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J. Biol. Chem., Vol. 277, Issue 27, 24120-24127, July 5, 2002
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From the Department of Biochemistry and Molecular Biology, Center for Mammalian Genetics, and Center for Nutritional Sciences, University of Florida College of Medicine, Gainesville, Florida 32610-0245
Received for publication, February 27, 2002, and in revised form, April 9, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Transcription from the asparagine synthetase
(A.S.) gene is increased in response to either amino acid
(amino acid response) or glucose (endoplasmic reticulum stress
response) deprivation. These two independent pathways converge on the
same set of genomic cis-elements within the
A.S. promoter referred to as nutrient-sensing response
elements (NSRE) 1 and 2, both of which are necessary for gene
activation. The NSRE-1 sequence was used to screen ATF/CREB family
members by electrophoresis mobility shift assays and supershift by specific antibodies. The results indicated that ATF4 binds to the
NSRE-1 sequence and that the amount of the ATF4 complex was increased
when extracts from amino acid-deprived or glucose-deprived cells were
tested. Using electrophoresis mobility shift assay experiments and a
probe that contained both NSRE-1 and NSRE-2, mutation of the NSRE-1
sequence completely prevented formation of the ATF4-containing
complexes, whereas mutation of the NSRE-2 sequence did not.
Overexpression of ATF4 increased A.S. promoter-driven transcription, whereas an inhibitory dominant negative ATF4 mutant blocked both basal and starvation-enhanced transcription. Collectively, the results provide both in vitro and in vivo
evidence for a role of ATF4 in the transcriptional activation of the
A.S. gene in response to nutrient deprivation.
Amino acid availability is an important factor in protein
biosynthesis and degradation. However, the role of amino acid
availability in controlling other fundamental cellular processes has
not been as extensively investigated (reviewed in Refs. 1 and 2). A
number of specific mRNAs, proteins, and cellular activities are
induced following amino acid deprivation (reviewed in Refs. 2 and 3),
and among these is asparagine synthetase
(A.S.),1 which catalyzes the
glutamine- and ATP-dependent conversion of aspartic acid to
asparagine. Arfin and co-workers (4, 5) showed that starvation
of Chinese hamster ovary cells for asparagine decreased the level of
asparaginyl-tRNAAsn with a concurrent increase in A.S.
enzymatic activity. Induction of A.S. activity also occurred in
temperature-sensitive Chinese hamster ovary cell mutants for
asparaginyl-, leucyl-, methionyl-, and lysyl-tRNA synthetases (5).
Similarly, starvation of several different cell types, for a wide range
of individual amino acids, causes the accumulation of A.S. mRNA,
illustrating that this sensing mechanism broadly detects amino acid
limitation (6-8).
Guerrini et al. (9) identified a region from nt Siu et al. (14) recently demonstrated that
CCAAT/enhancer-binding protein beta (C/EBP The present studies were designed to determine whether an ATF family
member binds to the NSRE-1 of the A.S. promoter, and if so,
to establish its role with regard to induction of the gene by the AAR
and ERSR pathways. Using electrophoresis mobility shift analysis
(EMSA), the data show that ATF4 binds this element in vitro,
but ATF1, ATF2, and ATF3 do not. Previous experiments had also
indicated that ATF2 does not bind to the NSRE-1 sequence (14).
Antibodies specific for ATF4 caused a supershift of an NSRE-1 complex,
and the abundance of that shifted complex was increased when extracts
from amino acid-deprived (AAR) or glucose-deprived (ERSR) cells were
used. Mutation of the NSRE-1 sequence, but not that of NSRE-2, caused
loss of ATF4 binding. Overexpression of ATF4 caused increased basal
transcription from the A.S. promoter, whereas expression of
a dominant negative of the ATF4 mutant in HepG2 cells blocked
transcriptional induction driven by the A.S. promoter
following activation of either the AAR or the ERSR pathway. Collectively, the results demonstrate that ATF4, along with C/EBP Cell Culture--
Human hepatoma HepG2 cells were cultured in
minimal essential medium (MEM), pH 7.4, supplemented to contain 25 mM NaHCO3, 4 mM glutamine, 10 µg/ml streptomycin sulfate, 100 µg/ml penicillin G, 28.4 µg/ml
gentamycin, 0.023 µg/ml
N-butyl-p-hydroxybenzoate, 0.2% (w/v) bovine
serum albumin, and 10% (v/v) fetal bovine serum. Cells were maintained
at 37 °C in a 5% CO2/95% air incubator.
Transient Transfection and Northern Analysis--
For HepG2
cells, a batch transfection protocol was performed (11). The cells
were seeded on 60-mm dishes (2.65 × 106 cells)
for 24 h before transfection. Transfection was performed with
Superfect reagent (QIAGEN, Valencia, CA) at a ratio of 6 µl of
Superfect to 1 µg of DNA. For each transfection, 5 µg of the
A.S.
Total cellular RNA was isolated using an RNeasy Mini Kit according to
the procedure described by the supplier (QIAGEN).
32P-Radiolabeled cDNA probe synthesis for growth
hormone, glutamate dehydrogenase, and LacZ, as well as the Northern
analyses, were performed as described (18). The cDNA probe for
human ATF4 (nt 853-1933) was generated by reverse transcript-PCR and
then subcloned into the pcDNA3.1 vector.
ATF4 Expression Vectors--
The plasmids containing rat ATF4
wild-type sequence and a mouse dominant negative ATF4 mutant were
generously provided by Dr. Jawed Alam (21). The rat ATF4
cDNA was cloned into the pcDNA3.1/Myc-His vector (Invitrogen),
and its expression was driven by the cytomegalovirus promoter. The
mouse dominant negative ATF4 expression vector was created by first
cloning the wild-type sequence into the pEF/Myc/mito vector
(Invitrogen), and then six amino acid substitutions
(292RYRQKKR298 to
292GYLEAAA298) were made within the DNA-binding
domain (19).
Electromobility Mobility Shift Assay--
Nuclear extracts were
prepared from HepG2 cells incubated for 16 h in complete MEM, MEM
lacking glucose, or MEM lacking histidine. Nuclear extracts were
prepared from four 150-mm dishes of cells, and all steps were performed
at 4 °C. The nuclear extract isolation procedure and the EMSA
protocol have been described previously (14). Antibodies were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). All
experiments were repeated with at least two independently prepared
nuclear extracts.
Polyacrylamide Gel Electrophoresis of EMSA Complexes--
EMSA
was performed as described previously (14), using
32P-labeled oligonucleotide containing the A.S.
promoter sequence Introduction--
Two separate cis-elements, NSRE-1 and NSRE-2,
are both required for induction of the A.S. gene in response
to activation of the AAR or ERSR pathways (12). The NSRE-1 sequence is
similar to an element within the chop gene termed a
C/EBP-ATF composite binding site (16, 17). Therefore, it was likely
that an ATF family member also binds to and functions through
NSRE-1.
Screen for ATF4 Binding by Electrophoresis Mobility Shift
Analysis--
EMSA was performed to determine whether ATF4 was
present in human HepG2 hepatoma cells and, if so, whether it exhibited
affinity for the A.S. NSRE-1 sequence. The radiolabeled NSRE-1
oligonucleotide probe (nt ATF4 Binding Is Also Increased Following ERSR Activation--
To
determine whether the abundance of the ATF4-containing NSRE-1 complex
was increased following activation of the ERSR pathway, HepG2 cells
were incubated with or without glucose for 16 h, and then nuclear
extracts were prepared (Fig. 2). EMSA illustrated that these extracts,
when incubated with 32P-labeled NSRE-1 oligonucleotide,
formed the same nutrient-regulated complexes (labeled A-C)
as those observed with extracts from histidine-deprived cells (Fig. 2,
compare lanes 6 and 9). Furthermore, ATF4
antibody caused a supershift of complex A (Fig. 2, compare lanes
9 and 11) in the same manner as that seen for the
histidine-free extracts (Fig. 2, lanes 6 and
8).
ATF4-Containing DNA-Protein Complexes Are Subject to
Competition--
To compare competition between NSRE-1 and a consensus
CREB/ATF sequence, EMSA experiments were performed with radiolabeled NSRE-1 and increasing concentrations of unlabeled oligonucleotide as
inhibitor (Fig. 3). To permit easier
detection of all three complexes, extracts from histidine-deprived
cells were used throughout. Increasing concentrations of unlabeled
NSRE-1 blocked formation of all three. A 100-fold excess of unlabeled
NSRE-1 also completely blocked formation of the ATF4-containing
complexes (Fig. 3, compare lanes 12 and 13). In
contrast, an oligonucleotide containing a consensus CREB/ATF sequence
exhibited some competition for binding but was not nearly as effective
as NSRE-1 itself (Fig. 3, lanes 7-11 and lanes
13 and 14). These data, along with our previous results
for C/EBP Mutagenesis of Individual Nucleotides within the
NSRE-1--
In vivo footprinting of the A.S.
promoter region revealed that the guanine nucleotides at Dependence of ATF4 Binding on de Novo Protein Synthesis--
The
increase in A.S. mRNA content following amino acid deprivation is
dependent on de novo protein synthesis (6, 7), consistent
with the hypothesis that production of one or more upstream regulators
is required prior to transcriptional induction of the A.S.
gene itself. To determine whether ATF4 binding activity was dependent
on de novo synthesis, nuclear extracts were prepared from
HepG2 cells that had been incubated in complete MEM or MEM lacking
histidine, each with or without 0.1 µM cycloheximide. These extracts were then tested by EMSA for the formation of
starvation-induced and ATF4-containing protein-DNA complexes (Fig.
5). Inhibition of protein synthesis was
completely blocked in the formation of the amino acid-regulated
complexes (e.g. in Fig. 5, compare lane 3 with
lane 7). Furthermore, when the nuclear extracts derived from
control and cycloheximide-treated cells were incubated with ATF4
antibody and then monitored for the presence of shifted complexes, the
results clearly document that de novo protein synthesis is required for formation of the ATF4-containing complex (Fig. 5, compare
lane 4 with lane 8).
ATF4 and C/EBP NSRE-2 Is Not Required for ATF4 Binding--
When an
oligonucleotide probe (NSRE-1/2) was prepared that corresponded to the
A.S. promoter region containing both the NSRE-1 and NSRE-2
sequences (Fig. 1), several complexes could be detected by EMSA (Fig.
7). One of these complexes, labeled
A, was enhanced in abundance when extracts from
histidine-deprived cells were tested (Fig. 7, compare lanes
1 and 2 with lanes 4 and 5), and anti-ATF4 caused a supershift of complex A that was readily visible in
the histidine-depleted cell extracts (Fig. 7, lane 6).
Incubation with ATF2 antibody served as a negative control. To
investigate whether or not NSRE-2 contributed to ATF4 binding, a
block of nucleotides within either the NSRE-1 or the NSRE-2 core
sequence was mutated to a sequence that previous functional assays had shown would block regulated transcription (12). Mutation of the NSRE-1
sequence, in the presence of a wild-type NSRE-2 sequence, completely
eliminated the starvation-inducible complexes and consequently, the
anti-ATF4 supershifted complexes (Fig. 7, lanes 7-12). In contrast, mutation of the NSRE-2 site and retention of the wild-type NSRE-1 sequence still permitted formation of the amino acid-regulated and ATF4-containing complexes (Fig. 7, lanes 13-18).
Therefore, although both cis-elements are absolutely
required for nutrient-regulated transcription (12), a non-functional
NSRE-2 sequence does not prevent ATF4 binding to the NSRE-1 site
in vitro.
ATF4 mRNA Content Is Increased by Either Amino Acid or Glucose
Deprivation--
The EMSA analysis suggested the possibility that the
amount of active ATF4 in the nucleus is increased by amino acid or
glucose deprivation. This is consistent with the observation that the translation of the ATF4 mRNA is enhanced by nutrient starvation (20), but the effect of amino acid deprivation on ATF4 mRNA content
has not been reported. HepG2 cells were incubated in MEM, MEM lacking
either histidine or glucose, or amino acid-free Krebs-Ringer bicarbonate buffer (KRB) for 16 h, and then RNA was isolated for Northern analysis (Fig. 8). The ATF4
mRNA content was increased within 2 h of removing all amino
acids or just histidine from the medium, and it reached a value of
2.5-3-fold at 12 h (Fig. 8, A and B).
Glucose deprivation was less effective in stimulating the ATF4 mRNA
level, but the content was elevated by about 2-fold. To document that
these increases were statistically significant, in a separate set of
experiments, multiple plates of cells were incubated in each of the
four media for 12 h, and then the mRNA was analyzed (Fig.
8C). Results similar to the time course were obtained with
statistically significant increases of 2-3-fold for amino acid
limitation. Parallel experiments using immunoblotting showed that the
amount of ATF4 protein also increased following nutrient deprivation
(data not shown), confirming earlier reports (20).
The Role of Protein Synthesis on the Induction and Decay of ATF4
mRNA Content--
As mentioned, the ATF4 protein level is
increased following amino acid deprivation through translational
control (20), an observation that is consistent with the fact that
increased transcription from the A.S. gene is dependent on
de novo protein synthesis (6, 7). If ATF4 synthesis
represents an upstream step in the AAR pathway, as the data from this
report and others suggest, it was important to further characterize the
increase in ATF4 mRNA. To determine whether de novo
protein synthesis was required, cells were incubated in amino acid-free
medium with or without cycloheximide (Fig.
9A). The
time-dependent increase in ATF4 mRNA was not affected by blocking protein synthesis. To investigate the role of de
novo protein synthesis on the turnover of the ATF4 mRNA, cells
were incubated in amino acid-free medium for 6 h to elevate the
ATF4 mRNA content and then transferred to amino acid-complete MEM
with or without 0.1 mM cycloheximide (Fig. 9, B
and C). A relatively rapid decay of the mRNA was
observed in the absence of the inhibitor, but blockade of protein
synthesis completely prevented the mRNA decay, demonstrating the
need for one or more newly synthesized proteins and suggesting the
potential involvement of additional regulatory steps in ATF4
expression.
Induction of ATF4 mRNA Content by Histidine Deprivation Is Not
Due to mRNA Stabilization--
To establish an estimate of the
half-life for the ATF4 mRNA during the decay process that occurred
after refeeding, HepG2 cells were incubated for 18 h in amino
acid-free medium. Then after transfer of these starved cells to amino
acid-complete MEM, RNA was collected at specific time points and
analyzed for ATF4 mRNA (Fig.
10A). Consistent with the
data of Fig. 9, refeeding resulted in a rapid decay of ATF4 mRNA
with an estimated half-life of less than 2 h (Fig.
10B). To evaluate mRNA stability as a possible mechanism
for the starvation induction, cells were incubated in histidine-free
medium to induce ATF4 mRNA content and then transferred to either
fresh histidine-free medium or amino acid-complete MEM, both containing
5 µM actinomycin D (Fig.
11). The rate of decay was much less in
the presence of the RNA synthesis inhibitor than in its absence
(compare Figs. 10 and 11), but analyzing the results graphically, it
was clear that the rate of turnover was no different in the presence or
absence of histidine. These results suggest that histidine deprivation
does not cause an elevation in the ATF4 mRNA content by increasing
mRNA stability. If not stabilization, then de novo RNA
synthesis would be the likely mechanism for ATF4 mRNA induction. To
test this possibility, HepG2 cells were incubated for 0-12 h in medium
lacking all amino acids or just histidine in the presence or
absence of 5 µM actinomycin D (Fig. 11C). The RNA synthesis inhibitor prevented completely the increase in ATF4 mRNA accumulation in response to amino acid limitation.
Activating or Inhibitory ATF4 Forms Regulate A.S. Expression
Accordingly--
HepG2 cells were transiently co-transfected
with a GH reporter gene driven by the A.S.
proximal promoter ( The data described in this report demonstrate that ATF4 binds to
the NSRE-1 cis-element within the proximal promoter of the human A.S. gene and activates transcription in response to
nutrient deprivation. Two cis-elements, NSRE-1 and NSRE-2,
are required for increased transcription of the A.S. gene
following activation of either the AAR or the ERSR pathways (10-12).
We have coined the phrase nutrient-sensing response unit to describe
this combination of elements that function together to sense nutrient
availability and modulate the transcription of this gene accordingly.
The ATF family of transcription proteins represents a subclass of the
basic leucine zipper family, and ATF members are known to
heterodimerize with the C/EBP basic leucine zipper subgroup. The human
chop gene 5' upstream region contains an AARE
(5'-TGATGCAAT-3') that differs from the A.S. NSRE-1 sequence by only
two nucleotides, and this element has been shown to be a C/EBP-ATF
composite site (16, 17). Fawcett et al. (17) reported ATF4
binding to this site in response to arsenite-induced stress, but Bruhat
et al. (15) demonstrated that although both C/EBP The known characteristics of A.S. regulation are consistent with the
present observations indicating a role for ATF4. In vivo footprinting documented that nutrient limitation caused an increase in
protein binding at NSRE-1 (12), and the results in this report show
that ATF4 binding is increased when nuclear extracts from either
histidine-deprived (AAR pathway) or glucose-deprived (ERSR pathway)
cells are tested. Following nutrient limitation, there is a lag of
about 4 h prior to a significant increase in A.S. mRNA content
(7, 10), and the increased abundance of A.S. mRNA is dependent on
de novo protein synthesis (7, 22). Those results indicate
that synthesis of a regulatory protein is required prior to activation
of A.S. gene transcription. Consistent with those
observations, inhibition of protein synthesis blocked the starvation-dependent enhancement in protein-DNA complex
formation as assayed by EMSA and completely prevented the increase in
nuclear extract ATF4 binding activity. These results are also
consistent with the observation that synthesis of ATF4 is
translationally enhanced by amino acid or glucose deprivation (20).
Furthermore, the present data extend our knowledge of how ATF4
expression is controlled by documenting: 1) that ATF4 mRNA is also
elevated in response to amino acid and glucose limitation and 2) that
the increase in ATF4 mRNA is likely the result of transcriptional activation of the gene rather than mRNA stabilization.
Two observations indicate that ATF4 functions in vivo to
modulate transcription of the human A.S. gene. First, the
basal rate of A.S. promoter-driven transcription was induced
significantly in ATF4 overexpressing cells, and the induction by
nutrient deprivation was enhanced further when exogenous ATF4 was
expressed. Second, expression of a dominant negative ATF4 mutant caused
an inhibition of basal transcription and blocked activation of the
A.S. promoter following either histidine or glucose
deprivation. Collectively, the data provide strong support for the
proposed role of ATF4 as a transcriptional regulator for the NSR
pathway that, in the case of the A.S. gene, represents a
convergence of the AAR and the ERSR pathways.
The basis of metabolite control of transcription in mammalian
cells is not well understood for most molecules, including amino acids.
Through the identification of the corresponding transcription proteins
responsible for regulation of specific nutrient-regulated target genes,
one can progress backwards up the signal transduction pathway to reveal
the individual steps required. For the human A.S. gene, this
strategy has demonstrated the interesting observation that two
independent metabolic sensing pathways, one that detects amino acid
limitation and one that detects endoplasmic reticulum stress, converge
at some point and ultimately act on the A.S. promoter
through a common set of genomic elements (11, 12). A previous report
documented that one of the transcriptional regulators associated with
this process is C/EBP
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 to 62
within the human A.S. promoter that functioned as an amino
acid response element (AARE). More recently, Barbosa-Tessmann et
al. (10-12) have demonstrated that the human A.S. gene
is also induced by glucose deprivation and that this activation is
mediated by the endoplasmic reticulum stress response (ERSR), also
known as the unfolded protein response pathway in yeast (13). In
vivo footprinting and mutagenesis demonstrated that the promoter
sequence 5'-TGATGAAAC-3' (nt 68 to 60), the region first identified
by Guerrini et al. (9), was also responsible for induction
of the A.S. transcription following activation of the ERSR
pathway (12). The ERSR activation demonstrates that this
A.S. promoter sequence serves in a broader capacity than
simply as an AARE and, to reflect this broader substrate-detecting
capability, this sequence is referred to as the nutrient-sensing
response element-1 (NSRE-1). A second element (5'-GTTACA-3', nt 48 to
43), 11 nucleotides downstream from NSRE-1, is also required for
activation and is referred to as NSRE-2. To underscore the collective
function of these two sequences, the term nutrient-sensing response
unit has been coined.
) binds to the NSRE-1
sequence in vitro and when overexpressed activates
transcription of an A.S. promoter-driven reporter.
Furthermore, expression of a dominant negative C/EBP isoform blocked
both basal and nutrient-regulated transcription. The NSRE-1 sequence is
highly similar to an element in the human chop gene
(5'-TGATGCAAT-3', 301 to 310) that functions as an AARE (15) and
has been termed a C/EBP-ATF composite site (16, 17). These sites appear
to bind to C/EBP-ATF heterodimers. For the human chop gene,
ATF2 binds to this site and mediates the amino acid responsiveness
(15). The ATF member that binds to the A.S. NSRE-1 sequence has not
been identified previously.
, acts via the NSRE-1 sequence to regulate A.S. gene
transcription in response to nutrient stress.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
173/+51 promoter/growth hormone reporter
plasmid was used along with 5 µg of the co-transfection control
plasmid, which was the pcDNA3.1 vector containing LacZ
gene driven by the cytomegalovirus promoter. Transfection was
performed as described previously (11), and after 24 h,
transfected cells from each 60-mm dish were divided by passage into
multiple 60-mm dishes and cultured for another 24 h before
treatment. The cells were then transferred to fresh complete MEM,
glucose-free MEM, or histidine-free MEM for 12 h, each
supplemented with 10% dialyzed fetal bovine serum. Using this batch
transfection protocol, cells exposed to the different media conditions
arose from the same initial transfection dish. Each experiment was
repeated with multiple batches of cells.
79 to 53, nuclear extract from histidine-deprived
cells, and antibodies for ATF1-4 and C/EBP. After exposure to film,
a parallel gel was aligned with the autoradiographic film, and a
1.5 × 1.5 cm2 gel piece was excised for each area of
the gel that aligned with the supershifted complexes of ATF4 and
C/EBP. For ATF4, two gel pieces were obtained, one each for the
upper and lower shifted complexes. Given that the ATF2 antibody did not
produce any supershifted complexes, a gel fragment aligning to the
upper complex of ATF4 was cut from the ATF2 EMSA lane to be used as a
negative control. The EMSA-derived gel pieces were placed into
individual wells of a 7.5% SDS-polyacrylamide gel along with 50 µl
of sample dilution buffer (0.125 M Tris (pH 6.8), 1% (w/v) SDS, 20%
(v/v) glycerol, 30 mg/ml bromphenol blue, and 715 mM
2-mercaptoethanol). The supershifted complexes were then subjected to
electrophoresis, electrotransferred to a nitrocellulose membrane, and
immunoblotted with C/EBP antibody at a dilution of 1:5000
(Santa Cruz Biotechnology). Goat anti-rabbit IgG conjugated with
horseradish peroxidase was used as the secondary antibody and was
detected with an enhanced chemiluminescence kit (Amersham Biosciences).
The membrane was then exposed on Biomax MR film (Eastman Kodak
Co.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
79 to 53 of the human
A.S. proximal promoter) containing the NSRE-1 sequence (Fig.
1) resulted in three distinct complexes (Fig. 2, A-C). All three complexes appeared to be specific
(Fig. 2, compare lanes 1 and
2) and were increased in amount when extracts from
histidine-deprived cells were tested (Fig. 2, compare lanes 3 and 6). Complex A was at or below a detectable level
in the MEM-incubated control cells (Fig. 1, lane 3) but was
increased substantially by amino acid limitation (Fig. 2, lane
6). Complex B was only slightly increased by histidine starvation,
whereas complex C was increased significantly. Antibodies specific for ATF1 caused no supershift, and for ATF3 antibody, there was a small but
detectable amount of shift of the starvation-induced complex (data not
shown). ATF2 is responsible for binding to the amino acid response
element in the human chop gene and mediates activation
following amino acid starvation (15). Confirming earlier work,
incubation with antibody specific for ATF2 did not cause any of the
NSRE-1 complexes to be supershifted (Fig. 2, compare lane 3 versus lane 4 for MEM and lane 6 versus lane 7 for His). Given that ATF4
mRNA translation is enhanced by amino acid deprivation (20), ATF4
was a potential regulator of A.S. transcription. Inclusion of ATF4
antibody resulted in a nearly complete loss of complex A that ran as
two new supershifted complexes (Fig. 2, contrast lanes 6 and
8).
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Fig. 1.
The nutrient-sensing response unit sequence
of the human asparagine synthetase gene. Two cis-acting
elements within the A.S. promoter are necessary for
increased transcription following activation of the AAR or unfolded
protein response pathways (11, 12). These include NSRE-1 (nt 68 to
60) and NSRE-2 (nt 48 to 43). Below the promoter
sequence, the two oligonucleotide sequences used for the EMSA studies
(Figs. 2-7) are illustrated. As the name implies, the NSRE-1
oligonucleotide contains the NSRE-1 sequence only, whereas the NSRE-1/2
oligonucleotide contains both the NSRE-1 and NSRE-2 sequences.
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Fig. 2.
The human A.S. promoter
NSRE-1 sequence binds ATF4, but not ATF2, transcription proteins.
Nuclear extracts prepared from HepG2 cells maintained for 16 h in
complete MEM (MEM), histidine-free MEM ( HIS),
or MEM lacking glucose (GLC) were incubated with
32P-radiolabeled NSRE-1 oligonucleotide (see the
legend for Fig. 1). Lane 1 (NS)
illustrates the effect of including an unrelated sequence
(5'-TTGTCGACCTCACAGTGGCTGCTATGTATGC-3') as a nonspecific, unlabeled
competitor at a 100-fold excess. Lane 2 (SP)
represents a 100-fold excess of the unlabeled NSRE-1 oligonucleotide
itself. Where indicated, the incubation protocol included no antibody
(None) or an antibody specific for either ATF2 or ATF4. The
arrows labeled A-C denote specific complexes
that were increased in amount when extracts from histidine- or
glucose-deprived cells were tested, and the asterisks
indicate two supershifted complexes arising from the ATF4 antibody. The
autoradiographic film shown is representative of at least two separate
experiments using independently prepared nuclear extracts.
(14), indicate that the C/EBP-ATF4 heterodimer has a
strong affinity for the NSRE-1 site and are consistent with NSRE-1
being a C/EBP-ATF composite site rather than specific for the ATF
family.
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Fig. 3.
Specificity of protein-DNA complex formation
by the NSRE-1 oligonucleotide. Nuclear extracts prepared from
HepG2 cells maintained for 16 h in histidine-free MEM were
incubated with 32P-radiolabeled NSRE-1
oligonucleotide. Lane 1 (Competitor = 0) shows the complexes (A-C) induced by amino acid
deprivation. As marked above lanes 2-6,
competition of the radiolabeled NSRE-1 probe was tested at an excess
(5-200-fold) of unlabeled NSRE-1 oligonucleotide. Competition by an
excess of a CREB/ATF consensus sequence
(5'-AGAGATTGCCTGACGTCAGAGAGCTAG-3') is illustrated in lanes
7-11. For lanes 12-14, incubation with anti-ATF4 was
included in the protocol to effect a supershift, and the impact of
including a 100-fold excess of either unlabeled NSRE-1 oligonucleotide
(lane 13) or the CREB/ATF consensus sequence (lane
14) was monitored. The arrows labeled A-C
denote the starvation-induced complexes that were increased in amount
when the extracts from histidine-deprived cells were assayed, and the
asterisks indicate the ATF4 supershifted complexes. The data
shown are representative of at least two independent experiments using
separately prepared nuclear extracts.
67 and 64
within the NSRE-1 sequence showed enhanced protection from dimethyl
sulfate modification in cells incubated in either histidine- or
glucose-depleted media (12). Consistent with those results, mutation of
individual nucleotides from 68 to 60 blocked
nutrient-dependent transcription of a transiently transfected
A.S. promoter/reporter construct (12). Therefore, if ATF4
action at NSRE-1 is critical for nutrient control, then mutation of
these nucleotides should prevent ATF4 protein binding. To test this
hypothesis, the 32P-labeled wild-type NSRE-1 sequence was
compared with four other radiolabeled probes that each had a single
nucleotide mutated, nt 67, 64, 62, or 60, respectively (Fig.
4). Using extracts from
histidine-deprived cells, it was observed that mutation of any one of
these four nucleotides within the NSRE-1 sequence completely prevented
formation of the inducible ATF4-containing protein-DNA complex A (Fig.
4, compare lane 2 with lanes 3-6).
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Fig. 4.
Single nucleotide mutagenesis within the
NSRE-1 site. Nuclear extracts prepared from HepG2 cells maintained
for 16 h in histidine-free MEM were incubated with
32P-radiolabeled NSRE-1 oligonucleotide to monitor
formation of induced (arrows labeled A-C)
DNA-protein complexes (lane 1). For the incubations
represented in lanes 2-6, anti-ATF4 was included to
supershift the corresponding complexes (shown by the
asterisks). In lanes 1 and 2, the
wild-type (WT) sequence was used as the radiolabeled probe,
whereas for the incubations in lanes 3-6, the binding site
within the radiolabeled NSRE-1 probe contained a nucleotide mutation as
follows: G67A, G64A, A62G, and C60T. The results shown are
representative of several separate experiments using independently
prepared nuclear extracts.
View larger version (103K):
[in a new window]
Fig. 5.
Formation of ATF4-containing NSRE-1 complexes
requires de novo protein synthesis during amino acid
deprivation. Nuclear extracts were prepared from HepG2 cells
maintained for 16 h in complete MEM (MEM) or
histidine-free MEM ( HIS) with or without 0.1 mM cycloheximide (CHX). These extracts were
incubated with 32P-radiolabeled NSRE-1
oligonucleotide to assay formation of
starvation-dependent DNA-protein complexes. As indicated
above the lanes, incubations included either no antibody
() or anti-ATF4 (+). The arrows labeled A-C
denote specific complexes. The asterisks indicate
ATF4-containing complexes. The results shown are indicative of at least
two independent experiments using different batches of nuclear extract.
Ab, antibody.
Are Both Present in the Same NSRE-1
Complex--
To provide evidence that ATF4 and C/EBP can bind
simultaneously to the NSRE-1 site, an EMSA reaction that included ATF4, ATF2, or C/EBP antibody was used to generate shifted complexes (Fig.
6A). The shifted bands induced
by ATF4 or C/EBP were cut out of the gel, and then each gel plug was
placed into separate wells of an SDS-polyacrylamide gel. After
electrophoresis and transfer, the blot was probed with antibody
specific for C/EBP (Fig. 6B). Although the lower
ATF4-containing complex did not react with the anti-C/EBP (Fig.
6B, lane 2), C/EBP immunoreactivity was
clearly evident for the upper ATF4-containing complex (Fig. 6B, lane 1). The EMSA complex supershifted by
C/EBP antibody was also cut out (Fig. 6A) and run on the
same SDS gel as a positive control (Fig. 6B, lane
4). In contrast, the gel region corresponding to where the upper
ATF4 band would be was cut out from an EMSA lane resulting from an ATF2
antibody incubation (not shown) and used as a negative control. As
expected, this gel region showed no C/EBP content (Fig.
6B, lane 3).
View larger version (61K):
[in a new window]
Fig. 6.
ATF4 and C/EBP are
contained within the same NSRE-1 complex. Nuclear extracts
prepared from HepG2 cells maintained for 16 h in histidine-free
MEM were incubated with 32P-radiolabeled NSRE-1
oligonucleotide and antibody (Ab) against ATF4, C/EBP
(positive control), or ATF2 (negative control) to cause a supershift of
ATF4- or C/EBP-containing complexes. After separating the supershift
complexes on a 5% acrylamide EMSA gel, gel plugs were excised from the
regions corresponding to the upper or lower ATF4-shifted bands
(panel A), the blank region of an ATF2 antibody incubation
corresponding to the region where the ATF4 upper band would be (not
shown), or the band shifted by including anti-C/EBP (panel
A). The entire EMSA-derived gel plug was placed into the well of
an SDS-PAGE and subjected to electrophoresis, as described under
"Materials and Methods" (panel B). After electrotransfer
onto nitrocellulose paper, the blot was probed with anti-C/EBP and
exposed to autoradiographic film.
View larger version (114K):
[in a new window]
Fig. 7.
Effect of mutating NSRE-1 or NSRE-2 on
DNA-protein complex formation. Nuclear extracts prepared from
HepG2 cells maintained for 16 h in complete MEM
(MEM) or histidine-free MEM ( HIS) were
incubated with 32P-radiolabeled NSRE-1/2 oligonucleotide
(containing both NSRE-1 and NSRE-2 sites, as described in the legend
for Fig. 1). As indicated above each lane, the incubations
included no antibody (None), anti-ATF2, or anti-ATF4. The
binding site that was mutated within the radiolabeled NSRE-1/2
oligonucleotide probe is indicated below the lanes. The
specific nucleotides mutated within each binding site were: for NSRE-1,
wild type = 5'ATGATGAAAC-3', mutant = 5'-ATGCGCTCAC-3'; for
NSRE-2, wild type = 5-GTTACA-3', mutant = 5'-GGACGA-3'. The
arrow labeled A denotes the major
starvation-induced complex that is shifted by ATF4 antibody (compare
lanes 5 and 6), and the asterisks
indicate the ATF4-containing complexes. The results shown are
representative of several separate experiments using independently
prepared nuclear extracts.
View larger version (40K):
[in a new window]
Fig. 8.
ATF4 mRNA content is elevated by amino
acid or glucose deprivation. HepG2 cells were incubated in MEM,
MEM lacking histidine ( HIS) or MEM lacking glucose
(GLC), or amino acid-free KRB. At the indicated times, RNA
was isolated and subjected to Northern analysis (20 µg of RNA/lane)
for ATF4 or the ribosomal protein L7a as a negative control.
Panel A shows a typical autoradiographic result, and the
data are quantified in panel B. To illustrate the
statistical significance of the starvation, the results in panel
C are the averages ± standard deviation for four independent
dishes incubated in the medium indicated for 12 h. The
asterisks denote a p value of < 0.005 (Student's t test) relative to the MEM condition.
View larger version (50K):
[in a new window]
Fig. 9.
Induction of ATF4 mRNA Content is
dependent on de novo protein synthesis. HepG2
cells were incubated in complete MEM, amino acid-free KRB, or KRB
containing 0.1 mM cycloheximide (CHX,
panel A). At the indicated times, RNA was isolated and
subjected to Northern analysis for ATF4 or glutamate dehydrogenase
(GDH) as a negative control. The samples in panel
B were incubated in amino acid-free KRB for 0-6 h and then
transferred to complete MEM without ( ) or with (+) 0.1 mM
cycloheximide. RNA was isolated at the time points indicated and
subjected to Northern analysis (20 µg of RNA/lane) for ATF4 and
glutamate dehydrogenase (GDH) mRNA. The data from
panel B were quantified and are shown graphically in
panel C. The results are representative of multiple
experiments.
View larger version (35K):
[in a new window]
Fig. 10.
Decay rate of the starvation-induced ATF4
mRNA after amino acid refeeding. HepG2 cells were incubated in
amino acid-free KRB for 18 h (panel A, first
lane; panel B, time = 0) and then transferred to
amino acid-complete MEM. After refeeding the cells, RNA was isolated at
the times indicated in panel A and subjected to Northern
analysis (20 µg of RNA/lane) to measure ATF4 mRNA content. The
blot was also probed with glutamate dehydrogenase (GDH) as a
negative control. The ATF4 band intensities from panel A
were quantified and plotted on a semi-log graph to estimate the
half-life of the decay process.
View larger version (47K):
[in a new window]
Fig. 11.
Effect of amino acid deprivation on ATF4
mRNA turnover. HepG2 cells were incubated in MEM lacking
histidine for 12 h (panel A, first lane;
t = 0 h) and then transferred to fresh MEM lacking
histidine or to amino acid-complete MEM, both containing 5 µM actinomycin D (Act D). At the times
indicated in panel A, RNA was isolated and subjected to
Northern analysis (20 µg of RNA/lane) for ATF4 or glutamate
dehydrogenase (GDH) mRNA. The data were quantified,
expressed as a percent of the value obtained for t = 0 (12 h of histidine deprivation), and then plotted as the semi-log to
estimate the decay rate (panel B). For the data in
panel C, HepG2 cells were incubated in complete MEM or in
MEM lacking histidine without or with 5 µM actinomycin D. At the times indicated, RNA was isolated, and Northern analysis was
performed to measure ATF4 or L7a mRNA content. The results shown
are representative of three independent experiments.
173/+51) and a plasmid containing either the
activating form of ATF4 or an inhibitory dominant negative mutant (21).
The control cells were transfected with empty pcDNA3.1 vector
(control), and the transfection efficiency was monitored by
co-transfection with the LacZ gene driven by the
cytomegalovirus promoter. After 36 h, the cells were transferred to complete MEM or MEM lacking either histidine or glucose for 12 h prior to the isolation of RNA for Northern analysis (Fig. 12A). Expression of ATF4 was
confirmed by Northern blotting. The data are expressed as the fold
induction relative to the pcDNA3.1-transfected cells (control)
incubated in complete MEM. Consistent with previously published
functional analysis (11), A.S. promoter-driven transcription was increased in both the histidine- (5.3-fold) and the
glucose-deprived (3.5-fold) control cells that did not receive
exogenous ATF4 (Fig. 12B). Overexpression of the ATF4
wild-type (Fig. 12B) caused an increase in the basal (MEM)
transcription (4.9-fold), and although relatively modest, a further
enhancement of the increase following histidine (5.3-fold
versus 8.3-fold) or glucose (3.5-fold versus 4.3-fold) starvation. In contrast, after transfection with the dominant
negative ATF4 mutant, a 50% inhibition of the basal (in MEM)
A.S.-driven transcription was observed, and there was a blockade of the
induction by either histidine (5.3-fold versus 1.6-fold) or
glucose (3.5-fold versus 1.8-fold) deprivation (Fig.
12B, DN). Collectively, these data are consistent
with the in vitro EMSA studies described above and document
that ATF4 serves in vivo as a mediator of the nutrient-sensing response
pathway.
View larger version (44K):
[in a new window]
Fig. 12.
ATF4 and an ATF4 dominant negative modulate
A.S. promoter activity accordingly. HepG2 cells
were transiently transfected with vector containing no cDNA insert
(Control), the cDNA for the wild-type ATF4, or the
cDNA for a dominant negative ATF4 mutant (DN). The cells
were co-transfected with a vector containing an A.S.
promoter/growth hormone reporter construct to monitor transcription in
response to histidine or glucose limitation and the pcDNA3.1 vector
containing the LacZ gene to correct for transfection
efficiency between dishes. After transfection and culture as described
in the text, the cells were incubated for 12 h in complete MEM
(MEM), MEM lacking glucose ( GLC), or MEM
lacking histidine (HIS), and then RNA was isolated and
subjected to Northern analysis to measure growth hormone
(GH) and L7a (panel A). The data were quantified,
corrected for L7a as a loading control, and then plotted as the fold
induction relative to control cells (transfected with empty vector)
incubated in complete MEM (panel B).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
ATF2 bind this sequence in vitro, when knockout cell lines
for these two proteins were tested, amino acid-dependent
expression of the chop gene was blocked in the
ATF2/ cells but not in those cells lacking C/EBP.
Furthermore, expression of a dominant negative form of ATF2 suppressed
the starvation-dependent transcription from a
chop promoter/luciferase reporter construct (15). Our EMSA
experiments document that ATF2 does not bind to the A.S.
NSRE-1 sequence despite the fact that a positive control with a
CREB/ATF consensus oligonucleotide demonstrates that the HepG2 nuclear
extracts contain ATF-2 (data not shown). However, the data do
illustrate that ATF4 contributes to the regulation of the
A.S. gene by signaling nutrient deprivation and endoplasmic reticulum stress.
(14). With the identification of ATF4 as the
C/EBP heterodimer partner and the observation that both the
ATF4 and the C/EBP genes are also
transcriptionally regulated by nutrient availability, additional steps
within this metabolite control pathway have been revealed.
| |
ACKNOWLEDGEMENTS |
|---|
We thank other members of the laboratory for technical advice and helpful discussion.
| |
FOOTNOTES |
|---|
* This research was supported by Grant DK-52064 (to M. S. K.) from the NIDDK, the National Institutes of Health.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.
To whom correspondence should be addressed: Dept. of Biochemistry
and Molecular Biology, University of Florida College of Medicine, Box
100245, Gainesville, FL 32610-0245. Tel.: 352-392-2711; Fax:
352-392-6511; E-mail: mkilberg@ufl.edu.
Published, JBC Papers in Press, April 17, 2002, DOI 10.1074/jbc.M201959200
| |
ABBREVIATIONS |
|---|
The abbreviations used are: A.S., asparagine synthetase; ATF, activating transcription factor; NSR(E), nutrient-sensing response (element); AAR(E), amino acid response (element); ERSR, endoplasmic reticulum stress response; C/EBP, CCAAT/enhancer-binding protein; CHOP, C/EBP homology protein; CREB, cAMP-response element-binding protein; nt, nucleotide(s); MEM, minimal essential medium; EMSA, electrophoresis mobility shift analysis; KRB, Krebs-Ringer bicarbonate buffer.
| |
REFERENCES |
|---|
|
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
| 1. | Jousse, C., Bruhat, A., and Fafournoux, P. (1999) Curr. Opin. Clin. Nutr. Metab Care 2, 297-301[CrossRef][Medline] [Order article via Infotrieve] |
| 2. | Fafournoux, P., Bruhat, A., and Jousse, C. (2000) Biochem. J. 351, 1-12[CrossRef][Medline] [Order article via Infotrieve] |
| 3. | Laine, R. O., Hutson, R. G., and Kilberg, M. S. (1996) Prog. Nucleic Acid Res. Mol. Biol. 53, 219-248[Medline] [Order article via Infotrieve] |
| 4. |
Arfin, S. M.,
Simpson, D. R.,
Chiang, C. S.,
Andrulis, I. L.,
and Hatfield, G. W.
(1977)
Proc. Natl. Acad. Sci. U. S. A.
74,
2367-2369 |
| 5. |
Andrulis, I. L.,
Hatfield, G. W.,
and Arfin, S. M.
(1979)
J. Biol. Chem.
254,
10629-10633 |
| 6. |
Gong, S. S.,
Guerrini, L.,
and Basilico, C.
(1991)
Mol. Cell. Biol.
11,
6059-6066 |
| 7. | Hutson, R. G., and Kilberg, M. S. (1994) Biochem. J. 303, 745-750 |
| 8. |
Jousse, C.,
Bruhat, A.,
Ferrara, M.,
and Fafournoux, P.
(2000)
J. Nutr.
130,
1555-1560 |
| 9. |
Guerrini, L.,
Gong, S. S.,
Mangasarian, K.,
and Basilico, C.
(1993)
Mol. Cell. Biol.
13,
3202-3212 |
| 10. | Barbosa-Tessmann, I. P., Pineda, V. L., Nick, H. S., Schuster, S. M., and Kilberg, M. S. (1999) Biochem. J. 339, 151-158 |
| 11. |
Barbosa-Tessmann, I. P.,
Chen, C.,
Zhong, C.,
Schuster, S. M.,
Nick, H. S.,
and Kilberg, M. S.
(1999)
J. Biol. Chem.
274,
31139-31144 |
| 12. |
Barbosa-Tessmann, I. P.,
Chen, C.,
Zhong, C.,
Siu, F.,
Schuster, S. M.,
Nick, H. S.,
and Kilberg, M. S.
(2000)
J. Biol. Chem.
275,
26976-26985 |
| 13. | Patil, C., and Walter, P. (2001) Curr. Opin. Cell Biol. 13, 349-355[CrossRef][Medline] [Order article via Infotrieve] |
| 14. |
Siu, F. Y.,
Chen, C.,
Zhong, C.,
and Kilberg, M. S.
(2001)
J. Biol. Chem.
276,
48100-48107 |
| 15. |
Bruhat, A.,
Jousse, C.,
Carraro, V.,
Reimold, A. M.,
Ferrara, M.,
and Fafournoux, P.
(2000)
Mol. Cell. Biol.
20,
7192-7204 |
| 16. | Wolfgang, C. D., Chen, B. P., Martindale, J. L., Holbrook, N. J., and Hai, T. (1997) Mol. Cell. Biol. 17, 6700-6707[Abstract] |
| 17. | Fawcett, T. W., Martindale, J. L., Guyton, K. Z., Hai, T., and Holbrook, N. J. (1999) Biochem. J. 339, 135-141 |
| 18. | Aslanian, A. M., Fletcher, B. S., and Kilberg, M. S. (2001) Biochem. J. 357, 321-328[CrossRef][Medline] [Order article via Infotrieve] |
| 19. |
Haze, K.,
Yoshida, H.,
Yanagi, H.,
Yura, T.,
and Mori, K.
(1999)
Mol. Biol. Cell
10,
3787-3799 |
| 20. | Harding, H. P., Novoa, I., I, Zhang, Y., Zeng, H., Wek, R., Schapira, M., and Ron, D. (2000) Mol. Cell 6, 1099-1108[CrossRef][Medline] [Order article via Infotrieve] |
| 21. |
He, C. H.,
Gong, P., Hu, B.,
Stewart, D.,
Choi, M. E.,
Choi, A. M.,
and Alam, J.
(2001)
J. Biol. Chem.
276,
20858-20865 |
| 22. | Hutson, R. G., Warskulat, U., and Kilberg, M. S. (1996) Clin. Nutr. 15, 327-331 |
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