From the
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.
Acyl-CoA synthetase (ACS)
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
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.
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.
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,
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
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
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.
We thank Dr. Tim Osborne for helpful advice and
discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
(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) .
-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) .
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
10
phages 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
10
cpm/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 Superscript
as
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
10
cpm) 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
10
cells 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 CaCl
and 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) .
An aliquot of the 14,000
-Galactosidase Assay
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 Na
CO
, 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.
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
10
phages. 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).
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).
(
)
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.
-oxidation.
-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) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.