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J Biol Chem, Vol. 274, Issue 44, 31139-31144, October 29, 1999
From the The gene for the amino acid biosynthetic activity
asparagine synthetase (AS) is induced by both amino acid and glucose
deprivation of cells. The data reported here document that the human AS
gene is induced following activation of the Unfolded
Response Pathway (UPR), also known as the
Endoplasmic Reticulum Stress
Response (ERSR) in mammals. Increased AS transcription
occurs in response to glucose deprivation, tunicamycin, or
azetidine-2-carboxylate, all known to activate the UPR/ERSR pathway.
Previously identified ERSR target genes contain multiple copies of a
single highly conserved cis-element. In contrast, the human
AS gene does not contain the ERSR element, as it has been described for
other responsive genes. Instead, AS induction requires an Sp1-like
sequence, a sequence previously shown to be associated with amino acid
control of transcription, and possibly, a third region containing no
consensus sequences for known transcription factors. Oligonucleotides
covering each of these regions form DNA-protein complexes in
vitro, and for some the amount of these complexes is greater when
nuclear extracts from glucose-starved cells are tested. These results
document that a wider range of metabolic activities are activated by
the UPR/ERSR pathway than previously recognized and that genomic
elements other than those already described can serve to enhance
transcription of specific target genes.
Many mammalian cells contain asparagine synthetase activity that
catalyzes the biosynthesis of asparagine from aspartate and glutamine
with concurrent hydrolysis of ATP (1). The expression of asparagine
synthetase (AS)1 activity is
enhanced by amino acid deprivation (2), and this regulation is
transcriptional in nature (3, 4). Barbosa-Tessmann et al.
(5) recently showed that the human AS gene is activated when mammalian
cells are incubated in the absence of carbohydrate as well. Although
the cellular significance of this carbohydrate-dependent control of the AS gene is not fully understood, it is known that for
cells lacking sufficient AS activity asparagine deprivation results in
cell cycle arrest (6, 7) and induction of apoptosis (8, 9). Maintenance
of asparagine levels via induction of AS activity may play a key role
in the response to the cellular stress of carbohydrate limitation.
A number of genes are increased in their transcriptional rate following
glucose deprivation including, GRP78, GRP94,
protein disulfide isomerase, calreticulin, and the transcription
regulatory factor C/EBP homology protein (chop)/growth arrest and DNA
damage protein 153 (gadd153) (10). Collectively, the changes following glucose starvation are the result of a cellular recognition of protein
accumulation within the endoplasmic reticulum (ER), called the Unfolded
Protein Response (UPR) in yeast (11, 12) or the ER Stress Response
(ERSR) in mammalian cells (13, 14). The transcription of this same set
of genes can be increased by treatment of cells with the glycoprotein
biosynthesis inhibitor tunicamycin or with amino acid analogs that
incorporate and cause improper folding, such as the proline analog
azetidine-2-carboxylate (Aze) (15). The consensus
cis-element responsible for the UPR in yeast (5'-CAGCGTG-3')
(16) and the corresponding ERSR element in mammalian cells
(5'-CCAAT-N9-CCACG-3') (13, 14) are significantly
different, although many, if not most, of the corresponding genes are
targets for both.
Given the observed increased transcription of the human AS gene
following glucose deprivation (5), the present experiments were
designed to test the hypothesis that AS is a target gene of the
UPR/ERSR. If AS is a UPR/ERSR target, transcription should be enhanced
by recognized activators of the pathway, such as tunicamycin or Aze
(15). The Northern analysis reported here indicates that either
tunicamycin or Aze are as effective as glucose starvation in increasing
AS mRNA content. Furthermore, tunicamycin was able to replace
glucose deprivation as a way to activate the transcription of a
reporter gene under the control of the AS 5'-flanking sequence. Thus,
these data document that the AS gene is responsive to several known
activators of the UPR/ERSR pathway. Electromobility shift analyses and
promoter deletions within the proximal 200 bp of the human AS promoter
indicate that there are distinct regions of sequence required for a
maximal UPR/ERSR response by this gene.
Cell Culture--
Human HepG2 hepatoma cells were obtained from
the American Type Culture Collection (ATCC) and maintained in minimal
essential medium (MEM) as described (5). To test for induction of AS mRNA content, cells were incubated in glucose-free MEM, complete MEM medium, MEM containing 5 µg/ml tunicamycin, or MEM containing 5 mM Aze for 12-18 h, during which the media contained 10%
dialyzed fetal bovine serum.
RNA Isolation and Northern Analysis--
Total cellular
RNA was isolated and subjected to Northern analysis on a 1% agarose
gel (5). The cDNA probes for human AS, GRP78, and ribosomal protein
L7a, described previously (5), were radiolabeled with
[ Transient Expression and Mutagenesis--
HepG2 hepatoma cells
were transfected with a reporter construct (p0GH), obtained from
Nichols Institute Diagnostics, Inc. (San Juan Capistrano, CA) that
contains the complete gene sequence for human growth hormone (GH),
including a potential transcription initiator-like (Inr) sequence (17,
18) at the known transcription start site, but lacking a promoter (19).
To investigate transcriptional control by AS-specific sequences, the
constitutive (UPR unresponsive) mouse metallothionein promoter (MTT)
(20) or portions of the human AS promoter (as indicated in each figure)
were subcloned in front of the GH sequence using the BamHI
site within the p0GH vector. With the exception of the promoter and the
first nucleotide of the transcript, the entire GH genomic sequence is
contained within the p0GH vector. The AS promoter sequences and the
site-directed mutations were generated by PCR using primers based on
sequence from a human genomic clone obtained and characterized by our
laboratory.2 Our sequence
agrees with the sequence of the entire AS gene and its flanking regions
obtained by sequencing of a PAC clone (clone DJ1090P18,
GenBankTM accession number AC00536) and submitted to
GenBank by R. Waterston as part of the Human Genome Project.
A batch transfection technique was employed using HepG2 cells grown to
about 75% confluence. A ratio of 10 µg of DNA per 60 µl of
Superfect reagent (Qiagen, Germany) per 6.6 × 106
cells/100-mm dish was constant in each transfection. A 10-µg aliquot
of DNA was incubated with 60 µl of Superfect for 10 min at room
temperature in MEM alone. The MEM was removed, and the cells were
washed once with phosphate-buffered saline and then incubated with the
transfection mixture for 3 h at 37 °C in 5.0 ml of MEM
containing fetal bovine serum, bovine serum albumin, and antibiotics.
After transfection, cells were washed once with phosphate-buffered
saline, fresh culture medium was added, and the cells were cultured for
24 h. Each 100-mm dish of HepG2 cells was then split to four or
five 60-mm dishes so that, within each experiment, cells incubated in
either glucose-containing, glucose-free, or tunicamycin-containing
medium came from the same transfected cell population. This procedure
eliminates the concern about transfection efficiency between the
experimental treatments. After another 24 h of culture, the cells
were incubated for 12-18 h in the media, described in each figure
legend, and then total cellular RNA was isolated.
The transfection efficiency between dishes was measured by
co-transfecting the pcDNA3.1 vector containing a lacZ
insert behind the cytomegalovirus CMV promoter (Invitrogen, Carlsbad,
PA) and then monitoring lacZ mRNA expression by Northern
analysis. The ability of the AS promoter fragments to promote
transcription of the GH reporter gene also was evaluated by Northern
analysis. More traditional enzymatic or protein reporter assays were
not used to avoid potential confounding effects of glucose deprivation or tunicamycin treatment on general protein synthesis or turnover of
the reporter protein.
Mutagenesis of the AS sequence 5'-TATAA-3' ( Electromobility Shift Assay--
Total nuclear extracts were
prepared from HepG2 cells incubated for 18 h in either complete
MEM (+Glc) or in MEM lacking glucose ( The purpose of this study was to test the hypothesis that the
human AS gene is a target for the UPR/ERSR pathway. The UPR/ERSR pathway is thought to be responsible for increased expression primarily
of ER-bound proteins associated with protein processing, although a
number of other potential target genes have been identified recently
(23). The identification of an amino acid biosynthetic activity such as
AS would be unprecedented but perhaps would lead to new insights into
this important cellular pathway and the role played by asparagine availability.
Transcription of the human AS gene has recently been documented to be
enhanced by glucose starvation (5). To test whether or not the AS gene
responds to other recognized activators of the UPR/ERSR pathway,
incubations were performed in the presence of tunicamycin or Aze (15,
24). When HepG2 cells were incubated in MEM medium lacking glucose or
complete MEM containing 5 µg/ml tunicamycin or 5 mM Aze
for 18 h, the cellular content of AS mRNA was increased
significantly (Fig. 1A). The
response to glucose deprivation is consistent with our previous report
(5) and served, in this context, as a positive control. The Northern
blots were stripped and reprobed with cDNAs specific for GRP78,
which served as a positive control for the UPR/ERSR pathway, and for the ribosomal protein rpL7a, which served both as a negative control and to document equal loading between gel lanes (Fig. 1A).
To determine whether or not the induction by tunicamycin treatment and
glucose deprivation were additive, HepG2 cells were incubated under
each condition separately or in MEM medium both lacking glucose and
containing tunicamycin (Fig. 1, B and C). When
the cells were exposed to both conditions simultaneously, the degree of
enhancement in AS mRNA was approximately the same as either condition alone (Fig. 1C). These results support, but do not
prove, the hypothesis that the two processes at least share a common step, if not identical mechanisms. Quantitation and reproducibility of
the response to these UPR/ERSR activators was accomplished by treating
three independent sets of cells, measuring the AS mRNA content by
hybridization of a dot blot, and quantitating the results with a
phosphoimager (Fig. 1C).
To establish that the observed increase in steady state AS mRNA
content following tunicamycin treatment was transcriptionally mediated
and to localize potential genomic cis-element regions, a
transient transfection protocol was used. HepG2 cells were transfected with a reporter construct containing the entire genomic sequence for
human GH (19) in conjunction with: 1) no promoter (p0GH), 2) the mouse
MTT promoter (20), or 3) specific fragments of the AS promoter (Fig.
2A). Transcription was
significantly enhanced by glucose deprivation when 10 kilobase pairs of
the human AS 5'-flanking sequence was present (data not shown), and
when the AS sequence was progressively deleted to nt
Activation of the Unfolded Protein Response Pathway Induces Human
Asparagine Synthetase Gene Expression*
,,,,¶
Department of Biochemistry and Molecular
Biology, and the § Department of Neuroscience, University of
Florida College of Medicine, Gainesville, Florida 32610
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-32P]dCTP using a random primers labeling kit
according to the instructions of the manufacturer (Life Technologies,
Inc., Gaithersburg, MD) and purified over a G-50 column (Amersham
Pharmacia Biotech). Autoradiograms were quantified by densitometry
after demonstrating that the absorbance units were within the linear
range of the film. All experiments were repeated with independent RNA
preparations to show reproducibility.
29 to 25) to
5'-ACTCA-3' was accomplished using PCR as described by Ho et
al. (21). For this four-base substitution, two primers (P2, P3) were prepared that were mismatched (shown with an underline) to the
native sequence at the substituted block of bases (P2 = 5'-TGGCGCTGAGTCCGACCTGGCTCCTGTAACGC-3', P3 = 5'-CAGGTCGGACTCAGCGCCAGCGGCCTCGCCGC-3'). Two additional
primers were prepared that corresponded to the 5'-most (Primer 1) and 3'-most (Primer 4) sequences of the 173/+51 AS genomic sequence plus
a BamHI restriction site (underlined) for subcloning
(P1 = 5'-NNGGATCCCAAAAGAGCTCCTCCTTGCGC-3', P4 = 5'-NNGGATCCTAAGCAGGTCAGGGTGATGTGGCGG-3'). Two PCR reactions
(P1 + P2 and P3 + P4 were performed to generate the two overlapping
products. The PCR amplicons were agarose gel-purified, and the two
overlapping fragments were used as DNA templates for a second PCR
reaction using P1 and P4 to amplify the entire 173/+51 sequence with
the TATA substitution (designated 173/+51/*TATA in Fig.
3B). The PCR product was subcloned and transferred to the
BamHI site of the GH expression vector (p0GH). The
directionality and substitutions were checked by DNA sequencing.
Glc) (22). Double-stranded
oligonucleotide probes were radiolabeled by extension of overlapping
ends with Klenow fragment in the presence of
[-32P]dCTP (22). A 5-µg aliquot of nuclear extract
was preincubated for 10 min at 4 °C in a total volume of 20 µl
containing 40 mM Tris, pH 7.5, 200 mM NaCl, 2 mM dithiothreitol, 10% glycerol, 0.05% Nonidet P-40, 2 µg of poly(dI-dC), 0.05 mM EDTA, and, as indicated, 6 ng
(100×) or 12 ng (200×) of unlabeled competitor oligonucleotide. Then
0.06 ng of radiolabeled probe (10,000 dpm) was added and the incubation
continued for 20 min at room temperature. The mixture was subjected to
electrophoresis on an 8% polyacrylamide gel and the results visualized
by autoradiography. All experiments were repeated twice, and at least
two independently prepared nuclear extracts were tested.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (28K):
[in a new window]
Fig. 1.
AS mRNA content is increased in response
to several independent activators of the UPR/ERSR pathway. HepG2
human hepatoma cells were incubated in MEM, MEM Glc, MEM + tunicamycin (5 µg/ml), or MEM + Aze (5 mM) (panel
A) or MEM, MEM Glc, MEM + tunicamycin, or MEM Glc + tunicamycin (panel B) for 18 h. Total cellular RNA
was isolated and subjected to Northern analysis (15 µg of RNA/lane).
The resulting blots were hybridized sequentially with radiolabeled
probes for AS, rpL7a, and GRP78. To assess the variation of the
response, in a separate series of experiments, hybridization of AS
mRNA by dot blot (20 µg/spot) was performed on triplicate samples
and quantitated by phosphoimager analysis (panel C).
3121/+51 or
615/+51, the high degree of glucose deprivation-induced activity was
retained (Fig. 2A). An additional series of 5' deletions
(475/+51, 376/+51, 274/+51, 173/+51) documented that following
glucose starvation, induction of transcription was maintained at the
level of the 615/+51 construct (for an example of the 173/+51
construct, see Fig. 3), until the
72/+51 fragment was tested and shown to be inactive. To obtain
supporting evidence that glucose removal was activating expression via
the ERSR, the glucose-responsive 173/+51 AS fragment was shown to
respond to tunicamycin treatment as well (Fig. 2B).
View larger version (31K):
[in a new window]
Fig. 2.
Sequences within the human AS proximal
promoter region mediate the transcriptional induction in response to
activation of the mammalian ERSR pathway. HepG2 human hepatoma
cells were transfected with a human GH reporter construct lacking a
promoter (p0GH), containing the ERSR-unresponsive MTT promoter, or the
indicated regions ( 3121/+51, 615/+51, or 173/+51) of the AS
5'-flanking sequence. After incubating the cells for 12 h in MEM,
MEM Glc (panel A), or MEM + tunicamycin (panel
B) to induce the ERSR pathway, expression of the GH reporter
mRNA was monitored by Northern analysis (20 µg of RNA/lane).
Panels A and B illustrate a representative
experiment, whereas panel C represents quantitation of four
independent experiments. In panel C, the construct showing
the highest expression level (3121/+51 for the Glc series and
173/+51 for the + tunicamycin series) was set to 100% and
averages ± S.D. for the other values were normalized. To confirm
the ERSR pathway activation and to assess the lane loading consistency,
the blots were rehybridized with radiolabeled probes to measure the
endogenous AS, GRP78, and rpL7a mRNA content. To illustrate the
transfection efficiency between constructs, lacZ mRNA
was measured.
View larger version (46K):
[in a new window]
Fig. 3.
The sequences that mediate the ERSR within
the human AS proximal promoter region are between nt 111 and
34. HepG2 cells were transfected with the GH reporter construct
lacking a promoter (p0GH), containing the MTT promoter, or the
indicated AS 5'-flanking sequence. After incubating the cells for
12 h in MEM, MEM Glc, or MEM + tunicamycin, GH reporter
mRNA was monitored by Northern analysis (20 µg of RNA/lane).
Panel A represents data obtained by deleting sequences from
the 5' direction of the 173 to +51 AS sequence, whereas panel
B illustrates deletions from the 3' direction (left hand
set of gels) and the TATA block mutation (right hand
set of gels). The locations of potential cis-element
sequences are shown in the diagrams to the left
of the bar graphs. In the right hand set of gels
for panel B, for the lanes marked with 173/+51/*TATA, the
wild-type sequence 5'-TATAA-3' (nt 29 to 25) has been mutated to
5'-ACTCA-3' within the 173/+51 construct.
Co-transfection with a plasmid containing lacZ allowed the
relative transfection efficiency between dishes to be monitored by
probing the blots for lacZ mRNA. As noted above, with
the batch transfection protocol used, the cells incubated in the
presence or absence of glucose (or tunicamycin) arose from the same
original transfected dish for each construct. Therefore, any
differences observed for an individual construct (e.g.
173/+51 with or without tunicamycin in Fig. 2B) may
reflect a minor effect of the treatment on the cytomegalovirus
promoter. Activation of the ERSR pathway was confirmed by reprobing the
blots to measure the endogenous AS and GRP78 mRNA content. Equal
loading of lanes was established by probing with the rpL7a cDNA.
To further define the region encoding the ERSR responsive cis-elements,
deletions were made to the 173/+51 bp sequence from both the 5' and
3' directions (Fig. 3). Deletion of the AS 5' sequence from nt 173 to
111 had no effect on either basal or ERSR-mediated transcription.
However, reduction of the sequence to nt 90 or less caused a decrease
in transcription, consistent with the presence of an Sp1 consensus
sequence located at nt 106 to 97 (25). Given that the genomic GH
reporter sequence lacks only one nucleotide from the native transcript
sequence (19) and retains a Inr-like sequence that appears to serve as
a transcription start site (17, 18), 3'-deletions of the AS promoter
were also possible (Fig. 3B). Sequential removal of AS
promoter sequence from the 3' direction back to nt 34 resulted in a
partial loss of basal transcription, but the ERSR-induced transcription
remained evident (Fig. 3B). Collectively, these deletion
studies indicate that, by transient transfection studies, the sequences
required for effective expression of the AS gene following ERSR
activation are contained within the 111 to 34 proximal promoter sequence.
Interestingly, the TATA sequence (5'-TATAA-3') at nt 29 to 25
appeared to be unnecessary for basal transcription, at least in the
context of the GH reporter gene. To investigate further the
functionality of this sequence in the context of the AS promoter, the
AS sequence 5'-TATAA-3' was mutated to 5'-ACTCA-3' within the 173 to
+51 construct (labeled 173/+51/*TATA in Fig. 3B). Neither
basal nor induced transcription was altered by mutation of the TATAA
sequence, suggesting that this sequence is not functionally required
for AS expression. Genes lacking a TATA box often use an Inr initiator
element in conjunction with multiple Sp1 sites to localize the
transcription start site (reviewed in Ref. 18). The human AS has an
Inr-like sequence (5'-TCAAGCT-3') at the proposed major transcription
start site (7), although the functionality of this element in
vivo has yet to be established.
To test in vitro for protein binding to these AS promoter
sequences, electromobility shift assays (EMSAs) were performed (Fig. 4). Nuclear extracts were prepared from
cells incubated for 18 h in complete MEM (+Glc) or MEM lacking
glucose (Glc) and incubated with each of three
32P-labeled double-stranded oligonucleotide probes that
spanned most of the 111 to 34 region (see Fig.
5). The 112/91 oligonucleotide probe,
designed to have 6 nt on each side of a consensus Sp1 sequence (nt
106 to 97) (25), exhibited a single primary complex that could be
blocked by an excess of cold competitor (Fig. 4). Preliminary experiments suggest that this band undergoes a so-called supershift when anti-Sp1 antibody is included in the incubation (data not shown).
The relative amount of complex formed was the same or slightly greater
when extracts from glucose-deprived cells were compared with those from
glucose-fed cells.
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At nt 70 to 64, the human AS promoter contains a palindromic core
sequence (5'-CATGATG-3') that has been proposed to function as an amino
acid response element (3). An oligonucleotide probe that contained nt
72 to 62 did not produce a product by EMSA. However, when the
oligonucleotide length was increased to cover nt 79 to 53, two
distinct complexes were formed. The upper complex was increased by more
than 7-fold when extracts from glucose-deprived cells were used (Fig.
4). The lower complex also increased in amount when the Glc extracts
were tested, but the change (3-fold) was less than that for the larger
complex. Consistent with the block substitution by Guerrini et
al. (3), who documented the amino acid response activity of this
region, our data show that deletion of the seven nucleotides comprising
the core sequence (5'-CATGATG-3') from the 3121/+51 AS genomic
fragment completely prevented the ERSR-dependent induction
when tested by transient transfection.2
The deletion analysis suggested that sequences within nt 111 to 34
were necessary for activation of the AS gene by the ERSR pathway. The
AS sequence from nt 49 to 30, on the top strand, contains the
sequence 5'-CGTTACAGGAGCCAGGTCG-3' which yields
3'-GCAAT-N9-CCAGC-5' on the bottom strand, (see Fig. 5).
This sequence is similar to the consensus ERSR element
(5'-CCAAT-N9-CCACG-3') proposed by Yoshida (13) and Roy and
Lee (14). However, the location of the AS ERSRE-like sequence on the
bottom strand and in the opposite orientation is unusual, relative to
all previously described occurrences (13, 14). The 9-base pair spacer
of AS also contains a GGC element which is observed in most of the ERSR
elements identified previously (14). An oligonucleotide covering the
55 to 26 AS sequence formed two complexes (Fig. 4B). The
relative amount of the lower complex was unchanged by the medium
glucose content, but formation of this complex exhibited sequence
specificity given that self-inhibition was clearly evident, whereas an
unrelated oligonucleotide sequence had no effect (Fig. 4B).
The presence of a 100-fold excess of the ERSRE sequence did not prevent
formation of the complex, suggesting that the protein(s) bound to this
region do not recognize this consensus sequence. Although the addition of a 100-fold excess of an oligonucleotide corresponding to the consensus ERSRE element blocked the formation of the upper complex, an
oligonucleotide containing an unrelated sequence was just as effective
(Fig. 4B). These data suggest that formation of this minor
complex is not sequence-specific. Therefore, although the deletion data
indicates that the AS region between 34 and 52 is necessary for
ERSR-dependent activation of the gene, identification of
the specific nucleotides responsible must be confirmed by more extensive analysis.
In summary, the data presented document transcriptional control of the human asparagine synthetase gene following activation of the UPR/ERSR pathway. This report is the first demonstration of a link between amino acid metabolism and the UPR/ERSR. The physiologic importance of asparagine biosynthesis to this signal transduction process remains open for investigation. Asparagine is the N-linked carbohydrate attachment site within ER-synthesized glycoproteins, so perhaps the UPR/ERSR activation of the AS gene is to ensure that asparagine limitation is not the cause of ER protein accumulation. Alternatively, both asparagine deprivation (6-9) and activation of the UPR/ERSR pathway (26) have been linked to cell cycle arrest and initiation of apoptosis. If the original signals that arise from asparagine deprivation and the UPR/ERSR represent two completely independent events, induction of AS expression may be a cellular attempt to provide sufficient asparagine so as to eliminate limiting amounts of this amino acid as the cause of the apoptotic signal.
Beyond documenting the novel observation that AS is a UPR/ERSR target,
the data also provide evidence that the transcriptional elements
required for control of some genes by the mammalian ERSR pathway is
more complex than previously thought. Known ERSR target genes, such as
the GRP family, appear to be fully activated by multiple copies of a
single cis-element termed the ERSRE (13, 14). The observation that an
Sp1 sequence (106 to 97) is required for full induction of AS by
the ERSR pathway is unprecedented. Interestingly, Sp1 also is reported
to play a key role in regulating genes that are carbohydrate-responsive
in the opposite direction, that is, that their expression is induced by
the presence of glucose (27). The second AS promoter region that is
implicated by the present data contains a sequence previously reported
to be necessary for the transcriptional response to amino acid
starvation (3). Deletion analysis shows that the core sequence (nt 70
to 64) also is necessary for activation of the AS gene by the
UPR/ERSR pathway.2 Furthermore, the EMSA results of Fig. 4
document that the region forms specific DNA-protein complexes in
vitro for which the amount is greater when nuclear extracts from
glucose-starved cells are tested. Additional research will be necessary
to fully understand the dual role of this regulatory cis-element in the
cellular response to both amino acid and glucose deprivation.
Future genomic analyses, both in vitro and in
vivo, will further define the specific nucleotides required for
this interesting transcriptional activation of the human AS gene by the
UPR/ERSR signal, whereas knockdown or knockout approaches will address the physiological role of asparagine biosynthesis in the pathway.
| |
ACKNOWLEDGEMENTS |
|---|
We thank Dawn Beachy for secretarial support and other members of the laboratories for technical advice and helpful discussion.
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FOOTNOTES |
|---|
* This research was supported by Grant DK-52064 from the NIDDK, the National Institutes of Health (to M. S. K.), and by the CNPq-Conselho Nacional de Desenvolvimento Científico e Tecnológico and Universidade Estadual de Maringá, Brazil (to I. P. B.-T.).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, JHMHC, Gainesville, FL 32610-0245. Tel.: 352-392-2711; Fax: 352-392-6511; E-mail: mkilberg@ufl.edu.
2 I. P. Barbosa-Tessmann, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: AS, asparagine synthetase; UPR(E), unfolded protein response (element); ERSR(E), endoplasmic reticulum stress response (element); GRP, glucose regulated protein; rp, ribosomal protein; nt, nucleotide(s); Aze, azetidine-2-carboxylate; MEM, minimal essential medium; GH, growth hormone; MTT, metallothionein promoter; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay.
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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