(Received for publication, November 4, 1994)
From the
L-type pyruvate kinase (L-PK) gene transcription is induced by
glucose through its glucose response element (GlRE) composed of two
degenerated E boxes able to bind in vitro ubiquitous upstream
stimulator factor (USF) proteins. Here we demonstrate in vivo,
by transient transfections in hepatoma cells, that (i) native USF
proteins synthesized from expression vectors can act as transactivators
of the L-PK promoter via the GlRE, stimulating transcription without
glucose and, therefore, decreasing the glucose responsiveness of the
promoter; (ii) expression of the truncated USF proteins, able to bind
the GlRE but devoid of the NH-terminal activation domain,
represses the activation of the L-PK promoter by glucose; and (iii) a
similar repression of the glucose effect is observed upon expression of
mutant USF proteins devoid of the basic DNA binding domain, able to
dimerize with endogenous USF but not to bind the GlRE. We conclude that
USF proteins are components of the transcriptional glucose response
complex assembled on the L-PK gene promoter.
Glucose is a major regulator of gene transcription in all forms
of life. In vertebrates, it is often difficult to discriminate glucose
effects on gene transcription from those of insulin, whose secretion is
stimulated by glucose, and those of glucagon, whose secretion is
inhibited by glucose. However, the use of cultured cells has
definitively demonstrated that glucose on its own is able to stimulate
transcription of genes encoding glycolytic and lipogenic genes in
hepatocytes and
adipocytes(1, 2, 3, 4) . Insulin is
permissive to glucose action in hepatocytes; in particular, it
stimulates glucose phosphorylation to glucose 6-phosphate through
transcriptional activation of the glucokinase
gene(4, 5) . Similar glucose response elements
(GlREs), ()also referred to as carbohydrate response
elements (ChoRE)(6, 7) , have been described in the
upstream region of the L-type pyruvate kinase (L-PK) gene and of the
spot 14 gene and in the first intron of the fatty acid synthase
gene(8) . These GlREs are composed of E boxes able to interact, in vitro, with upstream stimulatory factor (USF) proteins (8, 9, 10) and to confer, when multimerized,
a transcriptional response to glucose on minimal
promoters(7, 8) . Still, the role of the USF proteins
in the glucose response cannot be inferred only from their affinity for
the GlRE because these basic helix-loop-helix/leucine zipper
transcription factors(11, 12) (
)interact
with various promoters that are not regulated by glucose (13, 14, 15) . We demonstrate here that USF
proteins actually are part of the glucose response complex of the L-PK
promoter and are required for this response in hepatocyte-derived
cells.
The (MLP)4-54 PK/CAT and (L4)4-54 PK/CAT plasmids consist of four tandem repeats of either adenovirus USE-MLP or L-PK L4 motifs (see Fig. 2) inserted in front of the -54 PK/CAT construct.
Figure 2: Transactivation by USF proteins of L4 and USE-MLP motif-dependent promoters in mhAT3F cells cultured without glucose (solid box) or with 17 mM glucose (empty box). A, schematic maps of the USF expression vectors and CAT plasmids used and nucleotide sequences of the USE-MLP and L4 elements are shown. Both oligonucleotides contain BamHI sites (lowercase letters) at their extremities to enable their polymerization in the (MLP)4-54 PK/CAT and (L4)4-54 PK/CAT plasmids. CMV, cytomegalovirus. B, transactivation by USF proteins synthesized from co-transfected expression vectors is shown. mhAT3F cells were first cultured in a lactate culture medium for 24 h. After transfection, these cells were grown in lactate (solid boxes) or in 17 mM glucose (empty boxes) for 24 h prior to assaying CAT enzyme activity. mhAT3F cells were co-transfected with 5 µg of the reporter plasmids -54 PK/CAT, (MLP)4-54 PK/CAT, (L4)4-54 PK/CAT, and either 0.25 µg of the expression vector encoding USF1 and USF2a proteins or 0.25 µg of the control plasmid (devoid of any cDNA). Each bar represents the mean ± S.D. of five to six independent transfection experiments.
All expression vectors were driven by the cytomegalovirus immediate
early promoter/enhancer (see Fig. 1). The human USF1 (U1)
expression vector was a kind gift from G. Roeder(18) . Human
U2a and U2aH cDNAs were cloned in our laboratory as already
described.
Four mutant USF cDNAs were used. Two of them
lacked the putative NH
-terminal activation domains
(corresponding to the first 163 or 198 amino acid residues) of U1 and
U2a (TDU1 and TDU2, respectively). One cDNA is devoid of both putative
NH
-terminal activation and basic DNA binding domains
(corresponding to the first 207 amino acid residues) of U1
(
bTDU1). At last, a mutant cDNA lacked the first 8 amino acid
residues of the second helix of the U2a HLH (U2a
H).
All of these cDNAs were cloned at convenient sites of the pCMV
expression vector parent plasmid(19) .
Figure 1:
A, schematic maps of the cDNAs encoding
native and mutant USF proteins are shown. The cDNA regions encoding the
different NH-terminal domains of USF1 and USF2a proteins
are represented as hatched and grilled rectangles,
respectively. The cDNA regions encoding the highly conserved basic and
dimerization domains of USF1 and USF2 species are represented as shaded and empty rectangles, respectively. Details
about deletions of mutant USF proteins are described under
``Materials and Methods.'' B, the electrophoretic
mobility shift assay analysis of the different in vitro synthesized USF proteins is shown. Mobility shift assays were
performed using the USE-MLP oligonucleotide probe and USF proteins that
were synthesized by in vitro translation. On the right
panel, TDU (1 or 2) transcripts were co-translated with increasing
amounts (µl) of
bTDU1 transcripts.
Band shift assays
were performed with cell-free translation proteins essentially as
already described. 1-3 µl of reticulocyte lysate
were incubated in the presence of 1.5 µg of poly(dI
dC) and
0.1-0.5 ng of [
P]ATP phosphorylated
oligonucleotide corresponding to (-70 to -43) USE-MLP
(upper strand, AGGTGTAGGCCACGTGACCGGGTGTTCC) (see Fig. 2).
Figure 3: Influence of USF proteins on the transcriptional response to glucose. A, dose-response effects of USF1 and USF2a ectopic expression on the transcriptional response to glucose of the -183 PK/CAT plasmid are shown. Five µg of -183 PK/CAT plasmid were co-transfected with increasing amounts of expression vector encoding USF1 (left panel) or USF2a proteins (right panel) in mhAT3F cells in culture conditions described in the legend to Fig. 2. In these experiments, 0.25 µg of expression plasmid without cDNA were used as a control. Each bar represents the mean ± SD of at least six independent transfections. B, effects of USF1 and USF2a ectopic expression on the transcriptional response to glucose of the -183 PK/CAT (wild-type promoter), or -150 PK/CAT and L4 mi-L3-119 PK/CAT plasmids are shown. The L4 miL3-119 PK/CAT differs from the -183 PK plasmid by a mutation in the L4 box (7) while the -150 PK/CAT construct lacks the L4 box. The expression vectors encoding either USF1 or USF2a proteins were co-transfected with 5 µg of reporter plasmids in the same experimental conditions as those described in the legend to Fig. 2. The amount of co-transfected vector was determined from the dose effect curves represented in A: 0.25 µg of U2a and control (empty vector) and 0.5 µg of U1 vector. Each bar represents the mean ± S.D. of at least four to six independent transfections.
Figure 4:
Effect of mutant USF proteins on the
transcriptional response to glucose. Five µg of the -183
PK/CAT or (MLP)4-54 PK/CAT plasmids were co-transfected with
different expression vectors: 2.5 µg of empty vector (control), 2.5
µg of bTDU1 vector, 0.25 µg of either TDU2 or U2a
H
vectors. The different amounts of expression vectors were chosen from
dose effect curves similar to those shown in Fig. 3A and corresponded to the maximal amount, which still allowed
avoidance of the nonspecific ``squelching'' phenomenon. Cells
were grown as described in the legend to Fig. 2except for the
experiments performed with
bTDU1 expression vector and control
(empty vector). For these two conditions, transfected cells were grown
for 72 h in a lactate medium before glucose supplementation. Each bar represents the mean ± S.D. of four independent
transfections.
We and Towle's group (8, 10) have
demonstrated that the glucose/carbohydrate response element present in
the regulatory regions of genes encoding glycolytic and lipogenic
enzymes (or related protein in the case of spot 14) consists of
(CACGTG) E boxes able to bind USF proteins in vitro.
Nevertheless, there was no evidence for these proteins to be
functionally involved in the transcriptional regulation by glucose. The
experiments presented here reveal that USF1 and USF2 (USF2a)
transactivators have a stimulatory effect on the L-PK promoter activity
through the L4 motif (i.e. the GlRE) in the absence of glucose
but not in its presence and that a mutant truncated USF2 (TDU2) protein
devoid of the first 198 amino acids of USF2a strongly inhibits the
glucose response. These first two sets of results demonstrate that USF
proteins functionally interact, in living cells, with the element L4.
Indeed, an excess of transactivators (USF1 or USF2a) was able to
compensate for the absence of glucose, and isoforms (TDU1 and -2) that
are able to bind to L4 and are transactivation-defective impaired the
glucose-dependent transcriptional activation. Still, these results
could be explained by USF proteins supplanting glucose responsive
proteins that normally take place in the transactivation complex.
However, the use of the bTDU1 negative transdominant expression
vector demonstrates that functional USF oligomers are indispensable to
the glucose responsiveness. Indeed, this mutant protein is able to
dimerize through its helix-loop-helix/leucine zipper motif but not to
bind DNA; consequently, in cells transfected with an expression vector
for the
bTDU1 mutant, functional USF oligomers are expected to be
progressively replaced by defective oligomers unable to interact with
the GlRE. This titration of the functional USF oligomers results in a
loss of the positive response to glucose as well as to a decreased
activity of the (MLP)4-54 PK/CAT and -183 PK/CAT constructs
in cells transfected 72 h ago. It is worth noting that the activity of
the -183 PK/CAT construct in the absence of glucose does not seem
to be affected by the
bTDU1-dependent titration of active USF
oligomers and that the activity in the presence of glucose is not
significantly further increased by overexpression of USF1 or USF2a
transactivators. These results suggest that, in the absence of glucose,
either L4 does not bind USF transactivators or it binds an inactive USF
complex and that glucose would allow either USF binding or
de-inhibition of the complex. In fact, the results of in vivo footprinting experiments rather stand for a process of in situ inhibition of a constitutively bound USF complex in the absence of
glucose and its activation in the presence of glucose since the in
vivo footprint on the L-PK GlRE is identical in fasted and
carbohydrate refed animals.
The negative effect of cyclic
AMP in hepatocytes, also mediated by the element L4 (7) , could
be to reverse such a glucose-dependent activation of the complex, most
likely through protein kinase A-dependent phosphorylation of one of its
components. The involvement of USF proteins in the glucose response of
glycolytic and lipogenic genes in the liver and probably fat cells is
reminiscent of the role of E12/E47 proteins recently evidenced by
German and Wang (21) in transcriptional response of the
insulin-1 gene to glucose in cultures of pancreatic beta cells. It is
striking that two types of b/HLH factors, i.e. USF and
E12/E47, could participate in the transcriptional regulation by glucose
in different cell contexts. The existence of partners of the USF
oligomers regulating their transactivating potential is highly probable
as the presence of USF proteins on the USE-MLP site or on other sites
is not a sufficient condition to confer by itself the glucose
responsiveness; USF proteins have been described to interact with
various gene promoters whose expression is not regulated by glucose (13, 14, 15) . The partnership between USF
oligomers and other factors could be governed by a special arrangement
of the E boxes in the GlREs/ChoREs described so far, namely the
presence of two palindromic E boxes separated by 5 base
pairs(7, 8) . (
)The next steps in the
elucidation of the transcriptional glucose signaling pathway should be
the identification of these partners of USF oligomers and the mechanism
of their regulation by glucose and cyclic AMP.