(Received for publication, September 25, 1996, and in revised form, December 3, 1996)
From the Department of Biochemistry and Institute of Human Genetics, Medical School, University of Minnesota, Minneapolis, Minnesota 55455
Hepatic expression of the genes encoding L-type
pyruvate kinase (L-PK) and S14 is induced in rats
upon feeding them a high carbohydrate, low fat diet. A carbohydrate
response element (ChoRE) containing two CACGTG-type E boxes has been
mapped in the 5-flanking region of both of these genes. The nature of
the ChoRE suggests that a member of the basic/helix-loop-helix/leucine
zipper family of proteins may be responsible for mediating the response
to carbohydrate. Indeed, the upstream stimulatory factor (USF), a
ubiquitous basic/helix-loop-helix/leucine zipper protein, is present in
hepatic nuclear extracts and binds to the ChoREs of L-PK and
S14 in vitro. We have conducted experiments to
determine whether USF is involved in the carbohydrate-mediated regulation of L-PK and S14. For this purpose, dominant
negative forms of USF that are capable of heterodimerizing with
endogenous USF but not of binding to DNA were expressed in primary
hepatocytes. Expression of these forms did not block either
S14 or L-PK induction by glucose. In addition, we have
constructed mutant ChoREs that retain their carbohydrate responsiveness
but have lost the ability to bind USF. Together, these data suggest
that USF is not the carbohydrate-responsive factor that stimulates
S14 and L-PK expression and that a distinct hepatic factor
is likely to be responsible for the transcriptional response.
The mammalian liver is capable of converting excess dietary carbohydrate into triglycerides for storage. Feeding of a diet high in simple carbohydrates and low in fats results in the induction of many hepatic enzymes involved in lipogenesis, including liver-type pyruvate kinase (L-PK),1 fatty acid synthase, malic enzyme, and acetyl-CoA carboxylase (for reviews see Refs. 1 and 2). In each case, the increased enzyme production correlates with an increase in mRNA levels, and in some of these cases the increase in mRNA has been shown to be due to elevated transcription of the corresponding genes (3-8). Little is known about the molecular mechanisms responsible for this transcriptional response.
In our efforts to characterize the regulation of lipogenic gene
expression, we have been studying the genes encoding L-PK and
S14 in rat liver. These genes are transcriptionally induced upon carbohydrate feeding (3, 9, 10). This induction can be reproduced
in cultured primary hepatocytes by varying the glucose concentrations
in the media (9, 11, 12). Increased glucose metabolism is thought to
generate the intracellular signal in the regulatory pathway. Although
the exact nature of this pathway remains unknown, several possible
intracellular signals have been proposed (13-15). While insulin is
necessary for the regulation, it has been shown to play only a
permissive role in promoting effective glucose metabolism (16, 17).
Sequences responsible for mediating the carbohydrate response have been
mapped in the 5-flanking region of both the S14 and L-PK
genes (18-22). Both genes contain a common regulatory element that
consists of two 5
-CACGTG-type E box motifs separated by 5 base pairs
(19, 23). Linking multiple copies of this element to a basal promoter
is sufficient to induce carbohydrate-responsive activity; this element is thus referred to as a carbohydrate response element (ChoRE). However, in the context of the natural promoter, each gene contains a
distinct accessory factor site adjacent to the ChoRE that is necessary
for the full extent of the response (22-24).
The presence of the CACGTG-like sites within the ChoRE suggests that a member of the basic/helix-loop-helix/leucine zipper (b/HLH/LZ) family of transcription factors may be involved in the carbohydrate-mediated regulation (25, 26). These proteins all contain a basic DNA binding region followed by helix-loop-helix and leucine zipper dimerization domains. The b/HLH/LZ class includes Myc and its related family members, along with other proteins such as TFE3, TFEB, SREBP1/ADD1, and the upstream stimulatory factor (USF) (27-33). Of these, USF appears to be the predominant b/HLH/LZ factor in hepatic nuclear extracts. USF was first identified by its ability to bind to the upstream stimulatory element of the adenovirus major late promoter (34, 35). In vitro electrophoretic mobility shift assays (EMSAs) using hepatic nuclear extracts produced one major shifted complex that bound specifically to the S14 and L-PK ChoREs and which was "supershifted" by the addition of an antibody against USF (21, 22, 24). However, the USF binding site from the adenovirus major late promoter was unable to substitute for the ChoRE in either the L-PK or the S14 gene, indicating that USF alone is not capable of mediating the response (21). Additionally, the ubiquitous nature of USF (36), coupled with its ability to interact with sequences in other genes that are not regulated by glucose (37-39), raised the question as to how the specificity of the response is achieved. We thus set out to determine whether or not USF is involved in the regulatory pathway induced by carbohydrate.
Primary Hepatocyte Culture and Transfection
The procedure previously described was followed (20). Primary hepatocytes were isolated from male Sprague Dawley rats (180-240 g) maintained on a 12-h light/dark cycle with free access to normal chow. Following a 3-6-h attachment period, cells were transfected using the Lipofectin reagent (Life Technologies, Inc.) in modified Williams E medium (lacking methyl linoleate and glucose) supplemented with 23 mM HEPES, 0.01 µM dexamethasone, 0.1 unit/ml insulin, 1 unit/ml penicillin, 1 µg/ml streptomycin, and 11 mM glucose for 12-14 h. Cells were subsequently incubated for 48 h in medium containing either 5.5 or 27.5 mM glucose and harvested for chloramphenicol acetyltransferase (CAT) assay. For studies using dominant negative forms of USF, Matrigel (Collaborative Biomedical Products) was added to the cells at a concentration of 0.5 mg/ml after the overnight transfection. This treatment has been shown to increase the time that cultured hepatocytes remain responsive to elevated glucose concentrations (40). After the addition of Matrigel, plates were incubated in low glucose medium for 24 h before the 48-h incubation with low or high glucose. Percentage conversion of chloramphenicol to its acetylated forms was determined by thin layer chromatography followed by phosphor screen autoradiography, with subsequent scanning and quantitation (Molecular Dynamics, Inc.)
Plasmid Constructs and Oligonucleotides
S14 MutantsThe
pS14CAT(An)-4316-mut1, -mut2, and -mut3 constructs were
prepared using PCR mutagenesis to engineer mutations at the ChoRE E
boxes and the accessory factor site (41). The exact location of each
mutation is shown in Fig. 1A (Mut1,
Mut2, and Mut3). Two primers were used for each
construct, one directed upstream from the mutation site and one
directed downstream. These primer pairs contained either an
XbaI site or a BamHI site in place of the motif
to be mutated. The polymerase chain reaction was carried out using
pS14CAT(An)-4316 (9) as a template. PCR products were
digested with HindIII and XbaI/BamHI
or XbaI/BamHI and SfiI to generate
fragments extending from 2111 through the mutation and from the
mutation to
767, respectively. These fragments were then ligated into
the HindIII and SfiI sites of
pS14CAT(An)-4316. Sequences of the mutations were confirmed
by DNA sequencing.
USF Expression Vectors
Plasmids psvUSF2 and psvUSF2B,
which encode mouse USF2 and USF2
b (39), were generously provided by
Dr. Michéle Sawadogo (M. D. Anderson Cancer Center, University of
Texas). USF2 coding regions were transferred into the CMV4 plasmid
(42). This was accomplished by ligating a BglII linker to
the EcoRI site and inserting the desired USF2 fragment into
the BglII site of CMV4. A similar construct containing
USF2
N
b was prepared by carrying out the polymerase chain reaction
on the USF2 construct using primers with appended MluI or
XbaI sites to generate a product encoding USF2 amino acids
1-6 and 249-346. The PCR product was cleaved with MluI and
XbaI and ligated into CMV4. A CMV/USF1 expression plasmid
was provided by Drs. Philippe Pognonec and Robert Roeder (Rockefeller
University). The bAP4/USF1 construct was prepared by using PCR to
amplify the N-terminal and C-terminal portions of USF1 separately.
Internal primers were directed upstream and downstream from either side
of the basic region; each had an extended overhang encoding the AP4
basic region containing an NspV site. The external primers contained
appended HindIII or XbaI sites. PCR products were
digested with HindIII and NspV or NspV and XbaI and inserted into the HindIII and XbaI sites of
CMV4 by triple ligation. The USF2/VP16 construct was made by first
amplifying the entire USF2 sequence to eliminate the stop codon and
replace it with a BglII site. The PCR product was cleaved
with HindIII and BglII and inserted into
pSJT1193, a plasmid containing the C-terminal activation domain of
VP16, which was kindly donated by Dr. Steve Triezenberg (Michigan State
University). The USF2/VP16 fusion was then removed from this plasmid by
digestion with KpnI and HindIII and inserted into
CMV4.
The parental PK(96)CAT plasmid into which
altered ChoRE sequences were inserted has been described (18).
Oligonucleotides were synthesized containing the sequences shown in
Fig. 4 flanked on each side by a HindIII site. These
fragments were cloned into the unique HindIII site in
PK(
96)CAT just upstream of the L-PK basal promoter. Plasmids were
subjected to sequence analysis to determine the orientation and copy
number of the insert.
Nuclear Extract Preparation and EMSAs
Rats were fasted overnight and refed a high carbohydrate, low fat diet (ICN Biomedicals, Inc.) for 4 h before sacrifice. Nuclear extracts were prepared by the procedure of Lavery et al. (43). EMSAs were performed essentially as described (20). A typical reaction contained 50,000 cpm of 32P-labeled oligonucleotide (0.5-1 ng) and 3 µg of nuclear extract. After 45 min at 23 °C, samples were subjected to electrophoresis on 4.5% nondenaturing polyacrylamide gels in a Tris-glycine buffering system (20). For competition experiments, unlabeled oligonucleotides were added to the radiolabeled probe prior to the addition of nuclear extract.
The sequences of the rat S14
gene involved in supporting a transcriptional response to carbohydrate
were previously mapped to the 5-flanking region between
1467 and
1422 (23). This region corresponds to a liver-specific DNase I
hypersensitive site that appears developmentally at the time the
S14 gene is first expressed (44). Within this regulatory
region, the ChoRE was localized to the sequences between
1448 and
1422, consisting of two CACGTG-type E box motifs; the 5
motif is a
perfect CACGTG, while the 3
CCTGTG site is a 4/6 match (Fig.
1A). In addition, an adjacent accessory
factor site was localized between
1467 and
1448. This site did not
support a response to carbohydrate on its own but greatly augmented the
response of the S14 ChoRE to glucose. The nature of the
factor hypothesized to bind to this accessory site is under
investigation. Mapping of the S14 regulatory sites utilized
plasmids in which the elements were linked directly to a basal
promoter, with large segments of the S14 5
-flanking region
deleted. We therefore felt it was important to determine the role of
these sites in the context of the natural S14 gene. Plasmids containing S14 sequences from
4316 to +18 were
linked to the CAT reporter gene. These plasmids contained clustered
mutations within the 5
E box, the 3
E box, or the accessory factor
site. The constructs were transiently transfected into primary rat
hepatocytes, which were cultured in low (5.5 mM) or high
(27.5 mM) glucose conditions for 48 h before being
harvested and assayed for CAT activity. As depicted in Fig.
1B, mutations at each of these sites dramatically decreased
the glucose response, as compared with the response of the wild type
S14 control plasmid. This experiment clearly demonstrated
the necessity for all three of these motifs for supporting the full
glucose response of the S14 gene.
Localization of the S14 and L-PK ChoREs
allowed us to investigate the identity of the carbohydrate-responsive
factor. Since USF is capable of interacting with either of these ChoREs
in vitro (21, 22, 45), we set out to determine whether it is
necessary for the response in hepatocytes. To test this, dominant
negative USF proteins were expressed in hepatocytes, and the effect on the glucose-mediated induction of the S14 and L-PK
promoters was examined. USF is present endogenously in two isoforms,
designated USF1 and USF2, which are capable of binding to DNA as
homodimers or as heterodimers with each other (33, 46, 47). Fig.
2 shows the various forms of USF used in our studies.
The dominant negative protein bAP4/USF1 contains the basic region of
the b/HLH/LZ protein AP4 in place of the USF1 basic region, thus
altering its DNA binding specificity (48). USF2b lacks the basic
region altogether, while USF2
N
b lacks both the basic region and
the N-terminal transactivation domain. Both of these proteins are therefore incapable of binding to DNA, and USF2
N
b further lacks transactivation ability. Since all three of these forms retain the
helix-loop-helix and LZ regions, they should be capable of heterodimerizing with USF1 or USF2 but incapable of binding to the
CACGTG motif.
Before testing these dominant negative forms of USF on the carbohydrate
response, it was important to demonstrate their effectiveness. Although
USF has been implicated in the expression of several hepatic genes
(e.g. Refs. 37, 39, and 49), to date no genes have been
shown to be directly induced by USF within the hepatocyte. In addition,
cotransfection of USF into hepatocytes with reporter constructs
containing USF binding sites has not led to
transactivation.2 To circumvent this
difficulty, we constructed a "superactivator" chimeric USF protein
in which the transcriptional activation domain of VP16 is linked to the
C terminus of USF2 (Fig. 2). Introduction of a plasmid expressing this
protein stimulated the activity of a reporter plasmid containing the
adenovirus USF binding site in a dose-dependent
manner.2 In addition, the USF2/VP16 construct stimulated
expression of a plasmid containing two copies of the S14
ChoRE linked to the L-PK basal promoter and CAT in low glucose
conditions (Fig. 3A). Thus, USF has the
ability to bind to the S14 ChoRE sequence when overexpressed in hepatocytes. When the construct expressing the USF2/VP16 activator was cotransfected with the dominant negative USF
plasmids, each of the three dominant negative forms was capable of
inhibiting the action of USF2/VP16 (Fig. 3B, stippled
bars). This indicates that the dominant negative forms of USF are
expressed and functional within the primary hepatocyte.
The effect of the three dominant negative forms of USF on carbohydrate
induction was subsequently evaluated. For this purpose, we used a
construct containing two copies of the S14 ChoRE linked to
the L-PK basal promoter in the absence of the accessory factor site
(Fig. 3A), since this was the simplest glucose-responsive construct that we had found. Following transfection of the reporter gene construct with or without constructs expressing the dominant negative USF forms, cells were incubated for 24 h in low glucose conditions to allow for accumulation of the dominant negative proteins.
Cells were then incubated in low or high glucose conditions for 48 h, at which time they were harvested and CAT assays were performed.
None of the three dominant negative forms were able to inhibit the
glucose response of the reporter construct (Fig. 3B).
Similar experiments were carried out using a reporter plasmid containing the L-PK ChoRE. In this case, the L-PK ChoRE was tested in
its natural context (197 to +12) with the accessory factor site (Fig.
3A). Again, we found no significant effect of expressing the
dominant negative USF forms on carbohydrate induction (Fig. 3C). Finally, we observed no effect of the dominant negative
USF proteins on the glucose response of a plasmid containing sequences from
4316 to +18 of the S14 gene (data not shown). These
experiments suggested that USF is not involved in the carbohydrate
induction of the S14 or L-PK promoter.
The results of the experiments with dominant negative
forms of USF were in contradiction to a published report by
Lefrançois-Martinez et al. (50) studying L-PK gene
induction in a hepatoma cell line. We therefore assessed the potential
role of USF by a second criterion. For this purpose, we examined the
bases of the ChoRE sequence that are necessary to confer a response to
glucose. The goal of this study was to determine whether mutations that
affect function also affect USF binding. As a starting point, we used an optimized ChoRE containing two perfect CACGTG motifs separated by 5 base pairs in the context of the S14 ChoRE sequences. Since this element is capable of supporting a glucose response in the absence
of an accessory site, we thought that interpreting the effects of
mutations should be more straightforward. We then altered individual
bases in each CACGTG motif to create palindromic mutations. The altered
ChoRE sequences are shown in Fig. 4. These elements were
cloned into a CAT reporter vector upstream of the L-PK basal promoter.
Following transfection and a 48-h incubation in low or high glucose
medium, cells were harvested for CAT assays. Mutation of positions 1-4
in each E box motif completely abolished the carbohydrate-mediated
regulation (Fig. 5). Motifs containing mutations at the
fifth or sixth position, however, were still able to confer a response
to glucose. We also prepared plasmids in which the altered ChoREs were
placed adjacent to the L-PK accessory factor site to test their effects
in a context more similar to the natural situation. The response
pattern of this series of plasmids, however, paralleled that of the
constructs with only the basal promoter.3
Thus, at least with the specific base changes tested, the first four
bases of the E boxes were more critical for the carbohydrate response
than the last two bases.
One of the more striking results of these experiments came from the plasmid containing mutations at position 5 of each motif. The mutations created two CACGgG sites (lowercase letters here and elsewhere represent differences from the consensus CACGTG motif), identical to those found in the natural L-PK ChoRE, but flanked by bases surrounding the S14 ChoRE. This plasmid consistently responded strongly to glucose treatment, with a -fold induction approximately twice as high as that of the plasmid containing two perfect CACGTG motifs. Perplexingly, mutational studies of the L-PK promoter had demonstrated that the ChoRE, in the absence of the accessory factor binding site, was not capable of supporting a response (19, 22). Two possibilities might explain these results. The first is that the minor difference in the positioning of the ChoRE relative to the L-PK basal promoter might dramatically affect the glucose response. The second is that the nature of the bases surrounding and separating the E box motifs could modulate activity. To distinguish between these possibilities, we constructed a plasmid containing the natural L-PK ChoRE (mutant 5(PK); Fig. 4) in the same context as the other plasmids used in these experiments. This plasmid supported only a 2-fold glucose response (Fig. 5), suggesting that the bases flanking and separating the E boxes are indeed relevant to the response level.
An additional sequence was also tested in these studies. Blackwell et al. (51) had identified a series of oligonucleotides with differential binding affinities for various members of the b/HLH/LZ family of proteins. One such sequence, CAtGcG, was capable of binding to b/HLH/LZ proteins such as Myc and Max, but was unable to interact with USF. We therefore felt that this sequence might be useful for assessing the role of USF and set out to determine whether it could function as a ChoRE. Transfection analysis of a plasmid containing two CAtGcG sites, again separated and flanked by S14 bases, showed that this sequence could indeed support a glucose response approximately equal to that of the plasmid containing two CACGTG motifs (Fig. 5).
Following these functional studies, we used two types of experiments to
test the ability of the mutant ChoREs to bind to USF. The first
involved cotransfecting primary hepatocytes with reporter plasmids
containing the various mutant ChoREs and the expression plasmid
encoding the USF2/VP16 chimeric protein. In this way, we could evaluate
the binding of USF under conditions present in the hepatocyte. As
expected, the USF2/VP16 protein was capable of stimulating expression
from the construct containing two "perfect" CACGTG motifs (Fig.
6). Constructs containing mutations at E box positions
1-4, which did not respond to glucose, were also not responsive to
USF/VP16 (Fig. 6 and data not shown). Interestingly, expression of
those plasmids containing altered ChoREs that were glucose-responsive
was also not stimulated by USF/VP16. This separation of the
carbohydrate response from activation by USF suggests that USF is not
involved in the glucose-mediated regulation.
A second approach to test the role of USF in carbohydrate induction was
to use in vitro EMSAs to determine the ability of the
altered ChoREs to bind USF. Mobility shift assays were performed in
which rat hepatic nuclear extracts were incubated with the radiolabeled
oligonucleotide containing two perfect CACGTG motifs. This probe gave
one primary complex that comigrated with a complex formed by an
oligonucleotide containing the adenovirus USF site (data not shown).
This complex was supershifted completely by the addition of an antibody
to USF. Furthermore, the band was identical in migration to that seen
when bacterially expressed USF was used instead of nuclear extract.
Thus, this complex represents binding of USF to the probe sequence.
Some of the altered ChoREs were then selected for use as competitors to
determine their relative binding affinities. For this study we used
only those sequences that retained carbohydrate responsiveness, with
the exception of one negative control (mutant 4) that did not respond.
Fig. 7A depicts an example of a
representative competition experiment, while Fig. 7B
demonstrates the quantified results of the overall study. The
"self-competitor" containing two CACGTG sites competed quite
strongly for the bound complex. Three of the other sequences showed
lesser degrees of competition. The oligonucleotide containing the
CACGgG motifs flanked by S14 bases, which was highly
responsive to glucose, competed approximately 20-25 times less
effectively than the CACGTG sequence. The competitor with the natural
L-PK ChoRE and the competitor with the CACGTt motifs, both of which were somewhat less glucose-responsive, competed approximately 40-50
times and 75-100 times less efficiently, respectively. The CAtGcG- and
CACtTG-containing competitors were essentially unable to compete for
binding. These results were reproduced when recombinant human USF1 was
used in place of nuclear extract (data not shown). These experiments
demonstrated a lack of correlation between carbohydrate-responsive activity and USF binding.
We have previously identified by deletional mutagenesis a
carbohydrate response element between positions 1448 and
1422 in
the 5
-flanking region of the S14 gene (23). This sequence is similar to the ChoRE of the L-PK gene (also called the
glucose/insulin response element (19)). In either case, two CACGTG-like
E boxes separated by 5 base pairs are present. In the case of the
S14 ChoRE, however, the 3
CCTGTG motif has only a 4/6
match with the consensus sequence. Two lines of evidence support the
importance of this motif. First, mutations introduced into the 3
E box
that make it a better fit to the consensus CACGTG resulted in stronger responses to glucose (23). Second, we have now shown that specific mutagenesis of the 3
E box in the context of the natural
S14 gene largely abolished the ability of the
S14 promoter to respond to glucose. Likewise, mutation of
the 5
E box eliminated the response. Previous work has shown that both
E boxes of the L-PK gene are also critical for control (19, 22). Thus,
the presence of two E box motifs is a critical feature for mediating
the glucose response of these two members of the lipogenic enzyme
family.
The S14 and L-PK ChoREs are sufficient to support a
response to glucose when linked in multiple copies to a basal promoter or mutated to give an optimal binding site. However, in either case an
adjacent accessory factor site is required for the full extent of the
response in the context of the natural gene. For the L-PK promoter, the
hepatic-enriched factor HNF-4 is capable of binding just 3 to the
ChoRE and greatly enhancing its activity (22, 24). In the case of the
S14 gene, the accessory site located immediately 5
to the
ChoRE binds an as yet unidentified nuclear factor (23). Mutating this
element in the context of the natural S14 gene greatly
diminished the glucose response. Hence, this site appears to play an
important role in supporting regulation. A similar situation has been
found in several other genes subject to transcriptional regulation by
hormonal and metabolic factors. For example, the gene encoding the low
density lipoprotein receptor is activated when sterol levels are low by
the action of the transcriptional factor SREBP (52). The action of
SREBP by itself is weak, but it is greatly enhanced by binding of the ubiquitous factor Sp1 to adjacent sites within this gene (53, 54). Sp1
also synergizes with SREBP in the activation of the fatty acid synthase
and acetyl-coenzyme A carboxylase promoters (55, 56). Interestingly,
Daniel and Kim (57) have recently reported that Sp1 binding is also
required for glucose activation of the acetyl-coenzyme A carboxylase
promoter. Whether this Sp1 binding site is serving as an accessory
factor to an undetected ChoRE or whether it is the binding site for the
actual carbohydrate-responsive factor remains to be determined. Neither
the S14 nor the L-PK promoter appears to contain binding
sites for Sp1.
The presence of CACGTG-type E boxes within the ChoRE strongly suggests
that a member of the b/HLH/LZ family of transcription factors is
involved in control. In particular, USF has emerged from this family as
a candidate for the carbohydrate-responsive factor, based largely on
its ability to bind to the S14 or L-PK ChoREs in
vitro (22, 45). However, we have found no evidence that USF is
involved in the glucose regulation of the L-PK or S14 genes
in vivo. The expression of three different dominant negative
forms of USF did not affect the carbohydrate response of either ChoRE
in primary hepatocytes, although the same proteins were able to block
the action of cotransfected USF2/VP16. This finding is in direct
contradiction with that of Lefrançois-Martinez et al.
(50). This group found that a dominant negative form of USF1 similar to
USF2N
b could block the L-PK glucose response in the
glucose-responsive, hepatocyte-derived cell line mhAT3F. It is
difficult at present to reconcile this contradiction. Several differences in the experimental strategies might be important. The use
of the mhAT3F cell line versus primary hepatocytes
constitutes a significant dissimilarity. While the extent of the
differences between these two cells is unclear, they are certainly not
identical. For example, as opposed to the primary hepatocyte, the
glucose response in the mhAT3F cell is insulin-independent. This is
apparently due to the fact that a hexokinase other than glucokinase is
responsible for initiating the glycolytic process. Although not
specifically known, it is also possible that the mhAT3F cells could
contain less endogenous USF than the primary hepatocyte. If this were true, less dominant negative USF would be needed to inhibit the endogenous USF. Another significant difference in the experimental protocols is the time between transfection of the dominant negative USF
forms and initiation of the glucose induction period. While we used
24 h in the present study, Lefrançois-Martinez et
al. allowed a 72-h period. It may be that 72 h is needed to
effectively sequester the endogenous USF pool and thus inhibit USF
action. On the other hand, it is also conceivable that over 72 h
the effective suppression of USF could lead to reduced expression of
another transcription factor (e.g. HNF-1 or HNF-4) that is
required for transcription of the L-PK promoter. In this way, the
action of the dominant negative form of USF on the glucose response
would be secondary rather than direct.
Given these conflicting results, we sought an additional way in which the role of USF in the glucose response could be addressed. The correlation between USF binding and glucose responsiveness was examined in a set of ChoRE mutations. The identification of mutations that discriminate between USF binding and function argues against a role for USF. In particular, mutation of the fifth position of the CACGTG motifs to give CACGgG resulted in an increased response to glucose when tested in the context of S14 ChoRE sequences. This mutation was not activated by USF2/VP16 in cotransfection experiments and bound USF less tightly than the element containing CACGTG motifs by EMSA. This decreased binding was observed both for nuclear extract, which predominantly consists of USF1/USF2 heterodimers (36, 47), and recombinant USF1, which should be present as homodimers. Even more strikingly, alteration of each E box motif to CAtGcG resulted in a ChoRE that responded nearly as well as the CACGTG-containing ChoRE. As reported by Blackwell et al. (51), this sequence did not effectively bind USF either in cotransfection assays or by EMSA. Thus, a role of USF homodimers or heterodimers in glucose activation appears unlikely.
It is worth noting that previous work from both our laboratory and others has shown that USF can bind to the PK ChoRE sequence (22, 45). This is consistent with the demonstration that USF activated expression of L-PK promoter constructs when cotransfected into mhAT3F cells. Our competition studies show, however, that this binding is of significantly lower affinity than that to the CACGTG motif. It is hard to reconcile a reduced binding affinity with a higher potential to activate transcription if USF is indeed the carbohydrate-responsive factor. While we cannot exclude the possibility that USF may function as a complex with another unidentified factor that alters its affinity for ChoRE sequences compared with USF alone, we consider this possibility to be less likely given that no other members of the b/HLH/LZ family have been found to heterodimerize with USF.
It is also worth noting that mutants 2 and 5 possess the exact same E box motifs but in different orientation relative to each other. These two constructs differ dramatically in their abilities to support a glucose response, implying that the relative orientation of the two E boxes is critical. To further explore this question, we have tested a construct in which the same E box motif is present in a direct repeat fashion: CACGgGnnnnnCACGgG. As with mutant 2, this construct was essentially incapable of supporting a glucose response.3 Previously, we observed that the spacing between the ChoRE E boxes is also critical for regulation (23). Together, these results indicate that a highly specific interaction between two binding factors must occur for an effective glucose response.
If USF is not the carbohydrate-responsive factor, then why do we not see another unique factor capable of binding to the ChoRE sequences in vitro? At present, we speculate that such a factor or complex may be present in liver but at a much lower concentration than USF, such that detecting this complex in crude nuclear extracts of liver may not be possible. A similar situation was encountered in the search for SREBP, which binds to the sterol response element of the low density lipoprotein receptor gene. Although proteins are present in crude nuclear extracts that bind the sterol response element, none proved to be the functional factor mediating the response to sterol (58). Only upon significant purification was the physiologically significant SREBP detected. We are currently using the CAtGcG element ChoRE, which is not bound significantly by USF, as a tool to search for such a complex involved in carbohydrate regulation.
We thank Dr. Michéle Sawadogo for providing the USF2 expression clones and for helpful discussion. We also thank Dr. Philippe Pognonec and Dr. Robert Roeder for the USF1 clone and Dr. Steve Triezenberg for the VP16 plasmid. Additionally, we are grateful to Deilee Calvert Minor for technical assistance.