From the
Expression of the gene encoding the mitochondrial fatty acid
Medium chain acyl-CoA dehydrogenase (MCAD)
To delineate transcriptional
regulatory mechanisms involved in the control of nuclear genes encoding
mitochondrial FAO enzymes, we have focused on the MCAD gene. As an
initial step in the identification of MCAD gene transcriptional
regulatory elements, genomic clones encoding the human MCAD gene and
5`-flanking region were isolated and characterized(6) .
Transfection of plasmids containing varying lengths of the MCAD gene
5`-flanking region fused to a reporter gene into human hepatoma G2
(HepG2) cells localized the functional promoter to a region between 362
base pairs upstream and 189 base pairs downstream of the transcription
start site. Recently, we identified a novel nuclear hormone receptor
response element (NRRE-1) within this promoter region located 343 base
pairs upstream of the transcription start site(7) .
In this
study, we sought to delineate the cis-acting regulatory elements within
the human MCAD gene promoter and to identify transcription factors that
interact with these sequences. To this end, DNA-protein binding studies
and mammalian cell gene transfer studies were performed. The results of
these studies revealed that the MCAD gene promoter contains a series of
cis-acting regulatory modules composed of Sp1 binding sites juxtaposed
to nuclear receptor response elements. The transcriptional regulatory
properties of the elements suggest a mechanism for modulation of MCAD
gene expression among distinct cell types in vivo.
Antibody supershift
experiments were performed with a polyclonal antibody to human COUP-TF
kindly supplied by Dr. Bert O'Malley (Baylor College of
Medicine)(12) , a rabbit polyclonal antibody against the
amino-terminal region of steroidogenic factor-1 (SF-1) kindly supplied
by Dr. Keith Parker (Duke University)(13) , and a rabbit
polyclonal Sp1 antibody raised to an epitope corresponding to amino
acid residues 520-538 of human Sp1 (Santa Cruz Biotechnology).
An MCAD promoter
deletion series was employed in transfection experiments to examine the
transcriptional regulatory properties of the DNase I protected regions
in the MCAD gene promoter. Deletion of the 50-base pair region from
-362 to -312, which contains NRRE-1, resulted in a modest
but statistically significant increase in transcriptional activity in
3T3 cells (1.4-fold, p = 0.006 based on four
independent experiments) but no significant changes in HepG2 or Y-1
cells (Fig. 2B). Removal of the 5`-promoter region
containing site A (from -312 to -170) resulted in a
significant decrease in transcriptional activity in HepG2 (81%), Y-1
(49%), and 3T3 cells (64%). As predicted by the DNase I protection
studies, further deletion of the region from -170 to -102,
which lacks footprinted regions, had no significant effect on
transcriptional activity in any cell line. These data confirm that
promoter module A functions as a cis-acting transcriptional activator
and suggests that NRRE-1 confers transcriptional repression in 3T3
cells.
The transfection experiments indicated that MCAD promoter
module A is a positive regulatory element in all three cell lines. To
evaluate the transcriptional regulatory properties of the site A
element in the absence of the NRREs and in the context of a
heterologous promoter, a single copy of a double-stranded
oligonucleotide containing site A was placed upstream of the TK
promoter in a CAT reporter plasmid (pTKCAT(A)) and transfected into the
three cell lines. The transcriptional activity of pTKCAT(A) was 2.3-,
3.3-, and 2.5-fold higher than that of pTKCAT lacking the site A
element (pTKCAT(-)) in HepG2, Y-1, and 3T3 cells, respectively (Fig. 2C). A DNA fragment containing site A conferred
orientation-independent transcriptional activation (8.6 ±
1.3-fold induction) when inserted at a remote distance upstream of the
SV40 promoter (data not shown). Thus, site A is a transcriptional
activator with enhancer-like properties.
To
confirm that Sp1 or a related protein was contained in complexes I and
II, antibody ``supershift'' experiments were performed with
anti-Sp1 antibody and the Y-1 cell nuclear protein extract. Addition of
anti-Sp1 antibody, but not pre-immune serum, resulted in a mobility
supershift of complex I, confirming that Sp1 was present in this
DNA:protein complex (Fig. 3C, lanes1-3). Identical results were obtained with an Sp1
consensus oligonucleotide probe and the Y-1 cell extract (Fig. 3C, lanes4-6). Incubation
of the site A probe with purified recombinant Sp1 formed a single
complex that comigrated with complex I. In contrast to the partial
supershift observed with the Y-1 nuclear extract, all of the
DNA:recombinant Sp1 complex was supershifted by the anti-Sp1 antibody (Fig. 3C, lane8). These data suggest
either that the anti-Sp1 antibody does not recognize all forms of Sp1
present in complexes I and II or that other proteins participate in
this DNA-protein interaction. Similar EMSA results were obtained using
HepG2 and 3T3 cell-derived nuclear protein extracts with site A, B, and
C probes (data not shown). Taken together, the transfection and
DNA-protein binding studies indicate that Sp1 or a highly related
transcription factor plays a major role in MCAD gene promoter function.
Analysis of the DNA sequences of sites A-C reveals the
presence of both typical and atypical Sp1 binding sites. Site C
contains three sequences that match the known core Sp1 binding
consensus (CCCGCCC, Fig. 1F). In contrast, sites A and B
lack the Sp1 binding consensus but do contain several related sequences
(CCCAGCC, CGCAGCG, and CCCTCCC) (Fig. 1F). Atypical Sp1
binding sites have been identified in promoters upstream of a variety
of other genes, including those encoding the mouse secretory protease
inhibitor P12(22) , the T-cell receptor
The DNase I protection studies shown
here (Fig. 1) identified two additional MCAD promoter sites
(NRRE-2 and NRRE-3) that contain hexamer sequences conforming to the
known consensus (RG(G/T)TGNA) for binding to members of the
thyroid/retinoid subgroup of the nuclear receptor superfamily (Fig. 1F). The DNA footprinting patterns suggested that
these NRREs interact with multiple nuclear receptors in a cell
line-specific and element-specific manner. We have shown previously
that the orphan receptor HNF-4 present in nuclear extracts derived from
HepG2 cells binds NRRE-1(15) . To identify the endogenous
nuclear receptors that interact with the NRREs in 3T3 and Y-1 cells,
double-stranded oligonucleotides containing the NRRE-1, NRRE-2, or
NRRE-3 sequences were employed in EMSA with nuclear protein extracts
prepared from these cells. With 3T3 cell nuclear extracts, NRRE-1 and
NRRE-3 probes both formed single prominent complexes with identical
mobilities (Fig. 4A). Formation of the complexes was
inhibited by addition of 100-fold molar excess specific unlabeled
oligonucleotide but not by an unrelated double-stranded DNA, confirming
that the NRRE-1-protein and NRRE-3-protein interactions were specific.
Cross-competition experiments revealed that the NRRE-1:protein complex
could be specifically inhibited by unlabeled NRRE-3, and conversely the
NRRE-3:protein complex was specifically inhibited by unlabeled NRRE-1 (Fig. 4A). Formation of the complexes formed with NRRE-1
or NRRE-3 probes was also inhibited by unlabeled NRRE-2, but to a
lesser extent, suggesting that the NRRE-2 binds the nuclear protein(s)
with lower affinity (Fig. 4A).
Mobility shift experiments were also performed
with the NRRE probes and Y-1 cell nuclear extracts. The NRRE-3 probe,
but not NRRE-1 or NRRE-2 probes, formed a prominent complex with Y-1
cell nuclear extract (Fig. 4C, complex III, and data not
shown). In addition, two faint complexes of lower mobility (I and II)
formed with all three NRREs (Fig. 4C and data not
shown). Competition experiments confirmed that complexes I, II, and III
represent specific NRRE-3-protein interactions. Cross-competition with
unlabeled NRRE-1 and NRRE-2 abolished complexes I and II but not
complex III, indicating that only NRRE-3 contained a recognition
sequence for the Y-1 cell nuclear protein present in this complex (Fig. 4C, lanes4 and 5). The
mobility shift pattern observed with the NRRE-3 probe and 3T3 cell
nuclear extract was distinct from that with Y-1 cell extract (Fig. 4C, lanes6 and 7).
Antibody supershift and competition experiments were employed to
identify the specific nuclear receptors present in the complexes
obtained with the NRRE-3 probe and Y-1 cell nuclear proteins (Fig. 4D). The same panel of antibodies employed for the
experiments shown in Fig. 4B were used. In addition,
because complex III was obtained exclusively with nuclear extracts
derived from Y-1 adrenal cells, a steroid-producing cell line derived
from a mouse adrenal tumor, an antibody to SF-1 (the rabbit homolog of Drosophila nuclear receptor FTZ-F1) was used in these
experiments(13) . This anti-SF-1 antiserum has been shown
previously to specifically abolish SF-1:DNA complexes (13) and
virtually abolished the formation of complexes I-III (Fig. 4D). Anti-COUP-TF supershifted complexes I and II
but had no effect on complex III, indicating that COUP-TF was also
present in the two minor complexes. In contrast to the results obtained
with 3T3 cell nuclear extract, the amount of COUP-TF:NRRE-3 complex
formed with Y-1 cell-derived extract was minimal. Neither anti-9-cis
retinoic acid receptor
These results demonstrate
that the adrenal-specific orphan receptor, SF-1, binds NRRE-3.
Comparison of the NRRE-3 sequence with the known SF-1/FTZ-F1 binding
consensus sequence (PyCAAGGPyCPu) reveals a potential SF-1 binding site
at +141 to +149 (TCAAGGCCG, Fig. 1F). The
interaction of both SF-1 and COUP-TF at a single site (complexes I and
II, Fig. 4D) has also recently been described for a
transcriptional regulatory element in the oxytocin
promoter(29) , suggesting that this may represent a general
transcriptional regulatory mechanism. The biological role of SF-1 in
regulating MCAD gene expression is not clear. SF-1 is an adrenal
cortex-enriched orphan receptor that plays a crucial role in
steroidogenesis and adrenal gland development(30) . Acetyl-CoA,
the major product of fatty acid oxidation, is necessary for the
biosynthesis of steroid compounds in the adrenal cortex. Accordingly,
it is possible that SF-1 coordinately regulates the expression of genes
encoding enzymes involved in fatty acid
Our previously published work with NRRE-1 and the
results of the transfection and DNA-protein binding studies shown here
suggested that the MCAD gene promoter NRREs function as cell-specific
transcriptional regulatory elements by interacting with multiple
nuclear receptor transcription factors. To determine whether NRRE-3
mediates transcriptional activation or repression in accordance with
cell-specific nuclear receptor-DNA interactions, a single copy of a
double-stranded oligonucleotide containing NRRE-3 was inserted upstream
of a TK promoter in a CAT reporter plasmid (pTKCAT(NRRE-3)) and
transfected into Y-1 and 3T3 cells. The transcriptional activity of
pTKCAT(NRRE-3) was compared to that of a pTKCAT lacking NRRE-3 (pTKCAT
(-)). As predicted by the DNA binding studies, NRRE-3 conferred a
2.7-fold transcriptional activation in Y-1 adrenal cells and a 70%
repression in 3T3 cells (Fig. 5). These data are consistent with
the binding studies and indicate that, as with NRRE-1 (16), either
activator (SF-1) or repressor (COUP-TF) orphan receptors interact with
NRRE-3 in a cell-specific manner to modulate transcriptional activity
differentially in a cell-specific fashion. In contrast, NRRE-2 binds
COUP-TF in all three cell lines.
In summary, we have demonstrated that the MCAD gene promoter
consists of a complex series of nuclear receptor response elements and
Sp1 binding sites. We propose that multiple nuclear receptor
transcription factors interact with the MCAD gene promoter in a
cell-specific and element-specific manner to influence transcriptional
activity in vivo. Our work strongly suggests that in cells and
tissues with high capacity for fatty acid
We thank Kelly Hall for expert secretarial
assistance.
Note Added in Proof-During review of this
manuscript, Krey et al. (42) reported that the acyl-coenzyme-A
oxidase gene promoter contains multiple Sp1 sites and nuclear receptor
response elements.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-oxidation enzyme, medium-chain acyl-CoA dehydrogenase (MCAD), is
regulated among tissues during development and in response to
alterations in substrate availability. To identify and characterize
cis-acting MCAD gene promoter regulatory elements and corresponding
transcription factors, DNA-protein binding studies and mammalian cell
transfection analyses were performed with human MCAD gene promoter
fragments. DNA:protein binding studies with nuclear protein extracts
prepared from hepatoma G2 cells, 3T3 fibroblasts, or Y-1 adrenal tumor
cells identified three sequences (nuclear receptor response element 1
or NRRE-1, NRRE-2, and NRRE-3) that bind orphan members of the
steroid/thyroid nuclear receptor superfamily including chicken
ovalbumin upstream promoter transcription factor and steroidogenic
factor 1. Sp1 binding sites (A-C) were identified in close
proximity to each of the NRREs. NRRE-3 conferred cell line-specific
transcriptional repression by interacting with chicken ovalbumin
upstream promoter transcription factor or activation via steroidogenic
factor 1. In contrast, the Sp1 binding site A behaved as a
transcriptional activator in all cell lines examined. We propose that
multiple nuclear receptor transcription factors interact with MCAD gene
promoter elements to differentially regulate transcription among a
variety of cell types.
(
)(2,3-oxidoreductase, EC 1.3.99.3) is a
nuclear-encoded mitochondrial enzyme that catalyzes the initial
reaction in the fatty acid
-oxidation (FAO) pathway(1) .
The pivotal role of MCAD in cellular energy metabolism is underscored
by the serious and often fatal clinical consequences of inherited MCAD
deficiency including hypoglycemia, liver dysfunction, and sudden death
(2, 3). MCAD expression is highly regulated in parallel with cellular
FAO rates during development, among tissues, and by dietary factors and
hormones(4, 5) .
DNase I Protection Analysis
Using
internal restriction sites, three overlapping MCAD gene promoter DNA
fragments were gel-isolated for the DNase I protection assay (all
numbers are relative to the transcription start site =
+1(6) ): -362 (HindIII) to -12 (SphI), -102 (PstI) to +134 (XbaI), and -11 (SphI) to +189 (XbaI). DNase I footprint analyses were performed by a
modification of a standard protocol(8) . In brief, 5-25
µg of nuclear protein was incubated with 3-5 pmol of labeled
DNA fragment and digested with DNase I. Samples were separated on an 8%
denaturing polyacrylamide gel followed by autoradiography. The location
of the footprinted sequences was determined by a Maxam-Gilbert sequence
reaction of the identical labeled fragment. Nuclear protein extracts
were prepared as previously described(9) .
Electrophoretic Mobility Shift and Antibody
``Supershift'' Assays
Electrophoretic mobility
shift assays (EMSAs) were performed as described(10) . The
following double-stranded oligonucleotides were prepared (sense strand
sequence only, lower case nucleotides indicate restriction site ends
added to allow labeling): site A (MCAD gene promoter region -282
to -258), 5`-gatccGGCCCCAGCCACGCCCTCTAACCCAg-3`; NRRE-1
(-343 to -311),
5`-gatccGGGTTTGACCTTTCTCTCCGGGTAAAGGTGAAGg-3`; NRRE-2 (-39 to
-18), 5`-gatccACGGCGCACGCAAGGGTCACGGg-3`; NRRE-3 (+134 to
+159), 5`-gatccGAGTATGTCAAGGCCGTGACCCGTGTg-3`; site B (-59
to -36), 5`-gatccCCCCTCCCCAGGTCGCAGCGACGGg-3`; site C (+39
to +86),
5`-gatccCCCCGTCCTTCCGCAGCCCAACCGCCTCTTCCCGCCCCGCCCCATCCCg-3`; myocyte
nuclear factor, 5`-ACCACCCCACCCCCTGTGGC-3`; AP-2,
5`-GATCGAACTGACCGCCCGCGGCCCGT-3`; nuclear respiratory factor-1,
5`-gatccTGCTAGCCCGCATGCGCGCGCACCTTg-3`; Sp1,
5`-ATTCGATCGGGGCGGGGCGAGC-3`. Human recombinant Sp1 protein (11) was obtained from Promega.
Reporter Plasmids
MCAD genomic DNA
fragments were inserted into a promoterless chloramphenicol
acetyltransferase (CAT) reporter plasmid (pCAT-Basic, Promega).
Construction of MCADCAT(-362/+189),
MCADCAT(-312/+189), pTKCAT, and pTKCAT(NRRE-1) have been
described(7) . Promoter fragments for
MCADCAT(-170/+189) and MCADCAT (-102/+189) were
generated by PCR. The PCR products were ligated into the pCAT-Basic
plasmid via HindIII (incorporated into the 5`-PCR primer) and
the internal SphI site. DNA sequence analysis was performed to
confirm that the PCR product contained no mutations. The pCATSV40
plasmid was obtained from Promega (pCAT-Control). pTKCAT(A) and
pTKCAT(NRRE-3) were constructed by ligating double-stranded site A or
NRRE-3 oligonucleotides into the BamHI site present upstream
of the thymidine kinase promoter.
Mammalian Cell Transient
Transfections
Human HepG2 cells, Y-1 mouse adrenal cells,
and NIH-3T3 mouse fibroblasts were maintained in an atmosphere
containing 5% CO in minimal essential medium supplemented
with 10% Nu-serum (Collaborative Biomedical Products) (HepG2), F-10
supplemented with 15% heat inactivated horse serum and 5% fetal calf
serum (Y-1), and minimal essential medium supplemented with 10% fetal
calf serum (3T3). Cell transfection protocol and CAT and
-galactosidase assays were performed as
described(6, 7) . In brief, 10 µg of MCADCAT plasmid
were transfected by the calcium-phosphate technique along with
1-3 µg of a murine sarcoma virus promoter-
-galactosidase
chimeric gene plasmid (pMSV-
gal) to control for transfection
efficiency. In experiments shown in Fig. 2A, in addition
to normalizing to pMSV-
gal activity, the transcriptional activity
of pCATSV40 was determined in parallel transfections to allow
normalization for differences in background CAT activity among
different cell lines. Cells were harvested 48 h after transfection.
Figure 2:
Transcriptional regulatory properties of
the MCAD gene promoter DNase I protected sites. A, the mean
(±S.E.) transcriptional activity (CAT/gal) of MCADCAT
(-362/+189) transiently transfected into HepG2, Y-1, or 3T3
cells is shown as a percentage of pCATSV40 activity in parallel dishes. B, MCADCAT(-362/+189) and several 5`-promoter
deletion constructs were transiently transfected into HepG2, Y-1, and
3T3 cells. A schematic of the MCADCAT fragments is shown to the left of the corresponding transcriptional activities. Mean
CAT/
-gal activities were normalized to that of the full-length
promoter MCADCAT(-362/+189) (=100%). C, the
mean transcriptional activity (CAT/
gal) of pTKCAT(A) in the three
cell lines is shown in comparison to that of pTKCAT(-). The data
shown is normalized to the CAT/
gal activity of pTKCAT(-)
(=1.0). All values shown in A-C represent the mean of at
least three independent experiments.
The MCAD Promoter Contains Multiple Nuclear Protein
Binding Sites
As an initial step in the identification of
cis-acting regulatory elements within the MCAD gene promoter region,
DNase I protection experiments were performed with nuclear protein
extracts prepared from cell lines derived from liver (human hepatoma
G2) and adrenal cortex (mouse Y-1 adrenal tumor cells), two tissues
with a high capacity for FAO and relatively abundant MCAD expression,
and a third cell line with low MCAD expression (mouse NIH 3T3
fibroblasts)(4, 14) . Overlapping end-labeled DNA
fragments spanning from -362 to +189 (relative to the
transcription start site = +1) of the MCAD gene promoter
region were employed in these studies. Compared to the published human
MCAD promoter sequence(6) , we identified two additional guanine
nucleotides (at -281 and -298) by Maxam-Gilbert sequencing.
Repeat dideoxy sequence reactions revealed that compression artifacts
in the dideoxy sequence accounted for this disparity. Accordingly, the
numbering system used here to describe the MCAD promoter constructs and
regulatory elements was revised to correspond to this corrected
sequence. Six distinct DNase I protected sites were identified between
-343 and +160 (Fig. 1, A-F). The
footprinted regions include three similar GC-rich sequences (designated
A-C) and three sites (NRRE-1, NRRE-2, and NRRE-3) containing
hexamer sequences that conform to the consensus (RG(G/T)TNA) for
binding members of the steroid/thyroid nuclear hormone receptor
superfamily of transcription factors (Fig. 1F). We have
shown previously that one of the latter sites, NRRE-1, is a functional
nuclear receptor response
element(7, 15, 16, 17) .
Figure 1:
Identification of multiple protein
binding sites in the MCAD gene promoter region by DNase I protection
analysis. A-E, DNase I protection analysis was performed
with each of three overlapping radiolabeled MCAD promoter DNA fragments
and nuclear protein extracts prepared from HepG2, Y-1, or 3T3 cells.
The lanelabels at the top indicate the
Maxam-Gilbert sequence reaction (M), control without protein
added (-), and the addition of 5 and 25 µg of nuclear
protein, respectively. The brackets denote the DNase I
protected regions (numbers are relative to the MCAD gene
transcription start site = +1). F, schematic of
the DNase I protected sites of the MCAD gene promoter. The DNA
fragments employed in the DNase I protection experiments are shown at
the top. The location of each of the footprinted regions is
indicated by the symbols. The DNA sequences of the footprinted
regions are shown at the bottom. The underlinedsequences indicate the homologous GC-rich motifs shared
by sites A-C. The arrows indicate the location and
relative orientation of the putative nuclear receptor binding
half-sites in NRRE-1, -2, and -3 (based on the consensus RG(G/T)NA).
The box identifies the sequence within NRRE-3 that conforms to
the SF-1/FTZ-F1 binding consensus.
Each of
sites A-C and NRRE-1 exhibited similar footprinting patterns with
nuclear proteins derived from all three cell lines (Fig. 1, A-D). In contrast, the DNase I protection patterns at
NRRE-2 and NRRE-3 were cell specific. At NRRE-2, DNase I protection was
strong with 3T3 nuclear extracts, weak with HepG2 extracts, and absent
with Y-1 nuclear proteins (Fig. 1C). In contrast, both
Y-1 and 3T3 nuclear proteins conferred a prominent footprint at NRRE-3,
whereas HepG2 nuclear extracts did not interact with this site (Fig. 1E).
MCAD Gene Promoter Protein Binding Sites Function as
Transcriptional Regulatory Elements
To determine if the
MCAD gene promoter regions delineated by the DNase I protection studies
function as transcriptional regulatory elements, the full-length MCAD
promoter and a series of mutant promoter fragments with various
deletions fused to a CAT gene were transiently transfected into HepG2,
Y-1, and 3T3 cells. The initial transfection experiments evaluated the
activity of the full-length MCAD gene promoter construct
(MCADCAT(-362/+189)) among the three cell lines. The
transcriptional activity of MCADCAT(-362/+189) was
normalized for transfection efficiency by cotransfecting pMSV-gal.
MCADCAT(-362/+189) activity was compared to that of an SV40
promoter in the identical CAT reporter plasmid backbone (pCATSV40) to
control for cell line-specific differences in background CAT activity.
MCADCAT(-362/+189) activity was high in HepG2 cells (44%
pCATSV40 activity) and Y-1 cells (38% pCATSV40 activity) but more than
10-fold lower in 3T3 fibroblasts (3.4% pCATSV40 activity, Fig. 2A). The steady-state MCAD mRNA levels in each of
the cell lines, as determined by Northern blot analysis (data not
shown), paralleled the CAT activities. A similar cell line-specific
pattern of MCADCAT(-362/+189) transcription was observed
when the data were compared to the activity of a herpes simplex virus
thymidine kinase (TK) promoter-CAT plasmid standard (data not shown).
These data indicate that elements involved in cell-specific control of
MCAD gene transcription are contained within the
MCAD(-362/+189) promoter fragment.
Sp1 or a Related Protein Binds Sites A-C in the
MCAD Gene Promoter
EMSAs were performed to identify the
protein(s) that bind MCAD promoter elements A-C. A
double-stranded oligonucleotide containing the site A element was
employed as a probe in EMSA performed with nuclear protein extracts
prepared from Y-1, HepG2, and 3T3 cells. Two DNA:protein complexes were
observed with each nuclear extract (Fig. 3A, lanes2-4, I and II). Complex I was the most prominent in
each case. Competition with 100-fold molar excess unlabeled site A
oligonucleotide or a size-matched, unrelated DNA fragment confirmed
that complexes I and II represented a specific protein-DNA interaction (Fig. 3A, lanes5 and 6).
Parallel experiments revealed that an identical mobility shift pattern
was observed with oligonucleotide probes containing site B or C
sequences and that a molar excess of unlabeled site B diminished or
site C abolished the formation of complexes I and II (data not shown).
These data suggest that identical or similar transcription factors bind
MCAD promoter sites A-C in all three cell lines.
Figure 3:
Electrophoretic mobility shift assays
designate Sp1 or a highly related protein as binding to sites A-C
in the MCAD promoter region. A, autoradiogram of the EMSA
performed with a P-labeled site A double-stranded
oligonucleotide probe. The probe was incubated with 1.0 µg of
nuclear protein extract (Ext) derived from HepG2 (H),
3T3 (T), or Y-1 cells. Competition (Comp) with
100-fold molar excess of unlabeled, size-matched, nonspecific (N), or specific (S) double-stranded oligonucleotides
is indicated at the top (lanes5 and 6). Complexes I and II are indicated. B, EMSA with
the site A probe using 1.0 µg of Y-1 nuclear extract. Lanes2-7 contained 100-fold molar excess unlabeled DNA
competitor as denoted at the top: nonspecific (N),
site A (S), Sp1, AP-2, nuclear respiratory factor-1 (NRF-1), and myocyte nuclear factor (MNF) (see
``Materials and Methods'' for sequences). C,
antibody gel mobility supershift with site A (A) or Sp1
consensus site (Sp1) oligonucleotide probes and Y-1 nuclear
extract or purified recombinant Sp1 protein (one footprint unit).
Antibody (Ab) was added to the reaction mixture as indicated
at the top. 1 µl of undiluted anti-Sp1 or pre-immune serum (PI) was added to the incubation as described under
``Materials and Methods.''
Analysis of
the DNA sequence of sites A-C revealed that each contained
several homologous GC-rich sequences, including the known core
consensus for binding members of the Sp1 transcription factor family
(CCCGCCC) or a related motif, C(C/G)C(A/T)(C/G)(C/G)(C/G) (Fig. 1F). Competition experiments were performed with
unlabeled oligonucleotides containing consensus binding sites for
factors known to bind GC-rich sequences including Sp1(11) ,
AP-2(18, 19) , nuclear respiratory factor-1(20) ,
and myocyte nuclear factor (21) in an attempt to identify the
transcription factor that binds sites A-C. Competition was
observed only with the oligonucleotide containing an Sp1 binding
consensus sequence (Fig. 3B and data not shown).
(23) , and
alcohol dehydrogenases 2 and 3(24) . The wide variance in Sp1
binding site sequences may define diverse binding affinities necessary
for competitive or synergistic interactions of Sp1 with other
transcription factors. Alternatively, sequence-specific binding by
related but distinct members of the Sp1 family may occur at sites
A-C in vivo. In support of the latter possibility,
several new Sp1 isoforms have recently been
identified(23, 25) .
Orphan Nuclear Receptor Transcription Factors,
COUP-TF and SF-1, Interact with MCAD Promoter NRRE Sites in Cell
Line-specific and Element-specific Patterns
We have shown
previously that multiple nuclear receptor-mediated regulatory pathways
converge on the complex MCAD gene promoter element NRRE-1 to
bidirectionally modulate MCAD gene promoter
activity(7, 15, 16, 17) . Our cell
culture cotransfection experiments revealed that NRRE-1 is activated by
9-cis retinoic acid and all-trans retinoic acid receptors in the
presence of ligand(7) . NRRE-1 also confers transcriptional
activation by the orphan receptor, hepatocyte nuclear factor 4
(HNF-4)(15) , and repression by COUP-TF and its isoform,
apolipoprotein regulatory protein 1(15, 16) . These
studies also demonstrated that additional unidentified
COUP-TF-responsive element(s) are present in the MCAD gene promoter
region downstream of NRRE-1.
Figure 4:
NRRE-1, NRRE-2, and NRRE-3 bind nuclear
orphan receptors in a cell-specific and element-specific manner. A, EMSA with labeled NRRE-1 (N1) and NRRE-3 (N3) probes and 3T3 cell nuclear extracts (1.0 µg).
Competition (Comp) with 100-fold molar excess of unlabeled
specific (S), nonspecific (N), NRRE-1 (N1),
NRRE-2 (N2), or NRRE-3 (N3) double-stranded
oligonucleotides is indicated at the top. B, gel
mobility supershift with anti-COUP-TF (coup) or pre-immune
serum (PI) using 3T3 nuclear extracts with N1, N2, or N3
probes. C, EMSA with the N3 probe and Y-1 cell extract (1.0
µg) in lanes1-6 or 3T3 cell extract (1.0
µg) in lane7. Competition was performed with
100-fold molar excess of specific (S), nonspecific (N), NRRE-1 (N1), or NRRE-2 (N2) unlabeled
double-stranded oligonucleotides. Complexes I-III are indicated. D, gel mobility supershift with the N3 probe and Y-1 nuclear
extracts incubated with nuclear extract alone (-), pre-immune
serum (PI), anti-COUP-TF (coup), or anti-SF-1 (SF1) as described under ``Materials and Methods.''
Note that the SF-1 antibody has been shown previously to abolish
SF-1-DNA interactions (13), whereas the anti-COUP antibody supershifts
a COUP-TF-containing complex. Ext, nuclear protein extract; Ab, antibody; Comp,
competition.
Antibody supershift
assays were performed with antisera raised to a variety of nuclear
receptors to identify the specific factor that bound NRRE-1, NRRE-2,
and NRRE-3 in 3T3 cells. Anti-COUP-TF antibody supershifted the entire
DNA:protein complex formed with each of the probes (Fig. 4B). In contrast, pre-immune serum, anti-HNF-4, or
anti-9-cis retinoic acid receptor had no effect (Fig. 4B and data not shown). Thus, the orphan nuclear
receptor COUP-TF, or a closely related protein, binds each of the NRRE
sites in 3T3 cells. The low MCAD gene expression in 3T3 fibroblasts is
consistent with the known transcriptional repressive effect of COUP-TF
via the majority of known COUP-TF-responsive elements, including NRRE-1
(16, 26-28).
nor anti-HNF-4 affected the mobility of
any of the complexes (data not shown).
-oxidation and
steroidogenesis.
Figure 5:
NRRE-3 confers cell line-specific
transcriptional activation or repression to a heterologous promoter.
Mean CAT/-gal activities of pTKCAT(NRRE-3) and pTKCAT(-)
transfected into Y-1 and 3T3 cells are shown. The bars represent mean CAT/
-gal activity normalized to that of
pTKCAT(-) (=1.0).
Evidence is emerging that Sp1
enhances cooperative interactions among multiple transcription factors
to juxtapose the transcriptional regulatory domains of the proteins
with the transcription initiation
complex(31, 32, 33, 34, 35, 36) .
The interposition of Sp1 binding sites between multiple nuclear
receptor response elements shown here suggests that Sp1-nuclear
receptor cooperative interactions may play a role in MCAD gene promoter
function and regulation. The lactoferrin (37) and Oct-4 (38)
gene promoters also contain multiple Sp1 binding sites in close
proximity to nuclear receptor response elements. In other gene
regulatory regions, such as the human immunodeficiency virus long
terminal repeat (39) and promoters of the genes encoding growth
hormone (40), chorionic somatomammotropin(40) , and the
epidermal growth factor receptor(41) , Sp1 binding sites overlap
thyroid hormone receptor binding sites, indicating that, in some
instances, Sp1 may compete with nuclear receptors in binding cognate
DNA elements. Thus, competitive and cooperative interactions among Sp1
and other transcription factors, including members of the nuclear
hormone receptor superfamily, are a potential mechanism, whereby
promoter activity is modulated via many diverse signaling pathways.
-oxidation, MCAD
promoter NRREs interact with tissue-specific expressed activator orphan
nuclear receptors. In contrast, in low MCAD expressing cells and
tissues, transcriptional repression is mediated by members of the
COUP-TF family of receptors. Moreover, as we have recently shown, fatty
acids regulate MCAD gene transcription via the orphan receptor PPAR
through NRRE-1. Thus, this complex promoter structure allows multiple
regulatory pathways to differentially regulate MCAD gene expression in
a variety of cell types and in response to diverse metabolic and
physiologic conditions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.