From Institut Cochin de Génétique Moléculaire,
U.129 INSERM Unité de Recherches en Physiologie et
Pathologie Génétiques et Moléculaires, and
U.257 INSERM Laboratoire de
Génétique Cellulaire et Moléculaire Université
René Descartes, 24 rue du Faubourg Saint-Jacques,
75014 Paris, France
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ABSTRACT |
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USF1 and USF2 are ubiquitous transcription
factors of the basic helix-loop-helix leucine zipper family. They form
homo- and heterodimers and recognize a CACGTG motif termed E box. In
the liver, USF binding activity is mainly accounted for by the
USF1/USF2 heterodimer, which binds in vitro the
glucose/carbohydrate response elements (GlRE/ChoRE) of
glucose-responsive genes. To assign a physiological role of USFs
in vivo, we have undertaken the disruption of USF1 and USF2
genes in mice. We present here the generation of USF1-deficient mice.
In the liver of these mice, we demonstrate that USF2 remaining dimers
can compensate for glucose responsiveness, even though the level of
total USF binding activity is reduced by half as compared with wild
type mice. The residual USF1 binding activity was similarly reduced in
the previously reported USF2 /
mice in which an impaired glucose
responsiveness was observed (Vallet, V. S., Henrion, A. A.,
Bucchini, D., Casado, M., Raymondjean, M., Kahn, A., and Vaulont, S. (1997) J. Biol. Chem. 272, 21944-21949). Taken
together, these results clearly suggest differential transactivating efficiencies of USF1 and USF2 in promoting the glucose response. Furthermore, they support the view that USF2 is the functional transactivator of the glucose-responsive complex.
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INTRODUCTION |
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Upstream stimulatory factors (USFs)1 belong to the basic helix-loop-helix leucine zipper (b-HLH-LZ) family of transcription factors characterized by a highly conserved C-terminal domain responsible for their dimerization and DNA binding (for review, see Ref. 1). USFs were originally described as a nuclear activity from HeLa cells able to bind and to transactivate the adenovirus major late promoter (AdML) through the canonical sequence CANNTG, referred to as an E-box motif (2). Purification of USFs revealed in fact two polypeptides of 43 (USF1) and 44 kDa (USF2), which were subsequently shown to be encoded by two distinct genes in human, rat, and mouse (3-6). These two genes are ubiquitously expressed in mammalian cells. However, it is noteworthy that the relative abundance of USF1 and USF2 transcripts, as well as USF1 and USF2 proteins, varies among different cell types (7, 8). Furthermore, minor forms of USF2 generated by differential splicing and alternative utilization of translational start sites have been described (8). USF1 and USF2 have been shown to bind DNA as dimers (homodimers as well as heterodimers) and to share very similar DNA binding properties (8, 9). We previously reported that, in vivo, although the relative abundance between USF1 and USF2 homo- and heterodimers varies according to the tissue, the heterodimer USF1·USF2 seems to always represent the predominant DNA binding species (8).
Since the demonstration of USF involvement in the transcriptional activation of the AdML promoter, USFs have been reported as potential regulators of a high number of cellular genes involved in different important cellular processes (see Ref. 4). However, the respective roles of either USF1 and USF2 homodimers, or of the USF1·USF2 heterodimer, have usually not been well determined. Furthermore, involvement of USF is generally demonstrated by artificial ex vivo transfection experiments, which do not always reflect the actual in vivo situation.
Therefore, to gain insight into the specific role of USF1 and USF2 and to evaluate the relative transcriptional activity of USF-dependent regulated genes in cells devoid of USF1, USF2, or both, we have undertaken the generation of USF1 and USF2 knock out mice. The USF2 null mice have been recently reported (10) and we describe here the generation of the USF1 gene knock out mice.
In an effort to characterize the molecular mechanisms underlying the
regulation of gene expression by glucose in the liver, we previously
investigated the transcriptional activation of the genes for L-type
pyruvate kinase (L-PK), a glycolytic enzyme, and Spot14 (S14), a
protein associated with lipogenesis (11). In the regulatory regions of
these two genes, a common glucose-carbohydrate response element (termed
GlRE/ChoRE) has been precisely characterized. This element consists of
two E boxes separated by 5 bp. It was shown to bind in vitro
a major complex containing predominantly the USF1/USF2 heterodimer (8,
12-16). The involvement of USFs in the glucose response was confirmed
by ex vivo experiments using expression vectors (17, 18) and
anti-USF2 antibody microinjections (19). Finally, we were able to show
that the transcriptional response of L-PK and S14 genes was altered in
USF2 /
mice (10). In this paper, we present the transcriptional
response of L-PK and S14 gene to glucose in the USF1
/
mice and
discuss the results considering the previously reported data obtained
in the USF2
/
mice.
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EXPERIMENTAL PROCEDURES |
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Gene Targeting and Generation of USF1 /
Mice--
The USF1
genomic fragments were all generated from
clones isolated from an
ES-129 embryonic stem cell line murine genomic library (4). ES culture
and embryo manipulation were performed as described (20).
Nutritional Treatment and Metabolic Parameters of the
Animals--
For metabolic studies, animals were fed a high
carbohydrate diet for 18 h after a 24-h fasting. Mice were
sacrificed between 10 and 12 a.m., and tissue samples were stored
at 80 °C. Blood samples were collected from the orbital sinus.
RNA Analysis-- RNAs were purified by a modified guanidium chloride procedure from frozen liver, and Northern blot analysis was conducted as described before (23). For detection of L-PK mRNAs, a rat cDNA clone (clone G4) encompassing the coding sequence was used (24). For S14 detection, a polymerase chain reaction-made fragment corresponding to the murine coding sequence was used as a probe (25).
Each Northern blot was stripped and reprobed with a ribosomal 18 S cDNA to check for the integrity and the amount of loaded RNAs. The amount of specific mRNA was quantified using a PhosphorImager (Molecular Dynamics).Protein Analysis-- Western blot analyses were performed as described previously, using USF1-specific peptide antibody (Santa Cruz Biotechnology, Inc.) and affinity-purified USF2 1-49 antibodies both at a 1:200 dilution (8).
For electrophoretic mobility shift assays (EMSA), the DNA binding reaction was performed as described previously (8) in the presence of 30 µg of whole cell extract proteins, 10 µg of poly(dI-dC), and 0.1-0.5 ng of end-labeled MLP oligonucleotide. For supershift experiments, USF1 and USF2 antibodies were included in the binding reactions (8). Quality and quantity of the extracts were monitored by analyzing the ubiquitous NFY binding activities. DNA binding complexes separated on nondenaturing polyacrylamide gels were quantified using a PhosphorImager (Molecular Dynamics).Data Analysis-- Statistical analysis was performed by the Student's t test for unpaired data using the StatView software. The significance has been considered at p < 0.05.
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RESULTS |
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Generation and Characterization of Homozygous USF1-deficient
Mice--
To inactivate the USF1 gene, a replacement vector was
constructed with 6.7 kb of ES-129-derived genomic fragment and a
selection cassette disrupting exon 4 (Fig.
1A). Ensured that the USF1
gene was active in ES cells, we carried out a promoter trap strategy to
maximize the targeting frequency. To this end, the promoterless IRES-geo cassette was chosen as the selection cassette. This cassette allows, via an IRES element, for efficient translation of the
geo fusion transcript. It leads to the synthesis of a fusion protein
with both
-galactosidase and neomycin activities (21). After
electroporation and selection of recombinant ES cells, homologous
recombinants were detected by Southern analysis as described under
"Experimental Procedures." Among 155 colonies screened, 76% were
appropriately targeted. This result confirms previous data showing that
the use of IRES-containing constructs may allow homologous
recombination events to be isolated at a very high frequency (21).
After transmission of the mutation, heterozygous progeny, which was
normal and fertile, was intercrossed to produce homozygous offspring.
The presence of the mutated gene in the F2 offspring was routinely
checked by Southern analysis of tail DNA. As shown in Fig.
1B, hybridization of EcoRI-digested genomic DNAs
with the indicated 5' external probe lead to wild type alleles of 10.8 kb and mutated alleles of 5.1 kb.
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Characterization of Homozygous USF1 /
Knock Out Mice--
Out
of the first 239 F2 offspring genotyped, 19.7% were homozygous (
/
)
for the disrupted allele, and 24.7% were wild type (+/+), which was
compatible with the expected Mendelian frequency. At birth, the
homozygotes seemed to be indistinguishable from wild type and
heterozygous littermates. Furthermore, in contrast to the severe
phenotype of the USF2
/
mice (10), USF1
/
mice developed and
reproduced without any obvious alteration. No behavioral defects or
anatomical abnormalities in any major organs were seen.
USF Binding Activity Is Accounted for by USF2 Homodimers in
USF1-deficient Mice--
To determine the changes in the composition
of USF binding activity, liver cellular extracts were used in EMSAs
with a double-stranded oligonucleotide containing the canonical E box
from the AdML promoter (Fig.
2A). As previously reported
(10), the major USF binding activity in the +/+ mice liver was
predominantly accounted for by USF1/USF2 heterodimer. USF1 and USF2
homodimers, visualized as weak bands after supershift with either
anti-USF2 or USF1 antibody, respectively, seemed to be minor complexes.
In contrast, in USF1 /
cellular extracts, USF binding activity,
visualized as a fainter and slightly upper migrating band, was only
accounted for by the USF2 homodimer. Indeed, USF2 antibody fully
displaced this complex, whereas the USF1 antibody had no effect.
Quantification of this residual USF binding activity was performed by
EMSA with an internal control consisting of the binding site for the
ubiquitously expressed NFY factor (Fig. 2B). This analysis
revealed that the level of USF2 binding activity was reduced to 42%
(n = 6, p = 0.0001) to that of the wild
type. This result demonstrates that, in vitro, the only
detectable binding activity on the E boxes, in cell extracts devoid of
USF1, is USF2 and that probably USF2 gene expression is not
up-regulated in the liver of USF1 KO mice. This result was further
confirmed by Western blot analysis showing no change in the amount of
USF2 in liver cellular extracts of either
/
or +/+ mice (Fig.
2C).
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Hepatic Content of L-PK and Spot14 mRNAs Is Not Modified in
USF1-deficient Mice as Compared with Wild Type Mice--
To determine
the impact of USF1 deficiency on glucose responsiveness of hepatic gene
expression, a series of metabolic analyses was performed on wild type
and USF1 /
mice. After a 24-h fasting period, animals were refed
with a high carbohydrate diet for 18 h. Glucose homeostasis was
assessed by measuring blood glucose level, and hepatic glucose
utilization was monitored by quantitating the level of glucose-6P. Both
values were identical in normal and mutated mice (glycemia of 1.43 ± 0.3 (n = 9) and 1.41 ± 0.23 (n = 11) g/liter glucose, for the +/+ and
/
groups, respectively; and
glucose 6-P concentration of 8.2 ± 2.0 (n = 9)
and 8.3 ± 2.6 (n = 11) nmol/mg of proteins for
the +/+ and
/
groups, respectively). This indicated an
apparent normal glucose uptake and utilization by the liver of USF1
/
mice. Total liver RNAs were then analyzed by Northern blot to
assess the content of L-PK and S14 mRNAs (Fig. 3A). Quantification of this
analysis (Fig. 3B) revealed that the amount of L-PK and S14
mRNAs in the liver of the USF1
/
mice was not statistically
different from that of the +/+ mice.
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Hepatic Content of L-PK and Spot14 mRNAs Is Not Altered in
Double Heterozygote Mice, Whereas USF Binding Activity Is Reduced
2-Fold--
The absence of hepatic alteration of L-PK and S14 gene
transcription in response to a hyperglucidic diet in USF1 /
mice suggested that the remaining USF2 homodimers in the liver cells were
able to substitute for the USF1/USF2 heterodimer. Interestingly, this
substitution took place while the amount of USF binding activity was
reduced to 42% of the total USF binding activity. We therefore asked
whether this observation was specific to USF2 or if reducing the
quantity of the heterodimer would have also led to unchanged L-PK and
S14 mRNA levels. To address this question, we submitted wild type
and double heterozygote USF1 +/
USF2 +/
mice to the above
described feeding protocol. We first compared by EMSA the amount of USF
binding activity in liver cellular extracts of wild type and USF1 +/
USF2 +/
mice. Fig. 4A shows
a representative EMSA experiment along with the quantification of USF
binding activity. This result established that the USF binding
activity, which was mainly accounted for by the heterodimer, was
reduced to 46% in the double heterozygote mice. We then measured, by
Northern blot analysis, the amount of L-PK and S14 mRNAs in the
liver of USF1 +/
USF2 +/
mice as compared with wild type mice. As
presented in Fig. 4B, the amount of L-PK and S14 mRNAs
was comparable in the liver of wild type and USF1 +/
USF2 +/
mice.
Thus, the response of the L-PK and S14 genes was not altered in USF1
+/
USF2 +/
mice despite the reduced USF binding activity.
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DISCUSSION |
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In the USF1 /
mice presented in this paper and in the
previously reported USF2
/
mice (10), we have investigated the regulation of two glucose-responsive genes, namely the L-type pyruvate
kinase and Spot 14. Both genes were shown to contain in their
regulatory region a common sequence responsible for mediating the
glucose response. This sequence consists of two 5' CACGTG type E box
motifs separated by 5 bp. It is able to bind in vitro members of the USF family (8, 12-16). Several lines of evidence from
previous ex vivo and in vivo studies support the
importance of this cis-acting sequence in promoting the glucose
responsiveness. It was of prime interest to test the involvement of the
trans-acting proteins described as binding this sequence in
vitro. In the liver of wild type mice, we previously reported that
the USF binding activity is mainly accounted for by the USF1/USF2
heterodimer (10). By EMSA studies, we demonstrated, here and in the
previously reported USF2
/
KO mice (10), that the hepatic USF
binding activity is accounted for by the remaining homodimer species, i.e. USF1 homodimer in USF2 KO mice and USF2 homodimer in
USF1 KO mice. In addition, the amount of this homodimer in the liver, as assayed by gel shift and Western blot experiments, is consistent with an unchanged expression of the remaining gene. In other words, neither liver USF1 gene expression in USF2 KO mice nor USF2 in USF1 KO
mice seems to be overexpressed consequently to the mutation.
These results in adult liver are different from those recently reported
by Sirito et al. (26) in fetal fibroblasts from USF2 /
and USF1
/
mice. The authors observed that the USF2
/
mice
contain a reduced level of USF1, whereas the USF1
/
mice seem to
compensate USF1 deficiency by increased USF2 synthesis. It seems
therefore that the asymmetrical cross-regulation between USF genes
during fetal life is replaced after birth, at least in the liver, by an
independent pattern of USF1 and USF2 gene expression.
We previously demonstrated that in USF2 /
mice refed a
hyperglucidic diet for 18 h, L-PK and S14 gene expression was
dramatically reduced (by 3.8- and 6.8-fold, respectively). Therefore,
the glucose responsiveness of glucose-responsive genes in the liver,
impaired in the presence of USF1 homodimers in USF2
/
mice, is
normal in the presence of USF2 homodimers in USF1
/
mice. Because
the glucose response of the L-PK and S14 genes is also normal in double heterozygous mutants, we can conclude that 40-50% of residual USF
binding activity is sufficient to sustain a normal glucose response
when consisting of either USF2 homodimers or USF1/USF2 heterodimers but
not of USF1 homodimers. In other words, USF1 seems to be intrinsically
less efficacious than USF2, at least as a transactivating factor of the
glucose-response complex. The weak transcriptional effect of USF1 is
reminiscent of the results reported by Desbarats et al.
(27). These authors demonstrate that the activity of USF1 on isolated E
boxes in vivo is often poor and that the distance between E
boxes and start sites of transcription can be a critical factor. This
observation could explain why response of the S14 gene to glucose is
much more affected than that of the L-PK gene in USF2
/
mice.
Indeed, the E boxes are located far upstream from the start site of
transcription (
1422 bp) in the S14 gene and in a very proximal
position (
144 bp) in the L-PK gene. Furthermore, Carter et
al. (28) recently called into question the traditional view of
USF1 homodimer as a transcriptional activator. In an effort to
determine the selective utilization of b-HLH-LZ proteins at the Ig
heavy chain enhancer, the authors surprisingly observed that USF1 had a
negative effect on the activity of the IgH. They hypothesize that
repression may be a consequence of the absence of a strong activation
domain in USF1. Different studies suggest that USF1 could act as a
repressor by competing in vivo with positive-transacting
factors (29, 30).
The transactivating activity of USF1 and USF2 dimers could be intrinsically different and/or it could be brought out by differential interaction of USF1 and USF2 with other regulatory proteins. The highly divergent USF1 and USF2 NH2-terminal domains, encompassing the transcriptional activation domain, could specify these interactions. Alternatively, a role for the b-HLH-LZ domain could be evoked. In this respect, it is interesting to note that a specific cross-talk between USFs and other transcription factors begins to be documented. Blanar and Rutter (31) have first reported the in vitro interaction of USF2 with c-Fos. More recently, by different cloning strategies, specific interactions have been reported, on one hand, between USF1 and the b-Zip Fra1 factor (32), the b-HLH-E47 protein (33), and the winged HTH Ets-1 factor (34); on the other hand, specific interactions have been reported between USF2 and the c-Maf1 factor (35). So far, however, further experiments are needed to assign a physiologic role to these physical interactions. Finally, Roy et al. (36) recently reported the molecular cloning of the TFII-I transcriptional factor that physically and functionally interacts with USF1.
It will be interesting to determine whether the differential transactivating properties of USF1 and USF2 dimers can occur on other USF-dependent genes and, in this case, to what extent they could target different sets of responsive genes in different cell types and in response to various stimuli.
Although the results discussed before suggest that the USF1 homodimers
are not required for normal embryonic development, postnatal growth,
and transcriptional response to glucose and that they cannot by
themselves totally compensate for USF2 deficiency, their role is
nevertheless attested by embryonic lethality of double knock out mice
deficient in both USF1 and USF2
(26).2 Preliminary data also
indicate that the phenotype is more severe in animals with only a
residual USF1 intact allele (USF1 +/ USF2
/
), which die earlier
during in utero development, than in those with a residual
USF2 allele (USF1
/
USF2 +/
), which generally die immediately
before or after birth.2 This observation is consistent with
a better efficacy of USF2 than of USF1. However, the underlying
mechanism is here uncertain because, as mentioned above, Sirito
et al. (26) have shown that USF1 concentration is decreased
in USF2-deficient mice, whereas USF2 is increased in USF1-deficient
mice.
In conclusion, the USF knock out mouse models have allowed us to demonstrate a differential role of USF1 and USF2 in the transcriptional response of liver genes to glucose. Indeed, a normal glucose responsiveness seems to require the presence of either USF1/USF2 heterodimers or USF2 homodimers, even in mice with total USF binding activity reduced by half, whereas USF1 homodimers give rise to a delayed response. This result could reflect an intrinsic difference in the transactivating efficiency of USF isoforms. Accordingly, consequences of USF2 deficiency on embryonic development are more severe than those of USF1 deficiency. Further investigations will determine how transactivation of glucose responsiveness by USF2-containing dimers is finely tuned by glucose.
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ACKNOWLEDGEMENTS |
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We thank M. Cognet and I. Leclerc for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Institut National de la Santé et de la Recherche Médicale.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work.
¶ Supported by a grant from the Ligue Nationale Contre le Cancer (Comité Départemental de la Vendée).
Supported by a grant from the European Commission.
** Present address: Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215.
§§ To whom correspondence should be addressed. Tel.: 33-1-44-41-24-08; Fax: 33-1-44-41-24-21; E-mail: vaulont{at}cochin.inserm.fr.
The abbreviations used are: USF, upstream stimulatory factor; L-PK, L-type pyruvate kinase; S14, Spot 14; MLP, major late promoter; NFY, nuclear factor Y; glucox 6-P, glucose 6-phosphate; b-HLH-LZ, basic helix-loop-helix leucine zipper; HTH, helix turn helix; bp, base pair(s); kb, kilobase pair(s); IRES, internal ribosomal entry site; ES, embryonic stem; KO, knock out; EMSA, electrophoretic mobility shift assay.
2 M. Casado, V. S. Vallet, A. Kahn, and S. Vaulont, unpublished data.
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REFERENCES |
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