-3 element (Inv-SRE-3) and the second contains an inverted Y-box (Inv-Y-box) sequence. A single mutation in any of these sequences reduced, but did not completely remove, the response to sterols. Combination mutation studies showed that the HSS promoter activity was abolished only when all three elements were mutated simultaneously. Co-expression of SRE-1- or SRE-2-binding proteins (SREBP-1 or SREBP-2) with HSS promoter-luciferase reporter resulted in a dramatic increase of HSS promoter activity. Gel mobility shift studies indicate differential binding of the SREBPs to regulatory sequences in the HSS promoter. These results indicate that the transcription of the HSS gene is regulated by multiple regulatory elements in the promoter.