(Received for publication, August 31, 1995; and in revised form, September 26, 1995)
From the
Fatty acid synthase (FAS) plays a central role in de novo lipogenesis in mammals. The concentration or activity of FAS in liver and adipose tissue changes dramatically when animals are subjected to nutritional and hormonal manipulations. We previously reported that due to changes in transcription, FAS synthesis declines and increases in an insulin-dependent manner during fasting and refeeding, respectively, and that insulin administration of streptozotocin-diabetic mice stimulates FAS transcription. We previously mapped the FAS insulin response sequence (IRS) to the proximal promoter region from position -71 to position -50, which contains an E-box DNA binding motif. Here, using competition gel shift assays and specific upstream stimulatory factor (USF) antibodies, we identified USF1 and USF2 as major components of complexes that bind to the FAS IRS. UV-cross-linking experiments further supported that USFs bind the FAS IRS. We also found that the amount of the 43-kDa USF1 was dramatically increased in liver of refed rats. In contrast, the amount of USF2 remained the same in liver of fasted or refed rats. Moreover, a 17-kDa protein in both fasted and refed rat liver was recognized by anti-USF1 antibodies, and this 17-kDa USF1-related protein was expressed in a manner opposite to that of the 43-kDa USF1, i.e. high in liver of fasted rats and decreased in liver of refed rats. These data suggest that the regulation of USF expression may play an important role in the regulation of FAS transcription.
Fatty acid synthase (FAS) ()plays a central role in de novo lipogenesis in mammals and birds. By the action of its
seven active sites, FAS catalyzes all the reaction steps in the
conversion of acetyl-CoA and malonyl-CoA to palmitate. FAS activity is
not known to be regulated by allosteric effectors or covalent
modification. However, the FAS concentration is exquisitely sensitive
to nutritional, hormonal, and developmental
status(1, 2) . The concentration or activity of FAS in
liver and adipose tissue changes dramatically when animals are
subjected to different nutritional and hormonal manipulations. The rate
of FAS synthesis declines when rats are fasted for 1-2 days while
refeeding a high carbohydrate, fat-free diet increases synthesis of
FAS(3) . Increased circulating insulin and decreased glucagon
levels may participate in regulation of FAS synthesis. We previously
reported that FAS mRNA was not detectable in liver of fasted mice and
that refeeding with a high carbohydrate diet dramatically increased the
level of FAS mRNA, due to changes in the rate of FAS gene
transcription(4, 5) . The stimulation of FAS gene
transcription by fasting/refeeding was not observed in liver of
streptozotocin-diabetic mice, in which FAS expression is detected at a
very low level(5) . Administration of insulin to
streptozotocin-diabetic mice stimulated the level of FAS mRNA and FAS
transcription rate(5) . We also reported that insulin increased
levels of FAS mRNA in 3T3-L1 adipocytes(4) . Sequences
mediating insulin induction of the FAS gene have been located to the
first 332 base pairs of the FAS promoter by transient transfection of
H4IIE hepatoma cells and 3T3-L1 adipocytes (6) . We further
mapped insulin response sequence (IRS) to the proximal promoter region
from position -71 to position -50 by chimeric constructions
of serial 5`-deletions of the rat FAS gene promoter ligated to the
luciferase reporter gene and transfection into 3T3-L1
adipocytes(7) . This IRS confers a stimulation of the FAS
promoter activity by insulin, at physiological concentrations, in a
dose-dependent manner. Moreover, we also demonstrated that three tandem
repeats of FAS IRS linked to a heterologous SV40 promoter were
responsive to insulin. Both liver and adipocyte nuclear proteins bind
to the FAS promoter, resulting in the IRS region being protected on
DNase I footprinting analysis and specific band shift on a gel mobility
shift assay(7) . Neither the insulin response sequence from the
amylase promoter (8, 9) nor that from the GAPDH
promoter (10) displaced the binding of FAS IRS to the liver
nuclear factor(s), suggesting that unique protein(s) are involved in
the binding of FAS IRS and in the regulation of FAS gene transcription
by insulin(7) . Since these studies, we have been attempting to
identify and isolate the transcription factors that bind to FAS IRS.
The upstream stimulatory factor (USF), belonging to the basic-helix-loop-helix (bHLH) family of transcription factors, was first identified for its involvement in transcription from the adenovirus major late (AdML) promoter(11, 12, 13) . Purification of USF from HeLa cells has revealed that at least two different polypeptides, i.e. the 43-kDa USF1 and the 44-kDa USF2, contribute to USF activity in human cells(14) . In addition to the human USF1 (15) and partial USF2 (16, 17) and murine USF2(18) , USF cDNA clones have been isolated from Xenopus(19) and sea urchin(20) . Like other bHLH transcription factors such as Myc, E12, E47, and MyoD, USF binds to the ``E-box'' DNA binding motif, consisting of a canonical CANNTG sequence. In addition to the bHLH motif, a leucine-zipper (LZ) is immediately adjacent to bHLH and is also important for USF dimerization and DNA binding(15) . The transcription activation domain of USF has been located at the N terminus of the protein and consists of two subregions(21) . USF1 and USF2 have been found to be ubiquitously expressed, at various levels, in mammalian cells and bind to an E-box as homo- and heterodimers(16, 18) . USF binding sites have been found in a number of genes(22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38) , and USFs have been shown to participate in the transcriptional regulation of several of them. Two examples are the rat S14 gene (34) and liver-type pyruvate kinase (L-PK) gene(38) , which are important in energy metabolism. Hepatic S14 mRNA in the rat can be increased by feeding a high carbohydrate, fat-free diet(39, 40) , and L-PK gene transcription in hepatocyte-derived cells can be induced by glucose(41) . Glucose-response elements, also referred as carbohydrate response elements(42, 43) , contain E-box sequences and are able to interact with USFs and to confer a transcriptional response to glucose(34, 38) .
In this paper, we describe the identification of USF1 and USF2 as major components of protein complexes that bind to IRS of the FAS promoter, and we report changes in the amount of USF1, but not of USF2, associated with fasting and refeeding treatments. The amount of the 43-kDa USF1 was dramatically increased in liver of refed rats. In contrast, the amount of USF2 remained the same in liver of fasted and refed rats. Moreover, a 17-kDa protein in both fasted and refed rat liver was recognized by anti-USF1 antibodies, and this 17-kDa USF1-related protein was expressed in a manner opposite to that of the 43-kDa USF1, i.e. high in liver of fasted rats and decreased in liver of refed rats. Because the body circulatory insulin level is decreased and increased by fasting and refeeding, respectively, and insulin closely regulates FAS transcription, changes of USF1, which binds to the FAS IRS, may play an important role in the regulation of FAS transcription by insulin.
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
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On-line formulae not verified for accuracy
FAS IRS probes were made by labeling with
[-
P]dCTP and Klenow fragment of Escherichia coli DNA polymerase.
Figure 1:
Gel mobility shift assay of RNE with
fatty acid synthase insulin response sequence. Each reaction in a total
volume of 20 µl contained 8 µg of RNE, 1 gel shift
reaction buffer (see ``Experimental Procedures''), 0.1 µg
of poly(dI-dC), 0.1 ng of
P-labeled oligonucleotide probe
(1
10
cpm, S14 probe in lane 1, L-PK probe
in lane 2, and FAS IRS probe in lanes 3-11),
and various cold oligonucleotides as competitors. Sequences of the
oligonucleotides are listed under ``Experimental
Procedures.'' Lanes 1-3, no competitors; lanes
4 and 5, 20
and 40
cold FAS IRS; lanes 6 and 7, 20
and 40
poly(dI-dC); lanes 8 and 9, 20
and 40
cold L-PK
sequence; lanes 10 and 11, 20
and 40
cold S14 sequence. The reaction mixtures were incubated at room
temperature for 1 h and applied to a 6% nondenaturing polyacrylamide
gel. The gel was dried and exposed to a x-ray film with an intensifying
screen.
To further demonstrate that USFs bind to the FAS IRS, we tested whether Band 1 and 2 in Fig. 1are affected by addition of USF-specific antibodies to the gel mobility shift assay. As shown in Fig. 2A, incubating RNEs with FAS IRS probe in the presence of anti-USF1 antibodies directed against the C-terminal 20 amino acids (position 291-310) completely disrupted these two major bands and resulted in ``supershifts.'' At the same time, a new protein-DNA complex (Band 3) appeared upon the addition of anti-USF1 antibodies. This newly appeared band did not appear to be caused by protein contaminants of the antibody preparation because gel shift assays with fixed amounts of anti-USF1 antibody and increasing amount of nuclear extract (2-8 µg) resulted in a proportionally increased amount of Band 3 (Fig. 2B). This result demonstrates that 1) USF1 is a major component of the protein complexes that bind to FAS IRS and 2) within the nuclear extract there is at least one additional protein component that binds to the FAS IRS, probably by forming heterocomplexes with USF1.
Figure 2:
A,
gel supershift assay of RNE with anti-USF1 antibodies. Each reaction
(20 µl) contained 8 µg of RNE, 1 gel shift reaction
buffer (see ``Experimental Procedures''), 0.1 µg of
poly(dI-dC), 0.1 ng of
P-labeled FAS IRS probe (1
10
cpm), cold FAS IRS competitor in 40
molar excess (lane 2) or 2 µg of anti-USF1 antibody (lane 3). B, gel shift assay with various amounts of RNE and a fixed
amount of anti-USF1 antibodies. Each reaction (20 µl) contained 1
gel shift reaction buffer (see ``Experimental
Procedures''), 0.1 µg of poly(dI-dC), 0.1 ng of
P-labeled FAS IRS probe (1
10
cpm),
various amounts of RNE, and a fixed amount of anti-USF1 antibodies as
listed below. Lane 1, 8 µg of RNE, no anti-USF1
antibodies; lane 2, 0 µg of RNE, 2 µg of anti-USF1
antibodies; lane 3, 2 µg of RNE and 2 µg of anti-USF1
antibodies; lane 4, 4 µg of RNE and 2 µg of anti-USF1
antibodies; lane 5, 6 µg of RNE and 2 µg of anti-USF1
antibodies; lane 6, 8 µg of RNE and 2 µg of anti-USF1
antibodies. The reaction mixtures in A and B were
incubated at room temperature for 1.5 h and processed as described in Fig. 1.
Within the USF transcription factor family, USF2 is known to be able to heterodimerize with USF1 and has the same binding affinity to E-box sequences as USF1(48) . Currently, no other proteins have been reported to heterodimerize with USF1 and regulate gene transcription. To test whether USF2 is the other protein involved in binding to FAS IRS, we performed the following two experiments. First, to test the E-box specificity of Band 3, a gel mobility shift competition assay was carried out in the presence of anti-USF1 antibodies and with S14 and L-PK oligonucleotides as competitors (Fig. 3A). Both S14 and L-PK carbohydrate response elements, at molar excesses of 20- and 40-fold, could compete for the formation of Band 3, as well as for the two supershifted bands. Compared to S14 sequence, L-PK sequence was found to compete less effectively. As expected, FAS IRS itself competed and nonspecific poly(dI-dC) did not. This result suggests that Band 3 represents an E-box-specific protein-DNA complex, which supports the probability that it contains USF2. The second experiment used rabbit polyclonal anti-USF2 antiserum in the gel shift assay to directly detect the presence of USF2 in Band 3. As shown in Fig. 3B, addition of anti-USF2, together with anti-USF1 antibody to the gel shift assay resulted in the total disappearance of Band 3, as well as the lower supershifted band (lane 4) formed by the addition of anti-USF1 antibody alone, demonstrating the presence of USF2 in Band 3. On the other hand, adding anti-USF2 antibody alone (lane 3, Fig. 3B) to the gel shift assay disrupted protein-DNA complex formation in both Band 1 and 2, suggesting USF2 is present in both of them. Taken together, we concluded that USF2 is the other major protein component that interacts and forms protein heterocomplexes with USF1 to bind FAS IRS.
Figure 3:
A, competition of the supershifted bands
and Band 3 with various competitors. Each reaction (20 µl)
contained 8 µg of RNE, 1 gel shift reaction buffer (see
``Experimental Procedures''), 0.1 µg of poly(dI-dC), 0.1
ng of
P-labeled FAS IRS probe (1
10
cpm), 2 µg of anti-USF1 antibodies, and various cold
competitors at molar excesses listed below. Lane 1, no
competitor; lanes 2 and 3, 20
and 40
of poly(dI-dC); lanes 4 and 5, 20
and 40
of FAS IRS; lanes 6 and 7, 20
and 40
of L-PK; lanes 8 and 9, 20
and 40
of S14. B, gel mobility shift assay with anti-USF1 and
anti-USF2 antibodies. Each reaction (20 µl) contained 8 µg of
RNE, 1
gel shift reaction buffer (see ``Experimental
Procedures''), 0.1 µg of poly(dI-dC), 0.1 ng of
P-labeled FAS IRS probe (1
10
cpm),
and anti-USF1 (2 µg) and/or anti-USF2 antibodies (2 µl of 1:50
dilution) as listed below. Lane 1, no antibodies; lane
2, anti-USF1; lane 3, anti-USF2; lane 4,
anti-USF1 and anti-USF2. The reaction mixtures in A and B were processed as described in Fig. 2.
To
provide further supporting data for the involvement of USF1 and USF2 as
major protein factors in the FAS IRS binding, we performed an
UV-cross-linking experiment. The protein-DNA complexes formed under the
same condition as in the gel shift assays with RNE were exposed to UV
light, and the protein components cross-linked to the P-labeled FAS IRS probe were separated on SDS-PAGE and
revealed by autoradiography. As shown in Fig. 4, major proteins
cross-linked to the FAS IRS probe migrated at the 49-kDa position, and
the free probe itself migrated at about 6 kDa. Subtraction of the probe
size (6 kDa) from the cross-linked protein-DNA complex (49 kDa) gave
the size of the protein to be 43 kDa, which is consistent with the size
of USF1 (43 kDa) and USF2 (44 kDa in mouse and human but probably 42
kDa in rat; see descriptions below). As expected, addition of either
FAS IRS itself or AdML promoter, but not poly(dI-dC), in 50- and
100-fold molar excesses to the UV-cross-linking reaction abolished the
formation of the 49-kDa signal, demonstrating the specificity of the
experiment.
Figure 4:
UV-cross-linking of RNE to P-labeled FAS IRS. Each reaction (20 µl) contained 8
µg of RNE, 1
gel shift reaction buffer (see
``Experimental Procedures''), 0.1 µg of poly(dI-dC),
P-labeled FAS IRS probe (1
10
cpm),
and cold oligonucleotide competitors. Lane 1, no competitor; lanes 2 and 3, 50
and 100
poly(dI-dC); lanes 4 and 5, 50
and 100
FAS IRS; lanes 6 and 7, 50
and 100
AdML. The reaction mixtures were incubated at room temperature
for 1 h, UV-cross-linked for 10 min, separated on a 12% SDS-PAGE and
exposed to x-ray films (see ``Experimental
Procedures'').
Figure 5:
Gel shift assay of FNE with anti-USF1 and
anti-USF2 antibodies. Each reaction (20 µl) contained 8 µg of
nuclear extract (RNE in lane 1, FNE in lanes
2-5), 1 gel shift reaction buffer (see
``Experimental Procedures''), 0.1 µg of poly(dI-dC), 0.1
ng of
P-labeled FAS IRS probe (1
10
cpm), and anti-USF1 (2 µg) and anti-USF2 (2 µl of 1:50
dilution) antibodies. Lanes 1 and 2, no antibodies; lane 3, anti-USF1; lane 4, anti-USF2; lane
5, anti-USF1 and anti-USF2. The reaction mixtures were processed
as described in Fig. 2.
To test this
hypothesis, we carried out a Western immunoblot analysis of RNE and FNE
with anti-USF1 and anti-USF2 antibodies to directly show any
differences in USF1 and USF2 in these nuclear extracts (Fig. 6).
Equal amounts of protein (80 µg) were applied to a 12% SDS
denaturing polyacrylamide gel separation and transferred onto
Immobilon-P membranes and detected with the two antibodies. Anti-USF1
antibody is directed against the C-terminal 20 amino acids of USF1 and
anti-USF2 antiserum is directed against the C-terminal portion of USF2.
Anti-USF1 antibody detected two proteins, one at 43 kDa and the other
at 17 kDa. The 43-kDa protein (USF1) is in a form of doublet with a
minor form at 41.5 kDa. The quantity of the 43-kDa protein is about
4-fold that of the 41.5-kDa protein. While readily detectable in RNE,
this 43-kDa doublet is barely detectable in FNE. On the other hand, the
17-kDa protein was readily detected in both nuclear extracts. It seems
to be a doublet in FNE and just a single band in RNE. The amount of the
17-kDa protein in FNE is about twice that in RNE. Since the anti-USF1
antibody is against the C terminus of USF1, we thought the 17-kDa
protein might be a smaller USF1 containing the C-terminal portion or a
USF1-related protein. Anti-USF2 antiserum detected a single major
protein at 42 kDa in both extracts. Although the reported size of USF2
from human and mouse is 44 kDa, the size of rat USF2 remains
undetermined. Since the anti-USF2 antiserum is highly specific for the
detection of USF2, we strongly believe that the 42-kDa
protein is the rat USF2. In contrast to the 43-kDa USF1, rat USF2
exists in equal amounts in RNE and FNE. The fact that the same amount
of USF2 was detected in both nuclear extracts argues against the
disappearance of 43-kDa USF1 and the greater amount of 17-kDa
USF1-related protein in FNE is caused by general protein degradation
during preparation of the nuclear extract. Overall, our data highly
suggest that USF1, but not USF2, is regulated during fasting and
refeeding, including the amount changes of the 43-kDa USF1 and the
17-kDa USF1-related protein.
Figure 6: Western immunoblot of RNE and FNE with anti-USF1 and anti-USF2 antibodies. 80 µg of RNE (lanes 1 and 3) and FNE (lanes 2 and 4) were separated on a 12% SDS-PAGE and transferred onto polyvinylidene difluoride Immobilon-P membranes, incubated with primary antibodies (anti-USF1, lanes 1 and 2; anti-USF2, lanes 3 and 4) and secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit IgG), and developed with a chemiluminescence kit (see Experimental Procedures).
To investigate whether the 17-kDa USF1-related protein binds to DNA, we carried out a UV-cross-linking experiment with FNEs (Fig. 7). In addition to the 49-kDa signal, as was found with the RNEs, an additional 23-kDa signal was also detected. Both the 49-kDa and 23-kDa signal could be specifically blocked by adding either cold FAS IRS or AdML promoter to the UV-cross-linking reaction, while addition of nonspecific poly(dI-dC) had no effect, demonstrating the specificity of the cross-linking experiment. Subtraction of the probe size (about 6 kDa) from the probe-linked protein size of the small band (23 kDa) gave the molecular mass of the protein to be about 17 kDa, which is consistent with the molecular mass of the small USF1-related protein.
Figure 7:
UV-cross-linking of RNE and FNE to P-labeled FAS IRS. Each reaction (20 µl) contained 8
µg of RNE (lanes 1-4) or FNE (lanes
6-8), 1
gel shift reaction buffer (see
``Experimental Procedures''), 0.1 µg of poly(dI-dC),
P-labeled FAS IRS probe (1
10
cpm),
and cold oligonucleotide competitors. Lanes 1 and 5,
no competitor; lanes 2 and 6, 100
poly(dI-dC); lanes 3 and 7, 100
FAS IRS; lanes 4 and 8, 100
AdML. The reaction
mixtures were processed as described in Fig. 4.
Previously, we reported that the first 322 base pairs of the FAS promoter contained the insulin response element by transient transfection of H4IIE hepatoma cells and 3T3-L1 adipocytes(6) . Subsequently, we mapped FAS IRS to the proximal promoter region from position -71 to position -50 by making chimeric constructions of serial 5`-deletions of the rat FAS promoter ligated to the luciferase reporter gene and transfection into 3T3-L1 adipocytes(7) . This IRS sequence confers about 3-fold increase of the reporter gene activity upon insulin treatment at physiological concentrations. Triple copies of FAS IRS fused to a heterologous SV40 promoter also exhibit insulin response. Since this identification of the cis-acting DNA element mediating insulin response of FAS, we have been making our efforts to identify and isolate the trans-acting factors. In this study, we have demonstrated that USF1 and USF2, which belong to the family of bHLH-LZ transcription factors, are major components of the protein complexes that bind to the E-box contained in FAS IRS. It was previously shown that USF1 can be functionally regulated in a redox-dependent manner(46) . Sulfhydryl groups of the two cysteine residues, both present within the HLH protein-protein interface domain of USF1 and conserved among all known USF sequences from various species including human(15, 16) , mouse(18) , Xenopus(19) , and sea urchin(20) , are targets of this regulation in the DNA binding capacity, which can be translated into the modulation of USF1's ability to activate transcription(46) . USF1 and USF2 are known to be expressed ubiquitously among various cells and tissues(18) , based mainly on Northern blot analysis. Distributions of USF1 and USF2 among various tissues and their regulation under different physiological conditions have not been shown previously. In this report, we have demonstrated that USF1 protein in liver of fasted rats is very low and is markedly increased in liver of rats refed a high carbohydrate, fat-free diet. In contrast, USF2 remains at the same concentration in liver of fasted or refed rats. This observation suggests that these ubiquitous transcription factors are regulated at the protein level in response to physiological changes to control downstream gene expression.
Structure-function studies of the human 43-kDa USF1 revealed that the HLH and LZ regions are both important for USF oligomerization and DNA binding(15) . Subsequently, transcription activation domain was localized to two regions at the N terminus(21) . The first region is between positions 16 and 59 with a core between 26 and 39, and the second is between positions 93 and 156 with a core between 103 and 130 (21) . This assignment of the functional domains allows us to analyze the possible role of the 17-kDa USF1-related protein, which is detected in rat liver under both fasting and refeeding conditions. There are two possible origins of the 17-kDa USF1-related protein. First, the 17-kDa protein could be a cleavage product of the 43-kDa USF1, or the translation product of an alternative start codon, containing C-terminal amino acids. Since USF sequences show a high degree of sequence conservation almost throughout the entire protein, we used the human USF1 sequence to estimate the amino acids that the 17-kDa rat USF1-related protein might contain. By comparing our 17-kDa protein to the sizes of different portions of human USF1 revealed on SDS-PAGE by Roeder and co-workers(15) , the 17-kDa rat USF1-related protein was estimated to contain the region from around position 175 to the C terminus, which includes the bHLH and LZ region but not the N-terminal transcription activation domain. In this case, the 17-kDa USF1 still dimerizes with USF2 and bind to DNA, but loses its transactivation function, resulting in no activation of the FAS gene transcription. Regulation of transcription by proteolysis of bHLH-LZ transcription factors, e.g. sterol response element-1-binding protein, has been reported(47) . Since alternative splicing also exists for both human and mouse USF1 and USF2 mRNAs(15, 18) , the second possibility is that the 17-kDa USF1-related protein is a translation product of an alternatively spliced mRNA. Although the nature of alternative splicing of rat liver USF1 mRNA precursors remains to be studied, the 17-kDa size would not allow the inclusion of the activation, bHLH, and LZ domains at the same time to form a fully functional but ``ultra-compact'' USF1. In this case, the 17-kDa USF1-related protein is hindered from either proper DNA-binding, USF2 interaction, or transactivation.
Rat liver FNE containing the 17-kDa USF1-related protein but not the full-size USF1 (Fig. 6) still shows strong DNA binding activity, and the gel mobility supershift pattern from anti-USF1 and anti-USF2 antibodies are similar to those obtained from RNE (Fig. 5). These results demonstrate that the DNA binding and dimerization capability of the 17-kDa USF1-related protein is not severely affected by its smaller size. The existence of a 17-kDa protein, which could be UV-cross-linked to the FAS IRS probe in FNE, reinforces the notion that the 17-kDa USF1-related protein can effectively bind to the E-box in FAS IRS. These observations make the second possibility discussed above the less likely. Nevertheless, the 17-kDa USF1-related protein would not be fully functional in transactivation of the FAS gene. Controlling the proper protein expression level of USF1 and USF2 by pre- and/or post-translational events might be an important mechanism for the cell to regulate transcription of FAS and other E-box-containing genes under different physiological conditions (Fig. 8).
Figure 8: A proposed model for the roles of USF1 and USF2 in regulation of FAS gene transcription. Upon fasting treatment, the 43-kDa USF1 protein is replaced by a 17-kDa USF1-related protein. As a result, USF1-USF2 heterocomplexes lose their transactivation function and no longer activate the FAS gene transcription. During the process of refeeding a high carbohydrate, fat-free diet, expression of the 43-kDa USF1 is stimulated and USF1-USF2 heterocomplexes restore their function, resulting in activation of FAS gene transcription.
Glucose and insulin can regulate a number of glycolytic and lipogenic enzymes. Their effects are dependent on each other in most cases. USFs have been reported to be involved in the glucose stimulation of L-PK and S14 transcription, and the involvement of USF1 and USF2 and their regulation have not been clearly addressed. Here we provide evidence for its binding to the FAS IRS. During fasting/refeeding treatment of animals, both glucose and insulin control glycolytic and lipogenic enzyme expression. Their signal transduction pathways are different, but may converge at some points. Using common transcription factors such as USF may be one such intersection. The specificity of which genes are regulated by glucose and insulin may be contributed by the context of the DNA response element and/or protein interaction of USF with other transcription factors. In the case of rat S14 gene, although mutated glucose response element binds to the same protein complex containing USF in the gel shift assay in a manner indistinguishable from the wild-type glucose response element, it confers no glucose response in vivo(38) . This observation suggests that other protein components are required to cooperate with USF and determine the glucose response specificity. In respect to the glucose response element sequence 5`-CACGTGNNNGCC-3`, it contains not only the E-box but also the downstream GCC, in which the CC dinucleotide in the E-box context are essential for a specific glucose response(38) . FAS IRS contains the E-box sequence but not the downstream GCC even read from both strands, and the surrounding sequences may specify the insulin response. Further investigation will be necessary to elaborate the molecular mechanisms underlying the regulation of the glycolytic and lipogenic enzymes.