(Received for publication, April 10, 1997)
From the Genetics and Microbiology Department, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom, and § Institut für Allgemeine Pharmakologie und Toxikologie, Klinikum der Johann Wolfgang Goethe-Universität Frankfurt am Main, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany
In vitro DNase I footprint analysis
of the rat fatty acid synthase (FAS) promoter from 568 to
468
revealed four protein binding sites: A, B, and C boxes and the FAS
insulin-responsive element 1 (FIRE1). As demonstrated by gel mobility
shift analysis and supershift experiments, FIRE1, located between
516
and
498, is responsible for binding NF-Y. The C box located
downstream of FIRE1 was shown by in vitro footprinting to
be a Sp1 binding site, and furthermore, competition with Sp1 also
abolished FIRE1 binding. Since the half-life of the Sp1·NF-Y·DNA
complex is significantly longer than the half-lives of the Sp1·DNA or
NF-Y·DNA complexes, the two transcription factors are deemed to bind
cooperatively in the FAS promoter at
500. It is unusual that NF-Y
binds at this distance from the start site of transcription. NF-Y
binding sites are found in the promoters of at least three other FAS
genes, viz. goose, chicken, and man. A second NF-Y binding
site is located in the FAS promoter at the more usual position of
103
to
87, and it too has a neighboring Sp1 site. CTF/NF-1 competes for
proteins binding to the B box. The A box binds Sp1 and contains a 12/13 match of the inverted repeat sequence responsible for binding the
nuclear factor EF-C/RFX-1 in the enhancer regions of hepatitis B virus
and the major histocompatibility complex class II antigen promoter. The
same relative positions of NF-Y and Sp1 binding sites in the promoters
of FAS genes of goose, rat, chicken, and man emphasize the involvement
of these transcription factors in the diet and hormonal regulation of
FAS.
Fatty acid synthase (FAS; EC
2.3.1.85),1 responsible for
fatty acid synthesis de novo, is one of the main lipogenic
enzymes (1, 2). Not surprisingly, this enzyme, which converts dietary calories into a storage form of energy, reacts to diet, the cognate mRNA undergoing a severalfold induction when a previously starved animal is refed (3, 4). This refeeding phenomenon is not encountered in
diabetic animals, suggesting that the peptide hormone insulin may have
a direct or indirect effect on the nutritional response (5). To study
the molecular basis of the nutritional response we embarked on a
systematic investigation of the promoter of the FAS gene of
Rattus norvegicus and have shown that its chromatin structure responds to a nutritional stimulus as demonstrated by its
altered sensitivity to DNase I (6). The distribution of DNase
I-hypersensitive sites in the rat FAS promoter changed and their number
increased when hepatic chromatin from refed animals was compared with
that of starved animals. In the region of the DNase I-hypersensitive
site located at approximately 500, we identified a tripartite
element, FIRE1, with strong sequence homology to the insulin-responsive
element of the human GAPDH gene (7). Using gel mobility shift assays we
showed that the protein binding properties of FIRE1 were dependent on
each of the three regions, viz. 5
-GCCT, a 6-nucleotide
spacer, and a 3
-palindrome. Transient transfection of the human
hepatoma cell line HepG2 with successively deleted FAS promoter
constructs fused to the chloramphenicol acetyltransferase gene has
shown that the promoter construct retaining the DNase I-hypersensitive
site mediates a 2.5-fold effect of insulin as measured by CAT activity
(8). Based on these results we propose that the nutritional effect on
the expression of the rat FAS gene may be insulin-mediated via
protein(s) binding to the FIRE1 element. A second insulin-responsive
element, FIRE2, located at
300 base pairs on the promoter of the rat
FAS gene, was revealed by these transfection studies. Yet a third
element, FAS-IRS-A, has been postulated by Sul and co-workers (9),
between nucleotides
68 and
52 and has been defined by gel mobility
shift assay and in vitro footprint analysis. This location
of FAS-IRS-A could well be the same as that of FIRE3, which we claimed
to be responsible for the 2-fold insulin-mediated stimulation of the
FAS promoter activity observed with the
179 FAS/CAT promoter
construct in H4IIE and HepG2 (8). Furthermore, three tandem repeats of
FAS-IRS-A conferred insulin responsiveness on a heterologous promoter
in 3T3-L1 adipocytes (9). However, the insulin response conferred by
three tandem repeats of FAS-IRS-A could not be repeated in our hands.
The ubiquitous basic helix-loop-helix leucine zipper-containing transcription factors USF1 and USF2 have been identified as major components of the protein complex(es) binding to FAS-IRS-A (10). USF
has also been shown to bind to carbohydrate-responsive elements (ChoRE)
found in the promoters of several lipogenic genes (11). In
vitro footprint analysis and the ability to confer glucose responsiveness on a heterologous promoter, permitted the definition of
a ChoRE at position
1448 and
1428 in the promoter of the S14 gene (11) and at
171 and
124 in the liver-type
pyruvate kinase gene (12). A similar sequence has been postulated to be
present in the promoter of the rat FAS gene within the first intron and
has been shown to confer glucose responsiveness on a heterologous
promoter. This sequence is not liver-specific since a footprint was
found at the same position when using extracts from spleen, and gel
mobility shift studies have shown that it binds the ubiquitous USF/MLTF
transcription factor and CTF/NF-1 (13).
Another member of the basic helix-loop-helix leucine zipper
transcription factor family, ADD1, was found by screening a rat adipocyte library with the EC oligonucleotide having the sequence 5-GATCCAATTGGGCAATCAGGA-3
(14). The artificial EC oligonucleotide has
a 9-nucleotide identity with part of the FIRE1 element we have defined
by DNase I hypersensitivity between
518 and
495 of the promoter of
the rat FAS gene. A CAT reporter gene containing multiple copies of the
relevant region of the FAS promoter could be stimulated when the ADD1
protein was coexpressed in NIH 3T3-L1 fibroblasts. ADD1 is the murine
counterpart of the human SREBP-1, which has been shown to bind to the
promoter of the rat FAS gene (15). ADD1/SREBP-1 is involved in the
sterol regulation of the FAS promoter, thus linking the regulation of
lipogenesis with that of cholesterol (16).
In this study we investigated the protein binding characteristics of
FIRE1, i.e. we undertook footprint analysis of the promoter region that contains a diet-induced DNase I-hypersensitive site and
whose sequence contains both E- and CAAT-box motifs (6). Using a series
of gel mobility shift analyses, we searched for the FIRE1 binding
factor by a process of elimination and identified the transcription
factor binding thereon as one which has been conserved from yeast to
mammals, namely NF-Y. The initial in vitro footprint
analysis indicates the presence of a Sp1 binding site next to FIRE1.
The cooperative binding has been demonstrated between NF-Y and Sp1 by
measuring the half-lives of the relevant DNA·protein complexes. We
also found a second NF-Y/Sp1 binding site, albeit with a different
spacer, at 103/
82 of the FAS promoter and will comment on the
remarkable conservation between the promoters of FAS genes of birds and
mammals (17-20).
The Drosophila melanogaster Schneider line 2 cells (21) were cultured at 27 °C in Schneider's Drosophila medium (Sigma) supplemented with 10% fetal calf serum, 120 mg/liter penicillin, 120 mg/liter streptomycin, and 25 µg/liter Fungizone. Transfections were done by the calcium phosphate co-precipitation method (22) using 3 µg of the appropriate plasmids and 100 ng of the expression plasmid PacSp1. The transfected cells were incubated for 48 h, and extracts were prepared as described previously (8). H4IIE cells were cultured and transfected exactly as described in O'Brien et al. (23). This protocol minimizes variability arising from differences in transfection efficiency. Where indicated 10 nM insulin was added for a period of 24 h. Luciferase activity was measured in a Lumat luminometer LB9501 (Berthold, UK) with luciferin as the substrate (Promega Biotech Inc.).
Plasmids and OligonucleotidesThe FAS minimal promoter was
created by using an oligonucleotide corresponding to the rat FAS
sequence from 50 to +12 relative to the transcriptional start site of
the FAS mRNA (17). The double-stranded oligonucleotide decorated
with NheI and BglII sites was cloned into pGL2
basic (Promega). Two copies of the A oligonucleotide representing the A
box were inserted in the NheI site upstream of the FAS
minimal promoter to create plasmid A. Plasmid C with two copies of the
C box was constructed in a similar manner. The promoter fragment for
footprint analysis was derived from the FAS/CAT construct
565/+65
(8). pGEM-NF-YA was constructed by inserting the entire NF-YA-coding
sequence contained on an EcoRI/XbaI fragment of
pYA-PB9 (24) into pGEM-3Zf(+) (Promega). The
SalI/XhoI herpes simplex virus thymidine kinase promoter fragment of CAT5 (25) was inserted into the XhoI
site of pGL2 basic to create LuciTK. LuciTK-IGFBP-1 contains the IRE of
the promoter of the IGFBP-1 gene (26) upstream of the tyrosine kinase
promoter. pGL(
816) contains the FAS promoter from
816 to +67
upstream of the luciferase gene of pGL2 basic. In the pGL(
816) derivative pGL(
816)
FIRE1 the FIRE1 core sequence
5
-TGTCCAATTGGTCT-3
has been replaced by a PstI restriction
site. The mutation was created by the Chameleon double-stranded,
site-directed mutagenesis kit (Stratagene). Similarly, pGL(
816)
C
box contains a HindIII site instead of the core sequence
5
-CCACGCCCC-3
.
Single-stranded oligonucleotides were synthesized using the Applied
Biosystem model 392 with appropriate restriction endonuclease sites
BamHI and XbaI (not shown) at their termini.
AP-1, AP-2, C/EBP, CREBP, MLTF-1, NF-1, and Sp1 (27) are consensus
binding sites for the corresponding transcription factors. FIRE1, FIRE2 (6), FAS-IRS-A (9), CCAATFAS, and A, B, and C
oligonucleotides (17) represent sequences in the FAS promoter. mTF
C/EBP (28) and IRE-AGAPDH (7) are derived from the mouse
transferrin and human GAPDH promoters, respectively. The EC box is a
synthetic sequence containing a binding site for C/EBP and an
overlapping E-box motif (14). The CCAATTPH is derived from
the human tryptophan hydroxylase promoter (29). IGFBP-1 IRE is an IRE
in the promoter of the IGFBP-1 gene (26). The core sequence of each
oligonucleotide is underlined. The sequences of oligonucleotides
were as follows (coding strand): AP-1 = 5-CGCTTGATGACTCAGCCGGAA-3
; AP-2 = 5
-GATCGAACTGACCGCCCGCGGCCCGT-3
; CCAATFAS =
1035
-CGCTCATTGGCCTGGGC-3
87;
CCAATTPH =
725
-TTCTCATTGGCCGCTGC-3
56;
C/EBP = 5
-TGCAGATTGCGCAATCTGCA-3
; CREB = 5
-AGAGATTGCCTGACGTCAGAGAGCTAG-3
; EC box = 5
-GATCCAATTGGGCAATCAGGA-3
; FAS A box =
5645
-TGCCTAGCAACGCCCACCCGCGCGCCACCATTGGGC-3
529;
FAS B box =
5345
-TTGGGCCACCGAGAACGGCCTCGGTGTCCAA-3
504;
FAS C box =
4895
-GAGCAGGCCACGCCCCTCGGCT-3
468;
FAS ChoRE = +2835
-GGCCGCTGTCACGTGGGCGCC-3
+303;
FAS- IRS-A =
715
-TCAGCCCATGTGGCGTGGCCGC-3
50;
FIRE1 =
5175
-GCCTCGGTGTCCAATTGGTCTC-3
496;
FIRE1/C box=
5185
-GGCCTCGGTGTCCAATTGGTCTCGATGTGGAGCAGGCCACGCCCCTCGGCT-3
468;
FIRE2 =
2695
-GAGCCCCGCGTGGCCCGCGGGAG-3
247;
IRE-AGAPDH =
4715
-AACTTTCCCGCCTCTCAGCCTTTGAAAG-3
444;
IGFBP-1-IRE =
1245
-CACTAGCAAAACAAACTTATTTTGAACA-3
97;
MLTF-1 =
705
-AGGTGTAGGCCACGTGACCGGGTGTTCC-3
43;
mTF C/EBP =
1055
-CGGGGTGATTGGGCAATTGGACTG-3
82;
NF-1 = +205
-TATTTTGGATTGAAGCCAATATGATAATGA-3
+49;
Sp1 = 5
-ATTCGATCGGGGCGGGGCGAGC-3
.
Whole cell extracts from HepG2 were prepared according to Jiang and Eberhardt (30). Nuclear extracts of HepG2, H4IIE, and 3T3-L1 were prepared according to the procedure of Schlokat (31).
Gel Mobility Shift AnalysisAssays were performed exactly as described previously (6). For heat treatment, FAS-induced rat liver extract was incubated for 5 min at 80 °C before proceeding with the gel mobility shift analysis. Recombinant human Sp1 prepared from Escherichia coli was purchased from Promega.
Gel Supershift1 µg of specific anti-rat C/EB,
C/EBP
(Santa Cruz Biotechnology), NF-YA, or NF-YB antiserum was
preincubated with 5 µg of nuclear extract for 15 min at room
temperature before analysis of DNA/protein binding in the standard
binding assay.
A 6-fold scale-up of the standard gel mobility shift reaction was allowed to come to equilibrium. A 1000-fold molar excess of competitor in a volume of 4% of the binding reaction was added and aliquots of the binding reactions were loaded onto a continuously electrophoresing gel (electrophoresis buffer: 50 mM Tris-HCl, pH 8.5, 375 mM glycine, 2 mM EDTA) at 0, 2, 4, 8, 16, and 32 min after the addition of the competitor. To determine the half-life of the NF-Y·Sp1 complex the FIRE1/C box oligonucleotide was used and the FIRE1·NF-Y·Sp1 complexes were obtained using FIRE1 and C box oligonucleotides, respectively. Half-lives of the complexes were determined from a plot of the fraction of total probe in the specific complex at times during the competition divided by the fraction of total probe in the specific complex at time 0 min versus time. Complexes were quantified by PhosporImager analysis.
In Vitro DNase I FootprintingThe FAS promoter fragment
between 451 and
566 was cloned into the
HindIII/BamHI-restricted pBluescript SK(+)
(Stratagene) to create pBS(
566/
451). Restriction of this plasmid
with ApaI and XbaI releases the promoter fragment
to be investigated. The sense strand was labeled by filling in the
XbaI site using Klenow fragment in the presence of
[
-32P]dATP, and the antisense strand released from
pBS(
566/
451) as a SalI/SacI fragment was
end-labeled at the SalI site. Labeled DNA (~20,000 cpm)
was incubated for 30 min with 2 µg of poly(dI-dC)·poly(dI-dC) and
60 µg of nuclear protein extract of induced rat liver (6) in a final
volume of 40 µl (20 mM HEPES, pH 7.9, 25 mM
NaCl, 1.25 mM MgCl2, 0.62 mM
CaCl2, 0.2 mM EDTA, 7 mM
-mercaptoethanol, 10% glycerol). For competition experiments
oligonucleotide competitors (100-fold molar excess) were included in
the binding reaction. To determine the optimal conditions a titration
was performed for each probe using increasing concentrations of DNase I
for the same amount of nuclear extract. 5-10 milliunits of DNase I in
DNase I buffer (1 mM MgCl2, 1 mM
dithiothreitol, 20 mM KCl) were added, and following a
2-min incubation at room temperature the reaction was terminated by the
addition of 160 µl of DNase I stop buffer (200 mM NaCl,
30 mM EDTA, 1% SDS, 100 µg/ml tRNA) and 2 µl of
proteinase K (25 µg/µl). After a 30-min incubation at 42 °C
reactions were extracted twice with phenol/chloroform/isoamylalcohol and ethanol-precipitated before analysis on a 5% polyacrylamide, 7 M urea sequencing gel.
After linearization at
the XbaI site 3 to the inserted cDNA pGEM-NF-YA (NF-YA)
and pCiteCBF-C (CBF-C) or at the BglII site in pYB-EM38
(NF-YB) the plasmids were transcribed and translated in
vitro as described in Roder et al. (32).
To examine the protein binding
properties around the diet-induced DNase I-hypersensitive site at 500
of the FAS promoter, a restriction fragment extending from
451 to
566 was subjected to in vitro DNase I footprint analysis.
Suitably labeled probes were incubated with a nuclear extract prepared
from the livers of rats fed a FAS-induction diet (4). As shown in Fig.
1A, there are four protected
areas on the sense strand designated A, B,
C, and FIRE1. The FIRE1 footprint on the sense
strand extends from
498 to
514 and on the antisense strand from
501 to
516. Competition studies with FIRE1 confirm the footprint
(Fig. 1B). Of the remaining three protected regions B and C
flank FIRE1 and the A box is located upstream of the B box. The
protected region in A extends from
536 to
-564 and in B from
520
to
534 on the sense strand. Identical footprints were obtained when
the analysis was performed using nuclear extracts prepared from the livers of rats fed a normal diet or starved (data not shown).
Since the FAS promoter has been shown to have a high GC content (17),
67% in this region, the presence of binding sites for the general
transcription factor Sp1 (33) are to be expected. The C box extending
from 470 to
489 on the antisense and
488 to
468 on the sense
strand is indeed the result of Sp1 binding (Fig. 1B), since
the footprint is abolished when an oligonucleotide representing the Sp1
consensus sequence is included in the assay. Furthermore, the addition
of the Sp1 oligonucleotide to the binding assay also removes the FIRE1
footprint, suggesting cooperativity between Sp1 bound to the C box and
any protein(s) binding to FIRE1.
The proteins binding to the A, B, and C boxes have been identified by gel mobility shift assays. Enhancement of protein binding to the A and C boxes was observed following the addition of Zn2+ to the assay (data not shown). With recombinant Sp1 we have confirmed that Sp1 binds to the C box and does so strongly, since a 30-fold molar excess of an oligonucleotide corresponding to the C box abolishes binding. On the other hand, the affinity of the A box for Sp1 is lower, since a 300-fold molar excess of an oligonucleotide corresponding to the A region is required to abolish binding (data not shown). These differences in binding to the Sp1 consensus sequence of the A and C boxes can probably be explained by the number of mismatches to the Sp1 consensus sequence; viz. 1/9 for the C box and 2/9 for the A box.
From preliminary studies we know that the B box does not bind Sp1.
Therefore, we tested cognate oligonucleotides for C/EBP, CREB, Sp1,
AP-1, AP-2, and MTLF-1 (27) DNA binding sites as competitors for B box
binding in gel mobility shift assays. Upon incubation with nuclear
extracts from H4IIE, the B box represented by the oligonucleotide
extending from 534 to
504 of the FAS promoter gives rise to two
DNA·protein complexes. In all but two instances the complexes
remained unchanged. The exceptions were the oligonucleotides
corresponding to CTF/NF-1 (34) and the B box itself. The NF-1
oligonucleotide competed out the lower migrating complex B2 suggesting
an implication of CTF/NF-1 or related factor together with one or more
unknown proteins responsible for the B box binding characteristics
(Fig. 2). Indeed the core B box shows a
6/9 match with the CTF/NF-1 (34) consensus sequence 5
-(T/C)GG(A/C)N5-6GCCAA-3
(27).
The relative contribution of Sp1 binding at the A and C boxes of the
FAS promoter has been demonstrated in the Schneider cell line derived
from D. melanogaster embryos. Tandem copies of the A and C
boxes were placed in front of the FAS minimal promoter linked to the
firefly luciferase gene. When these plasmids were co-transfected with
an expression plasmid for Sp1, a 4.5-fold increase in luciferase
activity was observed with the plasmid C, whereas a 2.5-fold effect was
found with plasmid A (Fig. 3).
FIRE1 Is Different from Known E-box-containing Elements
Initially, we tested the binding capacity of the FIRE1
oligonucleotide with nuclear extracts derived from different cell lines and nuclear extracts prepared from the livers of rats corresponding to
the three dietary regimes, FAS-induced, normal, and starved (4). In all
instances a single retarded band migrating at about the same position
was obtained and provides the first evidence that the FIRE1-binding
protein is found in several different cell types and is independent of
dietary status (Fig. 4A). A
signal with the same mobility could be detected using nuclear extract from differentiated mouse 3T3-L1 adipocytes (data not shown).
We know from sequence comparisons of FIRE1, FIRE2 (6), and FIRE3 (8)
(FAS-IRS-A (9)) in the promoter of the rat FAS and IRE-A in the
promoter of the human GAPDH gene (7) that they have in common a
sequence-independent, tripartite structure consisting of a GCCN motif,
a spacer, and a 3-palindrome. The 3
-palindrome of FIRE1 harbors the
E-box hexanucleotide CANNTG (35) capable of binding proteins containing
the basic helix-loop-helix leucine zipper motif. This hexanucleotide
overlaps with four nucleotides of the pentanucleotide CCAAT found in
several promoters and enhancers (36). We therefore looked to see if
FIRE1 binds any of the proteins binding to other E- and
CCAAT-box-containing promoter sequences. The experiment was performed
using nuclear extracts of H4IIE and labeled FIRE1 oligonucleotide. None
of the oligonucleotides tested corresponding to the sequences,
FAS-IRS-A (9), FAS-ChoRE (11, 13), EC box (15), FIRE2 (6), and
IRE-AGAPDH (7) challenged the protein(s) binding to FIRE1
(Fig. 4B). This lack of competition between FIRE1 and each
of the oligonucleotides was confirmed in a "reverse" experiment,
i.e. using labeled FIRE2, FAS-IRS-A, FAS-ChoRE, IRE-AGAPDH, and EC box as the probes and FIRE1 as the
competitor (data not shown). Further consensus binding sites for well
characterized DNA-binding factors, such as CREB, Sp1, AP-1, AP-2,
CTF/NF-1, and MLTF-1 (27) were not capable of competing out the complex with FIRE1 detected in H4IIE nuclear extract (Fig. 4C).
These experiments eliminate known E-box-binding proteins as candidates for FIRE1- binding protein(s).
We now turned our
attention to the CCAAT motif. In gel mobility shift assay with nuclear
extracts from H4IIE the DNA·protein complex(es) bound to FIRE1 can be
abolished by an oligonucleotide corresponding to a C/EBP (37) binding
site from the mouse transferrin promoter mTF C/EBP (28) and a sequence
of the FAS promoter itself between 103 and
87
(CCAATFAS) (Fig. 4D). FIRE1 and the FAS promoter region between
103 and
87 contain a common CCAAT motif that might
be responsible for the same protein (FIRE1-binding protein) binding to
both sequences. Indeed, a labeled probe corresponding to the FAS
promoter region between nt
103 and
87 showed the same retarded
complex in gel shifts as FIRE1 and could be competed out by unlabeled
FIRE1 (32). In a FAS-induced hepatic nuclear extract the gel mobility
shift pattern with mTF C/EBP is complex due to the binding of different
C/EBP isoforms. This is in contrast to the single band obtained with
FIRE1 (Fig. 5). Since C/EBP isoforms are
resistant to thermal or chemical denaturation (38) the FAS-induced liver nuclear extract was incubated for 5 min at 80 °C prior to gel
mobility shift assay with FIRE1 and mTF C/EBP (Fig. 5). The mTF C/EBP
shows binding of several heat stable proteins, whereas the
FIRE1-binding protein (BFIRE1) disappears. This
experiment shows the heat instability of the FIRE1-binding protein and
therefore its significant difference from the other C/EBP isoforms. The thermally unstable complex obtained with mTF C/EBP migrates at the same
position as the FIRE1-binding protein and can be competed out by FIRE1
(data not shown). The common core sequence CAAT of FIRE1 and mTF C/EBP
is probably responsible for the formation of this heat-labile
DNA·protein complex.
Immunological Characterization of FIRE1-binding Protein(s)
To
further clarify that FIRE1-binding protein is not identical to one of
the C/EBP isoforms, antibodies against rat C/EBP and C/EBP
were
used in supershift assays (Fig. 6).
FAS-induced liver nuclear extract was exposed to antibodies raised
against rat C/EBP
and C/EBP
prior to incubation with labeled mTF
C/EBP oligonucleotide or FIRE1. Despite the complex gel mobility shift pattern, the use of isoform-specific antibodies made it possible to
identify DNA·protein complexes containing C/EBP
and C/EBP
because they are shifted to a higher position in the gel. No such supershift was detectable with the FIRE1 oligonucleotide. Further evidence for a common factor binding to mTF C/EBP and FIRE1 is that a
complex of apparently the same size as the FIRE1 complex is revealed in
the two supershifted samples (Fig. 6, lanes 2 and 3). Antiserum against rat C/EBP
was also incapable of
supershifting the FIRE1 complex (data not shown). The experimental
results underline clearly that some of the members of the C/EBP family
involved in the binding to the mTF C/EBP are not binding to FIRE1 but
do not rule out that other members of the C/EBP family (37, 38) bind to
FIRE1.
NF-Y, also known as CBF, CP1, or YEBP, was originally identified as
binding to the conserved Y-box element in the human or mouse major
histocompatibility complex class II Ea promoter (39-42). This Y box
contains an inverted CCAAT sequence. As seen in Fig. 7 preincubation of NF-YA and NF-YB
antisera with HepG2 extracts results in a supershift of the FIRE1
complex. The CCAAT box-binding protein NF-Y is involved in a number of
systems, among them the mouse tryptophan hydroxylase (TPH) promoter
(43). We therefore tested the CCAATTPH as well as the
CCAATFAS oligonucleotides for competition of the FIRE1
complex with NF-YA. The successful competition with both
oligonucleotides strengthens the results of the supershift with
anti-NF-Y antisera.
Proof that NF-Y also binds to CCAATFAS was obtained by supershifts with anti-NF-YA and NF-YB antisera and competition with appropriate oligonucleotides. The results of this experiment are shown in Fig. 7B.
Next we demonstrated the direct interaction of NF-Y with FIRE1 using
in vitro synthesized NF-YA and NF-YB (Fig.
8). When unprogrammed rabbit reticulocyte
lysate was incubated with FIRE1, a weak band could be observed
indicating the presence of NF-Y in the lysate. There was no difference
in the band intensity with in vitro translated NF-YA or
NF-YB showing that neither alone binds to labeled FIRE1. However, the
binding signal was enhanced approximately 10-fold when both in
vitro translated NF-YA and NF-YB were included in the assay. The
retarded complex obtained migrates at the same position as the FIRE1
complex obtained with HepG2 extract and can be competed out with FIRE1
but not with AP-1 (27). Furthermore, incubation with NF-YB antiserum
supershifted the complex.
Neither FIRE1 nor C Box Plays a Role in the Insulin Response of FAS in H4IIE
Transient transfection of hepatoma cell lines with
successively deleted FAS/CAT promoter fusion plasmids (8) suggested the
presence of three IREs in the FAS promoter (FIRE1, 2, and 3). To test
whether FIRE1 or its neighboring C box is involved in the insulin
response of the FAS promoter in H4IIE cells, we constructed several
versions of the FAS/luciferase promoter fusion plasmid of pGL(816).
As a control we used the previously identified IRE in the promoter of
the human IGFBP-1 gene (26). This element has been shown to mediate an
inhibitory effect of insulin on a heterologous promoter in human and
rat hepatoma cell lines (23, 26). The plasmids listed in Table
I were transfected into H4IIE cells, and
their ability to confer insulin-modulated luciferase expression was
assayed. LuciTK-IGFBP-1 showed a 40% inhibition of activity after
insulin treatment for 24 h. Under the same conditions, the FAS
wild type promoter construct pGL(
816) showed a 2.3-fold stimulation
of luciferase activity, which was not altered upon deletion of either
FIRE1 or C box.
|
Gel
mobility shift assays were performed with rat liver nuclear extract and
an oligonucleotide (FIRE1/C box) containing FIRE1 and the C box (Fig.
9A). Four distinct complexes
were formed with the FIRE1/C-box oligonucleotide when both NF-Y and Sp1
were allowed to bind (lane 1). One complex
(NF-Y/Sp1) corresponds to NF-Y as it is almost completely
eliminated by FIRE1 competition (lane 3) and supershifted by
NF-YB antibodies (lane 5). The other two complexes are not
influenced by FIRE1 competition and are also found when nuclear extract
is incubated with labeled C box or Sp1 oligonucleotide (cf. lanes
3 and 7 and data not shown). Two complexes
(NF-Y/Sp1 and Sp1) correspond to Sp1 as they are
competed out by Sp1 (C box; lane 2) and
supershifted by Sp1 antibodies (lane 4). The supershifted
Sp1 (BS(Sp1)) complex migrates to the same position
as the NF-Y·Sp1 complex (cf. lanes 1 and 4 and
data not shown). The GC band is caused by an immunologically unrelated
GC box-binding protein, despite competition by the C-box oligonucleotide and an Sp1 consensus oligonucleotide (data not shown).
Interestingly, competition with the C box creates a new complex
(BNF-Y) not observed previously. In this case NF-Y
binds on its own to FIRE1/C box since no free Sp1 is available. This
complex is identical to the NF-Y·FIRE complex (cf. lanes 2 and 6). Since the upper band (NF-Y/Sp1) is
eliminated by both NF-Y and Sp1 competitors and supershifted by both
anti-NF-YB and Sp1 antisera, it must be caused by binding of both NF-Y
and Sp1. As expected from the footprint results, the formation of the
NF-Y·Sp1 complex sequesters the NF-Y and precludes the formation of
the DNA·NF-Y complex (lane 2), a further indication of
cooperative binding.
To measure their relative stabilities the half-lives, i.e.
the time at which 50% of the complex remains were determined for the
complexes NF-Y·Sp1, NF-Y, and Sp1. As shown in Fig. 9B,
NF-Y binding to FIRE1 has a half-life of 5.5 min. However, when Sp1 is
given the opportunity to bind simultaneously with NF-Y the half-life of
the NF-Y·Sp1 complex increases 4.4-fold. In the opposite experiment
examination of Sp1 binding to the C box also revealed a stabilization
of binding when in an NF-Y·Sp1 complex (Fig. 9C). The
half-life of the Sp1 complex alone was 8.0 min. In the presence of
NF-Y, the half-life of Sp1 increased approximately 2.5-fold. These
findings confirm that the adjacent binding sites for NF-Y and Sp1
stabilize the NF-Y·Sp1 complex. Thus we have demonstrated that in the
FAS promoter at 500 NF-Y and Sp1 bind cooperatively.
A prerequisite for the positioning and functioning of transcription factors modulating a gene's transcriptional activity is rearrangement of the chromatin structure in its promoter (44). We observed that the chromatin structure of the promoter of the rat FAS gene is sensitive to dietary signals, not unexpected for a lipogenic gene. The dietary signal was sent out upon refeeding a starved animal and was detectable as a 30-fold increase in the amount of FAS mRNA (4). Since this transcriptional reaction to refeeding was not observed in a diabetic animal (5), the amount of circulating insulin could be the humoral factor triggering the transcriptional response.
The discovery that the position of the diet-induced DNase
I-hypersensitive site at 500 coincided with a sequence of the rat FAS
promoter (17) having similarity to the postulated IRE of the human
GAPDH gene suggested that this may be a region of the FAS promoter
receiving insulin-induced signals. This is the first indication that
the region of the FAS promoter designated FIRE1 (6) might be the end
point of an insulin cascade. However, transfection studies in H4IIE
using FAS/Luc constructs suggest that neither FIRE1 nor its neighboring
C box is directly involved in insulin regulation, since no difference
in response to insulin could be detected between constructs carrying
the wild type promoter or the same promoter deleted for FIRE1 or C box.
This correlates with the results obtained with successively deleted
versions of the FAS promoter transiently transfected in H4IIE and
examined for their insulin response (6).
The results of the in vitro footprint analysis suggest that
that part of the FAS promoter surrounding the DNase I-hypersensitive site can be subdivided into at least four discrete protein binding regions. From the results of the gel mobility shift analysis we know
that the A and C boxes bind the general transcription factor Sp1, the B
box binds NF-1, and that FIRE1 binds NF-Y. A computer search has shown
that in the region between 900 and
300 there are no less than 26 sequence elements with 89% homology to the Sp1 consensus sequence and
therefore no lack of opportunity for bound Sp1 molecules to interact
with each other and other transcription factors causing looping of the
DNA (45) with all its implications for the activation of the
transcription machinery of the FAS promoter. Using Schneider cells we
showed that two tandem copies of the C box upstream of the FAS minimal
promoter were twice as effective as two copies of the A box in the same
plasmid. Interestingly, the sequence of the A box contains a 12/13
match of the inverted repeat sequence of the EF-C/RFX-1 binding site
5
-GTTGC(T/C)NG(G/A)CAAC-3
located in the
enhancer regions of hepatitis B virus, polyomavirus, and the major
histocompatibility complex class II antigen promoter (46), suggesting
that other factors may also bind to the A box. The Sp1 binding capacity
of the C box appears to be important for proteins binding to FIRE1,
since the inclusion of Sp1 as a competitor in the footprint analysis
closes not only the C box "window" but also the FIRE1 "window."
Indeed, measurements of the half-lives of Sp1, NF-Y, Sp1/NF-Y,
complexed with DNA showed significant differences. In the
NF-Y·Sp1·DNA complex, the interaction of both proteins with their
target sites is stabilized. Cooperative binding allows the formation of
NF-Y·Sp1·DNA complexes rather than NF-Y·DNA or Sp1·DNA
complexes. Sp1 is involved in the regulation of many housekeeping
genes, either alone or acting synergistically with another
transcription factor (15, 47). The juxtaposition of putative Sp1 and
IREs in the promoters of genes whose expression is regulated by insulin
may be coincidental but is nevertheless remarkable (48). For instance,
potential Sp1 binding sites are found in close proximity to postulated
insulin-responsive elements in the promoters of several genes including
FAS (8, 9), the human GAPDH gene (7), and rat gene 33 (49). Synergy of Sp1 and SREBP-1 is found in the promoters of the low density
lipoprotein receptor and FAS genes (15), suggesting a common regulatory mechanism in lipid and cholesterol metabolism.
NF-Y, also known as CBF or CP1, consists of three subunits, A, B, and
C, and the yeast homologues are heme activator proteins 3, 2, and 5, respectively (50, 51). CBF-C can in fact complex with CBF-A and -B. The
yeast homologue of NF-Y appears to have a general role in energy
metabolism since several genes involved in mitochondrial function,
gluconeogenesis, the glyoxalate cycle, and even fatty acid metabolism
are regulated by it (52). The cross-species nature of the protein(s)
binding to FIRE1 was vindicated by positive competition with mTF C/EBP.
This transcription factor has been shown to bind specifically to the
pentanucleotide 5-GCAAT-3
(28). This was the first experimental
evidence that the transcription factor binding to FIRE1 could be a
C/EBP-binding protein. Comparing the protein complex of FIRE1 with that
of mTF C/EBP using heat-treated nuclear extracts from the livers of
rats induced for FAS showed that one of the proteins in the smear
obtained with mTF C/EBP and the FIRE1 complex migrate at the same
position and both are heat-labile. However, following this up with
supershift analysis using antiserum directed against C/EBP
,
C/EBP
, and C/EBP
relegated these factors to those previously
tested and found not to bind to FIRE1.
Our analysis of the FAS promoter provides further evidence for the
interaction of the general transcription factors Sp1 and NF-Y.
Cooperativity between NF-Y and Sp1 has been demonstrated in the
regulation of the major histocompatibility complex class II-associated
invariant chain gene expression (53) and in the transcriptional
regulation of the farnesyl diphosphate synthase gene which is regulated
by cholesterol status. The authors of this study pose the question
whether or not the rodent equivalent of SREBP-1, ADD1, requires NF-Y
for normal function (54). While we cannot answer this question, our
results illustrate binding of NF-Y in HepG2 cells to the corresponding
region of the FAS promoter that binds ADD1 in NIH 3T3 fibroblasts.
However, some other factor must also be involved because the EC
oligonucleotide which pulled out ADD1 (14) does not compete for FIRE1
binding in rat hepatocytes. It could be that in differentiated
adipocytes ADD1 rather than NF-Y, or the mouse equivalent thereof,
binds to the DNA. Interestingly, the juxtaposition of NF-Y and Sp1
binding sites found at 500 in the FAS promoter is repeated at the
103/
87 FAS fragment, albeit with different spacing. On the basis of
competition studies the CAATFAS (
103/
87) element
competes for FIRE1 binding. There is also a supershift when this
oligonucleotide is incubated with HepG2 nuclear extract pretreated with
anti-NF-YB antiserum. Using in vitro translated NF-YA and
NF-YB we have been able to show that both NF-YA and NF-YB are required
to form the FIRE1 complex. This is in agreement with the hypothesis put
forward by Sinha et al. 1995 (51) that the three components
of NF-Y are necessary for binding to DNA. The position of FIRE1 is
unusual for a NF-Y binding site and is to our knowledge the first time that NF-Y has been shown to bind so far upstream of the transcription start site. Furthermore, it is also the first example of a repeat of an
NF-Y/Sp1 combination within the same promoter.
Inspection of the sequence of the promoter of the goose FAS gene (18)
reveals that it has a high degree of sequence similarity with the rat
FAS promoter (17). For instance, in the goose FAS promoter there is a
reverse CCAAT box and two nucleotides downstream a Sp1 binding site
between 513 and
508 with only two mismatches to the consensus
sequence. Continuing the analogy, A and B boxes are also found upstream
of the postulated FIRE1 in the goose FAS promoter between
577 and
545. Furthermore, the goose A box contains an inverted repeat of the
EF-C/RFX-1 binding site (46) The goose equivalent of the rat
CCAATFAS motif is located at
94 and
88. Although we
have no functional data for the goose (18), chicken (19), and human
(20) FAS promoters, the similarity at the nucleotide level and the
conserved relative positions of the cis-elements functionally tested in the rat FAS promoter is compelling.
It is known that NF-Y, like Sp1, introduces distortions in the double
helix (55) and creates an environment for the recruitment of
transcription factors which more than likely exert their effects via
protein/protein interactions rather than DNA/protein interactions. This
could mean that the NF-Y/Sp1 interactions set the stage for the
transcriptional activation of the FAS gene. Our experiments show that
the region of the diet-induced DNase I-hypersensitive site at 500 is
occupied by a set of general transcription factors whose orchestrated
performance may be the result of glucose/insulin-induced effect on the
activation of one or more of them.
We are grateful to Dr. R. Tjian (University of California, Berkeley) for the Sp1 expression plasmid used in these studies. We also thank Dr. M. Freeman (MRC, Cambridge, UK) for the Schneider cell line from D. melanogaster and Drs. R. Printz and D. K. Granner (Vanderbilt University Medical School, Nashville, TN) for H4IIE cells. We are particularly grateful to Dr. R. Mantovani (Università degli Studi di Milano, Milano) for providing the NF-YA and NF-YB antisera and Drs. D. Mathis and C. Benoist (CNRS, INSERM, Université Louis Pasteur, Illkirch) for giving us the expression plasmids pYA-PB9 (NF-YA) and pYB-EM38 (NF-YB). We thank John Payne for synthesizing the oligonucleotides and Angie Walker for photography.