From the
The gene encoding cytochrome P-450 4A6 (CYP4A6) is
transcriptionally activated by peroxisome proliferators. This response
is dependent on a strong enhancer element (Z) and weaker elements (X
and -27). The peroxisome proliferator response is mediated by the
binding of heterodimers containing the peroxisome
proliferator-activated receptor
Peroxisome proliferators are non-genotoxic carcinogens that
cause profound changes in the subcellular structure and lipid
metabolism of hepatocytes in rodents(1, 2) . They are a
structurally diverse group of chemicals that include hypolipidemic
drugs, plasticizers, and herbicides. A well characterized biochemical
marker for the action of peroxisome proliferators is the induction of
the enzymes that catalyze the peroxisomal
We have previously identified
three peroxisome proliferator response elements (PPRE) in the CYP4A6
gene(5, 6) . The most distal element (X) provides a weak
response in the presence of cotransfected PPAR
In this
study, we demonstrate that the sequence requirements for the binding of
PPAR
As previously shown(5) , cotransfection of CYP4A6
reporter constructs pLUCA6-880 and pLUCA6-663 (Fig. 1) with an expression vector for PPAR-G (a mutant form of
mPPAR
To test the effects of these mutations on
transcriptional responses to peroxisome proliferators,
oligonucleotide-directed mutagenesis was used to generate the above
mutations in the 5`-flanking regions of the CYP4A6 gene. Cotransfection
of mutant CYP4A6 reporter constructs and the expression vector for
PPAR
Oligonucleotides containing a perfect consensus
DR1 and either a consensus or divergent extended 5`-binding site (DR1P
and DR1M, respectively) were assayed for their ability to bind
RXR
The addition of ARP-1 to the
DR1P and DR1M oligonucleotide pairs results in the formation of a
single complex (Fig. 6A) that presumably consists of a
homodimer (D) of ARP-1. A complex of similar mobility is seen
for the other elements; however, complexes of higher mobility (M) are seen for the APOCIIIB, ACOXA, FABP, and
CYP4A6-27 elements. These complexes may consist of monomeric
ARP-1 binding to the single consensus half sites seen in these
elements. The binding of ARP-1 to the PPREs that contain two divergent
half sites (Fig. 6B, Z, BIF, HMG) is greatly reduced
compared to the other elements.
These results demonstrate that the
extended binding site is not required for the binding of
PPAR
To assess the specificity of these
elements in a complex mixture of transcription factors, mouse liver
total protein was assayed for binding to the Z element and the APOCIIIB
element (Fig. 7). The binding studies with the individual
receptors described above suggested that the APOCIIIB element would be
less likely to bind PPAR
Previous studies have identified three PPREs in the
5`-flanking sequences of the CYP4A6 gene; each of these contain
directly repeated imperfect copies of the nuclear receptor binding
sequence AGGTCA(5, 6) with a spacing of one nucleotide
(DR1). The Z element has been shown to mediate the major proportion of
the response to peroxisome proliferators by CYP4A6 reporter constructs
in transient expression assays. This element binds
PPAR
The additional sequence
immediately 5` of the DR1 motif is partially conserved between PPREs
and appears to differ from the AGGTCA motif, which constitutes the
recognition sites for other members of this family of receptors. The
consensus derived from the known PPRE sequences (C(A/G)(A/G)A(A/T)CT)
and the mutational analysis described here suggests that the
5`-extended binding site for PPAR
This analysis of the
effects of the extended binding site on the binding of
PPAR
The presence of one non-consensus and one consensus half site is
seen in the DR1 of many natural elements. These elements display
markedly reduced RXR
The presence of two non-consensus
half sites in the DR1 is a common feature of natural PPREs. The high
efficiency PPREs from the CYP4A6 (Z), bifunctional enzyme, and the
3-hydroxy-3-methylglutaryl-CoA synthase genes are examples of elements
that contain two non-consensus half sites. These elements display very
weak binding to RXR
The lower efficiency PPREs, X and -27,
have mismatches in the half sites that are not observed in other PPREs
and display reduced PPAR
From
the above observations, it appears that the upstream, extended binding
site sequence allows the maintenance of PPAR
We express appreciation to Dr. D. J. Mangelsdorf and
Dr. R. Evans (Salk Institute, La Jolla, CA) for providing the RXR
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(PPAR
) and the retinoid X
receptor
(RXR
) to these elements. These peroxisome
proliferator response elements (PPREs) contain imperfect direct repeats
of the nuclear receptor consensus recognition sequence with a spacing
of one nucleotide (DR1) (AGGTCA N AGGTCA). This DR1 motif is seen in
the binding sites for other nuclear receptor complexes, such as ARP-1,
HNF-4, and RXR
homodimers. Mutational analysis of the Z element
reveals that the DR1 motif is required for the transcriptional
activation of the CYP4A6 gene by peroxisome proliferators; however,
deletion of sequences immediately upstream of this motif also abolishes
this response. Oligonucleotides corresponding to truncated and mutated
Z elements were assayed by gel retardation for binding to RXR
,
PPAR
, and ARP-1. Deletions or mutations within six nucleotides 5`
of the DR1 motif dramatically diminish PPAR
RXR
binding
without reducing the binding of either RXR
or ARP-1 homodimers,
whereas mutation or deletion of the core DR1 sequences abolishes the
binding of PPAR
RXR
heterodimers and of RXR
or
ARP-1 homodimers. Thus, the DR1 motif in the Z element is not
sufficient to constitute a PPRE. Moreover, the binding of
PPAR
RXR
to the Z element requires sequences immediately
5` of the DR1. These sequences are conserved in natural PPREs and
promote binding of PPAR
RXR
heterodimers in preference
to potential competitors such as ARP-1 and RXR
.
-oxidation and
microsomal
-hydroxylation of fatty acids. Recent studies have
shown that peroxisome proliferators induce transcription of the rat
fatty acid acyl-CoA oxidase gene (3, 4) and the rabbit
CYP4A6 fatty acid
-hydroxylase gene (5) via an orphan
member of the nuclear receptor superfamily, the peroxisome
proliferator-activated receptor (PPAR
).
(
)PPAR
has been shown to act by binding to specific
sequences in the 5`-flanking sequence of the acyl-CoA oxidase and
CYP4A6 genes, and this binding requires the presence of accessory
proteins such as the retinoid X receptor
(RXR
)(4, 6) .
, and deletion of
this element from CYP4A6 reporter constructs results in a 2-fold drop
in response to peroxisome proliferators(5) . Deletion of the Z
element from CYP4A6 reporter constructs results in the abolition of
significant peroxisome proliferator responses in the presence of
cotransfected PPAR
. The proximal element(-27) is a cryptic
element in that it is only activated in cotransfection studies when
RXR
is overexpressed together with PPAR
. The cryptic nature
of the -27 element has been shown to be due to the fact that it
binds PPAR
RXR
very inefficiently(6) . These and
other PPRE sequences contain imperfect direct repeats of the consensus
binding site for the nuclear receptor superfamily (AGGTCA)(7) ,
with a spacing of one nucleotide
(DR1)(4, 5, 8, 9, 10) . These
direct repeats are known to bind potential competitors such as
homodimers of other nuclear receptors including RXR
, ARP-1,
chicken ovalbumin upstream promoter transcription factor-1, and hepatic
nuclear factor-4(11, 12, 13) . This would
suggest a level of promiscuity in the regulation of the genes
controlled by these elements, and, in fact, ARP-1 and chicken ovalbumin
upstream promoter transcription factor-1 have been shown to antagonize
peroxisome proliferator signaling(6, 14) .
RXR
to the CYP4A6 Z element are distinct from those
seen for potential competitors such as homodimers of ARP-1 and RXR
and that sequences 5` of the DR1 contribute to selective binding of
PPAR
RXR
. These additional sequences form an extended
binding site that is conserved within natural PPREs.
Transient Transfection Experiments
The
RK13 cell line was obtained from the American Type Culture Collection
and maintained in minimal essential medium containing Earle's
balanced salt solution (Life Technologies, Inc.) and 10% fetal calf
serum (Gemini, Calabasas, CA). Luciferase reporter plasmids
pLUCA6-880 and pLUCA6-155 (5) as well as the
expression constructs pCMV-PPAR, pCMV-PPAR
-G(5) ,
pRS-RXR
(15) , pMT-ARP-1(16) , and pSV
Gal
(Promega) have been previously described. The reporter and expression
constructs were introduced into cultured cells by a modification of the
calcium phosphate coprecipitation procedure(17) . After a 16-h
exposure, the DNA containing culture medium was removed, and the cells
were washed twice with minimal essential medium and then exposed to
culture medium with either Wy-14,643 (50 µM) or the
equivalent volume of solvent (ME
SO, 0.25% (v/v) final
concentration). This medium was replaced after 24 h. After another 24
h, the cells were harvested, washed with phosphate-buffered saline
(0.01 M sodium phosphate, pH 7.4, 0.15 M NaCl), and
then lysed by suspension in 0.1 M potassium phosphate buffer,
pH 7.8, containing 1 mM dithiothreitol and 0.05% Triton X-100
followed by 3 cycles of freezing and thawing. Insoluble material was
removed by centrifugation, and luciferase activity was determined using
a Monolight 2010 luminometer (Analytical Luminescence Laboratory, San
Diego, CA).
-Galactosidase activities were determined as described
(5). The luciferase activity obtained for individual cultures was
expressed relative to the
-galactosidase activity obtained for the
same preparation of lysate.
Expression of PPAR
The mPPAR/GST, RXR
/MBP, and
ARP-1/MBP Fusion Proteins and Non-fusion PPAR
in Escherichia
coli
, RXR
, and ARP-1 receptors were
expressed as fusion proteins and affinity purified as previously
described(6) . The mPPAR
cDNA was modified by PCR to
contain an NdeI site at the initiator ATG. This allowed the
cloning of the cDNA into the pCW vector (18) and subsequent
expression of the PPAR
protein. The PPAR
cDNA was also
modified at the second codon to encode an alanine residue (GCT), and a
silent mutation was also incorporated into the fifth codon. These
modifications have been previously shown to increase the expression of
mammalian proteins in E. coli(18) . The upper primer
corresponds to
5`-CCATCGATCATATGGCTGACACAGAAAGCCCCATCTGTCCT-CTCTCCCCACTGGAGGCAGATGAC-3`
(bold type indicates changes from the published sequence). The lower
primer was that used in the isolation of the mPPAR
cDNA(5) . The growth conditions, induction, and lysis of E.
coli containing this plasmid were as described for the fusion
proteins(6) . The 100,000
g supernatants from
the bacterial lysates were used directly in the EMSA.
EMSA
Oligonucleotides were end labeled
with P using T4 polynucleotide kinase, and the
complementary strands were annealed. Each double-stranded, labeled
oligonucleotide was then purified by electrophoresis in a 20%
polyacrylamide gel using 89 mM Tris borate buffer, pH 8.0,
containing 2 mM EDTA (1
TBE). Following excision of
the portion of the gel containing the labeled oligonucleotide, the
probe was eluted from the gel with 0.5 M NH
OAc, 1
mM EDTA, and 1% SDS and was collected following precipitation
with ethanol. For EMSA, the partially purified fusion proteins were
incubated with 1 µg of poly[d(I-C)] in 10 mM Tris-HCl, pH 8.0, containing 100 mM KCl, 10% glycerol,
and 1 mM dithiothreitol in a total volume of 29 µl. The
reaction was incubated for 10 min on ice, and after the addition of 1
µl of labeled probe, the incubation was continued on ice for
another 10 min. Following the addition of 1 µl of loading buffer
(30% glycerol, 5 mg/ml bovine serum albumin, 0.005% bromphenol blue), 4
µl of the reaction mixture was loaded onto a gel containing 4%
acrylamide, 0.05% bisacrylamide, 0.5
TBE, 1.25% glycerol.
Electrophoresis was performed at 160 V for 90 min at 4 °C. The gel
was then dried and exposed to Kodak X-AR film at -70 °C. In
some cases, the gel was analyzed using a Molecular Dynamics
PhosphorImager, model SF (Sunnyvale, CA).
Oligonucleotide-directed Mutagenesis
The
two complementary mutant Z element oligonucleotides were each used as
PCR primers to introduce the nucleotide substitutions shown in Fig. 4into the CYP4A6 promoter. This procedure employed the
pLUCA6-880 plasmid as template in two separate PCR reactions to
produce fragments corresponding to nucleotides -880 to -642
of CYP4A6 and -671 of CYP4A6 to nucleotide 3122 of p19dLUC that
each carried the mutations. Following the purification of the two
fragments, they were denatured, mixed, annealed, and amplified in a PCR
reaction employing the outer primers. This produced a fragment spanning
from -880 of CYP4A6 to the nucleotide 3122 of p19dLUC. The
fragment was digested with HindIII and KpnI, and the
resulting fragment was inserted with the HindIII-SacI
fragment of pLUCA6-155M or pLUCA6-155 into SacI-KpnI-digested p19dLUC. The PCR reactions
employed Taq polymerase (Promega) and reaction buffers
supplied by the manufacturer. The identity of the mutated fragments was
confirmed by sequencing.
Figure 4:
Effect of mutations on the binding of
PPARRXR
and ARP-1 to the CYP4A6 Z element. A,
three point mutations, indicated by lowercase
letters, were introduced into each half site on separate
oligonucleotide pairs. This arrangement of mutations has proven
effective in abolishing ARP-1 binding to the APOCIIIB (13) and the
CYP4A6-27 elements (6). Mutants Zm1c and Zm5 are mutations
designed to disrupt the consensus extended binding site derived from
the known PPREs (Fig. 3). B, EMSA was performed with labeled
mutant oligonucleotide pairs. These experiments employed 10 fmol of
each double-stranded oligonucleotide, together with 1.5 µg of
PPAR
/GST and 0.2 µg of RXR
/MBP fusion proteins. In the
ARP-1 assays, 0.5 µg of affinity-purified ARP-1/MBP fusion protein
was used.
PPRE Oligonucleotides Used in EMSA
The
double-stranded oligonucleotides corresponding to the CYP4A6 elements,
Z and -27, were as previously described(6) . The CYP4A6 X
element oligonucleotide corresponds to -746 to -724 of the
CYP4A6 gene as follows: 5`-GACAAGTAGGACAAAGGCCAGGG-3`. The acyl-CoA
oxidase oligonucleotide (ACOXA) include nucleotides -553
to -575 of the rat peroxisomal acyl-CoA oxidase gene (19) as follows: 5`-acgtGGACCAGGACAAAGGTCACGTTCtcga-3`. The
bifunctional enzyme PPRE corresponds to -2919 to -2942 of
the rat peroxisomal bifunctional enzyme gene (8) and does not
contain the overlapping DR2 structure that is not required for
PPARRXR
binding(14, 20) (5`-TCAAATGTAGGTAATAGTTCAATA-3`). The fatty acid binding
protein PPRE (FABP) corresponds to -48 to -71 of the rat
liver fatty acid binding protein gene (9) as follows:
5`-TCAAATATAGGCCATAGGTCAGTG-3`. The 3-hydroxy-3-methylglutaryl-CoA
synthase PPRE corresponds to nucleotides -82 to -107 of the
rat mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene (10) as follows: 5`-CAGAAAAACTGGGCCAAAGGTCTCAG-3`. The APOCIIIB
element corresponds to nucleotides -63 to -84 of the human
apolipoprotein CIII gene (16) as follows:
5`-AGGGCGCTGGGCAAAGGTCACC-3`. DR1P =
5`-ACAAAACTAGGTCAAAGGTCAGGG-3`, and DR1M =
5`-CGCGCGCCAGGTCAAAGGTCAGGG-3`.
Preparation of Liver Lysates and Supershift
Analysis
Frozen mouse liver (100 mg) was homogenized in 500
µl of phosphate-buffered saline solution containing 5 mM EDTA, 1 mM dithiothreitol, 0.2 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1 µg/ml
leupeptin, 1 µg/ml aprotinin, and 10% glycerol. The homogenates
were then sonicated 10 s, diluted to 1 ml with the above buffer, and
centrifuged at 200,000 g for 30 min. The protein
content of the supernatant was determined by the Bio-Rad protein assay
(Bradford Assay). For supershift analysis, 100 µg of lysate (20
µl) was combined with 1 µg (1 µl) of poly(dI-dC) and 1
µg (1 µl) of sheared salmon sperm DNA and incubated on ice for
10 min. Following the addition of 25 fmol of radiolabeled
double-stranded oligonucleotide, the incubation was continued on ice
for 10 min, after which 0.5 µl of either rabbit pre-immune or
rabbit anti-PPAR
serum was added, and the incubation on ice was
continued for an additional 30 min. Gel loading and electrophoresis was
performed as described for the EMSA protocol; however, the entire
binding reaction was loaded onto the gel.
that displays an enhanced response to peroxisome
proliferators) revealed that sequences 5` of the direct repeat (DR1)
were required to confer responsiveness to peroxisome proliferators.
pLUCA6-880 is activated 13.3-fold by the inclusion of a
peroxisome proliferator (Wy-14,643) in the culture medium, whereas
pLUCA6-663 does not respond to Wy-14,643. An oligonucleotide that
spanned the deletion site (Z) was found to confer a peroxisome
proliferator response to the non-responsive pLUCA6-155 construct.
This element contains four imperfect repeats of the nuclear receptor
binding site (AGGTCA) (half sites). We would anticipate that the
binding of a PPAR
RXR
heterodimer would require two such
half sites, with a zinc finger from each receptor interacting with one
of these repeats. The portion of the Z element found in
pLUCA6-663 contains an imperfect direct repeated sequence (DR1)
that is formed with half sites 2 and 3. DR1 motifs have been found in
the PPREs of other genes, including the acyl-CoA oxidase gene, and are
known to bind RXR homodimers. However, half sites 2 and 3 are retained
in the inactive pLUCA6-663 construct. It appears that it is the
disruption of half site 1 that leads to a loss of response in the
-663 construct. Half site 1 (3`-AGTTCA-5`) is known to bind
retinoic acid receptors with a high efficiency(21, 22) .
This sequence forms imperfect inverted palindromes with the directly
repeated half sites 2, 3, and 4. Nuclear receptors are known to bind
inverted palindromic sequences of different
spacings(22, 23) .
Figure 1:
CYP4A6 reporter
constructs and their activation by peroxisome proliferators. The
relative positions of the three CYP4A6 PPREs (X, Z, and -27) are
indicated schematically. The peroxisome proliferator-dependent
stimulation of transcription is also shown (Fold Induction). Deletion
of X has previously been shown to reduce the response to Wy 14,643 by
2-fold; however, deletion into the Z element at position -663
abolishes the response to peroxisome proliferators (-663
construct). The Z element acts as an enhancer element and confers
peroxisome proliferator responsiveness to the pLUCA6-155 promoter
construct (-155 versus -155Z). Cotransfection of
expression vectors for PPAR and RXR
with reporter constructs
that do not contain the X and Z elements still respond to peroxisome
proliferators. This is due to the utilization of the cryptic -27
element at higher concentrations of RXR
(6). The position of the
direct repeats of imperfect nuclear receptor binding sites (numbered 1-4) in the Z element are
shown.
To determine the minimal sequences
required for the binding of PPARRXR
to the Z element,
EMSAs of double-stranded oligonucleotides containing various
combinations of these repeated sequences were performed. For this
purpose, the murine PPAR
was expressed as a fusion protein with
GST, and the human RXR
was expressed as a fusion protein with the
MBP. As previously shown(6) , a complex is observed for
PPAR
RXR
with the Z element (Fig. 2B, leftpanel, Z1234). Truncation of the Z element by
six nucleotides on the 5`-end and three nucleotides on the 3`-end does
not reduce the binding of PPAR
RXR
(Fig. 2B, left panel, Z123). This result
demonstrates that half site 4 is not involved in the binding of
PPAR
RXR
. Truncations of the 5`-end by a further six
nucleotides, which disrupts half site 1, results in a loss of binding
of PPAR
RXR
(Fig. 2B, left
panel, Z234, Z23). Deletion of a further nine nucleotides from the
3`-end, which removes half site 3, results in a loss of binding of
PPAR
RXR
(Fig. 2B, left panel,
Z12). EMSA employing finer truncations of the Z123 oligonucleotide
revealed that progressive truncations that disrupt half site 1 result
in a progressive loss of PPAR
RXR
binding (Fig. 2,
center panel, Z123-Z23). Truncations 3` of half site 3 revealed the
requirement of one additional nucleotide (Fig. 2B, centerpanel, ZD6-ZVVS) for the binding of
PPAR
RXR
. Therefore, it appears that the minimal
PPAR
RXR
binding sequence spans the regions
corresponding to half sites 1, 2, and 3. Mutagenesis of half site 2 (Fig. 2B, leftpanel, Z13) results in
the loss of PPAR
RXR
binding. The requirement for three
half sites (1, 2, and 3) was unexpected as the binding of a heterodimer
should require only two repeats. As such, we decided to compare the
binding requirements of PPAR
RXR
with those for ARP-1.
EMSA was performed with the partially purified ARP-1/MBP fusion protein
and double stranded oligonucleotides corresponding to various
truncations of the Z element (Fig. 2B, rightpanel). ARP-1 forms a single complex with the Z element
in contrast to that seen with the -27 element of CYP4A6 and other
characterized ARP-1 binding sites where both monomeric and dimeric
binding is observed(6) . The mobility of the complex formed with
the Z element approximates that of the lower mobility complex seen with
the -27 element. The lower mobility complex is presumed to
consist of a dimeric rather than monomeric complex as the slower
mobility of the complex is likely to reflect a larger molecular weight.
The appearance of a single, dimeric complex with the Z element and
ARP-1 suggests that the monomer binds to the Z element with a very low
efficiency. Truncations that disrupt half site 1 and abolish
PPAR
RXR
binding do not affect ARP-1 binding (Fig. 2B, rightpanel, Z123-Z23).
ARP-1 binds readily to the Z23 oligonucleotide, indicating that only
half sites 2 and 3 are required for binding; however, ARP-1 binding is
abolished by the deletion of the 3`-nucleotide adjacent to the DR1 (Fig. 2B, rightpanel, ZD5 versus ZVVS). Therefore, the 3`-sequence requirements for the binding of
ARP-1 are identical to those seen for RXR
PPAR
.
Figure 2:
Analysis of PPARRXR
binding to oligonucleotides containing different repeated half sites. A, the Z element contains four imperfect repeats of the
nuclear receptor binding consensus sequence (AGGTCA) or ``half
sites'' that are indicated by the arrows. Half sites 2
and 3 constitute a direct repeat with a spacing of one nucleotide. The
sequences of truncated oligonucleotides used in panelB are shown. Changes from the wild type sequence are denoted in bold and lower case. B, EMSA analysis was
performed using double-stranded oligonucleotides that were truncated to
assess the contribution of flanking nucleotides to
PPAR
RXR
and ARP-1 binding of the Z element.
Affinity-purified PPAR
/GST (1000 ng), RXR
/MBP (200 ng), and
ARP-1/MBP (200 ng) fusion proteins were used in this
assay.
The
half site 1 region is required for the function of the Z element and
the binding of PPARRXR
but is not required for the
binding of ARP-1 (Fig. 2B). This region, along with half
site 2, constitutes an imperfect palindrome that is not conserved
between PPREs. Comparison with other known PPRE sequences reveals that
the region 5` of the DR1, which constitutes half site 1 of the Z
element, exhibits a loose consensus (C(A/G)(A/G)A(A/T)CT) (Fig. 3). This consensus does not appear to be similar to the
core nuclear receptor binding sequence (AGGTCA) but does resemble the
extended consensus binding site for the monomeric nuclear receptors
(Rev-ErbA
, NGFI-B, and ROR) that contains an A-T-rich region 5` of
a single AGGTCA motif (24, 25, 26) (Fig. 3).
Figure 3:
Conservation between known PPREs. The DR1
motif is highly conserved between the CYP4A6 Z element and the PPREs
found in the genes encoding the rat CYP4A1 (CYP4A1) (37), fatty acyl
CoA oxidase (ACOXA) (3, 19), enoyl-CoA hydratase/3-hydroxyacyl-CoA
dehydrogenase bifunctional enzyme (BIF) (8, 38), FABP (9), and the
3-hydroxy-3-methylglutaryl-CoA synthase (HMG) (10). A region of limited
conservation is present immediately 5` of the DR1 motif. This region is
similar to that seen in the binding site for the monomeric nuclear
receptor, Rev-ErbA (24).
To test the
nature of the sequence requirements for the binding of
PPARRXR
within the Z element, double-stranded
oligonucleotides containing grouped point mutations in each of the four
half sites were synthesized (Fig. 4A) and assayed by
EMSA for binding to PPAR
RXR
and ARP-1 (Fig. 4B). Equivalent grouped mutations have been shown
to abolish binding of ARP-1 to the apolipoprotein CIIIB element and the
CYP4A6 -27 element (6, 13) and have also been
shown to abolish PPAR
RXR
binding to the CYP4A6
-27 element(6) . Mutation of half sites 2 and 3 greatly
diminished the binding of PPAR
RXR
and ARP-1, confirming
the importance of the DR1 motif for binding of these receptors.
Mutations that disrupt the nuclear receptor binding motif in half site
1 but retain an A-T-rich motif similar to the monomeric nuclear
receptor binding site (Zm1b) did not affect the binding of
PPAR
RXR
to the Z element. In contrast, mutations (Zm1c
and Zm5) that disrupt the loose consensus in the extended binding site
region display diminished binding to PPAR
RXR
(Fig. 4B) but have no significant effect on ARP-1
binding. These results confirm the specific requirement for sequences
5` of the DR1 in the binding of PPAR
RXR
and demonstrate
the requirement for an extended binding site such as that seen for the
monomeric receptors.
revealed that the mutations in half site 3 that severely
reduced PPAR
RXR
binding to the Z element also reduced
the transcriptional response to peroxisome proliferators (Fig. 5,
880 versus Zm3). This reduction is not as complete as may have
been expected from the DNA binding studies, suggesting that the other
elements (X and -27) may partially compensate for the loss of
function of the Z element. Mutation of the -27 element in the
CYP4A6 reporter construct (880m) has little effect on its own (Fig. 5, 880 versus 880m), although previous studies
have shown that this mutation abolishes the binding of
PPAR
RXR
to this element(6) . The -880m
construct was used to analyze the other mutations of the Z element to
minimize the effects of the -27 element in this analysis. Using
these double mutant CYP4A6 reporter constructs, it is clear that
mutations that diminish binding of PPAR
RXR
to the Z
element also abolish the response to peroxisome proliferators (Fig. 5, Zm1c-d, Zm2-d, Zm3-d). Mutations that do not affect DNA
binding of PPAR
RXR
do not diminish the response to
peroxisome proliferators (Fig. 5, Zm1b-d, Zm4-d). Mutation of
half sites 2 and 3 (Fig. 5, Zm2-d, Zm3-d) results in a loss of
responsiveness to peroxisome proliferators, demonstrating the
requirement for the DR1 in the function of the Z element. The Zm1c-d
construct, which contains mutations in the extended binding site, also
shows no response to peroxisome proliferators. This result, along with
the deletion analysis of the CYP4A6 reporter constructs (Fig. 1),
demonstrates the requirement of the sequences 5` of the DR1 for the
function of the Z element.
Figure 5:
Transactivation of mutant CYP4A6 promoter
constructs by mPPAR-G. Oligonucleotide-directed mutagenesis was used to
mutate the CYP4A6 Z element in the reporter construct pLUCA6-880
to generate pLUCA6-880Zm3 (Zm3). This and the other mutants were
also cloned into pLUCA6-880m to generate the respective double
mutant clones pLUCA6-880Zm1b-d (Zm1b-d), pLUCA6-880Zm1c-d
(Zm1c-d), pLUCA6-880Zm2-d (Zm2-d), pLUCA6-880Zm3-d (Zm3-d),
and pLUCA6-880Zm4-d (Zm4-d). These reporter constructs were
cotransfected with pCMV-PPAR-G into RK13 cells. After incubation
with DNA for 16 h, the cells were washed and then either solvent
(ME
SO; no drug) or peroxisome proliferator (Wy-14,643) was
added to fresh medium for an additional incubation of 48 h. The bargraphs represent the mean values plus standard deviation
obtained from three independent transfections and show the activity
normalized to that obtained with pLUCA6-880 in the presence of
peroxisome proliferator.
The Z element binds RXR homodimers very
weakly; this is in contrast to certain other DR1-containing elements,
such as the rat cellular retinol binding protein II
RXRE(12, 27) . The rat cellular retinol binding protein
II RXRE contains a DR1 that is a perfect repeat of the AGGTCA motif,
which binds RXR homodimers efficiently and confers a response to
both all-trans and 9-cis retinoic acid(27) .
The natural PPRE elements, such as the Z element, are divergent from
the idealized DR1 sequence (Fig. 3). This divergence may reduce
the binding of RXR
homodimers, and the extended binding site may
stabilize the binding of PPAR
RXR
heterodimers to these
divergent sequences.
, PPAR
, PPAR
RXR
, and ARP-1 (Fig. 6).
These results were compared to those obtained with natural elements
that contain imperfect direct repeats. DR1P and DR1M display
significant binding to RXR
(Fig. 6A, complexesI and II). The 5`-extended binding site on the
DR1P element appears to diminish RXR
binding (Fig. 6B). The binding of RXR
to DR1P and DR1M is
about 20-fold greater than that seen with the APOCIIIB and ACOXA
elements and over 100-fold greater than that seen with the other
elements (Fig. 6B).
Figure 6:
Analysis of PPAR, RXR
, and ARP-1
binding to DR1 containing elements. A, EMSA analysis was
performed using double-stranded oligonucleotides that correspond to DR1
containing elements that have been shown to be regulated by nuclear
receptors. Binding assays used crude bacterial lysates containing
expressed PPAR
(6 µg) or RXR
/MBP(0.5 µg) fusion
protein or affinity-purified ARP-1/MBP fusion protein (70 ng). RXR
homodimer complexes are shown (I and II) as are the
putative PPAR
RXR
heterodimeric complexes (III)
and the ARP-1 monomer (M) and homodimer (D)
complexes. B, the sequences of the PPREs used in the above
binding assays are shown. Also shown is the number of matches the
5`-region has with the consensus sequence and a summary of the binding
results obtained from the phosphorimager analysis. The values shown
represent relative binding activities (% oligonucleotide bound)
normalized to those obtained with the DR1P oligonucleotide pair.
Perfect nuclear receptor binding sites are indicated in boldtype.
The addition of PPAR and
RXR
to DR1P and DR1M results in a complex of greater mobility than
that seen with RXR
alone (Fig. 6A, complexIII). The binding to DR1M demonstrates that the extended
binding site is not required for the binding of PPAR
RXR
to a perfect direct repeat of the consensus nuclear receptor binding
motif. The DR1P oligonucleotide binds the PPAR
RXR
more
efficiently than DR1M (Fig. 6B), suggesting a modulatory
role for the extended half site. A complex of similar mobility is
formed with the addition of both PPAR
and RXR
to the natural
elements. The degree of binding of PPAR
RXR
to the
natural elements is relatively similar to that observed for the
consensus DR1, except for the APOCIIIB element and the CYP4A6-X and
-27 elements (Fig. 6B). The APOCIIIB element has
not been shown to function as a PPRE, and the CYP4A6-X and -27
elements are known to be weak PPREs.
RXR
to idealized DR1 sequences. PPREs that contain
divergent half sites are less able to bind competitors such as RXR
and ARP-1 homodimers, while PPAR
RXR
binding is
maintained to response elements with divergent DR1 sequences 3` of the
conserved extended binding site.
RXR
than the Z element when in
competition with other nuclear receptors. Similar amounts of binding
activity were observed with liver extracts for both oligonucleotides (C). The addition of anti-PPAR
serum to the binding assay
results in the supershifting (SS) of a major proportion of the
Z binding activity. In contrast, no detectable supershifting of the
APOCIIIB binding activity is observed. Therefore, the Z element is
likely to be occupied by PPAR
RXR
heterodimers in liver,
and the APOCIIIB element is more likely to be occupied by other
proteins.
Figure 7:
Binding of mouse liver proteins to the
CYP4A6 Z element and the apolipoprotein CIIIB element. 100 µg of
total liver protein was assayed for binding to double-stranded
oligonucleotides corresponding to the CYP4A6-Z element and the APOCIIIB
element. Shown is the assay in the absence of added serum (-), in
the presence of preimmune serum (Pi), and in the presence of
anti-PPAR serum (I). The protein/DNA complex is indicated (C), and the protein/DNA/antibody complex (supershift) is
indicated (SS).
RXR
much more efficiently than the other CYP4A6
PPREs (6) (Fig. 6). A reporter construct containing the
direct repeat of the Z element, in which the 5`-extended half site was
deleted, did not display a response to peroxisome proliferators when
cotransfected with expression vectors for PPAR
, suggesting that
sequences 5` of the DR1 were necessary for the response to peroxisome
proliferators. In vitro DNA binding studies revealed the
requirement of six additional nucleotides 5` of the DR1 for the binding
of PPAR
RXR
. This additional sequence was not required
for the binding of RXR
or ARP-1 homodimers. Mutagenesis of the
CYP4A6 reporter constructs confirmed that the ability of the CYP4A6
gene to respond to peroxisome proliferators is predominantly determined
by binding of PPAR
RXR
to the Z element; however, it
appears that binding of PPAR
RXR
to the -27 and X
elements may contribute to this response.
RXR
may be similar to
the A-T-rich 5`-extended binding site seen for the monomeric nuclear
receptors Rev-ErbA
, ROR, and
NGFI-B(24, 26, 28) . This binding is thought to
involve a region that is immediately adjacent to the second zinc finger
of the receptor known as the AT box (28). The AT box of the PPAR
is highly conserved with that of Rev-ErbA-
. This is the first
observation of this type of sequence requirement for binding by a
putative heterodimeric receptor complex. It is tempting to speculate
that the novel second zinc finger(29, 30, 31) that is characteristic of the PPAR subfamily of nuclear
receptors may prevent the binding of PPAR monomers to single half
sites. The requirement of this extended binding site for the binding of
PPAR
RXR
heterodimers would imply that PPAR
occupies the 5`-half site of the DR1. Studies on the binding of
RXR
heterodimers with the thyroid hormone and retinoic acid
receptors have shown a strict requirement for RXR
to occupy the
5`-half site of direct
repeat(32, 33, 34, 35) . Further studies
are in progress to determine the structure of the
PPAR
RXR
DNA complex.
RXR
to the consensus DR1 demonstrates that the
extended binding site sequence is not absolutely required for the
binding of PPAR
RXR
. However, the presence of this
sequence does seem to diminish the formation of RXR
homodimers and
promote the formation of the PPAR
RXR
heterodimer.
binding. The binding of ARP-1 remains high to
such elements, although much of this binding appears to be monomers of
ARP-1 binding to the single consensus half site. The binding of
PPAR
RXR
to these elements is variable, and in some
cases (the APOCIIIB element) binding is very weak. The APOCIIIB
contains a T at the first position in the 5` half site that is also
seen in the weak -27 element. It appears that this change may
reduce PPAR
RXR
binding. The -27 element displays
marked PPAR
RXR
synergism in binding, whereas the
APOCIIIB element binds RXR homodimers and PPAR
RXR
heterodimers equally well. This difference may be attributed to the
fact that the -27 element contains a conserved 5`-extended
binding site (6/7 match), and the APOCIIIB element contains a divergent
5`-extended binding site (2/7 match). In contrast, the FABP element
binds very strongly to PPAR
RXR
and contains a 6/7 match
with the PPRE 5`-extended binding site consensus. Therefore, it appears
that there is a stricter requirement for the extended binding site in
elements with imperfect repeats.
homodimers and display greatly reduced ARP-1
binding when compared to the elements that contain perfect AGGTCA
motifs. These elements can be considered to be very specific for
PPAR
RXR
and contain a 5`-extended binding site
consensus match of 6/7.
RXR
binding. However, they
display selective PPAR
RXR
binding over RXR
homodimers as they contain conserved 5`-extended binding sites.
RXR
binding
in elements containing divergent repeated structures that bind poorly
to competitors such as ARP-1 and RXR
, thereby generating
specificity from a seemingly promiscuous DR1 response element
structure. This conclusion is supported by the finding that the CYP4A6
Z element binds mainly to PPAR
containing complexes in mouse liver
lysates, while the APOCIIIB element, which was shown to be more
promiscuous in the binding of recombinant RXR
and ARP-1, is mainly
bound by proteins other than PPAR
. It is therefore apparent that
the sequence of the half site and the upstream extended binding site
will modulate the specificity of a DR1 response element with respect to
competition for other members of this receptor family (possibly
including the monomeric receptors) and thus determine the regulation of
genes in vivo.
,
peroxisome proliferator-activated receptor
; RXR
, retinoid X
receptor
; PPRE, peroxisome proliferator-responsive element;
ARP-1, apolipoprotein regulatory protein-1; PCR, polymerase chain
reaction; EMSA, electrophoretic mobility shift analysis; FABP, fatty
acid binding protein; GST, glutathione S-transferase; MBP,
maltose binding protein; DR1, a direct repeat with a spacing of one
nucleotide; PPAR-G, a mutant form of PPAR
; Wy-14,643,
4-chloro-6(2,3-xylindino)2-pyrimidinylthiolacetic acid; individual
cytochrome P-450s are designated according a uniform system of
nomenclature (36), and the gene designations are preceded by the
letters CYP.
cDNA in the pRS expression plasmid and to Dr. S. K. Karathanasis
(American Cyanamid Co.) for providing the ARP-1 cDNA.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.