©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Multiple Promoters in Rat Acyl-CoA Synthetase Gene Mediate Differential Expression of Multiple Transcripts with 5`-End Heterogeneity (*)

Hiroyuki Suzuki , Mitsuhiro Watanabe , Takahiro Fujino , Tokuo Yamamoto (§)

From the (1) Tohoku University Gene Research Center, 1-1 Tsutsumidori-Amamiya, Aoba, Sendai 981, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Nucleotide sequence analysis of six independently isolated cDNAs for rat acyl-CoA synthetase (ACS) revealed three forms of ACS mRNA, designated form-A, -B, and -C mRNAs, which differ in their 5`-untranslated regions. Form-A mRNA was preferentially detected in normal and peroxisome-induced livers, whereas form-B mRNA was found in peroxisome-induced livers but not in normal livers and hearts, and form-C mRNA was preferentially found in normal hearts and peroxisome-induced livers. Analysis of two overlapping genomic clones for the rat ACS gene revealed that the three 5`-untranslated regions of the mRNAs are individually encoded by three different exons located within a 20-kilobase genomic fragment. The transcription start sites of the three forms of ACS mRNA were determined and nucleotide sequences of 5`-upstream regions of the three 5`-end exons were determined. The 5`-upstream regions were fused to the chloramphenicol acetyltransferase gene and transcription units of the three forms of ACS mRNAs were determined. These data indicate that the three forms of ACS mRNA with 5`-end heterogeneity are generated by alternative transcription from three promoters in the rat ACS gene.


INTRODUCTION

Acyl-CoA synthetase (ACS)() (EC 6.2.1.3) catalyzes the formation of acyl-CoA from fatty acid, ATP, and CoA. In previous studies, we have shown that ACS is a member of the luciferase superfamily (1, 2) . This enzyme family includes ACS from various origin (1, 2, 3, 4, 5) , acetyl-CoA synthetases (6, 7, 8, 9) , luciferases from fireflies (10, 11) and click beetle (12) , and 4-coumarate:CoA ligase from parsley (13) .

Production of acyl-CoA catalyzed by ACS is the initial reaction in fatty acid metabolism. In mammals, ACS plays a key role in the metabolism of fatty acid, since the final product of fatty acid synthase is nonesterified fatty acid. Tomoda et al. (14) have demonstrated that triacsins, potent inhibitors against ACS, inhibited Raji cell proliferation by decreasing the synthesis of cellular lipids, indicating that ACS plays an essential role in the proliferation of animal cells. Acyl-CoA produced by ACS is utilized in two major metabolic pathways; the pathway for anabolic conversion of fatty acids to cellular lipids and the pathway for catabolism of fatty acids via -oxidation system. In Candida lipolytica, a hydrocarbon-utilizing yeast, there are two distinct ACS genes (15, 16) . ACS I is responsible for the production of acyl-CoA to be utilized exclusively for the synthesis of cellular lipids, whereas, ACS II provides acyl-CoA destined exclusively for degradation via the -oxidation system (17) . ACS I is distributed among various subcellular fractions including microsomes and mitochondria, while ACS II is localized mainly in peroxisomes (18) . In rat liver, ACS is localized in microsomes, outer mitochondrial membrane, and peroxisomes. Identity of the enzymes in microsomes, mitochondria, and peroxisomes were demonstrated by purification and characterization of the enzymes in microsomal and mitochondrial fractions (19) and immunochemical studies (20) .

The mRNA for ACS is abundant in liver, adipose tissue, and heart. In rat liver, the levels of the mRNA are markedly induced by feeding a diet containing high carbohydrate or fat (1) . In Zucker fatty rats (fa/fa), genetically obese rats, the levels of the mRNA were markedly increased in liver and adipose tissue as compared with those in lean litter mates (Fa/-), whereas those in heart were unchanged (21) . ACS mRNA expression is also regulated by treatment of fibric acid derivatives. Administration of fenofibrate, a hypolipidemic drug and potent peroxisomal proliferator, markedly increased both ACS activities and ACS mRNA levels in rat liver, whereas they are almost unchanged in heart (22) . These data suggest that the expression of ACS mRNA in heart, liver, and adipose tissue is regulated by different mechanisms.

As an initial approach to elucidate the mechanisms regulating the ACS gene expression, we have characterized transcription units of rat ACS gene. Interestingly, three forms of ACS mRNAs with heterogeneous 5`-ends were shown to be transcribed in different tissues and differentially regulated. In this paper, we describe the expression of rat ACS gene mediated by three independent promoters.


EXPERIMENTAL PROCEDURES

Materials

All restriction enzymes, exonuclease III, large fragment of Escherichia coli DNA polymerase (Klenow fragment), mung bean nuclease, S1 nuclease, T4 DNA ligase, T4 DNA polymerase, T4 polynucleotide kinase, terminal deoxynucleotidyl transferase, and random primer labeling kit were obtained from Takara Shuzo Corp. (Kyoto, Japan); calf intestinal alkaline phosphatase from Boehringer Mannheim; AmpliTaqDNA polymerase from Perkin-Elmer; Moloney murine leukemia virus reverse transcriptase (Superscript) from Life Technologies, Inc.; T7 DNA polymerase (Sequenase) from U. S. Biochemical Corp.; phage cloning vector EMBL3 and pBluescript vectors from Stratagene; [-P]ATP (7000 Ci/mmol) from ICN Biomedicals; [-S]dCTP (1000 Ci/mmol) and [-P]dCTP (3000 Ci/mmol) from Amersham Corp.; and Zeta-Probe nylon membrane from Bio-Rad. Oligonucleotides were synthesized with an automated DNA synthesizer (model 381A, Applied Biosystems, Inc.).

Animals and Treatment

Male Wistar rats weighing 200-300 g were used. Rats were fed ad libitum a laboratory powder diet (Clea Japan Inc., Tokyo). To induce peroxisomes in liver, rats were fed a laboratory powder diet containing di-(2-ethylhexyl)phthalate (DEHP) for 2 weeks (23) . All rats were exposed to 12 h of light (6 a. m. to 6 p. m.) and 12 h of darkness (6 p. m. to 6 a. m.) daily for 2 weeks prior to use.

General Methods

Standard molecular biology techniques were carried out essentially as described by Sambrook et al. (24) . Genomic DNA fragments were subcloned into pUC vectors in both orientations and sequenced by the dideoxy chain termination method (25) performed with T7 DNA polymerase and [-S]dCTP. To sequence DNA fragments carrying the 5`-untranslated regions, the DNA fragments were shortened successively by exonuclease III (26) and subcloned into pUC vectors.

Blot Hybridization of RNA

Total RNAs were prepared from livers and hearts of normal rat as well as livers of DEHP-fed rats according to the procedure as described (27) . 15 µg of total RNA denatured with glyoxal was electrophoresed on 1.5% agarose gel and transferred to a nylon membrane for hybridization. 5`-P-Labeled oligonucleotide probes were hybridized with the membrane according to the procedure provided by the supplier (Bio-Rad). Oligonucleotides used for Northern blotting were 25-mers with the following sequences: oligo-ACS, 5`-GAAACCCTTCTGGATCAGCGCCGAG-3`; oligo-1A, 5`-CTCCGCAGGCGGCTGTCACTGCAAT-3`; oligo-1B, 5`-ACATTCATGTCCAAGTCTTGTTAGG-3`; and oligo-1C, 5`-ATCTGTGCCACCGACAGCTGACTGC-3`. These oligonucleotides were labeled with [-P]ATP using T4 polynucleotide kinase.

Screening of Rat Genomic Library

Genomic DNA was prepared from livers of male Wistar strain rats. Genomic library was constructed in EMBL3 vector from a partial Sau3AI digest of the genomic DNA according to the procedure provided by the supplier (Stratagene). Approximately 1 10phages were screened by plaque hybridization with a mixture of 5`-P-labeled oligonucleotides, oligo-1A, -1B, and -1C. Two positive clones (RACS1 and RACS3a) were isolated and subjected to further analysis. DNA fragments carrying the 5`-untranslated regions were identified by restriction mapping and Southern blotting of the genomic clones. After subcloning into pUC vectors, the sequences of the 5`-untranslated regions were determined.

Anchored PCR to Clone the 5`-End of ACS mRNA

To obtain cDNA fragments for the 5`-end of the mRNA, anchored PCR (28) was carried out. cDNA was synthesized from 10 µg of total RNA using an antisense primer (oligo-ACS) and 200 units of Superscriptin 20 µl of reverse transcription buffer (50 m M Tris-HCl, pH 8.3, 10 m M MgCl, 50 m M KCl, 3 m M dithiothreitol, 0.1% Nonidet P-40, and 0.45 m M dNTP) at 37 °C for 1 h. A poly(A) tail was then added to the 3`-ends of the cDNAs in a 20-µl reaction mixture containing 30 m M Tris-HCl, pH 6.8, 140 m M potassium cacodylate, 1 m M CoCl, 0.1 m M dithiothreitol, and 0.25 m M dATP with 25 units of terminal deoxynucleotidyl transferase at 37 °C for 10 min. The poly(A) tailed cDNA was amplified with the oligo(dT)-linker primer (5`-CTCTAGAGGCGGCCGC(T)-3`) and an antisense primer (oligo RO6: 5`-GCATGGACAGATCACATGGTGGCTT-3`) that located upstream of the extension primer. One-tenth of the poly(A)-tailed cDNA was amplified in standard PCR buffer containing 25 pmol of each primer and 0.75 units of Taq DNA polymerase in 100 µl of standard PCR buffer (10 m M Tris-HCl, pH 8.8, 50 m M KCl, 1.5 m M MgCl, 0.1% Triton X-100, and 0.2 m M each dNTP). The thermal profile used was 94 °C for 30 s, 55 °C for 1 min, then 72 °C for 2 min. After 30 cycles, the PCR products were loaded onto a 5% polyacrylamide gel. A major product was eluted from the gel and subcloned into pBluescript vectors for sequencing.

Primer Extension Analysis

Primer extension was carried out using 5`-end-labeled oligonucleotides, oligo-PA (5`-CCCGGGCGCCTCCGCAGGCGG-3`), -PB (5`-CATTCATGTCCAAGTCTTGTTAG-3`), and -PC (5`-ATCTGTGCCACCGACAGCTG-3`). The labeled primer (approximately 6 10cpm/pmol) was mixed with 20 µg of total RNA in 100 µl of 50 m M Tris-HCl, pH 7.5, containing 150 m M NaCl and 1 m M EDTA. This mixture was heated at 65 °C for 20 min and was then precipitated with ethanol and redissolved in 100 µl of standard reverse transcriptase buffer. cDNA synthesis was carried out by adding Superscriptas described above. Following extraction with phenol/chloroform/isoamyl alcohol (50/49/1), the resulting cDNAs were ethanol precipitated, redissolved, and analyzed by electrophoresis on an 8% sequencing gel.

S1 Nuclease Mapping

Antisense DNA probes were prepared as described by Greene (29) . Genomic DNA fragments containing exon 1A ( PstI- SmaI, 172 bp), 1B ( EcoRI- BalI, 326 bp), and 1C ( NcoI- XbaI, 316 bp) were subcloned into pUC119. A 5`-P-labeled specific primer was hybridized with a denatured double stranded plasmid containing each exon and extended with Klenow fragment. Oligo-PA, -PB, and -PC were used as primers. The primer extended products were digested with appropriate restriction enzymes and purified on a denaturing gel. P-Labeled S1 probe (3 10cpm) was mixed with 20 µg of total RNA, heated at 80 °C for 10 min, and hybridized at 60 °C overnight. Other experimental conditions were as described by Sambrook et al. (24) . The reaction products were analyzed by electrophoresis on an 8% sequencing gel.

Promoter-CAT Constructs

To test for promoter activity, the 5`-upstream regions of the three first-exons were each fused to upstream of the CAT gene present in pCat-Enhancer vector (Promega). To fuse the 5`-upstream region of exon 1A (-788 to -49) with the CAT gene, restriction fragment derived from RACS1 was inserted into HindIII- XbaI sites of pCat-Enhancer vector using HindIII and XbaI linkers. This CAT gene construct was designated pCatEA(-788). Similarly, the fragment containing the 5`-upstream region of exon 1B (-728 to -132) and the fragment containing the 5`-upstream region of exon 1C (-981 to -53) were each inserted into the vector to create pCatEB(-728) and pCatEC(-981), respectively. To create a series of 5`-deleted mutants, DNA fragments containing the promoter regions were successively shortened by exonuclease III (26) and inserted into the CAT vector. The sites of the deletion were determined by nucleotide sequencing.

DNA Transfection

Mouse hepatoma Hepa 1 cells (kindly provided by Dr. C. B. Kasper, McArdle Laboratory for Cancer Research, University of Wisconsin) were grown in monolayers in medium A (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin). The cells were cultured at 37 °C in a humidified atmosphere of 5% COand 95% air. On day 0, Hepa 1 cells were plated at 3 10cells per 60-mm Petri dish and incubated for 24 h. On day 1, each dish received 3 ml of fresh medium A and was then transfected with calcium phosphate-precipitated DNA according to the transfection protocol described by Chen and Okayama (30) . Briefly, 8 µg of test plasmid and 2.7 µg of the -galactosidase expression plasmid pCH110 (Pharmacia) in 171 µl of Tris-HCl, 1 m M EDTA at pH 8.0 were added to 19 µl of 2.5 M CaCland mixed with 190 µl of BES-buffered saline solution (30) . The precipitate was allowed to form for 45 min at room temperature, after which 450 µl was added dropwise to each monolayer. The cells were incubated for 16 h with the DNA and then washed twice with 4 ml of warm Dulbecco's phosphate-buffered saline and refed with 3.5 ml of medium A. After incubation for 48 h (day 4), the cells were harvested for measurement of CAT and -galactosidase activity. In each transfection experiment, parallel plates of Hepa 1 cells were transfected with pCat-Enhancer vector and pSV2-cat plasmids that serve as negative and positive controls, respectively. The pCat-Enhancer vector contains an SV40 enhancer but lacks eukaryotic promoter. Apparently no CAT activity was detected in Hepa cells transfected with pCat-Enhancer vector. The pSV2-cat plasmid contains the SV40 early promoter driving the expression of CAT mRNA transcript (31) .

CAT Assay

Transfected cells were washed three times with phosphate-buffered saline, scraped into 250 µl of Tris-HCl, pH 7.5, 150 m M NaCl, and 1 m M EDTA, frozen and thawed five times, and centrifuged at 14,000 g for 20 min at 4 °C. An aliquot of the supernatant was incubated for 1 h at 37 °C in a standard CAT assay (31) in a final volume of 150 µl containing 0.9 n M [C]chloramphenicol (50 nCi) and 0.53 m M acetyl-CoA. All assays were linear with respect to time and concentration of extract protein. The protein content of cell extract was measured by the Lowry method (32) .

-Galactosidase Assay

An aliquot of the 14,000 g supernatant from the lysed transfected cells was incubated at 28 °C for 1 h with 0.67 mg/ml o-nitrophenyl-- D-galactopyranoside in a final volume of 1.2 ml (33) . The reaction was stopped with 0.5 ml of 1 M NaCO, and the amount of o-nitrophenol formed was measured spectrophotometrically at 420 nm. To normalize the transfection efficiency for each individual transfection, the total counts/minute of the acetylated forms of chloramphenicol were divided by the -galactosidase activity expressed as units/milligram of protein.


RESULTS AND DISCUSSION

Heterogeneity in the 5`-Untranslated Region of ACS mRNA

Prior to isolating genomic clones for rat ACS gene, we analyzed nucleotide sequences of the 5`-ends of the six independent cDNA clones isolated in the previous study (1) . Fig. 1 shows the nucleotide sequences of the 5`-untranslated regions of the ACS cDNAs, pRACS 10, pRACS 11, pRACS 12, pRACS 14, pRACS 15, and pRACS N1. All of these clones contain a unique EcoRI site at 33 nucleotides upstream of the initiator AUG codon and the downstream sequences past the EcoRI site are identical. However, upstream of the EcoRI site sequences are different and classified into three groups. These data suggested the presence of three forms of ACS mRNAs that differ in their 5`-untranslated regions.

Expression of Three Forms of ACS mRNAs

To analyze the expression of the three species of ACS mRNAs by Northern blotting, we used three antisense oligonucleotide probes specific to each of the 5`-untranslated regions. Northern blot analysis with oligo-ACS, specific to the coding region of ACS cDNA, detected the 3.8-kilobase pair ACS mRNA in normal and DEHP-fed (peroxisome-induced) livers and normal hearts (Fig. 2 A). Oligo-1A, an antisense oligonucleotide specific for the 5`-end of pRACS 10, pRACS 14, and pRACS 15, detected the 3.8-kilobase pairs ACS mRNA both in normal and DEHP-fed livers and, to a much lesser extent, in normal hearts (Fig. 2 B). On the other hand, oligo-1C, an antisense oligonucleotide specific for the 5`-end of pRACS 11 and pRACS 12, detected the mRNA in normal hearts and DEHP-fed livers and, to a much lesser extent, in normal livers (Fig. 2 C). Oligo-1B, an antisense oligonucleotide specific to the 5`-end of pRACS N1 did not detect any mRNA in normal and peroxisome-induced livers and normal hearts (data not shown). To detect mRNA containing complementary sequence to that of oligo-1B, anchored PCR was carried out using oligo-1B as a primer. The expected 240-bp cDNA fragment was detected when anchored PCR was performed with oligo-1B and total RNA from peroxisome-induced livers (Fig. 2 D). No amplification was observed with oligo-1B when total RNAs of normal hearts and livers were used as templates. Nucleotide sequence of the 240-bp cDNA fragment was full matched with that of the 5`-end of pRACS N1 (data not shown). These results indicate the presence of three forms of ACS mRNA that differ in their 5`-untranslated regions. The mRNAs detected by oligo-1A, -1B, and -1C were designated form-A, -B, and -C mRNA, respectively. To determine if the three forms of mRNAs were transcribed from distinct promoters, we isolated genomic clones that contain sequence corresponding to the three 5`-untranslated regions of ACS mRNAs.


Figure 2: Expression of three forms of ACS mRNA with heterogeneous 5`-untranslated regions. Total RNA (15 µg) from normal rat livers ( lane 1), DEHP-fed rat livers ( lane 2), and normal rat hearts ( lane 3) were electrophoresed on a 1.5% agarose gel, transferred to a nylon membrane, and hybridized with P-labeled oligo-ACS ( A), oligo-1A ( B), or oligo-1C ( C). The same samples were used for anchored PCR as described under ``Experimental Procedures'' using oligo-1B as a primer ( D). The resulting PCR products were separated on a 5% polyacrylamide gel and stained with ethidium bromide. HinfI-digested pBR322 DNA was used as a molecular size marker. The data shown is a representation of three independent experiments which gave essentially identical results.



Genomic Clones for the 5`-Untranslated Regions of ACS mRNAs

A rat genomic library was screened with a mixture of oligonucleotides, oligo-1A, -1B, and -1C. We isolated two overlapping phage clones, RACS1 and RACS3a from screening of approximately 1 10phages. Restriction mapping, Southern blotting, and nucleotide sequence analysis of the two clones revealed that the three 5`-untranslated regions are individually encoded by three different exons located within a 20-kilobase pair genomic fragment (Fig. 3). The first exons corresponding to the 5`-untranslated regions of form-A, -B, and -C of ACS mRNAs were designated exon 1A, 1B, and 1C, respectively.

Characteristics of the 5`-Flanking Regions of Rat ACS Gene

In describing the 5`-flanking regions of rat ACS gene, we use a numbering scheme in which the A of the AUG initiator codon of the ACS protein is designated +1. The transcription start sites of form-A and -B ACS mRNA were determined by both primer extension analysis and S1 nuclease protection mapping with total RNAs from normal and DEHP-fed rat livers. The transcription start site of form-C ACS mRNA was determined by primer extension analysis using total RNA from DEHP-fed rat livers and normal hearts (data not shown). In the 5`-flanking region upstream of exon 1A, potential Sp1 binding sequences (34) are present at -206 to -215, -269 to -278, -301 to -310, -334 to -343, and -368 to -377. AP2 (34) (-169 to -178, -419 to -426, -452 to -459, and -531 to -538) and fat-specific element (FSE) (35, 36) (-411 to -417) are also located in this region (Fig. 4 A). The 5`-flanking region of the exon 1B also contains a DNA motif for AP2 (-637 to -644) (Fig. 4 B). The 5`-flanking region upstream of exon 1C contains multiple copies of AP2 consensus binding sequences (-103 to -110, -145 to -152, -311 to -318, -346 to -353, -413 to -420, and -633 to -640). An AP1 sequence (34) is also found at -124 to -130 (Fig. 4 C).


Figure 4: Nucleotide sequences of 5`-flanking regions in rat ACS gene. The nucleotide sequences of 5`-upstream regions of exon 1A ( A), 1B ( B), and 1C ( C) are shown. Nucleotide 1 corresponds to the A of the translation initiator methionine codon AUG, and residues preceding it are indicated by negative numbers. The arrowheads above the sequence indicate transcription initiation sites identified by S1 mapping; and those below are the transcription initiation sites identified by primer extension analysis. Exons are boxed and introns ( IVS) are indicated by arrows. Location of potential transcription factor binding sites are underlined. Sequences for AP1, AP2, and Sp1 are taken from Ref. 34 and FSE2 from Refs. 35 and 36. E1- and E3-like sequences are identified by comparing the conserved E1 (GGGAC-CAG) and E3 (GGTCTCCT-G) in the 5`-flanking regions of inducible peroxisomal -oxidation genes, acyl-CoA oxidase (23), enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (40), and peroxisomal 3-ketoacyl-CoA thiolase (39).



Functional Analysis of Three Promoters in Rat ACS Gene

To verify that the 5`-upstream regions of exons 1A, 1B, and 1C each contain functional promoter elements, the corresponding genomic DNA fragments were fused to the CAT gene in the pCat-Basic vector which contains no eukaryotic promoter or enhancer elements. When all of these chimeric genes were transfected into mouse Hepa 1 cells, no expression was detected above the background. Therefore, we inserted the same ACS gene fragments into the pCat-Enhancer vector which contains the strong SV40 virus enhancer but does not contain a eukaryotic promoter element. Following transfection into Hepa 1 cells, all three genomic fragments produced substantial CAT activity relative to the vector indicating that each fragment contains a functional eukaryotic promoter (Fig. 5 A).

The promoter A (pCatEA(-788)) plasmid contains the sequence from -788 to -49 of the exon 1 flanking DNA and it produced comparable levels of normalized CAT activity as the positive control plasmid pSV2-cat which contains the SV40 enhancer and promoter fused to the CAT gene (Fig. 5 A). A series of 5` deletions were then engineered into the promoter A region. Fig. 5B shows that deletion of sequence from -354 to -226 resulted in a 75% loss of promoter activity and a further deletion to -182 resulted in a 2-fold recovery of activity. These data suggested that the boundary of a positive promoter element is contained between -354 and -226 and a negative element is contained between -226 and -182. A further deletion to -130 resulted in a severe loss in promoter activity.


Figure 5: Analysis of promoter activities of rat ACS promoters A, B, and C fused to the CAT reporter gene. The 5`-flanking regions of exon 1A (-788 to -49), exon 1B (-728 to -132), and exon 1C (-981 to -53) were each inserted into pCat-Enhancer vector to create pCatEA(-788), pCatEB(-728), and pCatEC(-981), respectively. pCatECRv contains the same insert of pCatEC(-981) in reverse orientation. The chimeric genes were cotransfected with a -galactosidase expression plasmid (pCH110) into Hepa 1 cells and assayed for CAT activity as described under ``Experimental Procedures.'' The acetylated forms of [C]chloramphenicol ( 1AcCM and 3AcCM) were separated from unreacted [C]chloramphenicol ( CM) by thin layer chromatography and detected by autoradiography ( A). Various fragments of promoter A ( B), B ( C), and C ( D) of rat ACS gene were fused to the CAT gene, introduced into Hepa 1 cells together with the -galactosidase expression plasmid, and assayed for CAT and -galactosidase activities as described under ``Experimental Procedures.'' CAT activity in an individual experiment was corrected for variation in transfection efficiency by normalizing the value to the -galactosidase activity in the same extract. The data represent the mean of triplicate transfection experiments for each plasmid.



Similar studies were also performed with promoter B (pCatEB(-728)) and promoter C (pCatEC(-981)) and the results are presented in Fig. 5 , C and D, respectively. Each promoter displayed significant promoter activity and the 5` deletion analyses suggest that minimal promoter B is contained within 126 bp (between -374 and -248) of exon 1B mRNA start site and minimal promoter C is contained within 357 bp (between -416 and -59) of exon 1C mRNA start site.

Alternative Transcription of Rat ACS Gene

The current studies indicate that the 5`-end heterogeneity of ACS mRNA is generated by alternative transcription from three promoters A, B, and C in the rat ACS gene (Fig. 6).

Form-A mRNA derived from promoter A is the major species in the liver and is expressed, to a much lesser extent, in the heart. Since liver is a major organ for the synthesis of complex lipids such as triglyceride and cholesterol ester, promoter A may mediate the expression of the enzyme that produces acyl-CoA utilized for lipogenesis. In the promoter A region, there are several DNA motifs that are similar to known transcriptional factor sites. In particular, the presence of the FSE2 site is noteworthy. FSE2 is involved in the regulation of genes whose expression is closely linked to adipocyte differentiation (37) . These include the putative fatty acid binding protein termed adipocyte P2 (aP2), glycerophosphate dehydrogenase, and adipsin (35, 36) . During the adipocyte differentiation, the transcription of mouse ACS gene is also markedly increased in 3T3 L1 cells (38) . Form-A mRNA transcribed from promoter A is also abundant in adipose tissue in rat,() therefore the transcription of ACS gene in adipocytes may be mediated by FSE2. The FSE2 site is located at -411 to -417 and deletion of this site did not drastically alter ACS promoter activity in mouse Hepa 1 cells. Further studies are required in 3T3 L1 cells to determine if the FSE2 site is involved in adipocyte specific expression of ACS. The minimal promoter A was defined by a 5`-deletion study to include the proximal 97 bp (between -182 and -85) relative to the exon 1A mRNA initiation site. This fragment contains one potential AP2 recognition site and no other known promoter type element.

The exon 1B containing ACS mRNA is barely detectable in livers of DEHP-treated rat but not in normal livers. Therefore, promoter B may be related to the regulation of fatty acid -oxidation.

Form-C mRNA transcribed from promoter C is the major transcript in the heart and is expressed, to a much lesser extent, in the liver. The hepatic levels of form-C mRNA were markedly induced after DEHP treatment. DEHP and fibric acid derivatives are known to be potent peroxisomal proliferators in rodents and are able to activate the fatty acid -oxidation system in liver. It has been demonstrated that DEHP induces many of enzymes involved in the -oxidative pathway including acyl-CoA oxidase (23) . Promoter C contains E1 and E3 sequences that are commonly found in the 5`-flanking regions of the inducible peroxisomal -oxidation genes (39) . These are genes of acyl-CoA oxidase (23) , enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (40) , and peroxisomal 3-ketoacyl-CoA thiolase (39) . Heart is active in -oxidation of fatty acid for the production of contracting energy. Therefore promoter C may be responsible for the production of the enzyme that produces acyl-CoA for fatty acid -oxidation. In the promoter deletion studies, removal of the E1 and E3 sites did not affect expression. It will be interesting to determine if promoter C is induced by peroxisomal proliferators and if the E1- and E3-like elements bind the peroxisome proliferator-activated receptor (41) .


FOOTNOTES

*
This research was supported in part by research grants from the Ministry of Education, Science and Culture of Japan, the HMG-CoA Reductase Research Foundation, the Ono Medical Research Foundation, and the Takeda Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence data bases with accession number(s) D38587, D38588, and D38589.

§
To whom reprint requests should be addressed: Tohoku University Gene Research Center, 1-1 Tsutsumidori-Amamiya, Aoba, Sendai 981, Japan. Tel.: 81-22-271-2815; Fax: 81-22-263-9295.

The abbreviations used are: ACS, acyl-CoA synthetase; BES, N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid; bp, base pairs; CAT, chloramphenicol acetyltransferase; DEHP, di-(2-ethylhexyl)phthalate; FSE, fat-specific element; PCR, polymerase chain reaction.

H. Suzuki, M. Watanabe, and T. Yamamoto, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Tim Osborne for helpful advice and discussions.


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