Differentiation-induced Gene Expression in 3T3-Ll Preadipocytes A SECOND DIFFERENTIALLY EXPRESSED GENE ENCODING STEAROYL-CoA DESATURASE’

Previously we isolated and characterized a differ- entially expressed gene from mouse 3T3-Ll preadipocytes that encodes stearoyl-CoA desaturase (SCD1; Ntambi, J. M., Buhrow, S. A., Kaestner, K. H., Chris-try, R. J., Sibley, E., Kelly, T. J., Jr., and Lane, M. D. (1988) J. Biol. Chem. 263, 17291-17300). Genomic Southern blot analysis indicated the existence of an- other closely related gene. Here we report the isolation and characterization of this gene and the correspond- ing cDNA which encode a second stearoyl-CoA desaturase, SCD2, in 3T3-Ll adipocytes. SCDS cDNA is 5 kilobase pairs in length and encodes a protein of 358 amino acids with >87% amino acid sequence identity to SCDl. RNase protection analysis reveals a 10-fold increase in the expression of SCDB mRNA during 3T3- L l preadipocyte differentiation. SCDB mRNA is expressed constitutively at a high level in brain, is not expressed in liver, and its expression in kidney, adipose, and lung tissue is increased greatly by shifting mice from a diet containing unsaturated fatty acids to a diet devoid of fat. The tissue distribution and the dietary alteration of SCDl mRNA expression differs markedly from that of SCDB mRNA being absent from brain, constitutive

sequence for the glucocorticoid regulatory element at position -500 and a potential CCAAT boxlenhancer binding protein sequence at position -540. The SCD gene family provides a new model system for the study of differentiation-induced as well as tissue-specific metabolite controlled gene expression.
When appropriately induced, 3T3-Ll preadipocytes differentiate in culture into cells possessing the morphological and biochemical characteristics of adipocytes (1)(2)(3). Accompanying acquisition of the adipocyte phenotype is a dramatic rise in the cellular levels of lipogenic and lipolytic enzymes (3)(4)(5)(6)(7)(8), as well as other proteins, e.g. the insulin receptor (9), that are expressed at high levels in adipocytes. Where investigated the increased expression of these proteins has been shown to result from an increased rate of transcription of their specific mRNAs (10) and a corresponding increase in their translation (11)(12)(13). Analysis of the regulatory elements of these genes should advance understanding of the factors that govern adipocyte differentiation.
Recently, we isolated the differentiation-induced gene encoding stearoyl-CoA desaturase 1 (SCD1,' 14) from 3T3-Ll preadipocytes. The function of this enzyme is the synthesis of unsaturated fatty acids as well as the regulation of this process. SCDl catalyzes the Ag-cis desaturation of fatty acyl-CoAs (15), the major products being palmitoleoyl-and oleoyl-CoA. Palmitoleic and oleic acid are the major (58% of total) storage form of unsaturated fatty acids in the triacylglycerols found in 3T3-Ll adipocytes (16). SCD activity is induced 20-100-fold during the differentiation of 3T3-Ll preadipocytes (16).
Genomic Southern analysis using SCDl cDNA fragments as radioactive probes indicated the existence of related genes (14). In the present paper, we report the isolation and characterization of both the cDNA and the gene for a second differentially expressed mouse stearoyl-CoA desaturase (SCD2). The availability of these two genes should facilitate studies on the basis of their distinct tissue-specific expression as well as their regulation during preadipocyte differentiation.

EXPERIMENTAL PROCEDURES
Materials-Restriction enzymes and other nucleic acid modifying enzymes were obtained from Boehringer Mannheim, Bethesda Research Laboratories, Pharmacia P-L Biochemicals, New England Biolabs, and United States Biochemical Corp. Radionucleotides were obtained from Du Pont-New England Nuclear. Nylon membranes were from Amersham Corp.

Structure and Regulation
of the Stearoyl-CoA Desaturase 2 Gene pocytes were as described by Bernlohr et al. (10). Briefly, confluent cell monolayers were incubated for 48 h in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, methylisobutyixanthine, dexamethasone, and insulin. After 48 h the cells were washed free of methylisobutylxanthine and dexamethasone and then maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and insulin. Adipocyte morphology was monitored by the appearance of cytoplasmic triacylglycerol droplets which is closely correlated with the acquisition of other markers of the adipocyte phenotype (4).
Isolation and Analysis of RNA and DNA-RNA was isolated from 3T3-Ll cells as described by Chirgwin et al. (17). RNase protection analysis was performed according to Melton et al. (18). Antisense RNA probes for SCDl and SCD2 were synthesized using T7 or SP6 RNA polymerases, linearized plasmid templates, and [a-32P]CTP. The probes (200,000 cpm each) were hybridized to the RNA samples (10 fig) a t 54 "C in 80% formamide followed by digestion with RNases A and TI. The protected probe fragments were isolated by ethanol precipitation, analyzed on 6% acrylamide, 8 M urea gels, and visualdensitometry. When hybridized with SCDl or SCD2 RNAs synthe-ized by autoradiography. Some gels were analyzed quantitatively by sized in uitro, all probes used were shown to be specific for only their cognate mRNA species. Genomic DNA from 3T3-Ll cells was prepared as described (19) and DNA blot analysis was performed according to Southern (20). DNA probes were labeled to lo9 cpmlpg by the method of Feinberg and Vogelstein (21).
Construction and Screening of cDNA and Genomic Libraries-A cDNA library in the XZAP vector was prepared by Stratagene. The poly(A)' mRNA for this library was prepared from 3T3-Ll adipocytes 5 days after initiating differentiation. This library was screened using various genomic and cDNA fragments as radioactive probes as described (22). Two of the 55 cDNA clones obtained are illustrated in Fig. 1, and the sequence of the full-length SCD2 cDNA is shown in Fig. 2. A genomic library was constructed and screened as described (14) using a 0.5-kb intron fragment of XE14 (14) as radioactive probe. Two positive clones were obtained from lo6 plaques and were purified. The clones were found to be identical and designated XS18 (Fig. I).
Phage DNA was prepared (19), and cloned genomic DNA fragments were subcloned into pGEM plasmids. The restriction patterns of cloned DNA were compared to those of 3T3-Ll DNA by Southern blot analysis using various cDNA probes.
Distribution of mRNA in Mouse Tissues-Three-to four-week-old C57B1 mice were fed according to a modification of the protocol of Thiede and Strittmatter (23) to induce expression of hepatic stearyol-CoA desaturase. Briefly, mice were starved for 36-40 h, refed a normal lab chow diet (Purina) containing both unsaturated and saturated triacylglycerols, starved for an additional 40 h, and then refed a fatfree high carbohydrate diet (United States Biochemicals COW.) for 24 h. RNA from mouse tissues was isolated as described above for isolation of RNA from tissue culture cells, except that the tissues were first treated briefly with a Polytron in 5 M guanidinium thiocyanate containing 5% 8-mercaptoethanol.
DNA Sequencing and Mapping of the Transcriptional Start Site and Intron-Exon Boundaries-Genomic and cDNA fragments, generated by digestion with various restriction enzymes, were ligated into pGEM-blue (Promega ). Intron-exon boundaries were identified by comparing the genomic and cDNA sequences. Primer extension analysis was carried out as described previously (14) using a synthetic oligonucleotide (5"GATAAGGATAGAAAAT-GAG-OH-3'). T o obtain the nucleotide sequence of the primerextended product, dideoxy sequencing of the first exon of the gene was performed using the same oligonucleotide as a primer.

RESULTS
Cloning of the cDNA Encoding Stearoyl-CoA Desaturuse 2 (SCD2)"During cloning of the mouse stearoyl-CoA desaturase gene (SCDl), a related genomic clone was isolated. Nucleotide sequence analysis indicated that this clone designated XE14 (Fig. l), was not part of the SCDl gene (14), but contained five exons which were very similar to those of the SCDl gene. To determine whether XE14 represented an expressed gene, we attempted to isolate the corresponding cDNA. A 235-bp PstIIPstI fragment of XE14 (Fig. l), which contains parts of the first exon (corresponding to nucleotides 205-324 in Fig. 2) hybridized to a 5-kb mRNA on Northern blots (not shown). This probe shares less than 55% identical nucleotide sequence with SCDl and was shown to be specific for SCD2 by hybridization to in vitro transcribed SCD2 and SCDl RNA under the stringent conditions used (data not shown). This fragment was used as a radioactive probe to screen a mouse 3T3-Ll adipocyte cDNA library (14). Out of 20 clones purified, X32 was the longest (2.7 kb, see Fig. 1). The 3'-most 200-bp BarnHJ/EcoI fragment of X32 was used in a subsequent screen of the same library to obtain clone X53 (4.2 kb, see Fig. l ) , which contains a poly(A)-track. Together, clones X32 and X53 represent the complete mRNA of approximately 5 kb. After restriction endonuclease mapping and subcloning into pGEM plasmids the entire cDNA was sequenced by the dideoxy chain termination method. The sequence obtained (Fig. 2) contains a single open reading frame of 1074 nucleotides that encodes a protein of 358 amino acids and a calculated molecular weight of 41,070. The cDNA contains 300 nucleotides of 5'-untranslated region and, like the previously described SCD cDNAs from rat liver (25) and mouse 3T3-Ll adipocytes (14), contains an unusually long 3'untranslated sequence of 3658 nucleotides. The protein sequence is strikingly similar to those of the other stearoyl-CoA desaturases, SCDB having overall amino acid sequence identities of 86 and 87% with respect to the rat and mouse enzymes. We suggest, therefore, that the newly cloned cDNA encodes a second murine stearoyl-CoA desaturase, i.e. SCD2. The three stearoyl-CoA desaturase sequences are compared   in Fig. 3. The amino acid substitutions are concentrated in were found to be identical and designated AS18 (Fig. 1). The the amino-terminal one-third of the protein. All three SCD two genomic clones, hE14 and XS18, were shown to be oversequences exhibit virtually superimposable hydropathy plots lapping by restriction endonuclease mapping and sequencing (data not shown). and together encompass the entire gene. The predicted re-Cloning of the SCD2 Gene-In order to obtain the complete striction pattern shown in Fig. 1 agrees with the genomic gene for SCDP, we used a 0.5-kb intron fragment of hE14 to Southern blots using SCDP cDNA probes (Fig. 4), indicating screen a 3T3-Ll genomic library (14). The two clones isolated that no rearrangements occurred during the cloning proce- Comparison of the amino acid sequence of SCDB with those of mouse SCDl and rat SCD sequences. The translated SCDB cDNA sequence is aligned with those of the mouse SCDl (14) and rat SCD sequences (25). Only amino acids diverging from the SCDZ sequence are shown for SCDl and rat SCD. Overall, the SCDZ and SCDl sequences are 87% identical; the SCDS and rat SCD sequences are 86% identical.  The nucleotide sequences of the exon-intron junctions were determined as described under "Experimental Procedures." The exonintron junctions were positioned by comparing the nucleotide sequence of the genomic DNA with that of the cDNA. The GT and AG dinucleotides of the introns which correspond to the splice consensus sequences are underlined. The sizes of the exons were determined by sequencing. dure. The great similarity between SCDZ and SCDl extends to the structure of the genes. The intron/exon organization for SCD2 is given in Table I together with the size of the exons. All intronlexon boundaries occur at the same sites as in the SCDl gene (14) except for the 5' donor of exon 3, which is shifted by one base. The transcription initiation site for SCD2 was mapped by sponding to the inverse sequence of the sense strand from nucleotide 50 to 69) was end-labeled with 32P to a specific activity of 10' cpm/ pg. Primer extension analysis was carried out as described under "Experimental Procedures" using 10 pg of tRNA ( l a n e 1 ), or 3 pg of poly (A)+ RNA from differentiated 3T3-Ll cells ( l a n e 2). Marker lanes contain the sequences of the 8-kb BamHI/BamHI fragment of XE14 using the same primer. The arrow indicates the major start site of transcription. primer extension analysis (Fig. 5) using poly(A)+ RNA from fully differentiated 3T3-Ll adipocytes. The strongest band corresponds to base 1 in the cDNA sequence (Fig. 2). A similar primer extension analysis revealed that the same initiation site is used in brain (data not shown). A SCDB primer extension product could not be detected in mRNA isolated from 3T3-Ll preadipocytes suggesting that expression of SCD2 mRNA is induced upon differentiation into adipocytes (see below). The 5"untranslated leader of SCD2 markedly diverges in sequence and length from that of SCDl (14), the former being 120 nucleotides longer than the latter. Differential Expression of SCDB mRNA-To determine whether SCD2, like SCD1, is induced during differentiation of 3T3-Ll preadipocytes into adipocytes, we isolated total RNA every 24 h during the course of differentiation. Message levels for SCDl and SCD2 were analyzed by RNase protection using the divergent untranslated 5' or 3' regions of each cDNA as probes. As shown in Fig. 6, both expression of SCDl and SCDZ mRNAs are induced during differentiation. Confirming previous observations (14), SCDl mRNA is expressed on day 3 (following induction of differentiation), peaks on day 5, and remains at high levels through day 7. SCD2 RNA is detectable on day 2, peaks on day 4, and is not as abundant as SCDl mRNA. At day 5, SCDl mRNA is approximately 5 times as abundant as SCDZ mRNA.

Exon
It has been well documented that hepatic stearoyl-CoA desaturase levels in rats can be increased by a shift from a natural diet containing unsaturated triacylglycerides to a regimen of fasting followed by refeeding a fat-free diet (23), conditions under which the animal is required to synthesize  (lanes A-C, 1000 or 5000 cpm) were synthesized and used for RNase protection analysis as described under "Experimental Procedures." The protected fragments (lanes D-F) were obtained using probes A, B, or C hybridized to total RNA from day 5 fully differentiated 3T3-Ll adipocytes. Lanes A , probe A, synthesized from a 350-bp BamHI/EcoRI 3' end subclone of the SCDZ cDNA. all monounsaturated fatty acids. We have demonstrated previously that hepatic SCDl mRNA levels in mice are induced by a similar feeding protocol (14). To assess the tissue distribution of SCDS mRNA and to determine whether SCDB is induced by dietary conditions as well, 3-to 4-week-old mice were starved and then refed a fat-free diet (see "Experimental Procedures") or fed ad libitum. Total RNA from seven tissues was isolated and SCDl and SCD2 mRNA levels were measured by the RNase protection method (Fig. 7). As observed previously (14) SCDl is expressed constitutively (Fig. 7B) in adipose tissue, induced substantially in liver and to a lesser extent in kidney and lung, and is absent in brain, heart, and spleen under either dietary condition. On the other hand, SCD2 mRNA (Fig. 7A) is constitutively expressed in brain, is induced by refeeding a fat-free diet in adipose, kidney, and lung and is present at low levels in heart and spleen, but is not detected in liver from both groups of mice.
The 5"Flanking Sequence of the SCD2 Gene-Part of the 5"flanking region of the SCD2 gene was sequenced by the dideoxy chain termination method after subcloning the appropriate fragments into pGEM plasmids. The promoter region (Fig. 8) contains a weak "TATA" nucleotide sequence homology at position -25 relative to the major start site of transcription. Two CCAAT boxes are found at positions -90 and -135, and a potential Spl binding site is located at position -175. Further upstream, at nucleotide -500, is a nucleotide sequence similar to core glucocorticoid regulatory elements, and at position -540 is a sequence closely related to the suggested consensus sequence for binding of the nuclear transcription factor C/EBP (26).
Of particular interest is the fact that 146 bp of promoter sequence (nucleotide -201 to -54) in the SCD2 gene possesses  were analyzed for SCDZ mRNA levels ( A ) or SCDl levels ( B ) by RNase protections. In A , probe C (see Fig. 6) was used to assay SCDZ mRNA expression; in B, probe B (see Fig. 6) was used to measure SCDl mRNA expression. 77% nucleotide sequence identity to the promoter sequence (nucleotide -472 to -325) in the SCDl gene (see Fig. 9). The conserved sequence includes two CCAAT boxes which are located, only in the SCD2 gene, at the typical position in proximity to the start site of transcription. The Spl consensus binding site (GC-box) at position -175 in the SCD2 gene is not found at the corresponding location (-450) in the conserved segment of the SCDl promoter. The SCDl promoter does, however, possess a GC-box at nucleotide -210.

DISCUSSION
Stearoyl-CoA desaturase is the key regulatory enzyme of unsaturated fatty acid biosynthesis, the major form of fatty acid stored in the triacylglycerols of mouse adipocytes. It was shown previously that the activity of this enzyme increases dramatically during differentiation of 3T3-Ll preadipocytes into adipocytes (16). More recently, we isolated the gene for a stearoyl-CoA desaturase (SCD1) from 3T3-Ll preadipocytes. Here we describe the isolation and characterization of a previously unrecognized closely related gene, SCD2, which encodes an isoform of this differentially expressed enzyme.
The SCDB gene spans about 15 kb and has six exons and five introns. The cDNA is a 5 kb in length and contains a single open reading frame of 358 amino acids, followed by an unusually long 3658-bp 3"untranslated sequence. This arrangement is reminiscent of that for SCDl (14). In contrast to other genes with large 3"untranslated regions that often contain multiple polyadenylation signals allowing for the synthesis of mRNAs of varying length, the SCD2 gene has only one such sequence (AAUAAA) 24 bases upstream from the poly(A) track. The functional role of the large 3"untranslated region is unknown. From genomic Southern blots both in this (Fig. 4) and a previous paper (14), it appears that the mouse SCD gene family may contain a third closely related  gene, as the SCDl and SCD2 genes do not account for all bands detected in high stringency genomic Southern blots.

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Comparison of the three SCD amino acid sequences reported thus far (14,25) and that in this report shows a highly conserved carboxyl-terminal half of the enzyme molecule, suggesting that this region may contain the catalytic site. This is supported by a recent report in which a deletion of the first 26 amino acids did not affect the catalytic activity of bacterially expressed rat SCD (27). Although the aminoterminal half of the protein contains most of the amino acid substitutions, the overall hydropathy of this segment is conserved. The region from amino acid 70 to 120 is quite hydrophobic and may facilitate the well known membrane association of the protein. SCD is though to function as a part of a multienzyme complex in the endoplasmic reticulum, together with cytochrome b5 and cytochrome b5 reductase (15). The functional cooperation between SCD and two other proteins may necessitate conservation of amino acid sequence.
In mice fed a normal diet containing unsaturated fatty acids, SCD2 mRNA is found at high levels only in brain. Presumably, the major function of SCDB in brain is to supply unsaturated fatty acids constitutively for the synthesis of membrane phospholipids, particularly for myelination. Expression of SCDB mRNA is induced in adipose, kidney, and lung by refeeding a diet devoid of unsaturated fatty acids. Under these conditions fatty acids are synthesized de novo and are desaturated for membrane phospholipid synthesis and adipose storage in the monounsaturated form (16). Strikingly, SCDB mRNA is absent from liver regardless of the dietary condition, whereas SCDl mRNA is markedly inducible in this tissue. This difference in tissue-specific expression and dietary induction demands distinct control mechanisms for the expression of each of the two genes. It is not yet clear whether the control of expression of the SCD genes occurs primarily at the transcriptional level. Although certain regions of the promoter of the two genes differ markedly, there is one region with high sequence identity. A nucleotide sequence comparison of the 5"flanking regions of the SCD2 and SCDl genes revealed a stretch of 146 bp of striking sequence homology. The sequence between -201 and -54 bp in the SCDB gene is 77% identical to the sequence between -472 and -325 in the SCDl gene, including conservation of the two CCAAT boxes. This marked sequence homology suggests that duplication occurred during evolution of the SCD gene family. Presumably after gene duplication, either a sequence insertion or deletion occurred to yield the different promoters for SCDl and SCD2.
The SCD2 promoter lacks the potential CAMP core regulatory sequence present in the SCDl promoter (14), but contains a potential binding site for the CCAAT/enhancer binding protein (26). The SCD gene family provides a model system for the study of tissue specific as well as differentiation-induced gene expression.