(Received for publication, September 2, 1994; and in revised form, October 20, 1994)
From the
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.
Glycerol-3-phosphate acyltransferase (EC 2.3.1.15) (GPAT) ()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.
Complementary strands were annealed by combining equal amounts
of each oligonucleotide in 25 mM Tris-HCl, pH 8.0, 5 mM MgCl, 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
, 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
-mercaptoethanol, 1 µg of
poly(d(I
C)), 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.
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.
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.
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--galactosidase. Cells were
harvested 48 h after transfection and assayed for LUC and
-galactosidase activity. Luciferase activity has been normalized
to
-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.
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
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
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.
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U11680[GenBank].