©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Upstream Stimulatory Factor Proteins Are Major Components of the Glucose Response Complex of the L-type Pyruvate Kinase Gene Promoter (*)

(Received for publication, November 4, 1994)

Anne-Marie Lefrançois-Martinez Antoine Martinez Bénédicte Antoine Michel Raymondjean Axel Kahn (§)

From the Institut Cochin de génétique Moléculaire, U.129 INSERM, Université René Descartes 24, rue du Faubourg Saint-Jacques, 75014 Paris, France

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(2)-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.


INTRODUCTION

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), (^1)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) (^2)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.


MATERIALS AND METHODS

Cell Cultures and Lipofection CAT Assays

The hepatocyte-derived mhAT3F cells have been previously described(16) . They were cultured either in glucose-free lactate conditions or in the presence of 17 mM glucose(4) . CAT constructs, expression vectors and luciferase plasmid, were co-transfected by lipofection as described previously(4) . Results were expressed as a ratio of the percentage of KSV2 CAT activity versus luciferase activity to compensate for the transfectability variability. The KSV2 CAT expression was similar in all conditions.

Tested Plasmids and Expression Vectors

The different L-PK CAT constructs (named -183 PK/CAT, -150 PK/CAT, -54 PK/CAT, L4 mi-L3-119 PK/CAT) have been previously described(7, 17) .

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 U2aDeltaH cDNAs were cloned in our laboratory as already described.^2 Four mutant USF cDNAs were used. Two of them lacked the putative NH(2)-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(2)-terminal activation and basic DNA binding domains (corresponding to the first 207 amino acid residues) of U1 (DeltabTDU1). At last, a mutant cDNA lacked the first 8 amino acid residues of the second helix of the U2a HLH (U2aDeltaH).^2 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(2)-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 DeltabTDU1 transcripts.



In Vitro Synthesis of USF Proteins and Gel Shift Assays

In vitro transcriptions and translations of USF proteins were realized from a linearized plasmid using T7 RNA polymerase in the presence of m^7G(5`)ppp(5`)G (Boehringer, Mannheim) and reticulocyte lysate according to the recommendations of the manufacturer (Promega). Each translation was monitored in the presence of [S]methionine and analyzed through a 11% (w/v) SDS-polyacrylamide gel electrophoresis.

Band shift assays were performed with cell-free translation proteins essentially as already described.^2 1-3 µl of reticulocyte lysate were incubated in the presence of 1.5 µg of poly(dIbulletdC) and 0.1-0.5 ng of [P]ATP phosphorylated oligonucleotide corresponding to (-70 to -43) USE-MLP (upper strand, AGGTGTAGGCCACGTGACCGGGTGTTCC) (see Fig. 2).


RESULTS

In Vitro Binding Activity of the Different Native and Mutant USF Proteins Used

As expected, the 43-kDa USF1 and the 44-kDa USF2a wild-type proteins (Fig. 1B, left panel) and the TDU1 and TDU2 NH(2)-terminal truncated mutant proteins (Fig. 1B, right panel) were all shown by an electrophoretic mobility shift assay to bind the USE-MLP oligonucleotide. On the contrary, USF2aDeltaH (which lacks the second helix of the HLH dimerization domain) and DeltabTDU1 (which lacks the basic DNA binding domain) were unable to bind DNA (Fig. 1B, left panel). The ability of DeltabTDU1 to prevent the DNA binding activities of USF proteins by its heterodimerization was ascertained in the same experiment by co-translation of either TDU1 or TDU2 transcripts with increasing amounts of DeltabTDU1 transcripts (Fig. 1B, right panel). Co-translation with the lowest amount of DeltabTDU1 transcripts (2 ml) was sufficient to prevent almost all TDU2 (consisting of the USF2 core binding domain) from binding DNA, whereas greater amounts of DeltabTDU1 could only lead to a partial decrease of the DNA binding of TDU1 species. The same results were obtained with full-length USF proteins (data not shown). These results could illustrate the preferential association of USF1 and USF2 species as heterodimers as it was observed in nuclear extracts (20) .^2

USF Proteins Act through the GlRE to Stimulate Activity of the L-type Pyruvate Kinase Promoter in a Glucose-free Medium

The ability of USF proteins to function as transcriptional modulators via the GlRE was analyzed by transient transfections in mhAT3F cells, a hepatoma cell line allowing the glucose responsiveness of the L-PK gene expression(4) . The (L4)4-54 PK/CAT plasmid accounted by itself for a weak CAT activity that was stimulated 3-fold by glucose, while the activity generated by the -54 PK/CAT plasmid was barely detectable and was insensitive to glucose. As a control experiment, when the oligomerized L4 element was replaced by an oligomerized element of four tandem repeats of the USE-MLP motif, glucose was ineffective on the (MLP)4-54 PK promoter activity. These results confirm that the L4 element is, on its own, a glucose response element (6, 7, 9) and that the USE-MLP motif is unable to confer this property(8) . Co-transfection of the (L4)4-54 PK/CAT construct with expression vectors encoding the USF1 and USF2a proteins strongly stimulated the CAT activity with or without glucose (Fig. 2B). None of these expression vectors had any effect on the minimal -54 PK/CAT plasmid.^2 Therefore, these experiments demonstrate that USF1 and USF2a are transactivators that act through the L4 element and that their overexpression suppresses the slight glucose responsiveness of the (L4)4-54 PK/CAT construct. These results were then confirmed in the context of the wild-type -183 PK promoter (Fig. 3, A and B). Overexpression of USF1 and USF2a resulted in an 8.5- and 7.8-fold stimulation, respectively, of the CAT activity generated by the -183 PK/CAT plasmid in mhAT3F cells cultured without glucose; in cells cultured with glucose, this stimulation was only 1.2- and 1.3-fold, respectively. Thus, the level of glucose activation of the CAT activity was strongly reduced from 9.5-fold with the empty expression vector to 1.2-fold with U1 and 1.6-fold with U2a expression vectors (Fig. 3B). Moreover, Fig. 3B confirms that USF protein overexpression had no effect on constructs that lack the box L4 (-150 PK/CAT) or that possess a mutant L4 box (L4 mi-L3-119 PK/CAT).


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.



USF Transdominant Negative Mutants Inhibit the Glucose Response of the L-PK Promoter

Fig. 4shows that the expression of DeltabTDU1, which can dimerize with USF proteins but is unable to bind DNA, dramatically reduced the glucose responsiveness of the -183 PK/CAT construct by suppressing the activation by glucose. Moreover, the mutant DeltabTDU1 protein resulted in a decreased activity of the (MLP)4-54 PK/CAT construct (Fig. 4) regardless of the presence of glucose in the culture medium (data not shown). Expression of the truncated TDU2 (Fig. 4) or TDU1 (data not shown) mutant proteins, devoid of the NH(2)-terminal activation domain and possessing a normal DNA binding domain, similarly reduced the glucose responsiveness of the -183 PK/CAT construct by inhibiting the glucose-dependent stimulation and also reduced activity of the (MLP)-54 PK promoter. As expected, the expression of USF2aDeltaH mutant protein, devoid of an intact HLH domain and therefore unable to dimerize and to bind DNA, had no effect on the glucose responsiveness of the -183 PK/CAT construct.


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 DeltabTDU1 vector, 0.25 µg of either TDU2 or U2aDeltaH 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 DeltabTDU1 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.




DISCUSSION

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 DeltabTDU1 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 DeltabTDU1 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 DeltabTDU1-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.^3 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) . (^4)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.


FOOTNOTES

*
This work was supported by the Institut National de la Santé et de la Recherche Médicale and by grants from the Fondation pour la Recherche Médicale, the Ligue Nationale contre le Cancer, and the Ministère de l'Enseignement Supérieur et de la Recherche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 33-1-44-41-24-24; Fax: 33-1-44-24-21.

(^1)
The abbreviations used are: GlRE, glucose response element; CAT, chloramphenicol acetyltransferase; ChoRE, carbohydrate response element; USF, upstream stimulatory factor; HLH, human luteinizing hormone; L-PK, L-type pyruvate kinase.

(^2)
A. Martinez, A. M. Lefrançois-Martinez, A. Henrion, A. Kahn, and M. Raymondjean, submitted for publication.

(^3)
S. Lopez, personal communication.

(^4)
H. C. Towle, personal communication.


ACKNOWLEDGEMENTS

We thank Dr. R. G. Roeder for the gift of the pET3d-USF plasmid. We are grateful to Drs. Marie-Hélène Cuif, Bruno Doiron, Alexandra Henrion, Mireille Vasseur-Cognet, and Sophie Vaulont for helpful discussions and advice. We especially thank Dr. Alexandra Henrion for careful revision of the text.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.