(Received for publication, January 14, 1997, and in revised form, May 14, 1997)
From the Department of Biochemistry, University of Adelaide, Adelaide, South Australia 5005, and the § Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Queensland 4072, Australia
Pyruvate carboxylase (EC 6.4.1.1) is a
biotin-containing enzyme that plays an important role in
gluconeogenesis and lipogenesis. Here we report the structural
organization of the rat pyruvate carboxylase gene, which spans over 40 kilobases and is composed of 19 coding exons and 4 5-untranslated
region exons. From this data, it is clear that alternative splicing of
the primary transcripts from two promoters is responsible for the
occurrence of the multiple mRNA species previously reported
(Jitrapakdee, S., Walker, M. E., and Wallace, J. C. (1996)
Biochem. Biophys. Res. Commun. 223, 695-700). The proximal
promoter, which is active in gluconeogenic and lipogenic tissues,
contains no TATA or CAAT boxes but includes a sequence that is typical
of a housekeeping initiator protein 1 box while the distal promoter
contains three CAAT boxes and multiple Sp1 binding sites. Several
potential transcription factor binding sites are found in both
promoters. A series of 5
-nested deletion constructs of both promoters
were fused to a firefly luciferase reporter plasmid and transiently
expressed in COS-1 cells. The results show that the 153 and 187 base
pairs, preceding the transcription start sites of the proximal
and distal promoters, respectively, are required for basal
transcription. Insulin selectively inhibits the expression of the
proximal promoter-luciferase reporter gene by 50% but not the
distal promoter in COS-1 cells, suggesting the presence of an
insulin-responsive element in the proximal promoter. A half-maximal
effect was found at ~1 nM insulin.
Pyruvate carboxylase (PC)1 (EC. 6.4.1.1) is a biotin-containing enzyme that catalyzes the ATP-dependent carboxylation of pyruvate to form oxaloacetate. Native enzyme from a variety of sources has a quasi-tetrahedral arrangement of four identical subunits of Mr 130,000 (1). Each subunit consists of three functional domains: the biotin carboxylation domain, the transcarboxylation domain, and the biotinyl domain (2). The prosthetic group biotin, which is covalently attached to lysine 35 residues from the carboxyl terminus of the enzyme, acts as a mobile carboxy-group carrier between the two catalytic domains (2). In vertebrates, PC is located in the mitochondrial matrix where it plays an anaplerotic role in intermediary metabolism (3). In liver and kidney, PC is an essential enzyme in gluconeogenesis catalyzing the first regulated reaction in the conversion of pyruvate to glucose. Conversely, in adipose tissue and lactating mammary gland, PC is a key lipogenic enzyme that enables the export of acetyl groups from the mitochondria as citrate for the de novo biosynthesis of fatty acids (3). PC is subject to both short and long term regulation. Short term regulation can be achieved by an allosteric regulator, acetyl-CoA (4). In liver, kidney, and adipose tissues, changes in the total amount of PC through alterations in the rate of enzyme synthesis is a key mechanism for long term regulation. These two types of mechanism permit an increase in the rate of gluconeogenesis during starvation and diabetes, in periods of enhanced cellular metabolism induced by thyroid hormone, in neonatal development and permit an increase in the rate of lipogenesis in differentiating adipocytes (4). It has been shown that regulation of PC gene expression during the differentiation of 3T3-L1 mouse fibroblasts to adipocytes may be exerted at a pretranslational level (5, 6). However, little is known about the mechanism of hormonal regulation of PC expression at the molecular level.
Genes encoding this enzyme in bacteria (7), yeast (8-10), and
cDNAs from mosquito (11), mouse (12), rat (13, 14), and human (15,
16) have been isolated and characterized. We have also identified and
described multiple transcripts of rat and human PC mRNAs, which
within the same species encode the same protein but diverge in their
5-untranslated regions (5
-UTR) (17). The rat PC mRNAs are
expressed in a tissue-specific manner and are very likely to be under
the control of two different promoters, thus allowing independent
regulation of each mRNA isoform (17). In the yeast
Saccharomyces cerevisiae, there are two genes encoding two
isoenzymes, PC1 and PC2, which are differentially expressed (18).
Unlike yeast, only one gene has been identified in rat (17), mouse
(19), and human (20). However, the genomic organization of PC in these
organisms has not been reported. Here we report the first genomic
structure of a mammalian biotin-carboxylase gene. We have isolated and
characterized the structural organization of the rat PC gene and
present evidence consistent with alternative transcription from two
distinct promoters being responsible for the production of different
primary transcripts, which then are differentially spliced to five
species of mature transcripts. In addition, insulin has been shown to
selectively inhibit the expression of the reporter gene when fused to
the rat PC proximal promoter.
The rat PC genomic clones were isolated from two
genomic libraries constructed in EMBL3 Sp6/T7 using liver DNA
(CLONTECH, Palo Alto, CA) and in Charon 4A
(prepared from HaeIII partially digested DNA and kindly
supplied by Dr. J. Bonner, Phytogen Corp., CA). Approximately
5 × 105 to 1 × 106 plaques were
plated using Escherichia coli LE 392 host cells and screened
with a nick-translated insert of the cDNA clone, RL 1.1 (14).
Plaque hybridization was carried out according to standard procedures
(21). The filters were washed in 0.5 × SSC, 0.1% SDS at
65 °C. The positive clones were plaque purified and characterized
(21). Screening of other overlapping clones was performed by replating
the libraries and hybridization with insert DNA from positive clones
under the same conditions as described above. A rat cosmid genomic
library constructed in pWE15 (CLONTECH) was also
screened. About 2 × 106 clones were plated and
screened by standard colony hybridization (21) with randomly primed
fragments synthesized with the most 5
7-kb BamHI fragment
of the
RG 2 clone or with a 3.6-kb PCR product (see Fig. 1, fragment
E) as template. The filters were washed under the same
conditions as described above.
Isolation of Rat PC Gene by Long Distance PCR
In addition to screening rat genomic libraries, we used long distance PCR (LD-PCR) (22) to isolate the rest of the gene. LD-PCR was performed both with genomic DNA as template and with the Rat GenomeWalker kit (CLONTECH). For LD-PCR using genomic DNA as template, the reaction was carried out in a total volume of 50 µl containing 1 × Tth PCR buffer (40 mM Tris-HCl, pH 9.3, at 25 °C, 15 mM potassium acetate), 1.1 mM magnesium acetate, 200 µM of each dNTP, 0.25 µM each primer, 100 ng of genomic DNA and 1 µl of 50 × AdvantageTM Tth polymerase mix (CLONTECH). The reaction mixture was subjected to 42 rounds of PCR amplification. The PCR profile consisted of an initial denaturation at 94 °C for 1 min followed by 7 cycles of denaturation at 94 °C for 30 s, annealing and extension at 72 °C for 6 min, 35 cycles of denaturation at 94 °C for 30 s, annealing and extension at 68 °C for 6 min, and followed by the final extension at 68 °C for 12 min. For the reactions performed with the Rat GenomeWalker kit, the PCR was carried out as follows. Primary PCR was carried out in a total volume of 50 µl containing 1 × Tth PCR buffer, 1.1 mM magnesium acetate, 200 µM each dNTP, 0.25 µM AP1 primer and the first gene-specific primer (GSP1) (see Table I), 1 µl of DraI, EcoRV, PvuII, ScaI, or SspI library, and 1 µl of 50 × AdvantageTM Tth polymerase mix. The reactions were submitted to PCR amplification. The PCR profile consisted of 7 cycles of denaturation at 94 °C for 25 s, annealing and extension at 72 °C for 6 min, 36 cycles of denaturation at 94 °C for 25 s, annealing and extension at 67 °C for 6 min, and followed by the final extension at 67 °C for 12 min. One microliter of primary PCR product was diluted to 1:50, and 1 µl was used as template for the secondary PCR using the conditions as described above except that the primers were AP2 and the second gene-specific primer (GSP2) (see Table I). The PCR program consisted of 5 cycles of denaturation at 94 °C for 25 s, annealing and extension at 72 °C for 6 min, 22 cycles of denaturation at 94 °C for 25 s, annealing and extension at 67 °C for 6 min, and the final extension at 67 °C for 12 min.
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Genomic clones were characterized by restriction enzyme digestion and PCR. To localize the positions of exons and introns, Southern blot analysis was performed using DNA probes derived from different regions of the published cDNA sequence (14). The fragments that hybridized to probes were subcloned and subjected to a series of nested deletions using the Sequenest IITM (Gold BioTechnology) transposon deletion system. The DNA sequences were determined from both strands with T7 Sequenase (U. S. Biochemicals) or PCR sequencing using fmoleTM DNA sequencing system (Promega).
Construction of Promoter-Reporter Fusion PlasmidsSix
constructs containing different lengths of the proximal promoter were
fused to the luciferase reporter gene in pGL-3 basic vector (Promega).
Briefly, pDra I plasmid containing the 1153 bp upstream from the
transcription initiation site and the first 40 bp of exon 1B was
isolated by digestion with BamHI and SalI. This
fragment was subjected to 5 nested deletion with HindIII, XhoI, and KpnI restriction digestions. The
resulting fragments were cloned in pBluescript II (SK) (Stratagene) to
provide a polylinker that is compatible with the polylinker of the
reporter plasmid. These constructs were then excised with
KpnI and BamHI and cloned into KpnI
and BglII sites of pGL-3 basic vector. Further 5
-deletions were carried out by PCR with either Del A primer (positions
116 to
93 relative to the transcription initiation site) and PC 24 primer
(positions +70 to +94 relative to the transcription initiation site) or
Del B primer (positions
179 to
153 relative to the transcription
initiation site) and PC 24 primer (see Table I) using genomic DNA as
template. The PCR products were then digested with KpnI and
BamHI, cloned into KpnI and BglII
sites of pGL-3 basic vector, and sequenced. Six constructs containing
different lengths of the distal promoter were also made and fused to
the luciferase reporter plasmid. pSSP1 plasmid, containing the 1151 bp
upstream and the first 50 bp of exon 1D, was progressively deleted from
the 5
direction by digestion with SacI, KpnI,
XhoI, SalI, and PstI. The resulting
constructs were cloned in pBluescript II (SK) and subcloned into pGL3
basic vector as described above. The resulting constructs were prepared
by the standard alkaline lysis method and purified by selective
precipitation with polyethylene glycol (21).
Cells of the
African green monkey kidney line (COS-1; ATCC: CRL 1750) were cultured
in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum, 100 µg/ml streptomycin, and 100 units/ml
penicillin. Cells were grown to 80-90% confluence in
175-mm2 flasks at 37 °C in a humidified atmosphere of
5% CO2. Cells were then trypsinized and transfected using
an electroporation method. Briefly, 5 × 106 cells
were suspended in 0.5 ml of cold buffer containing 20 mM Hepes, pH 7.5, 137 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 6 mM
dextrose and transfected with 10 pmol of each construct, 2 µg of
-galactosidase expression plasmid, pRSV-
Gal (kindly provided by
Dr. Brian May), and 250 µg of carrier DNA (salmon sperm DNA). Cells
and DNA mixture were transferred to a cuvette and subsequently
electroporated with a 250-volt pulse at 960 microfarads using the Gene
Pulser (Bio-Rad). Transfected cells were then maintained in the same medium at 37 °C for 24 h.
To investigate the effect of insulin on the regulation of the rat PC
promoters, COS-1 cells were transfected with either 10 µg of pGL-P1
or pGL-P2 and 2 µg of pRSV-Gal by the electroporation method
described above. The transfected cells were recovered in DMEM
supplemented with 10% fetal calf serum at 37 °C for 24 h and
then washed with serum-free medium (DMEM supplemented with 5 mM glutamine). Serum-free media containing different
concentrations of bovine pancreatic insulin (Sigma) were then added to
the cells, which were incubated at 37 °C for 24 h.
In each experiment, parallel plates of COS-1 cells were also transfected with pGL-3 basic vector and pGL-2 control vector containing SV 40 promoter and SV 40 enhancer (Promega) that served as negative and positive controls, respectively. Each transfection was performed with triplicate samples and repeated twice.
Luciferase andTransfected cells
were harvested and concentrated from Petri dishes by centrifugation at
13,000 × g for 3 min. Cells were washed with
phosphate-buffered saline before being suspended in 100 µl of 1 × cell culture lysis buffer (Promega) and frozen. Lysates were thawed
and centrifuged at 13,000 × g for 3 min. An aliquot of
supernatant equivalent to 100 µg of protein was assayed using a
luciferase assay system (Promega) in a Berthold model LB9502
luminometer. For -galactosidase activity, 100 µg of cell lysate
was assayed using
o-nitrophenyl-
-D-galactopyranoside as substrate (23). To normalize the transfection efficiency of each
experiment, the luciferase activity was divided by the
-galactosidase activity expressed as units per microgram of
protein.
To determine the positions of exons and introns within the PC gene, the DNA sequences obtained from genomic clones were compared with cDNA encoding PC (14) using the Gap program (24). Putative transcription factor binding sites within promoters of the gene were evaluated using the SIGNAL SCAN data base of the Australian National Genomic Information Service, and the TRANSFAC data base (25).
The initial screening of a rat genomic library (Charon
4A) with a cDNA probe (RL 1.1) encoding the carboxyl terminus of
PC (14) yielded two positive clones,
RG 1.2 and
RG 1.4. Restriction mapping revealed that these two clones overlapped by about
5 kb and spanned about 15 kb from the 3
-end of the gene. Southern blot
hybridization with different regions of cDNA sequence revealed that
these two clones represented approximately half of the coding region of
the gene. Attempts to isolate other clones from the same library either
with a cDNA sequence or the most 5
1.0-kb EcoRI-PstI fragment of
RG 1.2 failed to detect
any other positive clones. The genomic library constructed in EMBL3
Sp6/T7 was then screened with the same 5
-end of
RG 1.2, and this
yielded one positive clone,
RG 15. Further isolation of the rat PC
gene was continued using LD-PCR. The PCR primers were designed from
positions +61 to +81 (PC 9) (sense) and positions +1344 to +1368 (PC
18) (antisense) (Table I) of the cDNA
sequence (14) to amplify a segment of the gene from genomic DNA. Upon
amplification, an 8.0-kb product (fragment A) representing most of the
remaining coding exons was obtained (Fig.
1). The isolation of the 5
-end of the
gene encoding the 5
-untranslated region exons and the promoter regions
was carried out both by screening a genomic library and performing PCR
and long distance PCR with the Rat GenomeWalker kit. One
clone,
RG 2, and four overlapping PCR products, viz. B
(5.5 kb), C (1.5 kb), D (4.0 kb), and E (3.6 kb) generated from the
different pairs of primers shown in Table I were obtained. The
positions of the
clones and PCR products mapped on the rat PC gene
are shown in Fig. 1.
The location of individual
exons and the length of introns within the rat PC gene was determined
by a combination of Southern blot analysis, PCR, and DNA sequencing.
Comparison of the cDNA sequence with the nucleotide sequences of
clones and PCR products revealed that the rat PC gene consisted of
19 coding exons and spanned over 40 kb. Exon 2 was the first coding
exon starting immediately at the ATG initiation codon. This exon
spanned 138 bp downstream, encoding the mitochondrial targeting
sequence and part of the biotin carboxylation domain. The biotin
carboxylation domain and the transcarboxylation domain of the enzyme
(14) were encoded by exons 2-10 and exons 13-16, respectively. The last three exons, exons 18-20, encoded the biotinyl domain of the
enzyme (14). Exon 20 also encoded the 3
-untranslated region including
the polyadenylation signal. The polypeptide segment linking the biotin
carboxylation and transcarboxylation domains was encoded by exons 11 and 12, while that linking the transcarboxylation and biotinyl domains
was encoded by exon 17.
It has long been proposed that the biotin carboxylases represent a
group of enzymes that have evolved into complex multifunctional proteins from smaller monofunctional precursors through successive gene
fusions (26). This concept had been deduced from the common features
and mechanisms of reactions they catalyze and is now supported by the
high degree of sequence similarities among the three functional domains
(27). However, no complete structures of the genes for any of the
mammalian biotin carboxylases have yet been published. Only two studies
have reported the isolation of the 5 non-coding exons of rat and
chicken acetyl-CoA carboxylase genes (28, 29), thus limiting the
comparison of our data for genomic structures to those enzymes.
Although there are many proteins whose distinct structural or functional domains are encoded by single discrete exons (30), few conclusions can be drawn regarding the relation of exons to the domain structure of rat PC. Only the placement of a large intron between exon 10 and exon 11, separating the boundary of the biotin carboxylation and the transcarboxylation domains of the enzyme, suggested a close relationship between exon boundaries and protein domains. This region is also consistent with the highly susceptible chymotrypsin cleavage site that separates these two domains (14). Interestingly, the exons encoding the biotin carboxylation domain (the amino-terminal 490 residues) of rat pyruvate carboxylase yielded a primary structure closely corresponding to that of the biotin carboxylase subunit of E. coli ACC, which catalyzes the same first partial reaction (14, 31). The presumed biotinyl domain encompassing 211 amino acid residues, encoded by the last three exons of the gene, was also consistent with the length of the biotin carboxyl carrier protein subunit of E. coli ACC. However, the gene encoding the 210 residues of this subunit in E. coli is located upstream of the biotin carboxylase subunit gene, and both genes are cotranscribed as a single mRNA (31). Although, there are segments of the transcarboxylation domain of rat pyruvate carboxylase that share a high degree of amino acid sequence similarity to that of the 5S subunit of Propionibacterium shermanii transcarboxylase (32), the overall sequence similarity is not great. It would appear that the remainder of the PC gene is not modular, but more data regarding the functional and structural units within the enzyme are required.
All coding exon sequences were in agreement with our previously reported cDNA sequence (14). Three nucleotide differences were observed between the genomic sequence and the cDNA sequence reported by Lehn et al. (13). These resulted in three amino acid changes including Pro222 (Ser in genomic sequence), Asp866 (Ile), and Gly977 (Arg). These residues, inferred from the genomic sequence and our cDNA sequence, have been shown to be highly conserved across mammalian species (14). The sequences surrounding the intron-exon boundaries are shown in Table II together with the amino acid ranges encoded by these exons. The intron-exon splice junction sequences closely matched the consensus sequences: the 21 introns each begin with a GT dinucleotide and end with an AG dinucleotide, sequences thought to be necessary for correct RNA splicing (33).
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We have recently
identified and characterized multiple transcripts of rat PC mRNA.
These transcripts contained the same coding sequence but differed in
their 5-untranslated regions, suggesting that they were generated by
alternative splicing of the 5
-end of the primary transcripts (17). To
understand the origin of these heterogeneous PC mRNAs and to
determine the mechanisms of PC gene expression, we also isolated and
characterized a genomic fragment corresponding to the 5
-end of the
gene. Four oligonucleotides (UTR/A, UTR/B, UTR/C, and UTR/D) (Table I)
specific to the 5
-UTR of PC mRNAs isolated by RACE-PCR (17) were
used as probes to localize the position of the 5
-UTR exons within the
RG 2 clone and PCR products (Fig. 1). Two exons, viz.
exons 1A and 1B, were identified within a 1.3-kb region of DNA
occurring 4.3 kb upstream from the first coding exon (Fig. 1).
Extensive screening of several genomic libraries with probes
representing the 5
-UTR of class II transcript failed to detect other
positive clones. The isolation of upstream exons encompassing the
5
-UTR of class II transcripts was, therefore, carried out by PCR using
the primer designed from the 5
-UTR of rUTR D transcript (position
1
to
25) (17). Sequencing of the 1.2-kb PCR product (F) revealed that
it contains only one exon (1D) (corresponding to nucleotides
1 to
36 of rUTR D) interrupted by another intron. The most 5
exon (1E)
was finally obtained as a 1.1-kb fragment (G) by the same strategy but
with the primer designed from nucleotide
37 to
58 of rUTR D
transcript (17) (Fig. 1). However, the intron/exon boundaries at the
3
-end of exons 1C and 1D were missing due to an inability to get
overlapping fragments (Table II).
As indicated in Fig. 2, a direct
comparison of the nucleotide sequences of four 5-UTR exons to the
previously documented 5
-untranslated regions of PC mRNA isoforms
(17) allowed us to explain how the alternative splicing of these exons
can result in the production of multiple transcripts with 5
-end
heterogeneity. The class I mRNA (rUTR A, rUTR B, and rUTR C) (17)
were generated by joining exon 1B to one or more of the downstream
exons. rUTR A was generated by joining exons 1B, 1A, and 2, the first
coding exon, while rUTR B was generated by joining exon 1B directly to exon 2, and skipping exon 1A. Interestingly, rUTR C was generated by
the same mechanism as rUTR B except that an internal donor site within
exon 1B (Fig. 3) was used in joining to
exon 2 directly. These two functional 5
donor sites were each followed
by a dinucleotide GT and were thus consistent with the consensus splice
junction (see Fig. 2) (33). The use of an internal donor within the
5
-UTR exon has also been reported for the
3-hydroxy-3-methylglutaryl-CoA reductase gene in which four internal
donor sites in the same exon can be spliced to the same 3
acceptor
site (34). On the other hand, the class II mRNA (rUTR D and rUTR E)
were generated as follows: rUTR D was generated by joining exons 1D,
1C, and 2 while rUTR E was generated by joining exon 1D directly to
exon 2 (Fig. 3).
Our results suggested that two promoters regulated the production of different primary transcripts, which were then differentially spliced to five species of mature mRNAs. Class I mRNAs (rUTR A, rUTR B, and rUTR C), derived from the proximal promoter (P1), were expressed in liver, kidney, adipose tissues, and lactating mammary gland (17). Since liver and kidney are the gluconeogenic tissues while adipose tissue and lactating mammary gland are major lipogenic organs, P1 may mediate the transcript that is related to these metabolic pathways. In contrast, class II mRNA (rUTR D and rUTR E) transcripts appear to be transcribed from a distal promoter that is located more than 10 kb upstream from exon 1B. This transcript is expressed in a wide variety of tissues (17), suggesting that this form of transcript may be responsible for the synthesis of enzyme that is used in a more general anaplerotic role in cells.
Nucleotide Sequence Analysis of the Proximal and the Distal PromotersTo identify the putative promoter and
cis-acting elements that flank exon 1B, we sequenced the
1153-bp fragment upstream from this exon. The transcription initiation
site, previously identified by RACE-PCR (17), is designated as +1 in
Fig. 4A. No consensus TATA box
or CAAT box was present in the first 100 bp upstream from the
transcription initiation site although an inverted CAAT box was
observed at 220. This structure is frequently found in the promoter
of housekeeping genes and usually contains several transcription
initiation sites as well as Sp1 transcription factor binding sites
(35). Near the transcription initiation site, the motif
ATTCTGC+1GGGCCA very closely resembled the initiator
element HIP-1 (housekeeping initiator protein 1) with consensus
sequence ATTCN1-30GCCA (36). In the TATA-less promoter of
the dihydrofolate reductase gene, this motif has been shown to bind
housekeeping initiator protein 1 and direct RNA polymerase to bind and
initiate transcription (36). This initiator is also found in other
housekeeping genes (37). Computer-assisted analysis revealed several
potential transcription factor binding sites including AP2, Sp1, cAMP
responsive element binding protein (CREB), nuclear factor 1 (NF-1),
HNF-4, c-Myb, c-Myc, and PEA-3 (38). Interestingly, there was a
potential insulin-responsive element (IRE) that overlaps the Sp1
binding site located at position
138. This motif is found in the
promoter of glyceraldehyde-3-phosphate dehydrogenase gene (39). At
position
198, the sequence closely matched that of the fat-specific
element 1 (FSE1) of the fatty acid synthase gene (TCAGGGCCCAGGAACTG)
(40). The presence of the FSE1 in the proximal promoter suggested that this promoter may mediate the transcripts that are used under lipogenic
conditions as we have previously shown that these transcripts were
detected in both abdominal and epididymal fat tissues (17). FSE1 has
been shown to be involved in the regulation of genes whose expression
is closely linked to adipocyte differentiation. These include the
putative fatty acid binding protein (ap2) (41), glyceraldehyde-3-phosphate dehydrogenase (42), adipsin, acyl-CoA synthetase, fatty acid synthase (40). There were also eleven copies of
the unusual motif, TCCCC or TCCCCC arranged as direct or inverted
repeats (boxed) (Fig. 4A).
In contrast, the distal promoter (Fig. 4B) contained three
copies of CAAT boxes located at positions 64,
94, and
224
relative to the second transcription initiation site identified by
RACE-PCR (17), respectively. No TATA box was present in the first 100 bp of this promoter. Several putative transcription factor binding sites including c-Myc, Sp1, AP-1, AP-2, PPAR, and PuF (38) were found
within the 1151 bp upstream from exon 1D (Fig. 4B). A
potential IRE located at position
236, which overlapped the Sp1
binding site, was also found in this promoter.
The 5 region of the rat PC gene exhibits a structural arrangement
similar in some respects to that of the rat gene encoding the related
biotin-containing enzyme, acetyl-CoA carboxylase (ACC). In the ACC gene
two distinct promoters mediate the production of two primary
transcripts that are differentially spliced to five species of mature
mRNA with 5
-end heterogeneity (28). The distal promoter contains
TATA and CAAT boxes and is inducible under lipogenic conditions. On the
other hand, the proximal promoter lacks a TATA or CAAT box, thus
exhibiting features of a housekeeping promoter. Transcripts produced
from this type of promoter are expressed constitutively (43). In
addition, when transcripts are generated from two promoters, they have
been shown to have different translation efficiencies (44).
To verify that the 5-flanking sequence of exon 1B
contains functional promoter elements, the 1153-bp flanking region and the first 50 bp of this exon were progressively deleted from the 5
-end
and fused to the luciferase reporter gene. The resulting constructs
were transiently transfected into COS-1 cells, and their luciferase
activities were assayed (Fig.
5A). Expression of the longest
construct (pGL-P1) was substantially higher than the promoterless
construct (pGL-3-basic) confirming that this promoter fragment was
active. However, deletion of the region between
1153 to
785
(pGL-P1
HindIII construct) resulted in an increase in
luciferase activity up to 143% relative to pGL-P1. Further deletion of
the region between
785 to
686 (pGL-P1
XhoI) caused
only a small increase in relative promoter activity, whereas deletion
of the region to
323 (pGL-P1
KpnI construct) lead to a
dramatic increase of activity to 240% relative to pGl-P1. These results suggested that negative elements reside between
1153 and
323. Additional deletion between
323 and
153 led to a relative decrease in promoter activity to 195%. However, further deletion to
116 resulted in a marked decrease in relative activity to only 37%,
which was very close to that of the promoterless construct. Examination
of the DNA sequence in this region (
153 to
116) revealed the
presence of only one potential Sp1 binding site although the HIP-1 box
was still intact. This Sp1 site may be an important element for
transcription of the proximal promoter. The presence of TCCCC or TCCCCC
motifs or their inverted repeats (11 copies) throughout this promoter
raises the possibility that they might act as the repressor. As
deletions progressed, these motifs were removed. However, we have been
unable to identify any negative elements in the transcription factor
data bases that match to this motif.
Similar studies were also performed with the distal promoter (pGL3-P2)
(Fig. 5B). Expression of this construct was much higher than
the promoterless construct. Deletion of the region between 1151 to
658 (pGL-P2
SacI) resulted in a decrease in relative activity to 68%. However further deletions of the regions
550 (pGL-P2
KpnI) and
400 (pGL-P2
XhoI)
recovered the relative activities up to 117% and 161% respectively,
suggesting that repressor(s) may be located within these regions.
Progressive deletion to
187 (pGL-P2
SalI) led to a
decrease in relative activity to 85%. Further deletion to
35
(pGL-P2
PstI) caused a further loss of promoter activity
to only 3.5%, suggesting that the core promoter is located within the
first 187 bp. This included the two CAAT boxes and AP2 binding site
proximal to the transcription initiation site. Comparison of luciferase
activities detected from both promoters showed that the distal promoter
drives the expression of the reporter gene at a higher level than the
proximal promoter. However, this could reflect a difference in the
transfection efficiencies of COS-1 cells of different passage number.
Therefore, the highest expression constructs of the proximal promoter
(pGL-P1
KpnI) and of the distal promoter
(pGL-P2
XhoI) were transfected into COS-1 cells in the
same experiments. The expression of pGL-P2
XhoI was 8-9-fold higher than pGL-P1
KpnI (data not shown).
The presence of
a putative insulin-responsive element in both promoters suggests that
the promoters of the rat PC gene would be modulated by insulin. To
examine this, COS-1 cells were transiently transfected with pGL-P1
(proximal promoter) and pGL-P2 (distal promoter). The transfected cells
were cultured in serum-free media containing different concentrations
of insulin. We found that pRSV-Gal and pGL3-promoter (contains SV40
promoter) vectors were not regulated by insulin, a criterion we use to
monitor the specificity of the insulin response. As shown in Table
III, insulin decreased the promoter
activity of pGL-P1 construct in a dose-dependent manner. A
small response to insulin was seen at the lowest concentration tested
(0.1 nM), and progressively more inhibition occurred as the
insulin concentration was increased. The maximum inhibition (~50%)
of the promoter activity was achieved by 100 nM insulin with a half-maximum effect noted at 1 nM. Insulin was also
found to inhibit the promoter activities of phosphoenolpyruvate
carboxykinase (45) and the human insulin-like growth factor binding
protein 1 (46). In contrast, insulin did not affect the promoter
activity of pGL-P2 construct. Since both promoters contained identical putative IRE sequences, CCCGCCTCT (see Fig. 4, A and
B), but only the proximal promoter responded to insulin,
this suggests that insulin action may be directed through another
element located only in the proximal promoter. Alternatively, promoter
context may be important for functional activity of this sequence.
There are several different insulin-responsive elements identified in other genes whose products are regulated by insulin (47-49), but none
of those motifs is found in the proximal promoter. The selective response to insulin through the proximal promoter, which is active in
gluconeogenic tissues, is likely to be a key mechanism in controlling the expression of PC at the transcriptional level. It has previously been shown that in diabetic rats, in which the expression of PC activity was 2 times higher than in control animals, administration of
insulin decreased PC activity to the control level (50).
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The presence of alternate promoters in rat PC gene indicates that the regulation of PC expression is extremely complex. The finding of different and common putative transcription factor binding sites on both promoters suggests an interplay between these factors that results in an alternative usage of promoters. Further investigations of the regulation of PC expression will require more comprehensive studies to dissect each element of both promoters that interact with transcription factors and hormones. It will also be very interesting to investigate whether particular forms of the different transcripts are up- or down-regulated in parallel with the changes in PC activity in response to the nutritional, hormonal, and developmental stimuli as previously reported (4).
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U81515 and U95043.
We thank Dr. Brian May for helpful advice and critical reading of this manuscript.