From the Department of Cell Biology and Biochemistry,
Texas Tech University Health Sciences Center, Lubbock, Texas 79430, the
§ Howard Hughes Medical Institute, Department of
Biochemistry, University of Texas Southwestern Medical Center, Dallas,
Texas 75235-9050, and the
Dana Farber Cancer Institute, Harvard
Medical School, Boston, Massachusetts 02115
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ABSTRACT |
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A putative adipocyte-specific enhancer has been
mapped to approximately 1 kilobase pair upstream of the
cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene. In the
present study, we used transgenic mice to identify and characterize the
413-base pair (bp) region between The cytosolic isozyme of phosphoenolpyruvate carboxykinase
(PEPCK,1 EC 4.1.1.32)
catalyzes the interconversion of oxaloacetate and phosphoenolpyruvate
in adipose tissue and liver, yet PEPCK plays differing roles in the two
tissues (1, 2). In liver, PEPCK is a rate-limiting gluconeogenic
enzyme. However, white adipose tissue is not gluconeogenic because
adipocytes do not express glucose-6-phosphatase and
fructose-1,6-bisphosphatase, the enzymes that catalyze the two final
steps of gluconeogenesis (2). Radioactive tracer studies in white
adipose tissue indicate that PEPCK is involved in glyceroneogenesis,
the synthesis of glycerol 3-phosphate from gluconeogenic precursors
during fasting (1-3).
Both pathways are induced during fasting, and both are involved in fuel
release but in contrasting ways. During a fast, gluconeogenesis provides a supply of glucose, whereas glyceroneogenesis is believed to
prevent an all-out release of triglycerides liberated by lipolysis in
white adipocytes (3). The role of glyceroneogenesis can be illustrated
by comparing the sources of precursors for triglyceride synthesis
during feeding and fasting. During feeding, triglycerides are
synthesized from dietary fatty acids and glycerol-3-P, a metabolite of
dietary glucose. During fasting, a large fraction of the fatty acids
released from triglycerides by lipolysis is reesterified with
glycerol-3-P in what appears to be a futile cycle of triglyceride degradation and resynthesis (3). Indeed, lipolysis and fatty acid
reesterification are both induced during fasting, but a net release of
free fatty acids does occur. Because fat cells do not express glycerol
kinase they cannot phosphorylate the glycerol released from
triglycerides (4). Hence the glycerol-3-P needed for fatty acid
reesterification is derived from gluconeogenic precursors via
glyceroneogenesis. This reesterification of fatty acids probably
prevents ketogenesis during fasting (5). Diabetic ketoacidosis provides
an example of the problems that occur with an unregulated release of
fatty acids from adipocytes. Since PEPCK and glyceroneogenesis are
induced proportionately as a result of the hormonal milieu of fasting,
it was proposed that PEPCK is the rate-limiting glyceroneogenic
enzyme (2, 5).
Because PEPCK plays different metabolic roles in white adipose tissue
and liver we hypothesized that it might be regulated by distinct
tissue-specific mechanisms in these two tissues. Extensive work focused
upon PEPCK regulation in hepatocytes has revealed that transcription of
the gene is under developmental and hormonal control via a complex
upstream region within 600 bp of the promoter (1, 6). Much less effort
has been directed toward understanding how PEPCK is regulated in
adipocytes, but the available data indicate that it is quite different
from the mechanisms in hepatocytes. We reported previously that PEPCK
DNA upstream of The family of orphan nuclear receptors, the peroxisome
proliferator-activated receptors (PPARs), were discovered during the past decade (8). Briefly, these transcription factors are activated by
various hyperlipidemic drugs, xenobiotic compounds, fatty acids and
their metabolites, and they regulate genes encoding proteins involved
in lipid metabolism (8). In mammalian species there are three PPAR
subfamilies: PPAR Recent investigations have shown that PPAR The prototype adipocyte-specific enhancer is located 5 kilobase pairs
upstream of the aP2 promoter and is activated by PPAR The purpose of this study was to determine whether the region of PEPCK
DNA upstream of Transgene Constructs and Production of Transgenic Mice--
The
nine transgene constructs utilized in this study are illustrated in
Figs. 1A, 2A, and 3. All of the constructs were
prepared by standard techniques from the rat PEPCK genomic DNA clone,
Extraction and Measurement of mRNAs--
Mice were killed by
cervical dislocation, and tissues were removed, frozen immediately in
liquid nitrogen, and stored at
The cRNA probes were the same for the two different RNase protection
assays. The mouse PEPCK probe was transcribed from pmPCR10 using T3 RNA
polymerase to synthesize an antisense RNA from a HindIII-digested template. The cRNA product is 411 bp in
length, whereas the RNase-protected product is a 326-bp fragment. It
was necessary to construct pmPCR10 for these experiments because a mouse PEPCK cDNA had not previously been cloned, and the rat PEPCK cDNAs, although more than 95% identical to mouse PEPCK, were not suitable for RNase protection assays. Polymerase chain reaction primers
were designed by comparison of the sequences for rat, human, and
chicken PEPCK cDNAs and searching for identical regions. Forward
(5'-ATGCGGCCCTTCTTTGGCTA-3') and reverse (5'-TCCACCTCCTTCTCCCAGAA-3') polymerase chain reaction primers were selected and used to amplify mouse genomic DNA. A single product of the predicted size (326 bp) was
obtained and inserted into the EcoRV site of pBluescript IISK+ (Stratagene, La Jolla, CA). This sequence corresponds to nucleotides 394-719 in the translated sequence from exon 10 of the rat
PEPCK gene.
The cDNA, phGHex2, used for assay of hGH and hGX mRNAs was
constructed from a region of exon 2/intron 2 (nucleotides 833-1011, 989-1011 is intron 2). The hGH/hGX cRNA probe was transcribed from
phGHex2 using T3 RNA polymerase to synthesize an antisense RNA from a
XhoI-digested template. The hGH cRNA transcript is 272 bp in
length, whereas the RNase-protected product is a 156-bp fragment.
The Nuclear Extracts and in Vitro Translated Proteins--
In
vitro translation of RXR Electrophoretic Mobility Shift Assays--
DNA-protein binding
assays were performed in 15-µl incubations as described by Bretz
et al. (22). Briefly, nuclear extracts and/or recombinant
proteins were incubated in a reaction buffer consisting of 10 mM HEPES (pH 7.9), 1 mM EDTA (pH 8.0), 4%
Ficoll, 100 mM dithiothreitol, 1 µg of poly(dI·dC)
(Amersham Pharmacia Biotech), unlabeled double-stranded competitor DNA
(where specified), and 1 µg of sonicated salmon sperm DNA (Sigma).
These reactions were incubated at room temperature for 15 min before
the addition of 4 pmol of a 32P-labeled double-stranded DNA
probe followed by a second 15-min incubation.
The rat PCK2 probe was prepared as follows. A synthetic double-stranded
DNA oligomer (sense strand, 5'-GATCACAACTGGGATAAAGGTCTCG-3'; antisense
strand, 5'-GATCCGAGACCTTTATCCCAGTTGT-3') was end labeled by
filling the 3'-recessed ends with [
Human PCK2 sense: 5'-GATCACAACTGTTATAAAGGTTTCA-3'
Human PCK2 antisense: 5'-GATCTGAAACCTTTATAACAGTTGT-3'
Rat PCK1 sense: 5'-GATCCATGACCTTTGGCCGTGGGAG-3'
Rat PCK1 antisense: 5'-GATCCTCCCACGGCCAAAGGTCATG-3'
When antiserum was used, binding reactions were incubated with
antiserum during the 15 min prior to the addition of the labeled DNA.
Sera used included: 1) polyclonal rabbit anti-human RXR DNA Sequence Analysis--
The rat adipocyte-specific enhancer
was sequenced on an automated sequenator from Applied Biosystems in the
Institute for Biotechnology Core Laboratory, Texas Tech University.
Both DNA strands were sequenced in their entirety. All computer
analyses were performed using the Wisconsin Package Version 9.0 software, Genetics Computer Group (GCG), Madison, WI.
Effect of the Systemic Production of hGH on Transgene
Expression--
In a previous study using transgenic mice we utilized
the hGH gene as the reporter to define tissue-specific elements
upstream of the PEPCK promoter (7). A comparison of endogenous PEPCK expression in tissues of normal versus transgenic animals
indicated that the high serum concentrations of hGH did not affect
transgene expression (7). However, a similar study was reported by
McGrane et al. (25) who used bovine growth hormone (bGH) as
the reporter. Serum bGH, at high concentrations, caused
hyperinsulinemia and significantly repressed both endogenous PEPCK and
transgene expression in their mice. Although we did not observe a
similar effect with hGH, the use of a biologically active hormone as
reporter risks introducing artifacts. We therefore obtained hGX, a
reporter encoding a biologically inactive mutant form of hGH, from Dr.
Stanley McKnight (18). We used hGX to produce new transgenic mice in
which the rat PEPCK promoter was identical to the The Adipocyte-specific Element Is Located between
We concluded previously that an adipose-specific element is localized
between
The The Region between
During analysis of these "enhancer mice" we noted that the level of
expression of the transgene was only approximately 5% of the level
obtained with either the Function of the Adipocyte-specific Enhancer in Vivo Requires an
Intact PPAR Response Element(PCK2)--
We reported previously the
presence of two PPAR
We therefore asked whether mutations of PCK2 and/or PCK1 will block
transgene expression in transgenic mice. Three new groups of transgenic
mice, PCK2M, PCK1M, and PCK1+2M, were produced, and transgene
expression was compared with the wild type transgene counterpart,
Fig. 3C shows the same analysis of the four founder mice
bearing the PCK1 mutation. It appears that this mutation decreases, but
does not eliminate, transgene expression in either adipose tissue or
liver. Unfortunately, we obtained too few mice in this group to
determine with certainty whether a PCK1 mutation affects expression in
these two tissues. We can, however, conclude that PCK1, unlike PCK2, is
not essential for PEPCK expression in adipose tissue.
Fig. 3D shows the analysis of the final group of six founder
mice bearing the PCK1 + PCK2 double mutation. There was no transgene expression in adipose tissue, an effect that can be attributed to the
PCK2 mutation.
In Adipocyte Nuclear Extracts, Only PPAR PCK1 and PCK2 Bind Different Factors--
The experiment shown in
Fig. 3 shows that only one of the two PPAR The Adipocyte-specific Enhancer Region Is Conserved in Rats and
Humans--
It is likely that DNA-binding proteins in addition to
PPAR
To assist in dissecting the enhancer, several computer analyses were
performed: TESS with the Transfac 3.2 data base at
http://agave.humgen.upenn.edu; MAP (Wisconsin Package) with its
Transcription Factor Data Base (27); and the Institute for
Transcriptional Information object-oriented transcription factor data
base at www.ifti.org (28). Not surprisingly this produced a lengthy
list of candidate binding sites including elements for C/EBP family
members, CTF/NF-1, Sp-1, AP-1, and PPAR
Of the remaining candidate factors, the C/EBP family is particularly
relevant because they are intimately involved in adipocyte differentiation (30) and regulation of the PEPCK gene in hepatocytes (1). Several of the putative C/EBP sites (Fig. 6B) are
highly conserved between rats and humans, suggesting that they could be
involved in adipocyte-specific enhancer activity. We are currently investigating which, if any, of these putative sites are functional. It
will also be necessary to investigate whether any of the other candidate factors are involved.
The Adipocyte-specific PEPCK Enhancer Requires the Binding of
PPAR
The adipocyte-specific enhancer of the PEPCK gene is apparently
unimportant for tissue-specific expression of the PEPCK gene in liver
and kidney because we observed no differences in transgene expression
associated with the presence or absence of the enhancer in these
tissues. This major regulatory difference between adipose tissue and
liver or kidney is in keeping with the functional difference of PEPCK
between these tissues.
The Enhancer-Promoter Interaction May Be
Gene-specific--
Reporter expression in adipose tissue of the
Enhancer hGX mice was only 5-10% that of the Does PCK2 Play a Regulatory Role in Addition to
Development?--
Although PCK2 is not involved in the developmental
activation of PEPCK in liver, it may be involved in the modulation of
PEPCK expression by fatty acids in both liver and adipose tissue.
Indeed, various fatty acids induce PEPCK gene expression in adipocytes and cultured hepatoma cells (31). Thus PPAR Is There a Locus Control Region Upstream of
Chalkley and colleagues also suggested that the element at What Is the Function of PCK1?--
The data presented here suggest
that PCK1, the proximal PPAR What Do the Similarities between the Rat and Human PEPCK Promoters
and Flanking DNAs Suggest?--
The simplest suggestion is that the
conserved regions point to transcription factor binding sites that are
critical to some aspect of PEPCK expression. It is impossible to
ascribe adipocyte-specific functions to any region other than PCK2
because the PEPCK gene is regulated by numerous hormones and other
factors and is expressed in adipocytes, hepatocytes, intestinal
epithelium, and proximal tubule epithelium of the kidney. Based upon
the available evidence (1, 6) we suggest that the proximal 500-bp
region is responsible for liver- and kidney-specific expression and for
the complex regulation by cAMP and other factors. We also suspect that
this region interacts with the adipocyte-specific enhancer to cause the
effect shown in Fig. 2D. There is no information available to suggest a function of the intermediate region between Summary--
We have found a second example of an
adipocyte-specific enhancer; the first example is the aP2
gene (14). More importantly, we have provided the first in
vivo evidence that the function of an adipocyte-specific enhancer
is absolutely dependent upon the binding of PPAR1242 and
828 bp as a bona
fide adipocyte-specific enhancer in vivo. This
enhancer functioned most efficiently in the context of the PEPCK
promoter. The nuclear receptors peroxisome proliferator-activated receptor
(PPAR
) and 9-cis-retinoic acid receptor
(RXR) are required for enhancer function in vivo because:
1) a 3-bp mutation in the PPAR
-/RXR-binding element centered at
992 bp, PCK2, completely abolished transgene expression in adipose
tissue; and 2) electrophoretic mobility supershift experiments with
specific antibodies indicated that PPAR
and RXR are the only factors
in adipocyte nuclear extracts which bind PCK2. In contrast, a second
PPAR
/RXR-binding element centered at
446 bp, PCK1, is not involved
in adipocyte specificity because inactivation of this site did not
affect transgene expression. Moreover, electrophoretic mobility shift
experiments indicated that, unlike PCK2, PCK1 is not selective for
PPAR
/RXR binding. To characterize the enhancer further, the rat and
human PEPCK 5'-flanking DNA sequences were compared by computer and
found to have significant similarities in the enhancer region. This high level of conservation suggests that additional transcription factors are probably involved in enhancer function. A putative human
PCK2 element was identified by this sequence comparison. The human and
rat PCK2 elements bound PPAR
/RXR with the same affinities. This work
provides the first in vivo evidence that the binding of
PPAR
to its target sequences is absolutely required for
adipocyte-specific gene expression.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
888 bp is required for expression of the PEPCK
promoter in white adipose tissue of transgenic mice (7). In contrast,
normal expression of the PEPCK promoter in liver was obtained with DNA
elements downstream of
600 bp of the promoter (7). These observations
and the fact that the PEPCK gene is a single-copy gene that is
transcribed from the same promoter in both tissues indicate that there
are separate and distinct hepatocyte- and adipocyte-specific enhancers that control PEPCK gene transcription (6).
, PPAR
, and PPAR
. All three PPARs activate
genes through their binding (as heterodimers with retinoid X receptors
(RXR)) to single-spaced direct repeat (DR1) hormone response elements
(5'-AGGTCANAGGTCA-3') (8). PPAR
is most abundant in the liver and in
various cells of the immune system where leukotriene B4 has
been shown to be a natural activating ligand (9). PPAR
is ubiquitous
in its distribution, but its function and natural activating ligands
are unknown (8).
is most abundant in
adipose tissue and activated macrophages (10). It is a key factor
responsible for differentiation of the adipocyte lineage during
development (for review, see Ref. 10), and it is antiinflammatory and
causes apoptosis in macrophages (11). Its activating ligands include
the thiazolidinedione class of antidiabetic drugs and the natural
compound, 15-deoxy
12,14 prostaglandin J2
(12, 13).
/RXR via two
DR1 elements (14). We subsequently found two PPAR
/RXR binding sites,
designated PCK1 and PCK2, upstream of the PEPCK promoter (15). PCK1 is
centered at
446 bp, and PCK2 is at
992 bp, which is within the
region required for PEPCK activation in adipocytes. Transient
transfection assays in 3T3-F442A adipogenic cells and NIH-3T3
fibroblasts showed that PCK2 is essential for differentiation-dependent enhancer activity and for
transactivation by PPAR
/RXR (15).
888 bp constitutes a bona fide
adipocyte-specific enhancer and whether PCK2 is required for its
activity in transgenic mice. We also asked whether the proximal DR1
element, PCK1, is important for adipocyte-specific expression of the
PEPCK gene. We have identified the 413-bp region between
1242 and
828 bp as an adipocyte-specific enhancer. This enhancer requires an
intact PCK2 element, and it functions most efficiently in the context of the PEPCK promoter, suggesting that there are gene- and/or tissue-specific interactions between PPAR
and other factors involved in regulating transcription. This work provides the first evidence that
PPAR
is required for adipogenic gene expression in
vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PC112, described previously (16). The
5000 hGH construct is an
EcoRI-BglII fragment extending from
5000 bp to
+69 bp inserted into the hGH vector, p0GH (17), digested with
EcoRI and BamHI. The
2086 hGH construct, which
has been described before (7), is a
2086 bp to +69 bp
SstI-BglII fragment inserted into p0GH. The
888 hGH construct, also described previously (7), is a
888 bp to +69 bp
Bal-31 deletion end point BglII fragment inserted into p0GH.
The remaining six constructs all utilized hGX as the reporter (18).
This reporter was made by generating a frameshift mutation in the
translated sequence of hGH such that the encoded protein is
biologically inactive (18). The
2086 hGX construct is identical to
the
2086 hGH construct except for the hGX mutation in the reporter.
The
1330 hGX construct is identical except that it terminates at a
BsmI site at
1330 bp. The "Enhancer hGX" construct is
a
1240 to
830 bp TaqI-BamHI fragment joined
to the hGX gene at
90 bp. The promoter in this vector is therefore a
minimal promoter (extending to
90 bp) from the hGH gene. Finally, the three genes bearing PPAR
/RXR binding site mutations, PCK2M, PCK1M, and PCK1+2M, were all generated from the
2086 hGX construct. The PCK2
mutation (PCK2M) was inserted by swapping a
2086 bp to
14 bp
SalI-NheI fragment from a PCK2M-CAT
(chloramphenicol acetyltransferase) construct (15) for the same
fragment in
2086 hGX. The sequence of PCK2M was
5'-GGGATCCTGGTCT-3', whereas the wild type PCK2 sequence is
5'-GGGATAAAGGTCT-3' (the mutated sequences are underlined). PCK1M was
introduced via site-directed mutagenesis as described by Kunkel
et al. (19). The sequence of PCK1M was 5'-TGACGATCGGCCG-3', whereas the wild type PCK1
sequence is 5'-TGACCTTTGGCCG-3' (note that this DR1 is in the opposite
orientation from PCK2). In every case, the transgenes were digested and
gel purified from plasmid vectors prior to injection of DNA into
fertilized mouse eggs as we described previously (7).
70 °C. Total RNA was extracted from
the frozen tissues by the acid-guanidinium thiocyanate-phenol method
(20) using Tri-ReagentTM (Molecular Research Laboratories, Cincinnati,
OH) according to the manufacturer's instructions. PEPCK and hGH or hGX
mRNAs were then assayed by either one of two methods. The first
method was a solution hybridization-RNase protection assay described
previously (7, 21) which was designed to assess the average number of
mRNA molecules/cell. Briefly, the hybridized, RNase-resistant probe
was collected by filtration of a trichloroacetic acid precipitate.
Filter-bound radioactivity was then measured by scintillation counting.
The molar amount of mRNA was calculated by comparison with a
standard curve prepared using known amounts of pure target RNA (PEPCK
or hGH). The second method was a standard RNase protection assay
utilizing a kit purchased from Ambion (Austin, TX) according to the
manufacturer's instructions. This method is more sensitive and allowed
for the simultaneous assay of PEPCK, hGH (or hGX), and
-actin
mRNAs in a single tube.
-actin probe was supplied in the RNase protection assay kit from
Ambion. The
-actin cRNA transcript is 133 bp in length, whereas the
RNase-protected product is a 161-bp fragment.
-SPORT and PPAR
2-SPORT (15) plasmids
was performed with the TNT SP6-coupled reticulocyte lysate system
(Promega) as recommended by the manufacturer. 1 µl of the 50-µl
translation product was used in each binding reaction. 3T3-L1 preadipocyte and adipocyte nuclear extracts were prepared as described previously, except that extracts were not dialyzed but were added directly to the binding reaction mixture (15). Final protein concentrations were typically 1-2 µg/µl.
-32P] dGTP
(DuPont Easytides, 800 Ci/mmol) using the large fragment of
Escherichia coli polymerase I (Klenow fragment). The
unlabeled rat PCK2 double-stranded oligonucleotide probe thus includes
the 13-bp DR1 sequence, six 5'- and two 3'-flanking bp, plus
BamHI-compatible extensions at both ends. The human PCK2 and
rat PCK1 probes were prepared in the same manner. Their sequences were
as follows.
(directed
against amino acids 214-229) obtained from R. Evans, Salk Institute
(23); 2) polyclonal rabbit anti-mouse PPAR
2 described previously
(24); and 3) normal rabbit serum purchased from Sigma. DNA-protein
complexes were resolved on 5% polyacrylamide gels (30:1
acrylamide:bisacrylamide) in 0.5 × TBE (1 × TBE, 50 mM Tris borate (pH 8.3), 1 mM EDTA). The gels
were dried and exposed to a Phosphor screen overnight. Quantitation was
performed with a PhosphorImaging device (Molecular Dynamics).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2086 to +69 bp
construct that we used previously (7). The two "
2086" transgene
constructs are shown in Fig.
1A. Transgene expression in
white adipose tissue and liver of these two groups of mice was measured
by quantitative solution hybridization assay, and the results are shown
in Table I and Fig. 1, B and
C. There was no statistically significant difference in
transgene expression between the hGH and the hGX group in either liver
or adipose tissue (p = 0.65 and 0.28 for liver and
adipose tissue, respectively). This supports our previous conclusion
that hGH does not confound the conclusions and demonstrates that
comparisons between hGX and hGH transgenes are valid. It does appear
that there could be a slight, albeit statistically insignificant,
inhibitory effect of expression in adipose tissue, however. To avoid
potential unanticipated artifacts caused by hGH we have prepared all
subsequent transgenes with hGX.
View larger version (22K):
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Fig. 1.
An adipocyte-specific element is localized
between 1330 and
888 bp. Panel A, the five
transgene constructs analyzed are depicted. All constructs are
designated by their upstream deletion end points and the reporter gene
used. The two different reporter genes were hGH and hGX (designated by
the bullet,
), which encodes a biologically inactive
protein (18). The filled regions represent PEPCK DNA from
the indicated upstream deletion end point to +69 bp in exon 1. This
downstream end point is upstream of the initiator methionine codon. The
unfilled regions represent the reporter genes (hGH or hGX).
The wide lines and boxes represent transcribed
DNA (+1 is indicated by the arrow); the thick
lines represent upstream DNA. Details of these vectors are given
under "Experimental Procedures." Panel B, the number of
hGH or hGX transcripts/cell in white adipose tissue from each group of
transgenic mice was measured by a solution hybridization assay as
described under "Experimental Procedures." The results are
presented as the average ± S.E. for each group of mice. The
actual values for each mouse are shown in Table I. Panel C,
same as panel B except that the results for liver are
shown.
Transgene expression in individual mice
2086 hGH in WAT (p = 0.05); b, different from
1330 hGX in liver (p = 0.05); c, different from
1330 hGX in WAT (p = 0.02); d, different from all
other transgene groups (p = 0.0001); e, marginal
difference from
5000 hGH in liver (p = 0.06). There
were no other significant differences between groups.
1330 and
888
Base Pairs Upstream of the PEPCK Promoter--
In addition to the two
2086 bp constructs just described, Fig. 1 also shows the structures
and expression levels of two new constructs,
5000 bp hGH and
1330
bp hGX, which have not been reported previously. Moreover, the
888 bp
hGH construct described previously (7) is shown for comparison purposes.
888 bp and
2086 bp because the
2086 but not the
888
transgene (Fig. 1A) was expressed in adipose tissue (Fig. 1B, Table I, and Ref. 7). Considering this observation we prepared the
1330 bp hGX construct (Fig. 1A) and found
that it is expressed in both adipose tissue and liver (Fig. 1,
B and C). This localized the adipose-specific
element between
1330 bp and
888 bp.
5000 bp hGH construct was prepared to test whether important
regulatory elements are located upstream of
2086 bp, the most distal
end point we had tested previously. We used hGH as the reporter in this
construct because these animals were prepared prior to our having
tested the hGX reporter. Compared with the
2086 hGH mice, transgene
expression in the
5000 hGH animals was significantly higher in
adipose tissue (p = 0.05), but not liver
(p = 0.30), indicating that this upstream region
contains one or more stimulatory elements that function in adipocytes. However, transgene expression in the liver of the
5000 hGH animals was significantly higher than in the
1330 hGX mice (p = 0.05). This indicates the presence of a stimulatory "liver
element" somewhere between
5000 bp and
1330 bp.
1240 Base Pairs and
830 Base Pairs Is an
Adipocyte-specific Enhancer in Vivo--
Transient transfection assays
in cultured cells suggested the presence of a
"differentiation-dependent" enhancer between
460 bp and
2086 bp (15). This enhancer required a PPAR
/RXR-binding element,
PCK2, centered at
992 bp (15). In the present study we asked whether
this differentiation-dependent enhancer had the characteristics of an adipocyte-specific enhancer. To test this possibility we employed convenient restriction sites to generate the
enhancer construct illustrated in Fig.
2A. Analysis of a total of six
founder mice bearing the "enhancer" construct showed that it is
expressed only in white and brown adipose tissue and not in liver,
kidney, or intestine, the only other tissues known to express the PEPCK
gene at high levels (Fig. 2, B and C).
Panel B shows both PEPCK and transgene expression in nine
tissue samples of a single founder animal. The same pattern of
expression was seen in the other five founders, but only three tissues
are shown for all six animals in panel C. Because the
1240/
830 bp PEPCK region directs transgene expression from a
heterologous minimal promoter at a distance of only 90 bp from the
promoter, instead of the native 830 bp, we have designated this element
as an adipocyte-specific enhancer.
View larger version (50K):
[in a new window]
Fig. 2.
The upstream element is an adipocyte-specific
enhancer in vivo. Panel A, the two
transgene constructs analyzed are depicted as in Fig. 1. The Enhancer
construct consists of PEPCK DNA from 1240 to
830 bp fused at
90
bp of the basal hGH promoter; this fusion is indicated by the
thin bent line. The
1330 construct is the same as that
shown in Fig. 1. Panel B, PhosphorImage of an RNase
protection assay for both hGX and PEPCK mRNAs in total RNA samples
isolated from nine tissues of a representative enhancer mouse (founder
number 360-3). The RNase-protected products representing the respective
mRNAs are designated by labeled arrows. The tissues are:
liver (Liv), kidney (Kid), white adipose tissue
(WAT), brown adipose tissue (BAT), ileum
(Ile), skeletal muscle (SkM), jejunum
(Jej), colon (Col), and lung (Lun).
Details of the RNase protection assay are described under
"Experimental Procedures." Panel C, same as panel
B except that hGX mRNA in total RNA samples isolated from
liver (L), kidney (K), and white adipose tissue
(F) from all six enhancer mice is shown. The individual
mouse numbers are indicated. Panel D, same as panel
B except that hGX and PEPCK mRNAs in total RNA samples
isolated from white adipose tissue from all six enhancer mice and all
eight
1330 mice are shown. The far right lane
(P) shows the undigested PEPCK and hGX cRNA probes.
2086/hGX or the
1330/hGX mice. This
difference is illustrated in Fig. 2D, which shows the results of an RNase protection assay performed on white adipose tissue
RNA from the six enhancer founders and the eight
1330 founders. The
average PEPCK mRNA concentration was the same for both groups of
animals, but transgene expression was 10-20-fold higher in the
1330
mice. This could be a positional effect of moving the enhancer too
close to the promoter for optimal interaction of transcription factors
and cofactors. Alternatively, it could be that optimal transcription of
the PEPCK gene requires the interactions of enhancer-bound factors with
promoter-associated proteins.
/RXR binding sites, PCK1 centered at
446 bp,
and PCK2 centered at
992 bp, and we hypothesized that expression of
PPAR
, an adipocyte-specific transcription factor, is responsible for
turning on PEPCK gene expression via PCK2 during adipogenesis (15).
2086/hGX (Fig. 3, A-D).
Fig. 3A shows an RNase protection analysis of both PEPCK and
hGX mRNAs in white adipose tissue and liver from nine founder mice
with the wild type PEPCK construct. The relative level of transgene
expression in the liver corresponded to the level of expression in
adipose tissue in these mice. By contrast, in seven founder PCK2M mice,
the 3-bp mutation in PCK2 eliminated transgene expression in white
adipose tissue but not in liver (Fig. 3B). A similar pattern
of expression was also seen in brown adipose tissue (not shown). Thus
PCK2 is essential for expression of the PEPCK gene in both white and
brown adipocytes.
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Fig. 3.
PCK2 but not PCK1 is essential for activity
of the adipocyte-specific enhancer. The figure shows
PhosphorImages of RNase protection assays for both hGX and PEPCK
mRNAs (as in Fig. 2D) in total RNA samples isolated from
white adipose tissues and livers of nine 2086 hGX mice (panel
A; the same animals used in Fig. 1 and Table I), seven PCK2M mice
(panel B), four PCK1M mice (panel C), and six
PCK1+2M mice (panel D). The four transgene constructs
analyzed are depicted as in Fig. 1A at the top of
their respective panels. The two DR1 sites that bind
PPAR
/RXR heterodimers, PCK1 at
446 bp and PCK2 at
992 bp, are
indicated by open circles in the upstream DNA regions.
Mutations of these elements are depicted by an X over the
symbol. The RNase protection assays and PhosphorImage exposures for all
four panels were done simultaneously so that all panels are directly
comparable.
/RXR Binds PCK2--
We
previously used an RXR
-specific antibody to show by electrophoretic
mobility supershift analysis that RXR
is a component of the binding
species from adipocyte nuclear extracts. Since that time a
PPAR
-specific antibody has been produced (24). These two antibodies
were used in the experiment shown in Fig. 4 to demonstrate that virtually all of
the PCK2-binding species contain both PPAR
and RXR. Moreover, there
is no detectable PCK2 binding activity in preadipocyte nuclear extracts
which, along with the above data, supports the idea that binding of
PPAR
/RXR heterodimers to PCK2 activates the adipocyte-specific PEPCK
enhancer during adipogenesis.
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Fig. 4.
PCK2 is bound exclusively by
PPAR /RXR heterodimers in adipocyte nuclear
extracts. A PhosphorImage of electrophoretic mobility shift
reactions using PCK2 as the probe is shown. The sources of added
protein were: in vitro translated recombinant PPAR
and
RXR
(lanes 1-4), preadipocyte nuclear extracts
(lanes 5 and 6), and adipocyte nuclear extracts
(lanes 7-10). Other additions to the binding reactions
included: no additions, normal rabbit serum (NRS), antiserum
to PPAR
(PPAR
Ab), or antiserum to RXR
(RXR
Ab) as indicated. The arrow indicates the
migration of PPAR
· RXR
·PCK2 complexes. Free probe is seen
at the bottom of each lane. Details of the assay
are described under "Experimental Procedures."
/RXR binding sites, PCK2,
is involved in adipocyte-specific expression of the PEPCK promoter.
Because PPAR
/RXR heterodimers bind PCK1 and PCK2 with approximately
equivalent affinities (15) we asked whether the two sites differed in
their selectivities for different binding proteins. The mobility shift
experiment shown in Fig. 5 indicates that
this is indeed the case because: 1) with preadipocyte nuclear extracts
there was no detectable binding to PCK2, whereas there were several
species bound to PCK1; and 2) with adipocyte nuclear extracts the
banding patterns displayed both similarities and differences between
PCK1 and PCK2.
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Fig. 5.
PCK2 is selectively bound by factors in
adipocyte but not preadipocyte nuclear extracts, whereas PCK1 is bound
by factors in both extracts. PhosphorImages of electrophoretic
mobility shift reactions using PCK1 and PCK2 as probes are shown. The
sources of added protein were in vitro translated,
recombinant PPAR and RXR
(R); 3T3-L1 adipocyte nuclear
extract (A); and 3T3-L1 preadipocyte nuclear extract
(P). The probes were rat PCK1 (lanes 1-3) and
rat PCK2 (lanes 4-6). The arrow indicates the
migration of recombinant PPAR
·RXR
·PCK2 complexes. Free probe
is seen at the bottom of each lane. Details of
the assay are described under "Experimental Procedures."
/RXR are involved in making the region between
1242 and
828
bp an adipocyte-specific enhancer. Because important regulatory
sequences are often conserved evolutionarily we compared the rat with
the human PEPCK promoters and upstream DNAs as part of our strategy to
dissect and characterize the enhancer. The dot plot in Fig. 6A shows that there are three
significant regions of similarity: the promoter-proximal region of
approximately 500 bp; an intermediate region between approximately
600 and
800 bp; and a distal region upstream of
900 bp within the
adipocyte-specific enhancer. The exact sequence alignment of the
enhancer region is shown in Fig. 6B. This alignment revealed
a putative human PCK2 element. This element matches rat PCK2 at 10 of
the 13 nucleotides that constitute the DR1. It is clear that the
5'-flanking nucleotides are critical to PPAR
/RXR binding to DR1
elements (26). The rat and human PCK2s are identical at 11 of 12 5'-flanking nucleotides (whereas rat PCK1 and PCK2 are identical at
only 3 of 12, which may explain the differences seen in Fig. 5). To
determine whether PPAR
/RXR binds human PCK2 the mobility shift
experiment shown in Fig. 7 was performed.
The results of this experiment show that the binding affinities of the
rat and human elements are virtually identical.
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Fig. 6.
The rat and human adipocyte-specific
enhancers are highly homologous. Panel A, the human and
rat PEPCK promoters from 1300 to
1 bp were compared using COMPARE
and DOTPLOT in the Wisconsin Package. A dot was placed at every
coordinate containing identical nucleotides within windows of 21 bp
containing a minimum of 14 matches (stringency). The box
encloses the enhancer regions that are shown in panel B. The
locations of the PCK2 elements are marked with a solid box
on each axis. Panel B, the rat enhancer region defined by
the experiment in Fig. 2 was compared with the human PEPCK promoter
using GAP and PILEUP in the Wisconsin Package. The optimal alignment is
shown here. PCK2 and three additional potential PPAR
/RXR binding
sites on the rat DNA are shaded. Potential C/EBP binding
sites, sequences containing the ATTGCGCAAT consensus with up to four
mismatches, are overlined for the rat and
underlined for the human sequences. The GenBank Accession
numbers for the DNAs used in this figure were K03243 (rat) and U31519
(human).
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Fig. 7.
PPAR /RXR
binds
rat PCK2 and human PCK2 with equal affinity. A PhosphorImage of
electrophoretic mobility shift reactions using either rat or human PCK2
as probe is shown. The source of added protein is indicated at the
top of the figure: no lysate, unprogrammed rabbit
reticulocyte (RR) lysate, or lysate programmed with
mRNAs encoding PPAR
and RXR
. The probes were rat PCK2
(lanes 1-10) and human PCK2 (lanes 11-20).
Unlabeled competitors were added as indicated: no competitor;
unlabeled rat or human PCK2 at 10-, 50-, and 100-fold molar excess; or
a nonbinding mutant rat PCK2 (m) at 100-fold excess. The
arrow indicates the migration of PPAR
·RXR
·PCK2
complexes. Free probe is seen at the bottom of each
lane. Details of the assay are described under
"Experimental Procedures."
/RXR. Indeed there were three
putative PPAR
/RXR binding sites in addition to PCK2 (Fig.
6B). One might expect that factors bound at multiple sites
could strengthen the enhancer. In support of this idea is the
adipocyte-specific enhancer of the aP2 gene which has two PPAR
/RXR binding sites (29). However, mobility shift assays such as
those in Fig. 7 showed that none of these additional sites binds
PPAR
/RXR (data not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/RXR to PCK2--
In this study we have identified as an
adipocyte-specific enhancer a 410-bp region located 1 kilobase pair
upstream of the PEPCK promoter. Expression of the PEPCK promoter in
adipocytes is totally dependent upon the presence of this enhancer.
Like that of the aP2 gene (29), the adipocyte-specific
enhancer of the PEPCK gene contains a DR1 element, PCK2, that binds a
PPAR
/RXR heterodimer. More importantly, we show that PCK2 must be
intact for this enhancer to function. This latter observation, coupled with the observation that PPAR
and RXR are the only factors in adipocyte nuclear extracts which bind PCK2, provides strong evidence that PPAR
/RXR is essential for expression of the PEPCK gene in adipocytes.
1330 hGX construct.
One possible explanation for this is that the spacing between the
enhancer and the promoter, which has been modified, is critical for
optimal enhancer function. However, the explanation that we prefer is that the PEPCK adipocyte-specific enhancer functions most efficiently in the context of the PEPCK promoter because of gene and/or
adipocyte-specific interactions among proteins, perhaps PPAR
, bound
to the enhancer, with promoter-associated transcription factors or cofactors.
in adipocytes and other
PPAR isoforms might serve as fatty acid-regulated transcriptional activators through their binding to PCK2 in hepatocytes. PCK2 could
thus serve a dual function: as part of a developmental switch to
activate the PEPCK gene during adipogenesis and as a fatty acid-responsive switch to regulate the PEPCK gene in adipocytes (and
possibly hepatocytes) subsequent to embryonic and fetal development. It
has yet to be determined, however, whether PPAR
and/or PPAR
binds
to PCK2 in hepatocytes or other cell types.
2086 Base
Pairs?--
Chalkley and colleagues (32,33) concluded that a
hepatocyte-specific element that binds a CREB/ATF factor and Pep A is
located at
4800 bp. Our observation that there is a significant
difference in liver between the
5000 hGH and the
1330 hGX mice is
consistent with this conclusion; however, additional transgenic animals
would need to be produced to achieve the statistical power necessary to
confirm this issue. Our data do suggest that one or more elements upstream of
2086 bp are involved in PEPCK regulation in adipocytes because there was a significant increase in transgene expression when
5000 bp of upstream DNA was included.
4800 bp
may be a liver-specific locus control region that causes transgene
expression to be proportional to the number of integrated copies (32,
33). Linear regression analysis of the data in Table I revealed a
significant correlation between copy number and transgene expression in
both liver (r2 = 0.79) and white adipose tissue
(r2 = 0.81) of the
5000 hGH mice. There was a
weaker, albeit significant, correlation in the liver
(r2 = 0.62) and white adipose tissue
(r2 = 0.64) of the
2086 hGX mice. This finding
is consistent with but does not prove the presence of liver and adipose
tissue locus control regions upstream of the PEPCK gene.
/RXR binding site, is not required for
PEPCK expression in vivo, even though it is also bound by
PPAR
/RXR heterodimers in vitro (Figs. 3 and 5). A
possible explanation was suggested by electrophoretic mobility shift
experiments that revealed that PCK2 is highly selective in its binding
to PPAR
/RXR in adipocyte nuclear extracts, and it is unoccupied in
preadipocyte nuclear extracts (Figs. 4 and 5). PCK1, on the other hand,
is bound by several factors in both preadipocyte and adipocyte nuclear
extracts (Fig. 5). This difference in factor selectivity is probably
related to contextual differences in the flanking nucleotide sequences
and/or the nonconsensus half-site differences between the two DR1s. The
5'- flanking sequence of PCK2 is more similar to an optimal binding
sequence for PPAR
/RXR (26) compared with PCK1. Also, PCK1 is in the
opposite orientation from PCK2, and this inverted polarity could
potentially contribute to its function in vivo. Nonetheless,
the available data indicate that PCK1 and PCK2 have differing
functions. We previously pointed to the fact that PCK1 is the same as
the AF-1 site identified by Granner and colleagues (34) as part of a
complex glucocorticoid-responsive unit in hepatoma cells. They
demonstrated that with hepatoma cell extracts PCK1/AF-1 is occupied by
several factors including RAR, RXR, HNF4, and COUP-TF (35, 36). An
interesting enigma is the fact that glucocorticoid hormones induce
PEPCK transcription in hepatocytes but repress it in adipocytes (1, 2).
Perhaps it is the tissue-specific difference in the factors bound to
PCK1/AF-1 which renders the complex glucocorticoid response unit an
activator in hepatocytes and an inhibitor in adipocytes.
600 and
800 bp. This will require further investigation. The distal homology
we have defined here is an adipocyte-specific enhancer. This region
must also be the target of future studies.
/RXR to its cognate
binding site. Studies are now under way to define the minimal enhancer
and to understand the molecular mechanisms that modulate and mediate
the adipocyte-specific actions of PPAR
and RXR.
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ACKNOWLEDGEMENTS |
---|
We thank Ida Schaefer, Joan Clapper,
and Matthew Laverdiere for technical assistance; Drs. Walter
Wahli and Simon C. Williams for helpful comments on the manuscript; Dr.
John J. McGlone for guidance with statistical analyses; Dr. G. Stanley McKnight for the hGX vector; and Dr. Ronald M. Evans
for the antibody directed against RXR and the RXR
-SPORT
expression vector.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant R01GM39895 and Texas Advanced Research Program Grant 010674 (to E. G. B.) and by the Howard Hughes Medical Institute and Perot Family Foundation funds (to R. E. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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 GenBankTM/EMBL Data Bank with accession number(s) K03243 for rat PEPCK promoter with PCK1 and PCK2, K03248 for rat PEPCK exon 10, and M13438 for hGH exon 2/intron 2.
¶ Present address: Howard Hughes Medical Institute, Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75235-9050.
** Present address: The Salk Institute for Biological Studies, La Jolla, CA 92037.
To whom correspondence to should be addressed. Tel.:
806-743-2705; Fax: 806-743-2990; E-mail: bioegb{at}ttuhsc.edu.
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ABBREVIATIONS |
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The abbreviations used are: PEPCK, cytosolic phosphoenolpyruvate carboxykinase; bp, base pair(s); PPAR(s), peroxisome proliferator-activated receptor(s); RXR, 9-cis-retenoic acid receptor; DR1, single-spaced direct repeat; hGH, human growth hormone; hGX, frameshift mutant of hGH encoding a biologically inactive protein; bGH, bovine growth hormone.
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REFERENCES |
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