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-Demethylase Gene (CYP51)
Department of Biochemistry (S.K.H., M.R.W.), Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146; and Institute of Biochemistry (M.F., D.R.), Medical Center for Molecular Biology, Faculty of Medicine, University of Ljubljana, Sl-1000 Ljubljana, Slovenia
Address all correspondence and requests for reprints to: Damjana Rozman, Medical Center for Molecular Biology, Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia. E-mail: damjana.rozman{at}mf.uni-lj.si.
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ABSTRACT |
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-demethylase (CYP51) is involved in the cholesterol biosynthesis pathway, producing follicular fluid meiosis-activating sterol. The promoter region of the human CYP51 gene contains a cluster of regulatory elements including GC box, cAMP response element (CRE), and sterol regulatory element (SRE). To understand the mechanism of sterol-dependent regulation of this gene, several constructs of the promoter with the reporter gene have been tested in JEG-3 cells containing overexpressed human sterol regulatory element binding protein (SREBP)-1a. The wild-type construct showed maximal SREBP-dependent activation, most of which is retained when the GC box is mutated/deleted. Activation is abolished when either CRE or SRE are removed/mutated. Furthermore, mutation of CRE abolishes SREBP-dependent activation after overexpression of SREBP-1a and CRE binding protein (CREB). This shows that CRE is essential, and that under ex vivo conditions CREB and SREBP cooperate in transactivating CYP51. Interestingly, protein kinase A shows a marked stimulation of the CYP51 promoter activity when overexpressed together with SREBP-1a but not when overexpressed with CREB, suggesting phosphorylation of SREBP-1a. Using a DNA probe containing all three regulatory elements, it is found that SREBP-1a, a CREB-like factor, and specificity protein (Sp1) all probably bind the CYP51 promoter. While SREBP-1a and the CRE-bound proteins are essential for the SREBP-dependent response, Sp1 apparently functions only to maximize sterol regulation of CYP51. To date this is the first gene in which cooperation between SREBP and a CREB/CRE modulator/activating transcription factor family transcription factor is shown to be essential and sufficient for SREBP-dependent activation. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-sterol demethylase that is highly conserved in sterol-synthesizing eukaryotic phyla (1). As a result of its recent discovery in Mycobacteria species (2), it is considered to be an ancient P450 arising before the divergence of eukaryotes. Lanosterol 14-demethylase (CYP51) catalyzes the first step after cyclization in sterol biosynthesis, the three-reaction 14-demethylation of lanosterol. Cholesterol biosynthesis involves at least 30 reactions. Genes involved in the presqualene portion of the pathway are well characterized, their sterol-dependent transcription being well established. These include 3-hydroxy-3-methyl- glutaryl coenzyme A (HMG-CoA) reductase (3, 4) HMG-CoA synthase (5), farnesyl pyrophosphate synthase (6), and squalene synthase (7). All require the transcription factor sterol regulatory element (SRE) binding protein (SREBP), and at least one additional coregulatory transcription factor, although the combination of factors differs from gene to gene. In addition to cholesterol biosynthesis, transcription factors of the SREBP family regulate synthesis of the low-density lipoprotein (LDL) receptor (3, 8, 9) and caveolin (10), as well as fatty acid and triglyceride synthesis (11, 12, 13). SREBPs are a family of nuclear transcription factors that are synthesized as precursors localized in the endoplasmic reticulum. There are at least two isoforms of SREBP-1 (SREBP-1a and SREBP-1c) that are expressed from a single gene, whereas SREBP-2 arises from a distinct gene (14, 15, 16). In the absence of sterols, the full-length, membrane-bound SREBPs are proteolytically processed, leading to release of the basic helix-loop-helix-Zip amino-terminal portion of the protein that directly translocates to the nucleus and activates transcription of target genes via binding to SREs (14, 17, 18, 19). Both SREBP-1 and SREBP-2 have the ability to bind to SREs and activate promoters of cholesterogenic and lipogenic enzymes (20, 21, 22, 23, 24). SREBP-1a is the predominant form in cultured cell lines, whereas in tissues SREBP-2 seems to be more important for regulation of cholesterogenic genes (21). Although SREBPs are the key regulatory proteins for cholesterol-dependent activation of all these genes, they are not sufficient for transcriptional activation of any and require the simultaneous binding of other transcription factors to response elements found near the SRE (25). Some genes require a single SREBP coregulatory protein, such as specificity protein 1 (Sp1) for the human LDL receptor gene (8) and nuclear factor (NF-Y) for both farnesyl pyrophosphate synthase (6, 26) and squalene synthase (7). Several genes involved in either cholesterol or fatty acid biosynthesis require two SREBP coregulatory proteins. Sp1 and NF-Y are needed for the fatty acid synthase (11, 12, 27) and 7-dehydrocholesterol reductase (28) activation, whereas NF-Y and a CRE-binding protein (CREB)/activating transcription factor (ATF)-1 family factor are coregulators for the HMG-CoA reductase (3) and HMG-CoA synthase (29) genes. Recently, evidence has accumulated that different isoforms of SREBP are not only involved in cholesterol-regulated events but are themselves targets for intracellular signaling pathways. For example, activation of AMP- activated protein kinase, which is a major cellular regulator of lipid and glucose metabolism, suppressed expression of SREBP-1a (30), whereas glucagon dramatically decreased the expression of adipocyte determination and differentiation factor 1 (ADD1)/SREBP-1c through generating cAMP and activating protein kinase A (PKA) (31). Phosphorylation of SREBP-1a at Ser 117 of SREBP-1a by the Erk subfamily of MAPKs was found to be involved in SREBP-1a-mediated induction of the LDL receptor gene by insulin and platelet-derived growth factor (32).
The CYP51 gene encodes lanosterol 14-demethylase, which belongs to the postsqualene portion of cholesterol biosynthesis and metabolizes lanosterol, which is the first cyclic intermediate of the pathway. Beyond expression and regulation studies of CYP51 (33, 34, 35, 36), regulation of only one other gene of the late cholesterol biosynthetic pathway has been studied (7-dechydrocholesterol reductase in Ref. 28). Our previous studies have shown that SREBPs are not essential for a high-level expression of the lanosterol 14-demethylase gene in male germ cells, where CRE modulator-
(CREM) is essential, but are involved in transcription of CYP51 in somatic cells (35). However, the molecular mechanism of the SREBP-dependent activation has not been established. The CYP51 promoter contains both a GC box and a CRE in close proximity to SRE, indicating that either the Sp or CREB/CREM/ATF family of transcription factors, or perhaps a combination of both, is involved in the SREBP-dependent activation.
In the current study we have used JEG-3, a human choriocarcinoma placental cell line, as a model for somatic cells to understand sterol-mediated regulation of human CYP51. Results show that SRE is crucial for regulation but is itself not sufficient in transactivating the CYP51 promoter. Data suggest that the sterol response of the lanosterol 14-demethylase gene depends not only on the availability of SREBPs but also on proteins of the CREB/CREM/ATF family.
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). This SRE is not identical with SREs studied in other cholesterogenic genes, but it is 100% identical with SREs present in rat and mouse CYP51 promoters (37). A GC box (GGGGGCGG) also identical in all mammalian CYP51 promoters is located 40 bp upstream from SRE, and a highly conserved CYP51 CRE (TGACGCGA) is found at -252/-244, immediately upstream of the SRE.
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) and the plasmid cytomegalovirus (CMV)-hSREBP-1a that expresses the mature, transcriptionally active form of SREBP-1a (amino acids 1–460) are presented in Fig. 2. The reporter construct -282/+314 showed a similar level of activation as the WT promoter (Fig. 2B, lanes 1 and 2). Removal of the GC box (-272/+314) reduces activity by 20% (lane 3). Deletion of the next 20 bp (lane 4) drops the activation to 50% of WT, although no recognizable transcription binding sites are present within the -272/-252 region. However, this region is immediately upstream of the 8-bp CRE site that is essential for SREBP response. Construct -237/+314, which still contains an intact SRE element but lacks the CRE site, is unable to mediate the SREBP-dependent transactivation of the CYP51 promoter (lane 5).
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). Mutation of SRE shows, as expected, a very low level of activation (lane 3). Mutation of the GC box results in approximately 46% activity loss (lane 4). Mutation in the CRE sequence reduces the activity to a similar extent as does mutation of SRE (compare lanes 3 and 5). Experiments in which individual elements have been deleted from the promoter-reporter constructs are in complete agreement with the mutation data (not shown). In the presence of overexpressed SREBP-1a, deletion of SRE diminished the reporter gene activity to near basal level. Deletion of the 8-bp GC box reduced the chloramphenicol acetyltransferase (CAT) activity by only 30%, whereas deletion of both SRE and GC box led to basal regulation that was observed also after the deletion of the entire proximal promoter region. Therefore, SRE in the human CYP51 promoter is crucial for sterol-mediated regulation. Modest reduction in CAT activity observed after deletion of the GC box suggests that Sp family transcription factors do not play a major role in SREBP-dependent transcription of CYP51. Mutation and deletion data clearly show that an intact CRE element is essential for the SREBP-dependent activation of CYP51. The presence of an intact GC box is beneficial and optimizes transcriptional activation, but it is not essential for the SREBP-mediated transactivation of CYP51.
SREBP-1a, a CREB, and Sp1 Bind to Their Corresponding Elements
To investigate the DNA-protein interactions, we used in vitro gel shift binding assays (Fig. 3). The CYP51 SRE probe interacts efficiently with purified SREBP-1a to form a complex similar to that of the human LDL receptor SRE (Fig. 3A, lanes 2 and 4). Complexes are supershifted in the presence of anti-SREBP-1 (lanes 3 and 5). However, the mutation examined in Fig. 2C completely abolished formation of the DNA-protein complex (lane 6).
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, lane 1). We were unable to supershift the complex, but the addition of two different anti-CREB antibodies (lanes 2 and 3) and an anti-ATF1 antibody (lane 4) ablated the binding, whereas anti-ATF2 antibody shows no effect (lane 5). Presence of a complex formed between CRE and the bZip binding domain of CREB (lane 6) also confirmed that a nuclear protein of the CREB/CREM/ATF family binds to this site. Binding was reduced in the presence of cold CRE competitor (lane 7), and the complex did not form on the mutated CRE (lane 8). We have shown previously that this CYP51-CRE sequence (CYP51-CRE2 in Ref. 35) binds CREM, which is the predominant cAMP-dependent transcriptional activator in haploid male germ cells. The DNA-CREM complex was disrupted in the presence of both anti-CREB and anti-CREM antibodies (Rozman, D., unpublished). CREM is rarely present in somatic cells and its presence in JEG-3 cells is not documented.
The CYP51 GC box forms a complex with Sp1 similar to that of the consensus GC box probe, and the complex is supershifted with anti-Sp1 antibody (Fig. 3C, lanes 4, 6, and 8). Cold competition with either the consensus GC box or the CYP51 GC box competed formation of the complex (lanes 9 and 10). The mutated GC box (lane 11) was unable to compete the formation of the DNA-Sp complex. Furthermore, the DNA-Sp complex was also not observed when the mutated CYP51-GC box was taken as a probe and either nuclear extracts or the purified Sp1 protein were applied (data not shown). In addition to Sp1, Sp3 can bind to the CYP51-GC box; however, the intensity of Sp1 binding is much greater, indicating that Sp1 is the major Sp family transcription factor in JEG-3 cells.
SREBP-Dependent Activation of the Human CYP51 Promoter in the Presence of Overexpressed Sp1 and CREB
Results described in Fig. 2 show the essential role of CRE for the SREBP-mediated transcription of CYP51 and that the GC box is not essential but contributes to the maximal activation of the promoter. To further evaluate the functional role of Sp1 in SREBP-dependent activation, SREBP-1a and Sp1 were overexpressed in a Drosophila cell line SL-2, together with CYP51 or LDL receptor luciferase reporter constructs. SL2 cells do not express functional homologs of several mammalian transcription factors, including Sp1. These cells have been used previously to understand regulation of the human LDL receptor gene in which SREBP and Sp1 are involved in synergistic activation (8). SREBP-1a alone weakly activates the LDL receptor promoter (Fig. 4A, lane 3), and the addition of Sp1 (lane 4) results in a 10-fold increase of the SREBP-dependent activation. SREBP-1a alone also weakly activates the CYP51 promoter (lane 7), but the presence of Sp1 results in a much smaller increase of the SREBP-dependent activation (compare lanes 4 and 8).
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, black bars in lanes 2 and 4) and some SREBP-1a dose-response is still observed with the CRE-mutated construct (dashed bars in lanes 2 and 4). However, the CRE-mutated construct failed to show SREBP-dependent dose-response when CREB is overexpressed together with SREBP-1a (dashed bars in lanes 3 and 5). A SREBP-1a dose-dependent (black bars, lanes 3 and 5) and SREBP-1a/CREB cooperative action (black bars, lanes 2 and 3; and 4 and 5) is observed with the WT promoter when both transcription factors are overexpressed.
The Role of PKA in SREBP-1a-Dependent Transcription of CYP51
CREB as well as SREBP-1a were both shown to be activated by phosphorylation at specific serine residues (32, 39). Because CREB and SREBP-1a are involved in transactivation of CYP51 ex vivo, we wanted to evaluate whether the PKA signaling pathway influences CYP51 transcriptional activation. Overexpression of CREB stimulates the WT CYP51-CAT reporter (black bars; Fig. 4D, lane 2). Surprisingly, overexpression of PKA did not influence the CREB-dependent activation of CYP51 (Fig. 4D, compare lanes 2 and 3), although with the somatostatin-CAT reporter that served as a positive control, overexpression of PKA stimulated the CREB-dependent activation (Fig. 4C, compare lanes 2 and 3). Overexpression of SREBP-1a also stimulated the WT CYP51-CAT reporter (Fig. 4D, lane 4), and the addition of PKA resulted in a 3-fold increase of the SREBP-1a-dependent activation (Fig. 4D, lanes 5 and 4). As shown in Fig. 4B, lane 4, the CRE-mutated promoter exhibits a markedly diminished activity compared with the WT promoter after overexpression of SREBP-1a. Overexpression of PKA does not stimulate the residual SREBP-1a-dependent activation of the CRE-mutated CYP51-CAT reporter (Fig. 4D, compare dashed bars in lanes 4 and 5).
SREBP-Dependent Activation of CYP51 Is Mediated by a DNA-Multiprotein Complex Composed of SREBP-1a, a CRE-Binding Factor, and Sp1
To investigate whether transcription factors can bind at the same time to the GC box, CRE and SRE elements in the 58-bp region of the human CYP51 promoter, a longer probe covering the three binding regions, has been studied in mobility shift experiments. The DNA/protein interaction between the 99-bp CYP51 promoter probe and nuclear proteins from JEG-3 cells resulted in two major complexes (lane 1 in Fig. 5, A and B). The cold SRE probe removed both complexes and generated a new, intermediate complex (Fig. 5A, lane 3). A similar effect is observed with the GC box competitor (Fig. 5A, lane 4). Presence of both SREBP and Sp1 was confirmed by adding specific antibodies. The upper complex was supershifted in the presence of anti-Sp1 (Fig. 5B, lane 2), whereas anti-SREBP-1a disrupted both DNA/protein complexes. The cold CRE probe failed to show an effect in competition studies (Fig. 5A, lane 2). However, supershift analysis using the anti-CREB antibody suggests binding of a CREB-like protein to the 99-bp CYP51 promoter region (Fig. 5B, lane 4).
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DISCUSSION |
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- demethylase is the most conserved gene in the cytochrome P450 (CYP) superfamily. Not only do the coding regions of mammalian CYP51 genes share more than 90% identity, the proximal promoter region is also conserved, containing the same regulatory elements at identical positions in the mouse, rat, pig, and human promoters (37). This suggests that molecular mechanisms of CYP51 regulation are conserved across mammalian species and might be indispensable for mammalian development, because all nucleated mammalian cells synthesize cholesterol. The CYP51 proximal promoter contains three conserved elements: GC box, CRE, and SRE within 58 bp (Fig. 1). Additionally, a conserved GCAAT sequence is located 12 bp downstream of SRE but does not seem to be important in the SREBP-dependent regulation because a construct containing SRE and the GCAAT box failed to show a SREBP-dependent activation (Fig. 2).
We have shown previously that the CRE element (CYP51-CRE2 in Ref. 35) binds CREM, a transcription activator of the CREB/CREM/ATF family. CREM is the major cAMP-dependent activator in haploid male germ cells where it controls expression of multiple genes that are needed for normal sperm development (40). CREM is not present in most somatic cells, but other CREM isoforms are found in many cell types (41). Interestingly, studies in CREM -/- mice (42) revealed that CREM is sufficient to drive high-level expression of CYP51 in haploid male germ cells in vivo (35). In WT mice fed a normal chow diet, the CYP51 mRNA level in testis exceeds that in the liver by at least 1 order of magnitude, due to the presence of highly expressed testis-specific CYP51 transcripts (35). In contrast, testis of CREM -/- mice (42) shows a low level of somatic CYP51 mRNAs, which is comparable to CYP51 mRNA in the liver. Because DNA-bound SREBP proteins are below the limit of detection in rodent germ cells (35), it is proposed that the cAMP-dependent pathway and CREM can activate CYP51 promoter independently of SREBPs in a tissue- specific manner. Herein it is shown that this same CYP51-CRE site is essential also for sterol-regulated expression of the lanosterol 14-demethylase gene in somatic cells, yet in somatic cells SREBP is required as well. SREBP-dependent activation is lost when this CRE site is deleted or mutated. We had difficulties establishing precisely which nuclear protein(s) of JEG-3 cells binds to the CYP51-CRE site. Experiments with antibodies indicate that the CYP51-CRE-bound protein has a bZIP domain and is likely a member of the CREB/CREM/ATF family of transcription factors. The protein is closely related to CREB and ATF-1 because two different anti-CREB antibodies and the anti-ATF-1 antibody ablate the intensity of the DNA-protein complex. Indications of a close relation of the CRE-bound protein from JEG-3 cells and CREB arise also from promoter-reporter studies. When overexpressed, CREB can functionally interact with the overexpressed SREBP-1a and act as a coregulatory protein in the SREBP-dependent transcription of CYP51. The involvement of transcription factors of the CREB/ATF family in the SREBP-dependent activation has also been reported for the HMG-CoA synthase gene (29), where the 5'-portion of the CRE site partially overlaps with one of the two SRE sites. Mutations within CRE abolished sterol-regulated transcription of HMG-CoA synthase, but overexpression of SREBP-1a in combination with either CREB or ATF-2 did not lead to transactivation of the synthase promoter in a Drosophila SL-2 cell model. Only when NF-Y was expressed together with SREBP-1a and a CRE-binding factor could transactivation of the HMG-CoA synthase promoter be observed, suggesting that simultaneous action of SREBP, a CRE-binding factor, and NF-Y is essential for activation of the HMG-CoA synthase promoter (29). Similarly, in the case of HMG-CoA reductase, chromatin immunoprecipitation showed the efficient binding of both CREB and NF-Y under SREBP-induced conditions, whereas binding of both coregulatory proteins was diminished in cholesterol-loaded Chinese hamster ovary cells (3). In the case of CYP51, we were able to show a cooperative effect of SREBP-1a and CREB in transactivating the CYP51 gene in JEG-3 cells, activation being abolished when the CRE site was mutated (Fig. 4B). This functional assay seems to rule out the requirement of other potential coregulatory proteins in SREBP-dependent transactivation of the CYP51 gene. Thus, CYP51 is the first gene in which cooperation with a CREB/CREM/ATF family transcription factor is essential and sufficient for the SREBP-dependent response. Interestingly, PKA stimulates the SREBP-1a-induced promoter activity but does not stimulate the CREB-induced promoter activity. This suggests that nonphosphorylated CREB and phosphorylated SREBP might be involved in CYP51 transactivation complex, which would rule out CREB binding protein as a potential coactivator. Apparently the well known PKA phosphorylation site in CREB is blocked by interaction with bound SREBP-1a.
It is not clear to us why Sp1 is involved in the CYP51 transactivation complex because SREBP and a CRE-binding factor alone are able to drive up to 80% of the sterol-dependent activation. In the case of the LDL receptor promoter in which Sp1 has a defined role in SREBP-mediated transactivation, it was proposed that SREBP recruits Sp1 to the promoter (3). In the case of the fatty acid synthase I promoter, where the Sp1 site also seems to be largely dispensable, in a similar manner as found for CYP51, it was proposed that Sp1 recruits SREBP to the SRE (11). In another study, Magana et al. (12) established that while Sp1 is largely dispensable for sterol regulation in immortalized cell lines, it is required for the carbohydrate activation of the FAS promoter in primary hepatocytes. This difference is explained by different isoforms of SREBP transcription factors in cultured cells vs. in tissues, and by the fact that different SREBP isoforms utilize distinct coregulatory proteins to activate expression of sterol-responsive genes (12). Our data show Sp1 as a contributing factor to achieve a maximal promoter activity of CYP51. Because Sp1 seems to bind simultaneously with SREBP-1 and the CRE-binding factor to the promoter of CYP51, it could have a role in stabilization of the DNA/multiprotein transactivation complex.
In conclusion, we propose that the essential role of a CRE-bound factor and the nonessential role of Sp1 in CYP51 transactivation occurs in tissues as well as in established cell lines. This assumption is supported by the fact that a CRE-binding factor CREM alone is sufficient to drive high-level expression of the CYP51 gene in mouse germ cells, where Sp1 is abundant (35). Further studies are needed to determine which CYP51-CRE-bound transcription factors are required for SREBP-dependent activation in different cell lines and tissues and how they interact biochemically with SREBP, as well as to understand the physiological role of Sp1 in this process. Most recent data indicate that other genes of the cholesterol biosynthetic pathway are also regulated differently in somatic cells and in male germ cells (43). What is particularly interesting about CYP51, however, is that the same CRE is essential in both cases, even though the biochemical details of activation of transcription are clearly different.
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MATERIALS AND METHODS |
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The site-specific deletion constructs, delSRE, delGC box, and delSRE/GC box were prepared using QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) with the following oligonucleotides: 1) SRE deletion, sense 5'-GACGC GATGT AGGCC GAGGC GCTCG CGGTG CAATC ACAGA GC-3', antisense 5'-GTGAT TGCAC CGCGA GCGCC TCGGC CTACA TCGCG TCAGC GGGGC C-3'; 2) GC box deletion, sense 5'-GTACC CTGCG TCCGG ACATG CTCAC GCCCA AGGCC CCGC-3', antisense 5'-GGGCC TTGGG CGTGA GCATG TCCGG ACGCA GGGTA CTGGG GCACC-3'. The plasmid used as a template for mutagenesis was generated by digestion of the WT promoter CAT vector by KpnI and SmaI, followed by cloning the -334 to -121 insert into the Bluescript (Stratagene). Inserts were recovered from Bluescript using the same restriction enzymes and reinserted into the same sites of the WT promoter vector. Site-specific deletions were confirmed by sequencing.
The substitution mutation constructs, mutSRE, mutGC box, mutCRE, and mutGC/CRE, were prepared as above using oligonucleotides that contain the following mutations: SRE, ATCACCTACAG to TTTTTTTTTT; GC box, GGGGG CGG to GGGTTTTG; and CRE, TGACGCCA to AATGGCCA. An additional construct containing a CRE mutation (CYP51-CRE2-mut1) was prepared by changing TGACGCGA to TGATTCGA. Effects of the two CRE mutations were indistinguishable. All modifications were confirmed by sequencing.
The 5' nested deletion constructs, -282/+314, -272/+314, -252/+314, and -237/+314, were generated by PCR using Pfu turbo polymerase (Stratagene). Using individual sense primers having a KpnI site at the 5'-end and the same antisense primer, we amplified promoter fragments from the WT-CAT promoter construct. Each fragment was subcloned into Bluescript at the SmaI site and sequenced. Clones having the correct orientation were used in construction of 5'-deletion CAT-reporter constructs.
Cell Culture and DNA Transfection
JEG-3 cells were cultured in DMEM (Life Technologies, Inc., Gaithersburg, MD) containing 10% (vol/vol) bovine calf serum with or without the addition of 1% L-glutamine and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin sulfate) or fungizone (Life Technologies, Inc.). In some experiments 10% delipidated serum was applied (35). Cells were grown to 90% confluency in T75 tissue culture flasks and were plated at 1 x 106 cells/60-mm dish 24 h before transfection. At 50–70% confluency, cells were transiently transfected by a standard calcium phosphate coprecipitation method (44). The total amount of plasmid DNA was adjusted to 15 µg with a pCAT-basic vector. After transfection, cells were washed twice with PBS followed by addition of fresh medium and allowed to grow for 24 h at 37 C. Cells were then harvested and total cell extracts isolated by two freeze-thaw cycles. Protein concentration in each extract was measured using a protein assay kit [Pierce Chemical Co. (Rockford, IL) or Bio-Rad Laboratories, Inc. (Richmond, CA)] and 20 µg protein were used to measure the choramphenicol acetyl transferase (CAT) activity (44). pCAT-control plasmid (Promega Corp., Madison, WI) was used as a control of CAT activity.
In SREBP-1a/CREB and PKA/CREB/SREBP-1a cotransfection studies in JEG-3 cells the total amount of plasmid was adjusted to 21 µg: 10 µg of the human WT CYP51-CAT construct or CYP51-CRE2mut plasmid, 5 µg of rous sarcoma virus (RSV) ß-galactosidase (for normalization), 10 ng or 100 ng of pCMV SREBP, 3 µg pRSV-CREB, and 1 µg pSV PKA. pCAT basic was used as the DNA carrier up to 21 µg. Conditions for transfections with the somatostatin CAT reporter were the same. Cells were harvested 48 h after transfection and analyzed as described previously (35). Every transfection was performed at least three times with two parallel samples in each experiment. The average value and SEM were calculated with the Excel program (Microsoft Corp., Redmond, WA).
Drosophila SL2 cells were cultured at 27 C in Shields and Sang medium (Sigma, St. Louis, MO) containing 10% (vol/vol) fetal bovine serum and supplemented with 100 U/ml penicillin, 100 µg/ml each streptomycin sulfate, and fungizone (Life Technologies, Inc.). Cells were plated at 1 x 106 cells per well in six-well plates and 16 h later transfected by effectene transfection reagent according to the manufacturer’s instructions (QIAGEN, Chatsworth, CA). Each reporter (200 ng) was transfected alone or with 50 ng of pPAC-Sp1 or pPAC-SREBP expression vectors or a combination of both. Total DNA for each transfection was adjusted to 0.4 µg with pPAC empty vector. Forty-eight hours after transfection cells were harvested, and extracts were prepared by freezing and thawing. The -314/+343 CYP51 promoter region was recloned from pCAT-basic to pGEM-luc and used in transfections. Firefly luciferase activity was measured as described by the manufacturer (Promega Corp.).
Nuclear Extract Preparation and Gel Shift Assay
JEG-3 cells were divided into 10-cm dishes and allowed to grow to 80% confluency. Cells were harvested in PBS and nuclear extracts were prepared (35). Pairs of oligonucleotides that were used as probes in gel shift assay or as competitors are listed in Table 1. Oligonucleotides were purified on a 15%–20% acrylamide gel under denaturing conditions, and pairs were mixed in equimolar concentrations. Each double-stranded probe (0.1 µg) was end-labeled using [
-32P]ATP and T4 polynucleotide kinase. They were then allowed to anneal at 70 C for 10 min and gradually cooled to room temperature. Radiolabeled probes were purified on Sephadex columns or by electrophoresis followed by ethanol precipitation. The gel-shift binding buffer contained 20 mM HEPES (pH 7.9), 80 mM KC1, 5 mM MgCl2, 0.2 mM dithiothreitol, 0.1 mM EDTA, 2% ficoll, 5% glycerol, 0.1% Nonidet P-40, 10 µg aprotinin, 2 µg of yeast tRNA, 0.5 µg of poly dI-dC, and 5 µg of BSA in a final volume of 20 µl. Different antibodies have been used in supershift analysis. Anti-CREB and anti-SREBP-1a were prepared as described previously (35). Additional anti-CREB, anti-ATF-1, anti-ATF-2, and anti-Sp1 antibodies were purchased from New England Biolabs, Inc. (Beverly, MA) and from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Each antibody (2 µl) was added into the binding reaction and incubated on ice for 10 min. Labeled probe (10,000 cpm) was used for each incubation (5 min on ice), and the DNA-protein complex was resolved on 5% native acrylamide gels by electrophoresis in 0.5x Tris-borate-EDTA buffer at room temperature. In the experiments with antibodies, gels were run at 4 C. The gel was dried by vacuum and exposed to x-ray film at -80 C with an intensifying screen.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 S.K.H. and M.F. contributed equally to the content of this work. 
Abbreviations: ATF, Activating transcription factor; CAT, chloramphenicol acetyl transferase; CMV, cytomegalovirus; CRE, cAMP-responsive element; CREB, cAMP-responsive element binding protein; CREM, CRE modulator; CYP51, lanosterol 14-demethylase; HMG-CoA, 3-hydroxy-3-methyl-glutaryl coenzyme A; LDL, low density lipoprotein; NF-Y, nuclear factor Y; PKA, protein kinase A; Sp1, specificity protein 1; SRE, sterol regulatory element; SREBP, sterol regulatory element binding protein; WT, wild-type.
Received for publication October 5, 2001. Accepted for publication April 18, 2002.
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REFERENCES |
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  Y. Wang, P. M. Rogers, C. Su, G. Varga, K. R. Stayrook, and T. P. Burris Regulation of Cholesterologenesis by the Oxysterol Receptor, LXR{alpha} J. Biol. Chem., September 26, 2008; 283(39): 26332 - 26339. [Abstract] [Full Text] [PDF]
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 T. Lemberger, J. R. Parkitna, M. Chai, G. Schutz, and D. Engblom CREB has a context-dependent role in activity-regulated transcription and maintains neuronal cholesterol homeostasis FASEB J, August 1, 2008; 22(8): 2872 - 2879. [Abstract] [Full Text] [PDF]
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 S. Rome, V. Lecomte, E. Meugnier, J. Rieusset, C. Debard, V. Euthine, H. Vidal, and E. Lefai Microarray analyses of SREBP-1a and SREBP-1c target genes identify new regulatory pathways in muscle Physiol Genomics, August 1, 2008; 34(3): 327 - 337. [Abstract] [Full Text] [PDF]
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 G. Ning, H. Ouyang, S. Wang, X. Chen, B. Xu, J. Yang, H. Zhang, M. Zhang, and G. Xia 3',5'-Cyclic Adenosine Monophosphate Response Element Binding Protein Up-Regulated Cytochrome P450 Lanosterol 14{alpha}-Demethylase Expression Involved in Follicle-Stimulating Hormone-Induced Mouse Oocyte Maturation Mol. Endocrinol., July 1, 2008; 22(7): 1682 - 1694. [Abstract] [Full Text] [PDF]
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 T. Ohnesorg, B. Keller, M. H. de Angelis, and J. Adamski Transcriptional regulation of human and murine 17{beta}-hydroxysteroid dehydrogenase type-7 confers its participation in cholesterol biosynthesis. J. Mol. Endocrinol., August 1, 2006; 37(1): 185 - 197. [Abstract] [Full Text] [PDF]
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 M. Fink, J. Acimovic, T. Rezen, N. Tansek, and D. Rozman Cholesterogenic Lanosterol 14{alpha}-Demethylase (CYP51) Is an Immediate Early Response Gene Endocrinology, December 1, 2005; 146(12): 5321 - 5331. [Abstract] [Full Text] [PDF]
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B. M. Mayr, E. Guzman, and M. Montminy Glutamine Rich and Basic Region/Leucine Zipper (bZIP) Domains Stabilize cAMP-response Element-binding Protein (CREB) Binding to Chromatin J. Biol. Chem., April 15, 2005; 280(15): 15103 - 15110. [Abstract] [Full Text] [PDF]
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Y. Yamashita, M. Nishibori, T. Terada, N. Isobe, and M. Shimada Gonadotropin-Induced {Delta}14-Reductase and {Delta}7-Reductase Gene Expression in Cumulus Cells during Meiotic Resumption of Porcine Oocytes Endocrinology, January 1, 2005; 146(1): 186 - 194. [Abstract] [Full Text] [PDF]
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