(Received for publication, May 5, 1995; and in revised form, July 12, 1995)
From the
Regulatory sequences involved in the transcriptional induction
of the rat S gene in response to increased glucose
metabolism in the hepatocyte were investigated and compared with those
of the liver-type pyruvate kinase (L-PK) gene. The carbohydrate
response element (ChoRE) of the S
gene was found to
consist of two motifs related to the consensus binding site for the
c-myc family of transcription factors, CACGTG. These two
motifs are separated by five base pairs, a similar arrangement to that
found in the L-PK ChoRE. In its natural context, the S
ChoRE requires a novel accessory factor to support the full
response to glucose. This factor, as well as the factor hepatic nuclear
factor-4, are both capable of binding to the L-PK gene to enhance its
carbohydrate regulation. The need for an accessory factor for
supporting the glucose response can be overcome in two ways. First,
multimers of the ChoREs of either the L-PK or S
genes can
function independently to support the glucose response. Second,
mutations in the S
ChoRE that create a perfect match to
the consensus CACGTG motif at each locus no longer require an accessory
factor site. The spacing of the two CACGTG motifs, but not the nature
of the bases within the spacer, are critical for control. These
observations suggest that a carbohydrate responsive factor binds to
both motifs in a highly specific spatial orientation to confer the
response to increased carbohydrate metabolism.
Feeding of a high carbohydrate, fat-free diet to the rat induces the synthesis of a set of enzymes in the liver involved in the formation of triglycerides(1, 2, 3) . Included in this set of lipogenic enzymes are pyruvate kinase, fatty acid synthase, malic enzyme, and many others required for conversion of glucose to triglycerides. In several cases, the induction of enzyme synthesis has been correlated with changes in transcription of the corresponding genes(4, 5, 6) . Dietary effects of the high carbohydrate diet can be mimicked in cultured primary hepatocytes by changing the glucose concentrations in which cells are maintained(7, 8, 9) . Using such systems, it has been shown that altered enzyme synthesis correlates with increased metabolism of glucose or other glycolytic intermediates(10) . This observation led to the hypothesis that elevated carbohydrate metabolism is responsible through an unknown signaling pathway for increased gene transcription in the hepatocyte. While insulin is necessary, at least in part due to its role in stimulating glucokinase, insulin does not appear to be the direct mediator of the transcriptional response for most of the lipogenic enzymes(11, 12) .
We have been studying two genes
induced by carbohydrate feeding in the rat: the liver-type pyruvate
kinase (L-PK) ()and S
genes. Both of these
genes respond at the transcriptional level to increased glucose
metabolism(4, 6) . The DNA sequences responsible for
the glucose response in primary hepatocytes, which we have designated
as carbohydrate response elements (ChoREs), have been mapped. The
ChoREs of the L-PK and S
genes share a region with 9 out
of 10 bp identity(13) . This region is centered by a CACGTG
motif, the core recognition site for the c-myc family of
transcription factors. In liver, the predominant member of this family
that is detected by in vitro binding studies is USF (also
known as MLTF)(14, 15, 16, 17) .
This factor was first identified by its ability to bind to the upstream
stimulatory element of the adenovirus major late
promoter(18, 19) . Two forms of USF are expressed
widely in mammalian tissues (20) and have been implicated in
the expression of several hepatic genes(21) . The addition of
the upstream stimulatory element from the adenovirus major late
promoter into either L-PK or S
promoters in place of the
normal ChoREs did not support the glucose response, indicating that USF
binding alone is not sufficient for this control (15, 17) .
We have found previously that a 21-bp
segment of the S gene from -1448 to -1428
functions as a ChoRE(17) . This segment contains a 5`
CACGTGNNNGCC motif that is sufficient to confer glucose induction to an
unresponsive promoter construct in primary hepatocytes. Comparison of
the S
ChoRE with the corresponding regulatory region of
the L-PK gene revealed two apparently distinct features. First, for the
L-PK gene, two nuclear factor binding sites, the USF-binding site (
)and an adjacent HNF-4 site, are required to establish a
glucose response. In contrast, the S
gene required only a
single factor binding site related to the USF-binding site of the L-PK
gene. Second, the USF-binding site of the L-PK gene consists of two
imperfect CACGTG motifs separated by 5 bp (Fig. 1A),
whereas only a single CACGTG motif was recognized in the S
ChoRE. These apparent differences between the S
and
L-PK ChoREs perplexed us, as we anticipated that the control of these
two genes was likely to be coordinated through a common mechanism. In
this study, we have analyzed these differences through a closer
examination of the S
ChoRE region. The results indicate
that there is a great deal of similarity between the regulatory
sequences of these two genes and that a unique arrangement of CACGTG
motifs is responsible for determining the specificity of the
carbohydrate response.
Figure 1:
The
S ChoRE includes a second CACGTG motif. Panel A,
sequences of the L-PK ChoRE compared with several
S
-derived oligonucleotides used for defining its ChoRE.
S
(MluI) and S
(XhoI)
represent S
gene sequences from -1448 to -1428
with MluI or XhoI sites used for cloning indicated by underlining. S
(WT) represents the
wild-type S
sequences from -1448 to -1422. The boldface letters indicate the perfect and imperfect CACGTG
motifs present in each oligonucleotide. Panel B,
S
-derived oligonucleotides shown in A were
inserted into a glucose-unresponsive S
enhancer-promoter
construct and transfected into primary hepatocytes. Cells were cultured
in 5.5 (solidbars) or 27.5 mM (stripedbars) glucose for 48 h. CAT activity in cell extracts,
expressed as percentage conversion of chloramphenicol to its acetylated
forms, is shown. Each value represents the average from duplicate
plates of cells. Data are representative of three independent
experiments. Panel C, constructs containing one or two copies
of the S
(-1448/-1422) oligonucleotide linked
to L-PK promoter sequences from -96 to +12 were tested for
their ability to support a glucose response as described in B.
Data are representative of three independent
experiments.
We have previously reported a 9 out of 10 bp identity between the
S and L-PK ChoREs(13) . This comparison involved
alignment of the CACGTG motif of the S
ChoRE with the
downstream CCCGTG motif of the L-PK USF-binding site. If, instead, the
CACGTG motif of the S
gene is aligned to the upstream
CACGGG motif of the L-PK gene, as shown in Fig. 1A, the
S
sequence from -1428 to -1423, CCTGTG,
provides a 5 out of 6 bp match to the downstream motif of the L-PK
ChoRE. Furthermore, the distance between the two motifs is identical to
that found in the L-PK gene. This sequence similarity suggested that
the -1428 to -1423 sequence in the S
gene
might be involved in carbohydrate regulation. To test this possibility,
an oligonucleotide containing the natural sequence of the S
gene with both potential CACGTG motifs (-1448 to
-1422) was subcloned into the S
enhancer/promoter
construct and assayed in transfected hepatocytes. Introduction of this
oligonucleotide resulted in a response to elevated glucose (6.8-fold),
although much weaker than that observed in the construct containing the
artificial MluI site.
To verify the observation that the
natural S sequences from -1448 to -1422 are
capable of mediating the glucose induction, we subcloned this segment
upstream of the basal promoter of the L-PK gene containing sequences
from -96 to +12. One copy of the
S
(-1448/-1422) segment was ineffective in
rendering a response to glucose; however, two copies of this segment
functioned effectively (Fig. 1C). These results were
similar to observations made previously with the L-PK USF-binding
site(15, 24) and suggest that S
and L-PK
ChoREs are functioning in a similar fashion in mediating the
carbohydrate regulation. In both cases, the USF-binding site likely
serves as the recognition site for the factor(s) that is directly
modulated by the carbohydrate signaling pathway.
Figure 2:
Sequences from the S gene
upstream of the ChoRE function as an accessory site to enhance the
response to glucose. Oligonucleotides representing different portions
of the S
gene with coordinates indicated were inserted
into a glucose-unresponsive S
enhancer-promoter construct.
Each construct was tested for its ability to support a response to
elevated glucose as described in the legend to Fig. 1B. Data are representative of two independent
experiments.
Since HNF-4 binds to the accessory factor site for
the L-PK ChoRE, we questioned whether this same factor might also serve
the S ChoRE. Little sequence similarity is observed
between the -1467 to -1440 segment of the S
gene and the known consensus binding site for HNF-4. When an
oligonucleotide containing the S
accessory factor site was
used as a probe in a gel shift experiment with rat liver nuclear
extract, two closely spaced bands were observed (Fig. 3A). In order to detect these bands effectively,
a modified nuclear extraction buffer, which contains 1 M urea
and 1% Nonidet P-40, was used(23) . The two bands could be
competed with the homologous S
oligonucleotide or an
oligonucleotide comprising the L-PK gene accessory site
(-146/-124), but not an unrelated DNA sequence
(TRE
). However, these two bands were not competed by an
oligonucleotide from the
-antitrypsin gene containing
a well-characterized HNF-4 site. These two bands thus appear to be
distinct from HNF-4 and will be referred to as the S
accessory factor. This observation raised the question of whether
the L-PK -146 to -124 segment might bind to the S
accessory factor in addition to HNF-4. When this L-PK gene
segment was radiolabeled and used as a probe, multiple bands were
indeed observed from nuclear extracts prepared by the modified
procedure (Fig. 3B). The major band was shown
previously to represent HNF-4(15, 16) , and, as
expected, this band could be competed by the
-antitrypsin HNF-4 site. In addition, two less intense
bands that migrated with similar mobilities to the bands formed on the
S
-1467 to -1440 fragment were observed. These
two bands were competed by an oligonucleotide containing the S
accessory site. Thus, it appears that the L-PK gene segment from
-146 to -124 is capable of recognizing two (or more)
distinct factors, one of which is HNF-4 and one (or more) of which is
shared with the S
accessory site.
Figure 3:
Detection of a hepatic nuclear factor(s)
that binds to the S accessory site. Panel A, an
oligonucleotide comprising S
sequences from -1467 to
-1440 was radiolabeled and used to test for binding to hepatic
nuclear factors by the EMSA. The ability of various unlabeled
oligonucleotides to compete for binding when added in 50- or 100-fold
molar excess was evaluated. TRE
represents a control oligonucleotide containing a binding
site for the thyroid hormone receptor(32) . Arrows indicate the positions of two specific complexes formed. Panel
B, an oligonucleotide comprising L-PK sequences from -146 to
-124 was radiolabeled and used to test for binding to hepatic
nuclear factors by the EMSA. Various unlabeled competitors tested are
shown. HNF-3 represents a control oligonucleotide containing the
binding site for this nuclear factor(26) . The thick arrow indicates the position of the HNF-4 complex. The two thin
arrows indicate positions of two novel complexes that bind to this
oligonucleotide and are competed by the S
accessory
site.
To confirm that the
factor(s) binding to the S accessory factor site was
distinct from HNF-4, gel shifts were performed in the presence of
antibodies to HNF-4. These antibodies were capable of disrupting the
major complex formed on the PK -146/-124 probe, resulting
in the appearance of a novel supershifted complex (Fig. 4).
However, they did not alter binding of nuclear factors to the S
accessory site probe. Diaz Guerra et al.(16) have suggested that the related orphan receptor
COUP-TF is also capable of binding to the L-PK -146/-124
segment. However, we found that antibodies recognizing either COUP-TFI
or COUP-TFII (25) did not interact with the factors binding the
S
accessory factor. These antibodies also did not displace
the additional bands seen with the L-PK probe. The COUP-TF antibodies
were shown to be effective by their ability to compete complexes formed
on a consensus COUP-TF site (DR+1) using brain nuclear extract.
Thus, the factor binding to the S
accessory site is
distinct from HNF-4 and the related factor COUP-TF.
Figure 4:
The
S accessory factor is distinct from HNF-4 and COUP-TF.
Oligonucleotides comprising the S
accessory site, the L-PK
accessory site or a synthetic element recognized by HNF-4 and COUP-TF (DR+1) (33) were radiolabeled and used
as probes in the EMSA as indicated. Nuclear extracts were prepared from
liver (L) or brain (B). Antibodies to either HNF-4 or
COUP-TF were added to certain binding reactions as indicated. The thick arrow indicates the position of the HNF-4 complex, and
the two thin arrows indicate positions of the S
accessory factor complexes. Arrowheads indicate the
positions of supershifted complexes seen in the presence of
antibodies.
Since both HNF-4
and the S accessory factor can bind to the accessory site
(-146/-124) on the L-PK gene, it was of interest to
determine whether both factors were capable of supporting the glucose
response of this promoter. Previously we showed that an oligonucleotide
containing the
-antitrypsin HNF-4 site can substitute
for the L-PK accessory site in supporting a glucose
response(15) . Since the
-antitrypsin
oligonucleotide does not bind the S
accessory factor (Fig. 3A), HNF-4 is functioning as the accessory factor
for the L-PK ChoRE in this case. To assess whether the S
accessory factor can also function to support the L-PK ChoRE,
mutations were made in the L-PK accessory site that differentially
altered binding of the two factors. Comparison of the S
-1467/-1440 and L-PK -146/-124 segments
showed two regions of sequence similarity (Fig. 5). These two
regions were separated by 4 bp of dissimilar sequence. We reasoned that
mutating these 4 bp should not disrupt binding of the S
accessory factor to the L-PK oligonucleotide, but might disrupt
HNF-4 binding. This prediction was supported by gel shift analysis (Fig. 5). A mutant L-PK oligonucleotide (m1PK) in which the 4 bp
was changed to the sequence of the S
gene was no longer
capable of forming an HNF-4 complex. However, this mutant
oligonucleotide still formed the two bands representing the S
accessory factor with comparable intensity to the L-PK wild-type
oligonucleotide. Another mutant oligonucleotide (m2PK) disrupted
binding of both HNF-4 and the S14 accessory factor.
Figure 5:
Mutations in the L-PK accessory site that
disrupt HNF-4 binding, but retain binding to the S
accessory factor. EMSA was performed with hepatic nuclear extracts and
various oligonucleotides. Sequences of the oligonucleotides are shown above. An area of sequence similarity between S
and L-PK accessory sites is indicated by the thick line. m1PK is a mutation of the L-PK sequences designed to disrupt
binding of HNF-4, but retain binding to the S
accessory
factor. m2PK is a mutation that was designed to disrupt
binding of both HNF-4 and the S
accessory factor. Bases
mutated are indicated by italics. The thick arrow indicates the position of the HNF-4 complex, while the two thin arrows indicate positions of the two novel
complexes.
The abilities of
these oligonucleotides to support the glucose response of the L-PK
ChoRE were tested using constructs shown in Fig. 6. The
construct with the wild-type accessory site gave a 3.6-fold induction
comparing cells cultured in high glucose to those with low glucose. The
diminished responsiveness of this construct compared to the construct
containing L-PK sequences from -197 to +12 (see Fig. 1C) results from an increased basal activity
arising from the deletion of sequences -124 to -96, as
noted earlier (26) . With the construct containing the m1PK
oligonucleotide, the amplitude of the response was reduced compared
with the wild-type sequence; however, this construct still gave a
4.5-fold induction. On the other hand, the construct containing the
m2PK oligonucleotide was only marginally responsive. These experiments
suggest that both the HNF-4 and the S accessory factor are
capable of enhancing the glucose response. However, HNF-4 appears to
possess greater activity on the L-PK promoter.
Figure 6:
The S accessory factor can
stimulate the glucose response of the L-PK ChoRE. Oligonucleotides
containing the wild-type L-PK gene sequence between -146 and
-124 or mutations that disrupt binding of nuclear factors to this
sequence were inserted into constructs containing the L-PK ChoRE
(-171/-147) and promoter. In these constructs, a 10-bp
spacer is added between position -147 and -146 of the L-PK
sequence, and L-PK sequences between -124 and -96 are
deleted. The ability of these constructs to support a response to
glucose was monitored as described in the legend to Fig. 1B. Data are representative of two separate
experiments.
Figure 7:
Mutations that create two perfect CACGTG
motifs in the S ChoRE eliminate the requirement for an
accessory factor. Oligonucleotides containing S
wild-type
sequences from -1448 to -1422 or mutations in the imperfect
CACGTG motif were inserted into the S
enhancer-promoter
construct shown in Fig. 1B. Changes from the wild-type
sequence are indicated by dashedarrows. These
constructs were introduced into primary hepatocytes and tested for
their ability to support a glucose response as described in Fig. 1B. Data are representative of three separate
experiments.
Figure 8:
The spacing between the two CACGTG motifs
of the ChoRE is critical for carbohydrate regulation. Panel A,
primary hepatocytes were transfected with plasmids containing different
oligonucleotides inserted into PK(-146/-124)(-96)CAT
as indicated. Conditions used were described in the legend to Fig. 1B. Each mutated nucleotide is indicated by an underlined lower case letter. The solid and stripedbars represent the CAT activity for cells
cultured in 5.5 and 27.5 mM glucose, respectively. Data are
representative of three independent experiments. Panel B,
oligonucleotides containing variations in spacing between the two
perfect CACGTG motifs were prepared. Each oligonucleotide was inserted
into the glucose-unresponsive S enhancer-promoter
construct shown diagrammatically and assayed as described in the legend
to Fig. 1B. The solidline represents
the construct with no oligonucleotide inserted. Data are representative
of two independent experiments.
To test whether the distance between the two
CACGTG motifs is critical for regulation, we assayed oligonucleotides
containing the L-PK ChoRE with a single nucleotide either deleted or
inserted between the two motifs. Remarkably, both mutations completely
abolished the ability of this element to support the carbohydrate
regulation (Fig. 8A), suggesting that the relative
positioning of these two CACGTG motifs is a major determinant for
glucose induction. To substantiate this observation, constructs
containing variations in spacing between two perfect CACGTG motifs
derived from the S USF-binding site were also tested. As
shown above, the oligonucleotide containing two perfect motifs
separated by 5 bp conferred a strong glucose response. Similar to the
L-PK USF-binding site, a spacing of 4 bp between the CACGTG motifs
resulted in a complete loss of induction. On the other hand, spacing by
either 6 or 7 bp gave a weak response to glucose; much less than the
response seen with the 5-bp spacer (Fig. 8B). To see
whether phasing might be important to establish control, a mutant with
15 bp of spacing was tested. This construct was also only weakly
effective compared with the construct with the natural 5 bp of spacing.
These results clearly indicate that spacing between the two CACGTG
motifs is a critical determinant for carbohydrate regulation.
We have demonstrated that S gene sequences from
-1448 to -1422 function as a ChoRE. This DNA element, like
the L-PK ChoRE, consists of two CACGTG motifs separated by 5 bp. The
ability of either the S
or L-PK ChoRE by itself to support
a response to glucose is weak (S
) or absent (L-PK).
However, multimers of either USF-binding element linked to a basal
promoter render a response to glucose. In the context of the natural
gene, both elements require an accessory factor binding site for
enhancing the responsiveness to glucose. For the L-PK gene, previous
work has shown that HNF-4 can function as an accessory
factor(15, 16) . However, the accessory factor that
works in conjunction with the S
ChoRE is distinct from
HNF-4. Interestingly, this factor also binds to the same region of the
L-PK promoter as HNF-4. In the context of the L-PK promoter, it is
capable of enhancing the activity of the L-PK ChoRE, although to a
lesser extent than that observed with HNF-4. What is the reason for the
complex organization of this regulatory region? Perhaps these factors
serve to mediate the actions of other signals important in the
modulation of L-PK gene expression. In line with this view, Liimatta et al.(30) have recently shown that the L-PK
accessory site is the site through which repression of transcription by
polyunsaturated fatty acids occurs. Whether this repression is mediated
by HNF-4 or the S
accessory factor is under investigation.
We have recently explored the basis of the synergistic action of the
ChoRE and HNF-4 sites of the L-PK gene(26) . Mutations that
altered spacing between the ChoRE and HNF-4 sites did not have a major
impact on the ability to respond to glucose. These studies suggested
that the synergism of these two elements was not due to a direct
interaction of factors binding to these two adjacent sites. On the
other hand, the ability of the HNF-4 site to support the glucose
response could not be substituted by insertion of either an HNF-3 or
C/EBP binding site in its stead, suggesting that some level of
specificity was involved in achieving the functional synergism. We now
show that in addition to HNF-4, at least one other factor can
functionally synergize with the ChoREs of either the S or
L-PK genes. The nature of this factor is currently unknown.
Previously, we have shown that a CACGTGNNNGCC motif of the S gene (-1448 to -1428) is sufficient for conferring
carbohydrate regulation(17) . The ability of this shorter
sequence to respond resulted from an imperfect CACGCG motif
introduced by an MluI site inserted at its 3`-end (underlined
bases). Thus, this construct contained two CACGTG motifs separated by 5
bp, in which a natural C of the S
gene (GCC)
provided the first base of the downstream motif. In the natural
S
sequence, bases from -1428 to -1423 (CCTGTG)
provide an imperfect CACGTG motif with a 4 out of 6 match to the
perfect site. Converting the imperfect S
motif to
sequences with a 5 out of 6 or perfect match to CACGTG resulted in
progressively enhanced responsiveness to glucose. We have previously
reported that an element from the first intron of the fatty acid
synthase gene at position +292 was capable of supporting a glucose
response(17) . However, in this case, the oligonucleotide was
also cloned in the context of an MluI site that could provide
a second CACGTG motif. In the natural context, the sequence
corresponding to the downstream motif is 5` CGCGCC, and an
oligonucleotide containing this region of the fatty acid synthase gene
was ineffective in supporting the glucose response. (
)Thus,
its role in regulation of this gene is questionable.
In addition to
the presence of two CACGTG motifs, the distance between the two motifs
is critical for determining carbohydrate regulation. When the two
motifs were moved closer than 5 bp, a complete loss of glucose
responsiveness was observed for either S or L-PK elements.
At distances greater than 5 bp, the S
-derived element
showed a significantly blunted response, whereas L-PK was unresponsive.
This effect of relative positioning of the two CACGTG motifs argues for
an interaction between factors bound at each of these motifs. This
interaction could be a direct protein-protein interaction or mediated
by a third component that simultaneously contacts both CACGTG-bound
factors. It is worth noting that with a 5-bp spacing, the centers of
the two CACGTG motifs are spaced by 11 bp, thus orienting proteins
bound at these two sites on the same side of the DNA helix. The mutant
with a spacing of 15 bp between the CACGTG motifs would again orient
factors bound at each motif on the same side of the DNA helix. The fact
that this construct showed only a marginal response suggests that a
close proximity of the two factors is critical. This observation is
most compatible with a direct protein-protein interaction between the
two sites.
What is the nature of the carbohydrate responsive factor? Factors binding to the CACGTG motif have been shown to belong to the c-myc family. While many members of this family have been identified, USF is the predominant factor in hepatic extracts that binds to the ChoRE in vitro. However, the ubiquitous distribution of USF raises the question of how the specificity of carbohydrate regulation would be achieved through this factor. The observation that glucose responsiveness requires two specifically positioned motifs could provide an answer to this question. Perhaps, two USF molecules binding to the CACGTG motifs provide an interface for interaction with a signaling molecule or for binding a third component that interacts directly with the signaling pathway. Members of the c-myc family are capable of heterodimerization in unique combinatorial fashions. It is also possible that USF may heterodimerize with another family member in binding to the ChoRE. The possibility that USF is involved in carbohydrate signaling was supported by the recent observations of Lefrancois-Martinez et al.(31) . In this study, dominant negative forms of USF were shown to repress the ability of the L-PK ChoRE to respond in a hepatocyte-derived cell line. We have attempted to detect a novel carbohydrate responsive factor by comparing binding of liver nuclear extracts to responsive and unresponsive USF-binding oligonucleotides. No unique complexes were found (data not shown). The exact nature of the carbohydrate responsive factor thus remains an open question.