©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of the Murine Mitochondrial Glycerol-3-phosphate Acyltransferase Promoter (*)

(Received for publication, September 2, 1994; and in revised form, October 20, 1994)

Ann A. Jerkins (1)(§) W. Robert Liu (1) Sunjoo Lee (1) Hei Sook Sul (1) (2)(¶)

From the  (1)Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115 and the (2)Department of Nutritional Sciences, University of California, Berkeley, California 94720

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the acylation of sn-glycerol 3-phosphate to form 1-acyl-sn-glycerol 3-phosphate, a committed step in triacylglycerol and phospholipid biosynthesis. We have previously reported the cDNA cloning and transcriptional regulation of the murine mitochondrial GPAT (mGPAT). We now report the cloning of the 5`-flanking region of the murine mitochondrial GPAT. The transcription start site was identified by primer extension and RNase protection assays. A TATA box-like motif (TTATTAT) was located between -34 and -29 and a reverse CCAAT box (ATTGG) was located between -78 and -74, relative to the transcription start site. To begin studying mechanisms underlying transcriptional regulation of the mGPAT gene, chimeric luciferase (LUC) plasmids containing serial deletions, from -1447 to -38, of the 5`-flanking region of the murine mGPAT gene were prepared and transfected into 3T3-L1 cells. The fusion construct -1447 GPAT.LUC showed high promoter activity and deletions to -1353, -747, -322, and -86 did not markedly change the promoter activity. With all constructs, luciferase activity was 2-fold higher when plasmids were transfected into 3T3-L1 adipocytes. However, deletion of sequences between -86 and -55 resulted in a 9-fold decrease in LUC activity in both preadipocytes and adipocytes. Deletion of sequences between -55 and -38 did not alter promoter activity. DNase I footprint analysis revealed a protected region between -95 and -65 which included the putative CTF/NF1 binding site. Electrophoretic mobility shift assays demonstrated a single protein-DNA complex formation. Oligonucleotides synthesized according to the CTF/NF1 consensus sequence or the adenovirus NF-1 site showed a different and more complex pattern of protein-DNA interaction and were not able to compete away the mGPAT promoter-protein complex, indicating that a distinct protein was bound to -86/-55, a region important for the basal promoter activity in 3T3-L1 cells. Luciferase activity was increased 2.8- and 8-fold when adipocytes stably transfected with -322 GPAT.LUC were treated with 5 and 25 mM glucose, respectively, in the presence of 10 nM insulin. These results indicate that carbohydrate-responsive sequences are located within -322 base pairs of the mGPAT promoter.


INTRODUCTION

Glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (GPAT) (^1)catalyzes the initial and committed step of glycerolipid synthesis, the acylation of sn-glycerol 3-phosphate to form 1-acyl-sn-glycerol 3-phosphate. This step may be rate-limiting and GPAT, therefore, plays a pivotal role in the regulation of triacylglycerol and phospholipid biosynthesis(1) . There are two major forms of GPAT in mammalian tissues, microsomal and mitochondrial(1) . In liver, 50% of GPAT activity is found in the mitochondrial fraction, while in most other tissues microsomal GPAT activity is about 10 times that of the mitochondrial fraction(2, 3) . In spite of the important role it may play in the regulation of triacylglycerol and phospholipid biosynthesis, mammalian GPAT has not been purified or characterized. The partitioning of fatty acids between esterification and oxidation pathways is partly carried out by GPAT, and GPAT activity is thought to be under nutritional and hormonal regulation(4) .

We have previously reported the isolation of the cDNA for a 6.8-kb mRNA, which dramatically increased in livers of fasted mice refed a high carbohydrate, fat-free diet(5) . The open reading frame had a 30% identity and an additional 42% similarity in an approximately 300 amino acid stretch to Escherichia coli GPAT(6) . We recently reported the positive identification of this mRNA gene product as the murine mitochondrial GPAT (mGPAT), by correlating the mGPAT protein concentration with mGPAT activity in liver, 3T3-L1 cells, and in Chinese hamster ovary cells stably transfected with the cDNA sequence. In Chinese hamster ovary cells, the expressed GPAT revealed a substrate preference for saturated fatty acids, a characteristic of the mitochondrial enzyme(7) .

Mitochondrial GPAT mRNA was highly expressed in lipogenic tissues such as liver and adipose tissue and was under nutritional and hormonal control(6) . The mGPAT mRNA level increased 20-fold at 8 h when previously starved mice were refed a high carbohydrate, fat-free diet; this induction was blocked 70% by the administration of dibutyryl cAMP (6) . Insulin caused a marked and rapid induction of mGPAT mRNA in diabetic mice. When previously starved streptozotocin-diabetic mice were refed, no increase in the mGPAT mRNA levels was observed. However, in diabetic animals, mGPAT mRNA increased 2-fold 1 h after insulin injection and reached a maximum 19-fold after 6 h(6) . In 3T3-L1 adipocytes insulin increased the mGPAT mRNA levels 2.5-fold, while dibutyryl cAMP decreased the mGPAT mRNA by 80%(5) . The hormonal effects were on the transcriptional rate of the gene coding for mGPAT (6) .

In an effort to study the molecular mechanisms underlying the regulation of mGPAT gene expression by nutrients and hormones, we isolated genomic clones coding for mGPAT. The transcription start site was determined by primer extension and RNase protection analyses. Sequences important for basal promoter activity of mGPAT were determined by transient transfection of 3T3-L1 cells with GPAT-promoter luciferase fusion constructs, and by DNase I footprint analysis and gel mobility shift assay. Luciferase activity, in 3T3-L1 adipocytes stably transfected with mGPAT.LUC plasmids, was increased after cells were exposed to glucose. Carbohydrate-responsive sequences reside within 322 bp of the mGPAT transcription start site.


MATERIALS AND METHODS

Isolation of the Murine mGPAT Genomic Clone and Sequence Analysis

Chromosome walking techniques were utilized to obtain genomic clones of murine mGPAT(8) . A murine mGPAT genomic clone, GPAT-1, with an approximately 13.4-kb insert was isolated by screening a EMBL-3 murine genomic library with a random-labeled (9) 3.8-kb HindIII genomic fragment, which includes the 5` end of the mGPAT cDNA sequence. High stringency screening was utilized with the final wash conditions of 0.1 times SSC (15 mM NaCl, 1.5 mM sodium citrate, pH 7.0), 0.1% SDS at 65 °C. The 13.4-kb insert was further characterized by Southern blot (10) and restriction mapping. Overlapping restriction fragments from GPAT-1 were subcloned into the pBluescript II SK+ vector (Stratagene). The subclones were sequenced by the dideoxynucleotide chain termination method (11) using Sequenase 2.0 (U. S. Biochemical Corp.). Sequences of overlappng restriction fragments were used to confirm the restriction mapping of GPAT-1.

Primer Extension Analysis

Primer extension experiments were based on procedures described previously(12) . A 17-base synthetic oligodeoxynucleotide primer, 5`-GCTCCTCCAGAGGCCTA-3`, which is complementary to nucleotides +99 to +115 of the mGPAT sequence, was labeled at the 5` end with [-P]ATP (DuPont NEN) and T4 polynucleotide kinase (New England Biolabs). The P-labeled primer (5.5 times 10^6 cpm) was mixed with 5 µg of mouse liver poly(A) RNA and added to a 22-µl volume containing 250 mM KCl, 47.5 mM Tris-HCl, pH 8.3, and 1 mM EDTA, incubated 45 min at 65 °C followed by 45 min at 45 °C, and allowed to cool slowly to room temperature. The hybridized primer-RNA complex was extended in a 80-µl volume containing 7.5 mM KCl, 47.5 mM Tris-HCl, pH 8.2, 10 mM MgCl(2), 10 mM dithiothreitol, 0.5 mM dNTPs, 50 units of human placenta RNase inhibitor, and 53 units of avian myeloblastosis virus reverse transcriptase, and heated for 5 min at 37 °C followed by 1 h at 42 °C. Following phenol/chloroform extraction and ethanol precipitation, the extension products were separated on a 6% polyacrylamide, 8 M urea sequencing gel and visualized by autoradiography. A M13 single-stranded DNA sequencing product was run in parallel as a reference to determine the size of the primer extended product.

RNase Protection Assay

A 1.5-kb StuI genomic fragment, spanning the region from approximately -1447 to +102, was subcloned into the SmaI site of pBluescript II SK+. The orientation of the insert was determined by DNA sequence analysis using flanking T3 or T7 promoter sequencing primers. The resulting plasmid was then digested with NotI or MboII to generate the constructs for antisense RNA probe synthesis with T7 RNA polymerase (New England Biolabs) and [alpha-P]CTP. The P-labeled antisense riboprobe (5 times 10^6 cpm) was co-precipitated with 12 µg of total RNA from refed mouse liver and dried. The pellet was resuspended in 30 µl of hybridization buffer (80% (v/v) formamide, 40 mM PIPES, pH 6.4, 400 mM NaCl, and 1 mM EDTA) and hybridized overnight at 45 °C. The annealed products were digested with RNase A and RNase T1 (Sigma), for 1 h at 30 °C, in the presence of 350 µl of RNase digestion buffer (10 mM Tris-HCl, pH 7.5, 300 mM NaCl, and 5 mM EDTA). The digestion was stopped by adding 10 µl of 20% SDS and 2.5 µl of proteinase K (20 mg/ml) and incubated an additional 15 min at 37 °C. The RNase-protected products were electrophoresed on a 6% polyacrylamide, 8 M urea sequencing gel and visualized by autoradiography. A M13 single-stranded DNA sequencing product was run in parallel to determine the size of the RNase protected fragments. Twelve micrograms of yeast tRNA, which was also hybridized with the P-labeled antisense RNA probe and digested with RNase A and RNase T1 under the same conditions, served as a negative control.

Rapid Amplification of cDNA Ends (RACE)

A modification of the method described by Frohman et al.(13) was utilized to clone the 5` ends of the mGPAT mRNA. First-strand cDNA was reverse transcribed from 2 µg of poly(A) RNA, prepared from previously starved mice refed a high carbohydrate-fat free diet, using hexamer random primers (Boehringer Mannheim). The first-strand cDNA was tailed in a 20-µl volume containing tailing buffer (Life Technologies, Inc.), 200 µM dATP, and 10 units of terminal deoxynucleotidyltransferase (Life Technologies, Inc.) for 5 min at 37 °C, and then heated for 5 min at 65 °C. The tailed cDNA was ethanol-precipitated in the presence of yeast tRNA, and the washed pellet was resuspended in 50 µl of water. Five-microliter volumes were used to synthesize the second strand cDNA with 10 pmol of the primer 5`-CGTCGACGGATCCATCGATTTTTTTTTTTTTTTTT-3` and 25 pmol of the primer 5`-CGTCGACGGATCCATCGAT-3`, both of which included the restriction sites for SalI, BamHI, and ClaI, 25 pmol of the primer 5`-AGCTCTTTGGCTTGTGGCTTCTAGGATTCCCCTAG-3` complementary to nucleotides +288 to +322 of GPAT mRNA, 5.2 µg of yeast tRNA, 140 µM of each dNTP, Taq DNA polymerase buffer, and 2.5 units of Taq DNA polymerase (Promega). The polymerase chain reaction amplification program involved 30 cycles of denaturation at 94 °C for 2 min, primer annealing at 50 °C for 2 min, and extension at 72 °C for 3 min. A second amplification was performed under the described conditions except that the poly(T) primer was omitted and 1 µl of the initial PCR products was used in place of the tailed cDNA. After primer removal by Gene Clean (Bio 101, La Jolla, CA), the PCR products were ligated into pCR 1000 (Invitrogen) for cloning and sequencing using T7 and M13 -40 primers.

Construction of mGPAT-Luciferase Fusion Plasmids

A luciferase reporter gene system was utilized to study the relative transcriptional activity of the various 5`-flanking regions of the murine mGPAT gene. To generate murine mGPAT.LUC fusion constructs, the 1.5-kb StuI fragment, spanning -1447 to +102 of the genomic murine mGPAT sequence, was inserted in the sense orientation upstream of the firefly luciferase (LUC) gene at the SmaI site of the promoterless LUC vector pGL2-Basic (Promega, Madison, WI) to create -1447 GPAT.LUC. Plasmids -1353 GPAT.LUC, -747 GPAT.LUC, -322 GPAT.LUC, and -86 GPAT.LUC were generated from -1447 GPAT.LUC by utilizing the restriction sites PstI, XbaI, SacI, and HindIII, respectively. The plasmid -1353 GPAT.LUC was constructed by digesting pBluescript II SK+ containing the 1.5-kb StuI mGPAT genomic fragment with PstI, isolating the 1.4-kb PstI fragment, treating with T4 DNA polymerase, and inserting the blunt-ended PstI fragment at the blunt-ended NheI site of pGL2-Basic. Plasmid -747 GPAT.LUC was constructed by digesting -1447 GPAT.LUC with XbaI and BglII and inserting the fragment at the NheI/BglII site of pGL2-Basic. Plasmid -322 GPAT.LUC was prepared by inserting the SacI fragment of -1447 GPAT.LUC into the SacI site of pGL2-Basic. Plasmid -86 GPAT.LUC was constructed by digesting the SacI fragment with HindIII and inserting into the HindIII site of pGL2-Basic. Additional 5` deletion constructs -55 GPAT.LUC and -38 GPAT.LUC were generated from -1353 GPAT.LUC using an Erase-A-Base kit (Promega). All constructs were verified by sequencing.

Cell Culture and Transient Transfections

Murine 3T3-L1 cells were grown in monolayers in 60-mm diameter culture dishes in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine serum, 100 µM non-essential amino acids, 50 units/ml penicillin G, and 50 µg/ml streptomycin at 37 °C in the presence of 5% CO(2) in a humidified incubator. At confluence, cells were differentiated by 48 h of exposure to 0.5 mM 1-methyl-3-isobutylxanthine and 0.25 µM dexamethasone, after which cells were maintained in medium without drugs for an additional 2 days(14) . Transfections were carried out using the calcium phosphate-DNA coprecipitation method (15) . Each 60-mm dish was co-transfected with 15 µg of GPAT.LUC and 1 µg of pCMV-beta-galatosidase. Cells were harvested 48 h after transfection and extracts assayed for LUC activity as described (16) using a Bertholdt Luminometer (Nashua, NH). beta-Galactosidase activity was assayed spectrophotometrically as described (17) using commercially available beta-galactosidase (Sigma) as the standard. The pCMV-beta-gal was a gift from Dr. G. R. MacGregor (Houston, TX). Protein content of extracts was determined using the Bradford assay (18) using bovine serum albumin as the standard. Luciferase activity was corrected for transfection efficiency by expressing relative to beta-galactosidase activity. At least two different cesium chloride plasmid preparations for each construct were tested in transfection experiments. Transfections were performed in triplicate dishes per experiment. Data were analyzed by ANOVA and Student's t test. Numbers represent means ± S.E. for three to five experiments.

Stable Transfections

For stable transfections, exponentially growing 3T3-L1 cells were co-transfected with 15 µg of GPAT.LUC, 8 µg of Rous sarcoma virus-beta-galactosidase, and 1 µg of SV2NEO plasmid DNA per 100-mm dish, via the calcium phosphate DNA co-precipitation method(15) . Cells were selected for 3-4 weeks in 400 µg/ml Geneticin (G418) (Sigma). Resistant colonies were pooled and grown without G418 and differentiated. To test for carbohydrate responsiveness, adipocytes were maintained in serum-free, glucose-free medium for 24 h before the addition of glucose and 10 nM insulin. Cells were cultured an additional 48 h and harvested, and cell extracts assayed for luciferase activity and protein content. Data were analyzed by ANOVA and Student's t test. The numbers represent means ± S.E. for 5-6 dishes/treatment group.

Preparation of Nuclear Extracts

Hepatic nuclei were prepared from the livers of male CD-1 mice, by centrifugation through a 2.1 M sucrose cushion at 20,000 rpm in a Beckman SW-28 rotor for 60 min(19) . Nuclear extracts were prepared as described by Henninghausen and Lubon(20) . The protein concentration was determined by the Bradford procedure, using bovine serum albumin as the standard (18) .

DNase I Footprinting Analysis

DNase I footprinting was carried out using a modification of a previously described procedure (21) . The DNA fragments used in the footprinting assay were prepared by end-labeling specific DNA fragments prepared by PCR using synthetic oligonucleotides and GPAT.LUC plasmids. The forward primer used was GLprimer1 (Promega). The reverse primers used were either 5`-CTGCCAACCGAACGGACATT-3`, complementary to +249 to +268 of pGL2-Basic, or 5`-TGGTTTTGCAACAGTGGAGGAGG-3`, complementary to -7 to +16 of mGPAT. Amplified GPAT fragments were end-labeled using T4 polynucleotide kinase and [-P]ATP, digested with the appropriate restriction enzyme, isolated by agarose gel electrophoresis, and purified by Gene Clean. DNA binding reactions were carried out in 50 µl containing 50 mM NaCl, 0.1 mM EDTA, 20 mM HEPES, pH 7.5, 0.5 mM dithiothreitol, 10% glycerol, 1 µg of poly(d(IbulletC)) (Boehringer Mannheim), and increasing amounts of nuclear extracts (10-50 µg). After 20 min at 0 °C, 70,000 cpm of end-labeled probe (2 ng) was added. After incubation at room temperature for 10 min, 6 µl of 100 mM MgCl(2), 20 mM CaCl(2) was added, immediately followed by 2 µl of 0.015 unit of DNase I solution. Digestion proceeded for 30 s at room temperature and was followed by the addition of 20 µl of 100 mM EDTA, pH 8.0. Samples were extracted with phenol/chloroform, ethanol-precipitated, resuspended in 90% formamide containing xylene cyanol and bromphenol blue, and separated on 6% polyacrylamide, 8 M urea sequencing gels. DNase I cleavage sites protected from digestion were identified using dideoxynucleotide chain termination reactions of the template DNA using the appropriate primer. Gels were dried and exposed to x-ray film at -70 °C with an intensifying screen.

Mobility Shift Assays

The following pairs of single-stranded oligonucleotides were synthesized for use in the gel shift assay.

Complementary strands were annealed by combining equal amounts of each oligonucleotide in 25 mM Tris-HCl, pH 8.0, 5 mM MgCl(2), and 25 mM NaCl, heating to 70 °C for 10 min and cooling to room temperature. A consensus CTF/NF1 oligonucleotide 5`-CCTTTGGCATGCTGCCAATATG-3` (top strand shown, Promega) was also used in these studies. The double-stranded DNA was 5`-end-labeled using [-P]ATP and T4 polynucleotide kinase or used as unlabeled competing DNA in competition experiments. Gel shift reactions were performed for 30 min at room temperature in 30 µl of 47 mM NaCl, 7 mM KCl, 3.5 mM MgCl(2), 28.2 mM Tris-HCl, pH 7.5, 5.4 mM EDTA, 14 mM HEPES, pH 7.9, 0.05% Tween 20, 7% glycerol, 0.8 mM beta-mercaptoethanol, 1 µg of poly(d(IbulletC)), and the indicated amount of nuclear extracts. Each reaction received 20,000 cpm oligonucleotides (0.1-0.5 ng) and was incubated at room temperature for 30 min. During competition experiments, specific or nonspecific competitor DNA was added to samples before the addition of nuclear extract. Samples were electrophoresed at 30 mA constant current on a 4% nonreducing polyacrylamide gel at 4 °C in a high ionic strength buffer (0.38 M glycine, 50 mM Tris, 2.1 mM EDTA, pH 8.5). Gels were dried, followed by exposure to x-ray film at -70 °C with an intensifying screen.


RESULTS AND DISCUSSION

Determination of the Transcription Start Site of the mGPAT Gene

In order to examine and identify sequences responsible for transcriptional regulation of mGPAT, the genomic clone GPAT-1, containing the 5`-flanking region of murine mitochondrial GPAT gene, was isolated by screening a EMBL-3 library with a P-labeled genomic GPAT fragment that codes for the 5` end of the mGPAT cDNA(6) . GPAT-1 was analyzed by restriction mapping and Southern blot analysis. The resulting restriction map indicated that the isolated clone had an insert of 13.4 kb.

The transcription start site of the mGPAT gene was determined by primer extension analysis and RNase protection assay. For primer extension analysis, an oligonucleotide complementary to sequence +99 to +115 of mGPAT mRNA was synthesized. The oligonucletide was end-labeled with P, hybridized to mouse liver RNA, and extended by reverse transcription. Poly(A) RNA from mouse liver directed the synthesis of an extension product of 115 nt, with several other faint bands including 112- and 118-nt-long products (Fig. 1A), indicating the 115-nt product as the probable transcription start site. Similar results were obtained by RNase protection analysis using antisense riboprobes. The cRNA probe generated from the NotI linearized StuI fragment produced four bands of 98, 101, 103, and 104 nucleotides protected from RNase digestion (Fig. 1B). One major band of 101 nucleotides in length coincided with the transcription start site identified by the primer extension as shown in Fig. 1A. In addition, identical results were obtained when a shorter riboprobe, generated by restricting the StuI fragment with MboII, was used in the RNase protection assay (data not shown). No band was detected with the yeast tRNA negative control. mRNA secondary structure can give rise to bands from primer extension and ribonuclease protection analyses that are actually artifacts, leading to errors in the interpretation of such results. However, the fact that the major band from the primer extension agrees with a major band from the ribonuclease protection supports our interpretation of the transcription start site. On the other hand, the two minor bands 3 nucleotides apart from the transcription start site also coincide in the two different analyses and, therefore, cannot be ruled out as minor sites of initiation of transcription. The sequence surrounding the transcription start site included a weak CA motif (cap-like site)(22) . We have also cloned by RACE-PCR the 5` ends of mGPAT cDNAs employing mouse liver mRNA. The nucleotide sequence of the 5`-untranslated region was obtained from numerous overlapping RACE-PCR clones (data not shown). The size of the longest 5`-untranslated region was 371 bp long and corresponded to that predicted from the primer extension and RNase protection assays.


Figure 1: Identification of the transcriptional start site for the murine mGPAT gene. A, primer extension analysis; 5 µg of poly(A) RNA from mouse liver was hybridized to a 17-base P-end-labeled oligonucleotide and extended by reverse transcription. A primer-extended product of 115 nt is indicated by an arrow. The sequence is shown to the left with the start site identified with an asterisk. B, RNase protection analysis; 12 µg of total RNA prepared from livers of refed mice were hybridized with a P-labeled antisense riboprobe and digested with RNase A and T1. The protected products were separated on a 6% polyacrylamide sequencing gel. Four protected products of 98, 101, 103, and 104 nt are indicated by arrows on the left of the autoradiogram.



Sequence Analysis of the mGPAT 5`-Flanking Region

The sequence of approximately 1.9 kb of mGPAT 5`-flanking region and first exon is shown in Fig. 2; the transcription initiation site is designated as +1. The region upstream of the transcription start site, from -34 to -29, contained a sequence (TTATTA) similar to the TATA box found in approximately the same position in most eukaryotic genes(23) . Computer analysis revealed a CTF/NF1-like sequence containing a reverse CCAAT box (ATTGG) between -78 and -74. These structural features of mGPAT revealed similarities with other genes, known to be induced under conditions (e.g. fasting/refeeding) leading to increased lipogenesis, such as the acetyl-CoA carboxylase promoter P1, which directs the synthesis of a liver specific acetyl-CoA carboxylase mRNA(24) . The rat fatty acid synthase promoter also contains both a TATA-like motif (TTTAAT) and inverted CCAAT sequence. However, unlike the FAS promoter, the mGPAT gene does not contain G-C-rich regions containing putative Sp1 transcription factor binding sites(25) .


Figure 2: Nucleotide sequence of the murine mGPAT 5`-flanking region. The 1873-bp sequence of GPAT spans -1447 to +426 of the mGPAT gene. The numbers on the left represent the positions relative to the transcription start site, designated as +1. The TATA box-like motif at -34 to -29 and a CTF/NF1-like sequence at -78 to -74 are doubleunderlined. Areas protected from DNase I digestion are in boldface. Exon sequences are in uppercase letters. The ATG initiator is italicized. Primers used in PCR and primer extension analysis are underlined.



Deletion Analysis of the Basal mGPAT Promoter Activity in 3T3-L1 Cells

To test whether the 5`-flanking region of the mGPAT gene had promoter activity, a series of 5` deletions within the first 1.5 kb of the mGPAT 5`-flanking region ligated to the LUC reporter gene were generated. These deletions were between -1447 and -38 and shared a common 3` end at +102. Plasmids were transiently transfected into 3T3-L1 preadipocytes and adipocytes. The sequence from -1447 to +102 supported a high level of gene expression. In 3T3-L1 cells, 5` deletion constructs -1353, -747, -322, and -86 showed luciferase activity similar to that of the longer -1447 GPAT.LUC construct. As shown in Fig. 3, in all constructs studied, luciferase activity was greater when plasmids were transfected into 3T3-L1 adipocytes compared to preadipocytes (p < 0.05, Student's t test). Further deletion of sequences from -86 to -55 resulted in a 9-fold decrease in LUC activity in both preadipocytes and adipocytes (Fig. 3). The dramatic decrease in LUC activity suggested that cis-acting sequences between -86 and -55 were necessary for high mGPAT promoter activity in 3T3-L1 cells.


Figure 3: Transient transfection analysis of the murine mGPAT 5`-flanking region. 3T3-L1 preadipocytes and adipocytes were transiently transfected with 15 µg of GPAT.LUC fusion constructs and 1 µg of pCMV-beta-galactosidase. Cells were harvested 48 h after transfection and assayed for LUC and beta-galactosidase activity. Luciferase activity has been normalized to beta-galactosidase activity to correct for transfection efficiency. On the left are diagrams of the mGPAT promoter-luciferase fusion constructs. Plasmid -1447 GPAT.LUC was created by inserting a 1.5-kb StuI fragment of GPAT-1, spanning -1447 to +102, into the SmaI site of the promoterless LUC vector, pGL2-Basic. Verticalbars indicate restrictions sites used to make the constructs. Plasmids -55 GPAT.LUC and -38 GPAT.LUC were generated from -1353 GPAT.LUC using an Erase-A-Base kit. The constructs are aligned with the 5` mGPAT gene sequences numbered relative to the transcription start site. The data represent the means ± S.E. of three to five independent experiments. Triplicate transfections were performed in each experiment. The hatchedbars represent preadipocytes; solidbars represent adipocytes.



Binding of Nuclear Factor(s) to the mGPAT Proximal Promoter

To examine the potential regulatory sequences located between -86 and -55, mGPAT DNA fragments were used to determine the presence of proteins capable of binding to these regulatory elements. The results of DNase I footprint analysis of the murine mGPAT promoter, using increasing amounts of mouse liver nuclear extracts, are shown in Fig. 4. Liver nuclear proteins were found to interact with several regions of the mGPAT promoter in vitro. When the DNA fragment spanning -189 to +39 was labeled on the sense strand and used for DNase I footprinting, two regions corresponding to nucleotides -65 to -95 encompassing the CTF/NF1-like sequence and -136 to -161 were protected from DNase I digestion (Fig. 4A). A third region upstream of -136 to -161 was also protected from DNase I digestion. Similarly, when the DNA fragment spanning -121 to +14 was labeled on the antisense strand (Fig. 4B), footprints were evident in the region extending from -95 to -58 as well as a region near the putative TATA box. In addition, binding of liver nuclear proteins produced distinct hypersensitive sites adjacent to the protected regions (Fig. 4).


Figure 4: Binding of nuclear protein(s) to the murine mGPAT promoter region. PCR-amplified mGPAT fragments were 5`-end-labeled using [-P]ATP and T4 polynucleotide kinase, digested with SacII (coding) or KpnI (non-coding), isolated by agarose gel electrophoresis, and purified by Gene Clean. A DNA fragment containing the murine mGPAT promoter region, -189 to +39 (panelA, coding strand labeled), or -121 to +14 (panelB, non-coding strand labeled), was subjected to DNase I footprint analysis as described under ``Materials and Methods.'' Increasing amounts of liver nuclear extracts, as indicated, were incubated with the P-end-labeled DNA fragment. The regions of protection from DNase I digestion, demarcated by boxes, were identified by a dideoxy chain termination sequence ladder separated in parallel on a 6% polyacrylamide sequencing gel. Asterisks indicate hypersensitive sites.



To investigate the specificity of binding to the -95/-65 protected sequence of mGPAT observed in DNase I footprinting analysis, gel mobility shift assays were performed. End-labeled oligonucleotide GPAT -95/-65 was incubated with mouse liver nuclear extracts and subjected to non-denaturing polyacrylamide gel electrophoresis. One major DNA-protein complex was observed and was competed away by increasing amounts of unlabeled oligonucleotide GPAT -95/-65 (Fig. 5). These results demonstrated the specificity of the DNA-protein complex. A similar binding pattern was observed when nuclear extracts from 3T3-L1 adipocytes were used (data not shown). No differences were observed in the DNA-protein complex formed when nuclear extracts from fasted or refed mice were used, suggesting that the binding of transcription factors to DNA was not changed in vitro under these two metabolic states.


Figure 5: Gel mobility shift assays with nucleotides -95 to -65 of the murine mGPAT gene. Single-stranded oligonucleotides of GPAT -95/-65 were synthesized, annealed, and end-labeled with T4 polynucleotide kinase and [-P]ATP. End-labeled oligonucleotide GPAT -95/-65 was incubated with nuclear extracts prepared from mouse liver. The competitor, when used, was added at 0, 10, and 100 times the molar concentration of labeled oligonucleotide and incubated in the presence of 5 µg of mouse liver nuclear extracts. Samples were subjected to 4% non-reducing polyacrylamide gel electrophoresis in a high ionic strength buffer.



Since computer analysis showed that a 6-bp stretch of the -95/-65 GPAT sequence corresponded to a CTF/NF1-like sequence containing a reverse CCAAT box, a consensus CTF/NF1 sequence and an adenovirus NF1 sequence were used as cold competitors of P-labeled GPAT -95/-65 in gel mobility shift assays (Fig. 6). Increasing amounts of unlabeled GPAT -95/-65 competed away the complex, but neither of the NF1 sequences displaced binding of the GPAT -95/-65 sequence to liver nuclear extract protein(s) or 3T3-L1 adipocyte nuclear extract protein(s) (data not shown), suggesting that protein(s) other than CTF/NF1 are involved in the DNA-protein complex.


Figure 6: NF1 is not involved in DNA-protein complex formation of GPAT -95/-65. Competition experiments were carried out by incubating P-end-labeled GPAT -95/-65 with 15 µg of mouse liver nuclear extracts in the presence of unlabeled competitor at 0, 100, or 200 times the molar concentration of labeled oligonucleotide.



To further characterize the binding of liver nuclear extract protein(s) to CTF/NF1 sequences, adenovirus and consensus CTF/NF1 sequences were P-end-labeled and used in gel mobility shift assays (data not shown). The end-labeled adenovirus NF1 and CTF/NF1 sequences were incubated with increasing amounts of mouse nuclear extract. An identical but complex pattern of DNA-protein interaction was observed with both of these sequences. The DNA-protein complexes were competed away by increasing amounts of the respective unlabeled oligonucleotide; in addition, unlabeled CTF/NF1 sequence successfully competed away the adenovirus NF1-protein complex and unlabeled adenovirus NF1 sequence competed away the CTF/NF1-protein complex (data not shown). However, the NF1-protein complexes differed from the -95/-65 GPAT-protein complex and were not competed away by unlabeled GPAT -95/-65. These results confirm that a nuclear protein distinct from CTF/NF-1 interacts with the -95/-65 region of the GPAT gene and plays a role in basal promoter activity of the mGPAT gene.

The mGPAT Promoter Is Regulated by Carbohydrate

We have previously reported that the increase in steady-state mGPAT mRNA in livers of fasted mice refed a high carbohydrate fat-free diet was preceded by an increase in the rate of transcription of the mGPAT gene (6) . Towle and co-workers recently reported sequences responsible for carbohydrate regulation of the S(14) and L-PK genes. The E-box motif CACGTG has been implicated in mediating the carbohydrate responsiveness of the S(14) and L-PK genes(26, 28, 29, 30, 31) . A comparison of the mGPAT sequence revealed a region within the first 322 bp of the mGPAT promoter having sequence homology to the S(14)(26) and L-PK (27) carbohydrate response elements. An E-box motif present at -321 to -316 has a 5 out of 6 nucleotide match with the S(14) and L-PK genes. To examine if the mGPAT promoter is regulated by carbohydrate, stable transfectants of 3T3-L1 cells were generated using -322 GPAT.LUC. Stable pools of transfectants were first differentiated to adipocytes, by standard treatment as described, and subsequently incubated with various concentrations of glucose. Luciferase activity was increased 2.8- and 8-fold when treated with 5 mM and 25 mM glucose, respectively, in the presence of 10 nM insulin (Table 1). These results indicate that sequences responsible for carbohydrate regulation of mGPAT are located in the first -322 bp of the mGPAT promoter. Further studies are necessary to define sequences necessary for carbohydrate regulation of the mGPAT gene and to find whether the E-box found in the region is important. It would be interesting to see if common sequences or mechanisms are involved in the carbohydrate regulation of the mGPAT gene.



In conclusion, we have determined the transcription start site of the mGPAT gene. We showed that the region -86/-55, which contains a CTF/NF1-like sequence, is important for the basal promoter activity in 3T3-L1 cells; however, a distinct protein interacts with this region for basal promoter activity. We have also presented evidence that the mGPAT promoter is regulated by carbohydrate and that the sequence responsible for this regulation is present in the first 322 bp from the transcription start site.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK 36264. 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 GenBank(TM)/EMBL Data Bank with accession number(s) U11680[GenBank].

§
Present address: Dept. of Nutritional Sciences, University of Arizona, Tucson, AZ 85721.

To whom correspondence should be addressed: Dept. of Nutritional Sciences, University of California, Berkeley, CA 94720. Tel.: 510-642-3978; Fax: 510-642-0535.

(^1)
The abbreviations used are: GPAT, glycerol-3-phosphate acyltransferase; mGPAT, mitochondrial glycerol-3-phosphate acyltransferase; kb, kilobase pair(s); bp, base pair(s); nt, nucleotide(s); LUC, luciferase; CMV, cytomegalovirus; PIPES, 1,4-piperazinediethanesulfonic acid; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends.


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