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• W2154804960 abstract "To identify genes that are transcriptionally activated by sterol regulatory element-binding proteins (SREBPs), we utilized mRNA differential display and mutant cells that express either high or low levels of transcriptionally active SREBP. This approach identified stearoyl-CoA desaturase 2 (SCD2) as a new SREBP-regulated gene.Cells were transiently transfected with reporter genes under the control of different fragments of the mouse SCD2 promoter. Constructs containing >199 base pairs of the SCD2 proximal promoter were activated following incubation of cells in sterol-depleted medium or as a result of co-expression of SREBP-1a, SREBP-2, or rat adipocyte determination and differentiation factor 1 (ADD1). Electromobility shift assays and DNase I footprint analysis demonstrated that recombinant SREBP-1a bound to a novel cis element (5′-AGCAGATTGTG-3′) in the proximal promoter of the SCD2 gene. The finding that the endogenous SCD2 mRNA levels were induced when wild-type Chinese hamster ovary fibroblasts were incubated in sterol-deficient medium is consistent with a role for SREBP in regulating transcription of the gene.These studies identify SCD2 as a new member of the family of genes that are transcriptionally regulated in response to changing levels of nuclear SREBP/ADD1. In addition, the sterol regulatory element in the SCD2 promoter is distinct from all previously characterized motifs that confer SREBP- and ADD1-dependent transcriptional activation. To identify genes that are transcriptionally activated by sterol regulatory element-binding proteins (SREBPs), we utilized mRNA differential display and mutant cells that express either high or low levels of transcriptionally active SREBP. This approach identified stearoyl-CoA desaturase 2 (SCD2) as a new SREBP-regulated gene. Cells were transiently transfected with reporter genes under the control of different fragments of the mouse SCD2 promoter. Constructs containing >199 base pairs of the SCD2 proximal promoter were activated following incubation of cells in sterol-depleted medium or as a result of co-expression of SREBP-1a, SREBP-2, or rat adipocyte determination and differentiation factor 1 (ADD1). Electromobility shift assays and DNase I footprint analysis demonstrated that recombinant SREBP-1a bound to a novel cis element (5′-AGCAGATTGTG-3′) in the proximal promoter of the SCD2 gene. The finding that the endogenous SCD2 mRNA levels were induced when wild-type Chinese hamster ovary fibroblasts were incubated in sterol-deficient medium is consistent with a role for SREBP in regulating transcription of the gene. These studies identify SCD2 as a new member of the family of genes that are transcriptionally regulated in response to changing levels of nuclear SREBP/ADD1. In addition, the sterol regulatory element in the SCD2 promoter is distinct from all previously characterized motifs that confer SREBP- and ADD1-dependent transcriptional activation. Sterol regulatory element-binding proteins (SREBPs) 1The abbreviations used are: SREsterol regulatory elementSREBPsterol regulatory element-binding proteinADD1adipocyte determination- and differentiation-dependent factor 1HMG-CoA3-hydroxy-3-methylglutaryl coenzyme ALDLlow density lipoproteinbpbase pairLPDSlipoprotein deficient serumSCDstearoyl-CoA desaturaseSRDsterol regulatory defectivebHLH-Zipbasic-helix-loop-helix-leucine zipperPCRpolymerase chain reactionntnucleotidesCHOChinese hamster ovary.1The abbreviations used are: SREsterol regulatory elementSREBPsterol regulatory element-binding proteinADD1adipocyte determination- and differentiation-dependent factor 1HMG-CoA3-hydroxy-3-methylglutaryl coenzyme ALDLlow density lipoproteinbpbase pairLPDSlipoprotein deficient serumSCDstearoyl-CoA desaturaseSRDsterol regulatory defectivebHLH-Zipbasic-helix-loop-helix-leucine zipperPCRpolymerase chain reactionntnucleotidesCHOChinese hamster ovary. were purified based on their ability to bind to a 10-bp sequence, the sterol regulatory element-1 (SRE-1), that had been identified in the promoter of the LDL receptor gene (reviewed in Ref. 1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar). Subsequent isolation of the human SREBP cDNAs indicated that three proteins, SREBP-1a, SREBP-1c, and SREBP-2 are derived from two SREBP genes (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar). SREBP-1a and -1c mRNAs result from alternative splicing of exon 1 of the SREBP-1 gene (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar). Adipocyte determination and differentiation factor 1 (ADD1), the rat homologue of SREBP-1c, was cloned independently as a result of studies aimed at identifying a nuclear protein that bound the E-box motif in the promoter of the fatty acid synthase gene (2Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (534) Google Scholar). ADD1 expression was shown to be required for adipogenesis and for the induction of a number of mRNAs, including fatty acid synthase and glycerol-3-phosphate acyltransferase (2Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (534) Google Scholar, 3Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (838) Google Scholar, 4Ericsson J. Jackson S.M. Kim J.B. Spiegelman B.M. Edwards P.A. J. Biol. Chem. 1997; 272: 7298-7305Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), that occurs during this differentiation process. sterol regulatory element sterol regulatory element-binding protein adipocyte determination- and differentiation-dependent factor 1 3-hydroxy-3-methylglutaryl coenzyme A low density lipoprotein base pair lipoprotein deficient serum stearoyl-CoA desaturase sterol regulatory defective basic-helix-loop-helix-leucine zipper polymerase chain reaction nucleotides Chinese hamster ovary. sterol regulatory element sterol regulatory element-binding protein adipocyte determination- and differentiation-dependent factor 1 3-hydroxy-3-methylglutaryl coenzyme A low density lipoprotein base pair lipoprotein deficient serum stearoyl-CoA desaturase sterol regulatory defective basic-helix-loop-helix-leucine zipper polymerase chain reaction nucleotides Chinese hamster ovary. SREBPs/ADD1 are synthesized as 125-kDa proteins that contain two transmembrane domains that anchor the proteins to the endoplasmic reticulum (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar). When cellular sterol levels are low, two distinct proteolytic events release a mature 68-kDa NH2-terminal fragment of SREBPs/ADD1, which then translocates to the nucleus, binds to the promoters of target genes, and activates transcription (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar). Conversely, when levels of cellular sterols are high, proteolytic processing of SREBPs/ADD1 is diminished, nuclear levels of the mature proteins decline, and transcription of target genes is low (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar). A number of genes have been shown to be regulated at the transcriptional level by mature SREBPs/ADD1. These include genes involved in cholesterol homeostasis (e.g. HMG-CoA synthase, HMG-CoA reductase, farnesyl diphosphate synthase, squalene synthase, and the LDL receptor) (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar, 5Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 945-950Crossref PubMed Scopus (156) Google Scholar, 6Guan G. Dai P.-H. Osborne T.F. Kim J.B. Shechter I. J. Biol. Chem. 1997; 272: 10295-10302Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar), in fatty acid and triglyceride synthesis (e.g. fatty acid synthase, acetyl-CoA carboxylase, and glycerol-3-phosphate acyltransferase) (2Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (534) Google Scholar, 3Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (838) Google Scholar, 4Ericsson J. Jackson S.M. Kim J.B. Spiegelman B.M. Edwards P.A. J. Biol. Chem. 1997; 272: 7298-7305Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 7Magana M.M. Osborne T.F. J. Biol. Chem. 1996; 271: 32689-32694Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar, 8Lopez J.M. Bennett M.K. Sanchez H.B. Rosenfeld J.M. Osborne T.F. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1049-1053Crossref PubMed Scopus (248) Google Scholar) as well as the SREBP-2 gene itself (9Sato R. Inoue J. Kawabe Y. Kodama T. Takano T. Maeda N. J. Biol. Chem. 1996; 271: 26461-26464Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Recent studies have shown that ADD1/SREBP1 also activates peroxisomal proliferator-activated receptor γ through production of an endogenous ligand (10Kim J.B. Wright H.M. Wright M. Spiegelman B.M. Proc. Natl Acad. Sci. U. S. A. 1998; 95: 4333-4337Crossref PubMed Scopus (553) Google Scholar) and stimulates expression of the leptin gene (11Kim J.B. Sarrat P. Wright M. Yao K.M. Mueller E. Solanes G. Lowell B.B. Spiegelman B.M. J. Clin. Invest. 1998; 101: 1-9Crossref PubMed Scopus (611) Google Scholar). The mature, transcriptionally active SREBPs/ADD1 contain basic-helix-loop-helix-leucine zipper (bHLH-Zip) domains that are required for DNA binding and transactivation (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar, 2Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (534) Google Scholar). bHLH-Zip domains are found in a number of other transcription factors, including Myc and Max, that bind to E-box motifs (CANNTG) (12Murre C. Bain G. van Dijk M.A. Engel I. Furnari B.A. Massari M.E. Matthew J.R. Quong M.W. Rivera R.R. Stuiver M.H. Biochim. Biophys. Acta. 1994; 1218: 129-135Crossref PubMed Scopus (407) Google Scholar). In contrast, ADD1/SREBPs bind to both E-box and non-E-box motifs (13Kim J.B. Spotts G.D. Halvorsen Y. Shih H. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Scopus (295) Google Scholar). The ability of ADD1/SREBPs to bind to non-E-box motifs is a result of an atypical tyrosine replacing an arginine that is conserved in the basic domain of all other bHLH-containing proteins (13Kim J.B. Spotts G.D. Halvorsen Y. Shih H. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Scopus (295) Google Scholar). Promoters from a number of SREBP/ADD1-responsive genes have been analyzed to identify the nucleotide sequences necessary for SREBP/ADD1-DNA interaction. The results indicate that the nucleotide sequence of the different sterol response elements (SREs), vary considerably; many, but not all SREs, contain two half-sites (direct repeats of C/TCAC) separated by two nucleotides (1Brown M.S. Goldstein J.L. Cell. 1997; 89: 331-340Abstract Full Text Full Text PDF PubMed Scopus (2959) Google Scholar, 7Magana M.M. Osborne T.F. J. Biol. Chem. 1996; 271: 32689-32694Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). However, a number of SREs do not conform to this sequence motif (5Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 945-950Crossref PubMed Scopus (156) Google Scholar, 6Guan G. Dai P.-H. Osborne T.F. Kim J.B. Shechter I. J. Biol. Chem. 1997; 272: 10295-10302Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 7Magana M.M. Osborne T.F. J. Biol. Chem. 1996; 271: 32689-32694Abstract Full Text Full Text PDF PubMed Scopus (276) Google Scholar). A number of mutant cell lines have been isolated that do not regulate the expression/transcription of the HMG-CoA reductase, HMG-CoA synthase, and the LDL receptor genes in response to altered cholesterol levels (14Metherall J.E. Goldstein J.L. Luskey K.L. Brown M.S. J. Biol. Chem. 1989; 264: 15634-15641Abstract Full Text PDF PubMed Google Scholar, 15Evans M.J. Metherall J.E. Mol. Cell. Biol. 1993; 13: 5175-5185Crossref PubMed Scopus (28) Google Scholar, 16Yang J. Brown M.S. Ho Y.K. Goldstein J.L. J. Biol. Chem. 1995; 270: 12152-12161Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 17Hasan M.T. Chang C.C.Y. Chang T.Y. Somatic Cell Mol. Genet. 1994; 20: 183-184Crossref PubMed Scopus (52) Google Scholar, 18Panini S.R. Lutz R.J. Wenger L. Miyake J. Leonard S. Andalibi A. Lusis A.J. Sinensky M. J. Biol. Chem. 1990; 265: 14118-14126Abstract Full Text PDF PubMed Google Scholar, 19Schnitzer-Polokoff R. Torget R. Logel J. Sinensky M. Arch. Biochem. Biophys. 1983; 227: 71-80Crossref PubMed Scopus (16) Google Scholar). Such mutants include sterolregulatory defective (SRD) cell lines that constitutively express high (SRD-1, SRD-2) or low (SRD-6) levels of nuclear SREBPs irrespective of the sterol status of the medium or of the cells (14Metherall J.E. Goldstein J.L. Luskey K.L. Brown M.S. J. Biol. Chem. 1989; 264: 15634-15641Abstract Full Text PDF PubMed Google Scholar, 15Evans M.J. Metherall J.E. Mol. Cell. Biol. 1993; 13: 5175-5185Crossref PubMed Scopus (28) Google Scholar, 16Yang J. Brown M.S. Ho Y.K. Goldstein J.L. J. Biol. Chem. 1995; 270: 12152-12161Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Subsequently, it was shown that the 5′ part of the SREBP-2 gene, that contains the bHLH-Zip domains, had undergone a translocation event in SRD-1 and SRD-2 cells. This resulted in the synthesis of distinct fusion proteins that contain no transmembrane sequences and were translocated to the nucleus where they transactivate target genes (16Yang J. Brown M.S. Ho Y.K. Goldstein J.L. J. Biol. Chem. 1995; 270: 12152-12161Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In contrast, SRD-6 cells produce little or no mature SREBPs as a result of a defect at the site-2 cleavage (20Sakai J. Duncan E.A. Rawson R.B. Hua X. Brown M.S. Goldstein J.L. Cell. 1996; 85: 1037-1046Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). As a result, the mRNA levels of a number of SREBP-responsive genes are extremely low, even when SRD-6 cells are incubated in the absence of sterols (15Evans M.J. Metherall J.E. Mol. Cell. Biol. 1993; 13: 5175-5185Crossref PubMed Scopus (28) Google Scholar, 21Jackson S.M. Ericsson J. Metherall J.E. Edwards P.A. J. Lipid Res. 1996; 37: 1712-1721Abstract Full Text PDF PubMed Google Scholar). We hypothesized the existence of novel genes and/or known genes not previously ascribed to be under the transcriptional control of SREBPs and cellular sterols. To identify such genes, we utilized mRNA differential display (22Liang P. Pardee A.B. Methods Mol. Biol. 1997; 85: 3-11PubMed Google Scholar), using RNAs isolated from SRD-2 and SRD-6 cells. This approach identified a number of transcripts expressed at high levels in SRD-2 cells as compared with SRD-6 cells. Among these, we identified stearoyl-CoA desaturase 2 (SCD2), an enzyme involved in the synthesis of unsaturated fatty acids. SCD2 catalyzes the Δ9-cis desaturation of fatty acyl-CoAs such as stearoyl-CoA and palmitoyl-CoA to produce oleic and palmitoleic acid, respectively (23Ntambi J.M. Prog. Lipid Res. 1995; 34: 139-150Crossref PubMed Scopus (287) Google Scholar, 24Kaestner K.H. Ntambi J.M. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1989; 264: 14755-14761Abstract Full Text PDF PubMed Google Scholar). The current studies demonstrate that a gene (SCD2) encoding an enzyme involved in fatty acid desaturation is transcriptionally regulated by SREBPs/ADD1 or sterols. The finding provides a plausible explanation as to why certain mutant cells that are defective in SREBP processing require not only cholesterol for growth but also unsaturated fatty acids (17Hasan M.T. Chang C.C.Y. Chang T.Y. Somatic Cell Mol. Genet. 1994; 20: 183-184Crossref PubMed Scopus (52) Google Scholar). DNA modification and restriction enzymes were obtained from Life Technologies, Inc. 32P- and35S-labeled nucleotide triphosphates were obtained from Andotok Life Sciences Co. pCI-SREBP-1a encoding amino acids 1–490 of SREBP-1a and pCI-SREBP-2 encoding amino acids 1–485 of SREBP-2 were kindly provided by Dr. J. Ericsson. The 403 amino acids corresponding to the amino-terminal of ADD1 were cloned into pSV-SPORT (Life Technologies, Inc.) (11Kim J.B. Sarrat P. Wright M. Yao K.M. Mueller E. Solanes G. Lowell B.B. Spiegelman B.M. J. Clin. Invest. 1998; 101: 1-9Crossref PubMed Scopus (611) Google Scholar). The tyrosine at amino acid position 320 of ADD1 was mutated to an arginine to produce ADD1-R. ADD1-R was also cloned into pSV-SPORT (11Kim J.B. Sarrat P. Wright M. Yao K.M. Mueller E. Solanes G. Lowell B.B. Spiegelman B.M. J. Clin. Invest. 1998; 101: 1-9Crossref PubMed Scopus (611) Google Scholar, 13Kim J.B. Spotts G.D. Halvorsen Y. Shih H. Ellenberger T. Towle H.C. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 2582-2588Crossref PubMed Scopus (295) Google Scholar). Lipoprotein-deficient serum (LPDS) was purchased from PerImmune. The HMG-CoA synthase reporter gene (pSYNSRE) and all other reagents have been described elsewhere (4Ericsson J. Jackson S.M. Kim J.B. Spiegelman B.M. Edwards P.A. J. Biol. Chem. 1997; 272: 7298-7305Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 5Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 945-950Crossref PubMed Scopus (156) Google Scholar, 21Jackson S.M. Ericsson J. Metherall J.E. Edwards P.A. J. Lipid Res. 1996; 37: 1712-1721Abstract Full Text PDF PubMed Google Scholar). Oligonucleotides were synthesized by Life Technologies, Inc. Total RNA was isolated from SRD-2 and SRD-6 cells as described (21Jackson S.M. Ericsson J. Metherall J.E. Edwards P.A. J. Lipid Res. 1996; 37: 1712-1721Abstract Full Text PDF PubMed Google Scholar). Fifty μg of total RNA was DNase I-treated to remove any contaminating DNA (MessageClean, Genhunter Corp.). DNA-free RNA (0.2 μg) was reverse-transcribed utilizing a T11G primer. PCR was then performed using the T11G primer, a second primer, and [33P]dATP as described (RNAimage, Genhunter Corp.). The products were separated on a 6% denaturing polyacrylamide gel and exposed to x-ray film for 20 h. Differentially displayed bands were excised from the gel, eluted, and reamplified by PCR. DNA fragments generated by PCR were subcloned into the pCR2.1-TOPO cloning vector (Invitrogen) as described by the supplier. Escherichia coli, strain DH5α, were transformed and selected for growth on ampicillin-containing medium. β-Galactosidase-deficient colonies were screened for the presence of inserts following EcoRI digestion of the plasmid DNA and electrophoretic analysis on 1% agarose gels. Inserts were sequenced by the Sanger chain-termination method using the T7 Sequenase v2.0 kit (Amersham Pharmacia Biotech). SRD cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 (1:1), 5% LPDS and supplemented with either 25-hydroxycholesterol (0.5 μg/ml) (SRD-1 and SRD-2 cells) or cholesterol (5 μg/ml) and mevalonic acid (200 μm) (SRD-6 cells) (14Metherall J.E. Goldstein J.L. Luskey K.L. Brown M.S. J. Biol. Chem. 1989; 264: 15634-15641Abstract Full Text PDF PubMed Google Scholar, 15Evans M.J. Metherall J.E. Mol. Cell. Biol. 1993; 13: 5175-5185Crossref PubMed Scopus (28) Google Scholar, 21Jackson S.M. Ericsson J. Metherall J.E. Edwards P.A. J. Lipid Res. 1996; 37: 1712-1721Abstract Full Text PDF PubMed Google Scholar). HepG2 and CHO cells were cultured as described previously (25Spear D.H. Kutsunai S.Y. Correll C.C. Edwards P.A. J. Biol. Chem. 1992; 267: 14462-14469Abstract Full Text PDF PubMed Google Scholar). The 5′ end of the SCD2 gene (nt −588 to +81) was obtained by polymerase chain reaction using C57Bl/6J mouse genomic DNA as a template. The 5′ primer contained nucleotides that corresponded to −588 to −567 of the published SCD2 genomic sequence (24Kaestner K.H. Ntambi J.M. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1989; 264: 14755-14761Abstract Full Text PDF PubMed Google Scholar) and additional nucleotides (5′-TACCCTCGAG-3′) containing an XhoI restriction site. The 3′ primer contained nucleotides that corresponded to +81 to +60 of the published genomic sequence (24Kaestner K.H. Ntambi J.M. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1989; 264: 14755-14761Abstract Full Text PDF PubMed Google Scholar) and additional nucleotides (5′-ACTTAAGCTT-3′) containing an HindIII restriction site. The PCR product was cloned into the pGL2 basic vector, utilizing XhoI and HindIII restriction sites to produce pSCD2-588. This 670-bp sequence of the SCD2 gene (−588 to +81) was identical to that reported by Kaestneret al. (24Kaestner K.H. Ntambi J.M. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1989; 264: 14755-14761Abstract Full Text PDF PubMed Google Scholar). This DNA was used as template for additional PCRs that utilized 5′ primers corresponding to −199 to −167, −150 to −129, and −110 to −89 of the SCD2 genomic sequence in combination with the common 3′ primer. These reactions generated a series of SCD2 promoter fragments that were cloned into the pGL2 basic vector to produce pSCD2-588, pSCD2-199, pSCD2-150, and pSCD2-110, respectively. Oligonucleotides (28-mers) were used to introduce mutations into pSCD2-199 using QuikChange (Stratagene). The resulting reporter genes each contained three A to C mutations at nt −151, −148 and −146 (pSCD2-199mut A) or at nt −157 to −155 (pSCD2-199mut B), respectively. The promoter of each construct was sequenced to confirm sequence. Total RNA was isolated from SRD-1, SRD-2, SRD-6, and CHO cells using Trizol reagent (Life Technologies, Inc). Ten μg of each RNA sample was fractionated by agarose/formaldehyde gel electrophoresis, transferred to nylon membranes, and cross-linked by exposure to UV light as described (26Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Plainview, NY1989Google Scholar). [α-32P]dCTP-radiolabeled DNA probes were generated by the random priming labeling method (Amersham Pharmacia Biotech). Hybridization and quantification, using a PhosphorImager (Molecular Dynamics), were as described (5Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 945-950Crossref PubMed Scopus (156) Google Scholar). To correct for differences in RNA loading in each lane, blots were also hybridized to a probe, 36B4, that hybridized to a constitutively expressed mRNA. Transient transfections were performed using minor modifications of the transfection MBS kit (Stratagene). Details of the transient transfection of HepG2 cells with luciferase reporter genes, a plasmid encoding β-galactosidase, pCI-SREBP-1a (encoding mature SREBP-1a) pCI-SREBP-2 (encoding mature SREBP-2), appear in previous publications (5Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 945-950Crossref PubMed Scopus (156) Google Scholar, 25Spear D.H. Kutsunai S.Y. Correll C.C. Edwards P.A. J. Biol. Chem. 1992; 267: 14462-14469Abstract Full Text PDF PubMed Google Scholar). Following transfection, cells were incubated for 20 h in medium containing 10% LPDS supplemented with either 5 μmmevinolin (inducing medium) or sterols (10 μg/ml cholesterol and 1 μg/ml 25-hydroxycholesterol) (repressing medium). The cells were lysed and the luciferase activities determined and normalized to the β-galactosidase activity to correct for minor differences in transfection efficiency (5Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 945-950Crossref PubMed Scopus (156) Google Scholar). Double-stranded DNA fragments corresponding to −588 to −278, −299 to −239, −259 to −178, −199 to −130, −150 to −91, −110 to +1, and −259 to −91 of the murine SCD2 proximal promoter were generated by PCR. The primers used to generate these SCD2 DNA fragments were flanked by the same 10 nucleotides, containing either an XhoI or HindIII restriction site, as described above. Single-stranded DNA (−164 to −137) containing wild-type or mutated sequences were annealed and the double-stranded DNA isolated and used in EMSAs (5Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 945-950Crossref PubMed Scopus (156) Google Scholar). Recombinant SREBP-1a was purified from E. coli extracts as described previously (5Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 945-950Crossref PubMed Scopus (156) Google Scholar). The DNA was end-labeled with 32P (20,000 cpm; 1.5 fmol) and used in EMSAs as described (5Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 945-950Crossref PubMed Scopus (156) Google Scholar). A DNA fragment corresponding to nucleotides −259 to −91 of the proximal SCD2 promoter (24Kaestner K.H. Ntambi J.M. Kelly Jr., T.J. Lane M.D. J. Biol. Chem. 1989; 264: 14755-14761Abstract Full Text PDF PubMed Google Scholar) was used for DNase I footprinting. The fragment was cloned into the pCR2.1-TOPO cloning vector (Invitrogen). Purified plasmid DNA was cut with eitherBamHI or NotI and then end-labeled with [γ-32P]dATP. The labeled, linearized plasmid was digested with the reciprocal restriction enzyme, either NotI or BamHI, to liberate a fragment containing nucleotides −259 to −91, end-labeled at either end. The gel-purified, end-labeled fragments were incubated with purified SREBP-1a and treated with DNase I as described (5Ericsson J. Jackson S.M. Lee B.C. Edwards P.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 945-950Crossref PubMed Scopus (156) Google Scholar). The mRNAs for a number of SREBP-responsive genes, including the LDL receptor, HMG-CoA synthase, HMG-CoA reductase, and farnesyl diphosphate synthase genes are expressed at 5–10-fold higher levels in SRD-1 and SRD-2 cells as compared with SRD-6 cells (14Metherall J.E. Goldstein J.L. Luskey K.L. Brown M.S. J. Biol. Chem. 1989; 264: 15634-15641Abstract Full Text PDF PubMed Google Scholar, 15Evans M.J. Metherall J.E. Mol. Cell. Biol. 1993; 13: 5175-5185Crossref PubMed Scopus (28) Google Scholar,21Jackson S.M. Ericsson J. Metherall J.E. Edwards P.A. J. Lipid Res. 1996; 37: 1712-1721Abstract Full Text PDF PubMed Google Scholar). We hypothesized that the mRNA levels of other SREBP-responsive genes would also be differentially expressed in these mutant cells. Fig. 1 shows the results obtained using the technique of mRNA differential display utilizing the arbitrary upstream primer AP9, the downstream primer T11G, and RNA isolated from SRD-2 and SRD-6 cells. The portion of the gel shown contains a 700-bp band that was present when SRD-2 cells were the source of RNA but was absent when SRD-6-derived RNA was used (Fig. 1,arrow). This band was excised from the gel, reamplified by PCR, subcloned, and sequenced. The sequence revealed a >97% identity with the 3′-untranslated region of mouse stearoyl-CoA desaturase 2 (SCD2). The 700-bp insert was radiolabeled and used as a probe in a Northern blot analysis (Fig. 2). The probe hybridized to a mRNA of approximately 5 kilobases that was expressed at higher levels in SRD-1 and SRD-2 cells as compared with SRD-6 cells (Fig. 2). These latter studies confirm that the band identified by mRNA differential display corresponds to a mRNA that is more abundant in SRD-2 than in SRD-6 cells. Using the same methodology we have identified a number of other differentially regulated genes. 2D. E. Tabor and P. A. Edwards, unpublished data. Some, but not all, of these additional mRNAs are also expressed at higher levels in SRD1 cells as compared with SRD2 cells.2 The reason for this differential expression of mRNAs in the two cell types (SRD-1 and SRD-2) that constitutively overexpress different SREBP fusion proteins is unknown.Figure 2SCD2 mRNA levels are elevated in SRD-1 and SRD-2 as compared with SRD-6 cells. Ten μg of total RNA, isolated from duplicate dishes of SRD-1, SRD-2, and SRD-6 cells, was separated on a 1% agarose/formaldehyde gel and transferred to a nylon membrane as described under “Experimental Procedures.” The blot was hybridized with a radiolabeled probe generated from the cloned band indicated in Fig. 1 and then to a radiolabeled 36B4 probe. The SCD2 mRNA of approximately 5.0 kilobases and the control 36B4 RNA are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Increased expression of SCD2 mRNA in SRD-1 and SRD-2 cells (Fig. 2) might be a direct result of the elevated levels of transcriptionally active SREBP in these cells (16Yang J. Brown M.S. Ho Y.K. Goldstein J.L. J. Biol. Chem. 1995; 270: 12152-12161Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Alternatively, the effect may either be secondary, resulting from a cascade of effects initiated by elevated nuclear SREBP, or nonphysiological, because of the supraphysiological levels of transcriptionally active SREBP-2 fusion proteins. To distinguish between these possibilities, wild-type CHO cells were incubated in medium supplemented with 10% LPDS and either mevinolin (inducing conditions) or sterols (repressing conditions). These conditions are known to regulate the nuclear localization of SREBPs and to result in changes in the mRNA levels of SREBP-regulated genes (1Brown M.S. Goldstein J." @default.
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