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(Received for publication, August 29, 1995; and in revised form, October 12, 1995) From the
ATF3 gene, which encodes a member of the activating
transcription factor/cAMP responsive element binding protein (ATF/CREB)
family of transcription factors, is induced by many physiological
stresses. As a step toward understanding the induction mechanisms, we
isolated the human ATF3 gene and analyzed its genome
organization and 5`-flanking region. We found that the human ATF3 mRNA is derived from four exons distributed over 15 kilobases.
Sequence analysis of the 5`-flanking region revealed a consensus TATA
box and a number of transcription factor binding sites including the
AP-1, ATF/CRE, NF-
Transcriptional regulation plays an important role in both
differentiation and homeostasis (for reviews see (1) and (2) ). We have been studying the ATF/CREB ( Consistent with
this idea, ATF1 and CREB have been demonstrated to stimulate
transcription in response to cAMP and calcium influx (13, 14, 15, 16) , whereas
ATF2/CRE-BP1 has been demonstrated to stimulate transcription in
response to viral induction(17, 18, 19) .
ATF3, on the other hand, is not an activator; it represses
transcription when bound to DNA(20) . However, ATF3 can
heterodimerize with Jun proteins, and the ATF3/Jun heterodimers have
been demonstrated to activate transcription(21, 22) .
Therefore, depending on the cellular context, ATF3 may repress
transcription as homodimers or activate transcription as heterodimers. Recently, we found that the level of ATF3 mRNA greatly
increases both in tissue culture cells after serum stimulation (20) and in whole organisms after physiological stresses. ( In all types
of induction, ATF3 mRNA level greatly increased within 2 h
after stimulation. This quick induction of the ATF3 gene by
many physiological stressors suggests that ATF3 may play an important
role in stress responses. It is not clear, however, how stress signals
induce ATF3 gene. As a first step toward understanding the
induction mechanisms, we isolated the human ATF3 gene and
analyzed its 5`-flanking region. We also studied the regulation of the ATF3 gene in tissue culture cells by anisomycin, an approach
that has been used to study the stress responses in tissue culture
cells(24, 25, 26, 27) . In this
report, we present the genome organization of the ATF3 gene
and the sequence analysis of its 5`-flanking region. We also describe
the effects of anisomycin on the stability of ATF3 mRNA and
the activity of ATF3 promoter. The involvement of the
stress-inducible JNK/SAPK signal transduction pathway (for a review see (28) ) in the induction of ATF3 gene is discussed.
Figure 1:
Structure of the human ATF3 gene. Exons are indicated by boxes and designated as A, B, C, D, and E. EcoRI (R) and HindIII (H)
restriction sites are shown. pH2.8k contains the indicated 2.8-kilobase
fragment.
Figure 2:
Exon organization of ATF3 and
ATF3
As reported
previously(20) , there is an alternatively spliced isoform of
ATF3, ATF3
Figure 3:
Primer extension analysis of ATF3 mRNA. An ATF3-specific primer was annealed to 5 µg of total
RNA isolated from serum-induced HeLa cells. The cDNA was extended and
resolved on an 8% sequencing gel. The same primer was annealed to
pH2.8k, which contains the appropriate genomic fragment, to generate
the sequencing ladder. The arrow on the right indicates the transcriptional initiation site; the sequence around
this site is indicated on the left.
Figure 4:
Nucleotide sequences of the 5`-flanking
region of the ATF3 gene. The TATA box and several
transcription factor binding sites are boxed and labeled. Due
to the limited space, many transcription factor binding sites are not
indicated. The arrow marks the transcription start site
(+1). The GenBank
To
analyze the promoter, we sequenced the 5`-flanking region. Fig. 4shows the sequence of the 5`-flanking 1850 nucleotides.
Inspection of the 5`-flanking sequence revealed a consensus TATA
element around -30 and, interestingly, a consensus ATF/CRE site
around -90. We also noticed several other transcription factor
binding sites. Among them, two classes of binding sites are especially
interesting. One is the inducible site such as the ATF/CRE, AP1, and
NF-
Figure 5:
The ATF3 5` two-kilobase region has promoter activity and can be
stimulated by serum and anisomycin. A, in vivo transfection assay. CAT reporters driven by E1B TATA box (pEC),
the ATF3 promoter (pATF3-CAT), the adenovirus E4 promoter
(pE4SM-CAT), six SP1 sites (pG6TI-CAT), and the Rous sarcoma virus long
terminal repeat (pRSV-CAT) were transfected into HeLa cells and assayed
for CAT activity. A representative result of three experiments is
shown. B, in vitro transcription assay. pATF3-CAT,
pE4SM-CAT, and pG6TI-CAT were transcribed in vitro using HeLa
cell nuclear extracts. CAT mRNAs were analyzed by primer extension
using a CAT-specific primer. C, induction assay. NIH 3T6 cells
were transfected with either pATF3-CAT or pG6TI-CAT, starved for 72 h,
and then induced as indicated for 24 h. CAT activity was assayed, and
an average of four results is shown.
As reported
previously(20) , ATF3 gene is induced by serum
stimulation in tissue culture cells. We then examined whether pATF3-CAT
responds to serum stimulation in tissue culture cells. We transiently
transfected pATF3-CAT into NIH 3T6 cells, starved the cells in medium
containing 0% serum to arrest the cells in G
Figure 6:
Anisomycin increases the steady-state
level of ATF3 mRNA. A, anisomycin at 50 ng/ml
increased the ATF3 mRNA level as demonstrated by Northern blot
analysis. HeLa cells were starved for 48 h and induced by anisomycin at
50 ng/ml for 2 h. 30 µg of total RNA from untreated or treated
cells were analyzed by Northern blot using ATF3 or GAPDH cDNA as probe. B, anisomycin at 50 ng/ml increased the ATF3 mRNA level as demonstrated by in situ hybridization; HeLa cells were starved and then uninduced (left panel) or induced (right panel) as above and
assayed by in situ hybridization using ATF3 antisense
RNA as probe. Bar, 25 µm. C, anisomycin at 50
ng/ml did not significantly inhibit protein synthesis. Left
panel, HeLa cells were incubated with
[
Because both
Northern blot and in situ hybridization detect steady-state
mRNA levels, the increase could be due to the increase of mRNA
synthesis or the increase of mRNA stability. It is possible that
anisomycin treatment stabilizes ATF3 mRNA because the
3`-untranslated region of ATF3 mRNA contains several AUUUA
sequences, which have been demonstrated to destabilize mRNA (for
reviews see (34) and (35) ). To find out whether
anisomycin increases the stability of ATF3 mRNA, we compared
the stability of ATF3 mRNA in the absence and presence of
anisomycin as follows. We treated HeLa cells with 20% serum and 50
ng/ml of anisomycin for 2 h to increase the steady-state level of ATF3 mRNA. After removal of serum and anisomycin, we added DRB
to inhibit further RNA synthesis, allowing the existing RNA to turn
over. We then analyzed ATF3 mRNA by Northern blot at various
time points to assay for its stability. In one set of plates, we added
anisomycin back to determine whether it affects the stability of ATF3 mRNA. As shown in Fig. 7, anisomycin at 50 ng/ml
moderately increased the stability of ATF3 mRNA.
Figure 7:
Anisomycin moderately increases the
stability of ATF3 mRNA. HeLa cells were treated with 20% serum
and 50 ng/ml anisomycin for 2 h to increase the steady-state level of ATF3 mRNA. After removal of serum and anisomycin, DRB was
included in the medium at a concentration of 25 µg/ml to inhibit
RNA synthesis, allowing the existing RNA to decay. No anisomycin
(- Anisomycin, lanes 2-6) or 50 ng/ml of
anisomycin (+ Anisomycin, lanes 8-12) was
included in addition to DRB to examine the effects of anisomycin on ATF3 mRNA stability. Total RNA was isolated at the indicated
times and analyzed (30 µg/lane) by Northern blot using ATF3 or GAPDH cDNA as probe. RNAs from the uninduced cells
were also analyzed (lanes 1 and 7).
To find
out whether anisomycin increases the activity of ATF3 promoter, we examined pATF3-CAT in the absence and presence of
anisomycin. We transiently transfected pATF3-CAT into NIH 3T6 cells,
starved the cells for 72 h, and induced them for 24 h in medium
containing 50 ng/ml anisomycin. As shown in Fig. 5C,
pATF3-CAT was more active in the presence of anisomycin than in the
absence of anisomycin. A control reporter, pG6TI-CAT, was not activated
by anisomycin. We note that pG6TI-CAT was less active than pATF3-CAT in
NIH 3T6 cells (Fig. 5C) but was more active than
pATF3-CAT in HeLa cells (Fig. 5A). This discrepancy was
probably due to the differences between these two cell lines. The
observation that pATF3-CAT can be induced by anisomycin was
recapitulated by an in vitro transcription assay; nuclear
extracts made from anisomycin-treated HeLa cells transcribed the ATF3 promoter at a higher activity than nuclear extracts from
untreated HeLa cells (Fig. 8). These two extracts, however,
showed no difference in transcribing the control promoter composed of
SP1 sites (Fig. 8). These results suggest that the increase of
steady-state ATF3 mRNA level in the presence of anisomycin
was, at least partly, due to an increase of the ATF3 promoter
activity.
Figure 8:
Anisomycin treatment increases the ATF3 promoter activity in an in vitro transcription
assay. Nuclear extracts from HeLa cells or anisomycin-treated HeLa
cells were used to transcribe pATF3-CAT and pG6TI-CAT in
vitro. CAT mRNAs were analyzed by primer extension using a
CAT-specific primer. A representative result of three experiments is
shown.
As described earlier, anisomycin activates the JNK/SAPK
signal transduction pathway(32, 33) . Two
transcription factors, ATF2 and c-Jun, have been demonstrated to be
phosphorylated and activated by this
pathway(24, 25, 26, 28, 36, 37, 38, 39) .
They in turn regulate target promoters, presumably by binding to the
ATF/CRE- or AP-1-related sites. The observations that the ATF3 promoter is activated by anisomycin and that it contains the
ATF/CRE and AP-1 sites prompted us to ask whether the ATF3 promoter can be regulated by ATF2 or c-Jun. We transfected
pATF3-CAT with DNAs expressing ATF2 or c-Jun into HeLa cells. As shown
in Fig. 9, ATF2 by itself did not activate the pATF3-CAT
reporter; c-Jun, on the other hand, activated the reporter severalfold.
Interestingly, co-transfection of DNAs expressing ATF2 and c-Jun
greatly increased the CAT activity. Because the ATF3 promoter
contains potential binding sites for ATF2 and c-Jun (ATF and AP-1
sites), our result is consistent with the notion that ATF2 and c-Jun
act cooperatively on the ATF3 promoter to activate
transcription.
Figure 9:
Co-expressing of ATF2 and c-Jun activates
the ATF3 promoter. pATF3-CAT was transfected into HeLa cells
in the presence of pCG-ATF2 expressing ATF2, or pCMV-Jun expressing
c-Jun or pCG-ATF2 plus pCMV-Jun. In the reporter alone control,
pATF3-CAT was transfected with pCG, which carries the CMV promoter to
make sure that each transfection mix contained the same amount of
promoter. CAT activity was assayed, and a representative result of five
experiments is shown.
The activation of the ATF3 gene by anisomycin is, at least
in part, due to the stimulation of the ATF3 promoter, because
a CAT reporter driven by the ATF3 promoter can be activated by
anisomycin (Fig. 5C). Significantly, the ATF3 promoter can also be activated by the coexpression of ATF2 and
c-Jun (Fig. 9). Because these two transcription factors have
been demonstrated to be phosphorylated and activated by the JNK/SAPK
pathway(24, 25, 26, 28, 36, 37, 38, 39) ,
our preliminary evidence is consistent with the notion that the
JNK/SAPK pathway may be involved in the activation of the ATF3 promoter by anisomycin. This notion is reminiscent of the
observation that this pathway mediates the induction of the c-jun gene by genotoxic agents(39) . We emphasize that
although our results are consistent with the notion that the JNK/SAPK
signaling pathway may be involved in the induction of ATF3 gene by anisomycin, they do not prove it. We also note that
although the anisomycin approach has been used successfully as a model
system to study stress responses in tissue culture cells (24, 25, 26, 27) , it is not clear
whether the mechanisms by which anisomycin induces ATF3 gene
in tissue culture cells are the same as that by which physiological
stresses induce ATF3 gene in the whole organisms. Clearly,
many more experiments are required to clarify these points. In
summary, we analyzed the genome organization and promoter sequences of
the human ATF3 gene. We also studied the regulation of ATF3 gene by anisomycin. These results should aid future
studies of the induction of ATF3 gene by stress signals.
Volume 271,
Number 3,
Issue of January 19, 1996 pp. 1695-1701
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
GENOMIC ORGANIZATION, PROMOTER, AND REGULATION (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
B, E2F, and Myc/Max binding sites. As another
approach to understanding the mechanisms by which the ATF3 gene is induced by stress signals, we studied the regulation of
the ATF3 gene in tissue culture cells by anisomycin, an
approach that has been used to study the stress responses in tissue
culture cells. We showed that anisomycin at a low concentration
activates the ATF3 promoter and stabilizes the ATF3 mRNA. Significantly, co-transfection of DNAs expressing ATF2 and
c-Jun activates the ATF3 promoter. A possible mechanism
implicating the C-Jun NH
-terminal kinase/stress-activated
protein kinase (JNK/SAPK) stress-inducible signaling pathway in the
induction of the ATF3 gene is discussed.
)family
of transcription factors ((3, 4, 5, 6) ; for reviews see (7, 8, 9) ). Members of the ATF/CREB family
bind to a consensus DNA sequence (TGACGTCA), have a similar DNA binding
domain (the basic region/leucine zipper (bZip) domain), and form
selective heterodimers with each other via the leucine zipper region.
Although all ATF/CREB proteins share similarity in their bZip domains,
subgroups of proteins share additional similarity in other regions. For
example, ATF1(5) , CREB(4, 6) , and CREM (3) are similar in regions that contain the phosphorylation
sites. Similarly, ATF2/CRE-BP1 ((10) ; also named HB16 in (11) ) and ATFa (12) share similarity in regions
outside the bZip domain: the first 100 N-terminal residues and the last
13 C-terminal residues. It is possible that proteins within a given
subgroup have closely related functions. Proteins between subgroups,
however, are completely different from each other outside the DNA
binding domain, indicating that they may interact with different
proteins or ligands and have different functions.)Using rats as a model system, we demonstrated that ATF3 mRNA level increased in mechanically injured liver after
partial hepatectomy and in chemically injured liver treated with toxins
such as carbon tetrachloride or alcohol. ATF3 was also induced
in blood-deprived heart (ischemic heart) after coronary artery ligation
and in reperfusion-injured heart after coronary artery ligation coupled
with reperfusion. Furthermore, ATF3 was induced in postseizure
brain treated with pentylenetetrazole. Significantly, ATF3 was
not induced in the suprachiasmatic nuclei in entrained rats receiving
light stimulation during their subjective night. One difference between
light stimulation and the rest of the treatments is that light
stimulation does not elicit cellular injuries, whereas the others do.
Therefore, these results suggest a correlation between ATF3 gene expression and cellular injuries.
Plasmids
pH2.8k, a pGEM3 derivative, contains
the HindIII 2.8-kilobase fragment as shown in Fig. 1.
This fragment contains the following regions from the ATF3 gene: the 5`-flanking region (-1850 to +1), the first
exon (+1 to +167), and 680 base pairs from the first intron.
pATF3-CAT contains the -1850 to +34 region of the ATF3 gene. pE4SM-CAT contains the -330 to +17 region of the
adenovirus E4 promoter with an internal deletion from -138 to
-65(29) . pCG-ATF2 was described previously(20) .
Screening of the Genomic Library
Genomic DNA
isolated from a human placenta was partially digested with MboI; DNA fragments ranging from 16 to 20 kilobases were
isolated by sucrose density gradient and cloned into the -FixII
vector (Stratagene). Approximately 10
plaques were screened
by P-labeled ATF3 cDNA.
Cell Culture, Transfection, and Chloramphenicol
Acetyltransferase (CAT) Assay
Cell culture, transfection, and
CAT assays were performed as described previously (20) with the
following modifications. For comparison of promoter activities, HeLa
cells were transfected with 2 µg of the indicated reporter
construct in addition to 5 µg of pGEM4 (Promega) carrier DNA by the
calcium phosphate method. CAT assays were performed 36 h after
transfection. For induction experiments, NIH 3T6 cells were transfected
with 2 µg of pATF3-CAT or pG6TI-CAT by Lipofectamine (Life
Technologies, Inc.), followed by starvation for 72 h in medium
containing 0% serum and induction for 24 h in medium containing 20%
fetal bovine serum or in medium containing 0.5% fetal bovine serum and
one of the following reagents: 50 ng/ml anisomycin or 50 ng/ml
12-O-tetradecanoylphorbol-13-acetate (TPA). CAT assays were
performed after induction. For co-transfection experiments, HeLa cells
were transfected with 2 µg of pATF3-CAT in addition to 5 µg of
pCG-ATF2 expressing ATF2, pCMV-Jun expressing c-Jun, or 2.5 µg of
each DNA. In the reporter alone control, 5 µg of pCG was added.Cell Labeling and Immunoprecipitation
Cell
labeling and immunoprecipitation were described
previously(20) .In Vitro Transcription
In vitro transcription by crude nuclear extract was carried out as
described previously(30) . The CAT-primer,
5`-GCCATTGGGATATATCAACGG-3`, is complementary to the region from
+29 to +49 of the CAT mRNA. Nuclear extracts were made from
uninduced or induced HeLa cells according to Dignam et al.(31) . Induction was carried out for 2 h in medium
containing 50 ng/ml anisomycin.Northern Blot and Primer Extension
Northern blot
and primer extension were carried out as described
previously(20) . The primer (5`-CTCGGGCGGCCAGGG-3`) is
complementary to the region from +141 to +156 of the ATF3 mRNA. Extended products were resolved on an 8% polyacrylamide
sequencing gel, and the sequence ladder was generated from the same
primer using pH2.8k as a template.In Situ Hybridization
5 
10
HeLa cells were grown on Superfrost Plus glass slides (VWR
Scientific) and starved in medium containing 0% serum for 48 h. The
cells were then incubated with growing medium in the absence or
presence of 50 ng/ml anisomycin for 2 h. In situ hybridization
was carried out as described previously.Analysis of mRNA Half-life
HeLa cells were
induced with 20% fetal bovine serum and 50 ng/ml anisomycin for 2 h,
washed with phosphate-buffered saline, and incubated with medium
containing 10% fetal bovine serum and 25 µg/ml
5,6-dichloro-1-
-D-ribofuranosylbenzimidazole (DRB,
Sigma). In one set of plates, 50 ng/ml of anisomycin was also included.
Total RNA was isolated at the indicated time points and analyzed by
Northern blot using ATF3 or GAPDH cDNA as probe.DNA Sequencing
Dideoxy sequencing was carried out
using the regular method with Taq polymerase (Promega) or the
cycle sequencing method (Life Technologies, Inc.).
Isolation and Characterization of the Human ATF3
Gene
An unamplified -FixII genomic library prepared from
size-fractionated human placenta DNA was screened using ATF3 cDNA as probe. Two overlapping clones,
16kb-1 and
20kb-2, were further characterized using the N-terminal or
C-terminal cDNA as probe; only
16kb-1 contained the 5` end of the
gene. The
16kb-1 clone was further mapped, subcloned, and
sequenced to identify the mRNA coding region, exon/intron junctions,
and the 5`-flanking sequences. Fig. 1shows the restriction maps
for EcoRI and HindIII. The positions of the exons
were determined by Southern blot analysis using cDNA or
oligonucleotides as probes. DNA fragments containing the exons were
further characterized by detailed restriction mapping and sequencing.
Comparison of the genomic and cDNA sequences with the restriction map
suggested that the human ATF3 mRNA is derived from four exons
distributed over 15 kilobases, designated as exons A, B, C, and E in Fig. 1. Exon A encodes the 5`-untranslated region. Exon B
contains the AUG initiation codon and encodes the N-terminal 80 amino
acids. Exon C encodes 36 amino acids, which is mostly the basic region.
Exon E encodes 65 amino acids, which is mostly the leucine zipper (ZIP)
domain; it also encodes the 3`-untranslated region. This organization
indicates that exons B, C, and E each encode a functional domain,
consistent with the modular nature of many exons. Fig. 2summarizes the exon organization, and Table 1shows
the sequences around the exon/intron junctions.
Zip. Schematic representations of the mRNAs and
proteins for ATF3 and ATF3Zip are shown. Exons in mRNAs are
indicated by boxes labeled as A, B, C, D, and E. Nucleotide numbers are
indicated at the top. Functional domains of proteins are
indicated by boxes, with basic region and leucine zipper (ZIP)
domains labeled. Amino acid numbers are indicated at the bottom. The codons and amino acids at the border of each
domain are indicated.
Zip. ATF3Zip contains an additional exon between
exons C and E; this additional exon introduces an in-frame termination
codon, resulting in a truncated protein lacking the leucine zipper
region at the C terminus. In addition, the last three nucleotides (AAA)
in exon C were spliced out in this isoform, resulting in a glutamine
(Q) residue instead of a lysine (K) residue at the end of the
corresponding domain. The splicing event resulting in ATF3Zip was
shown previously(20) ; the exon organization of ATF3Zip is
shown in Fig. 2.Identification of the Transcriptional Start Site and
Analysis of the 5`-Flanking Sequences
To identify the
transcriptional start site of ATF3 gene, we performed primer
extension. We incubated HeLa cells with 20% serum to induce ATF3 gene, isolated total RNA, and annealed the RNA to an
oligonucleotide complementary to nucleotides +141 to +156 of
the ATF3 mRNA and extended the oligonucleotide by reverse
transcriptase. As shown in Fig. 3, one major extended product
was identified. When compared with the sequencing ladder generated by
the same oligonucleotide using pH2.8k which contains the appropriate
genomic DNA fragment as a template, the extended product co-migrated
with the T residue indicated by a box in Fig. 3.
Therefore, the complementary A residue on the mRNA-like strand is
indicated as the transcriptional start site in Fig. 4.
accession number is
U37542.
B sites; the other is the site implicated in cell cycle
regulation, such as the Myc/Max and E2F binding sites. It is not clear,
however, whether any of these binding sites are functionally important
for the promoter activity. The promoter region also contains other
transcription factor binding sites, such as the SP1, AP2, AP3, and
octamer binding sites, although they are not indicated in Fig. 4.Promoter Activity of the 5`-Flanking Two-kilobase
Region
To find out whether the 5`-flanking region contains
promoter activity, we constructed a CAT reporter driven by a fragment
containing the 5`-flanking two kilobases (pATF3-CAT), and compared its
activity with several that of CAT reporters driven by different
sequences: SP1 sites (pG6TI-CAT), adenovirus E4 promoter (pE4SM-CAT),
the Rous sarcoma virus long terminal repeat (pRSV-CAT), and the E1B
TATA box (pEC). As shown in Fig. 5A, when transfected into
HeLa cells, pATF3-CAT was much more active than pEC; it was similar to
pE4SM-CAT and 50% active compared with pG6TI-CAT but only 5% active
compared with pRSV-CAT. Consistent with this result, pATF3-CAT
displayed similar activity as pE4SM-CAT in an in vitro transcription assay but was less active than pG6TI-CAT (Fig. 5B). pRSV-CAT was not included in this assay,
because it was not active in vitro.
phase, and
induced the cells with 20% serum. We used NIH 3T6 cells instead of HeLa
cells, because serum starvation arrests NIH 3T6 cells better than HeLa
cells. As shown in Fig. 5C, pATF3-CAT was induced by
serum stimulation, whereas pG6TI-CAT was not. In addition, we examined
the inducibility of pATF3-CAT by the phorbol ester
tetradecanoyl-phorbol acetate, which also induced the endogenous ATF3 gene. (
)Fig. 5C shows that it
slightly induced pATF3-CAT, although the induction was not as high as
that of the endogenous ATF3 gene. Taken together, we conclude
that the 5`-flanking two-kilobase region contains promoter activity and
can confer, as least partly, the responsiveness to several inducing
agents.The Effects of Anisomycin on ATF3 Gene
Expression
As described earlier, the ATF3 gene is
induced by many physiological stresses. This observation
prompted us to ask whether the stress-inducible JNK/SAPK signal
transduction pathway (for a review see (28) ) is involved in
the induction of ATF3 gene by stress signals. As a first step
toward answering this question, we examined the induction of ATF3 gene by anisomycin, because anisomycin at subinhibitory
concentrations, concentrations that fail to inhibit protein synthesis,
can activate the JNK/SAPK signal transduction
pathway(32, 33) . We examined the expression of the
endogenous ATF3 gene in tissue culture cells by both Northern
blot analysis and in situ hybridization. As shown in Fig. 6, A and B, anisomycin at 50 ng/ml
greatly increased the level of ATF3 mRNA. That anisomycin at
this concentration did not significantly inhibit protein synthesis was
confirmed as follows. We incubated the cells with
[S]methionine to radiolabel the newly
synthesized proteins in the absence or presence of anisomycin and
examined the incorporation of [
S]methionine into
cellular proteins by an SDS-polyacrylamide gel. As shown in Fig. 6C, at 50 ng/ml (low dose), anisomycin had little
effect on the synthesis of total cellular proteins; at 10 µg/ml
(high dose), however, anisomycin completely abolished the synthesis.
Furthermore, immunoprecipitation using ATF3 antibody indicated that the
newly induced ATF3 mRNA was indeed translated to produce ATF3
protein in the presence of 50 ng/ml anisomycin (lanes 5-7).
Taken together, this information leads us to conclude that anisomycin
at a subinhibitory concentration induces ATF3.
S]methionine for 2 h in the absence of
anisomycin (-) or in the presence of low dose anisomycin (50
ng/ml, L) or high dose anisomycin (10 µg/ml, H).
Whole cell extracts (WCE) from approximately the same number
of cells were analyzed on an SDS-polyacrylamide gel. Right
panel, HeLa cells were incubated with
[
S]methionine in the absence (-) or
presence of low dose anisomycin for 2, 4, or 6 h. ATF3 from
approximately the same number of cells was immunoprecipitated by
antibody against ATF3 and analyzed on an SDS-polyacrylamide gel. The arrow on the right indicates
ATF3.
Genome Organization of the Human ATF3 Gene
In
this report, we describe the genome organization of the human ATF3 gene. We also describe the exon/intron junctions of two
alternatively spliced isoforms: ATF3 and ATF3Zip. However, the
physiological relevance of these isoforms is not clear at present.
Other ATF/CREB family transcription factors have also been demonstrated
to have alternatively spliced isoforms. Some examples are CREM (for a
review, see (40) ), CREB ((41, 42, 43) ; for a review see (9) ), ATF2(44) , and ATFa(12, 45) .
In light of the large and complex genome organization of both CREM and CREB genes, it is possible that the small,
15-kilobase genome region of the human ATF3 gene described in
this report represents only a portion of the gene. We are currently
further characterizing the ATF3 genome to clarify this point.The 5`-Flanking Region of the ATF3 Gene
Analysis
of the 5`-flanking 1.8-kilobase region revealed various transcription
factor binding sites. Two groups of binding sites are of special
interest: the inducible sites and the sites implicated in cell cycle
regulation. The inducible sites include the ATF/CRE, AP1, and NF-B
sites. Because these sites have been demonstrated to be induced by
signals such as cAMP, calcium influx, UV irradiation, and cytokines
(for reviews see (9) and (46) -52), their
presence is consistent with our observation that ATF3 gene is
induced by many stimulations. Their presence also supports
the observation described in this report that the induction is, at
least partly, due to the activation of the ATF3 promoter. In
addition, the presence of the ATF/CRE consensus sequence in the ATF3 promoter suggests that the ATF3 gene may be
regulated by the ATF/CREB family of transcription factors. The
observation that co-expression of ATF2 and Jun activates the ATF3 promoter is consistent with this notion. The presence in the ATF3 promoter of the Myc/Max and E2F sites, sites implicated
in cell cycle regulation, implies that the expression of ATF3 gene may also be regulated in an cell cycle-dependent manner. We
are currently pursuing this possibility.The Effects of Anisomycin on the Stability of ATF3
mRNA
As shown in Fig. 7, 50 ng/ml of anisomycin
moderately increased the stability of ATF3 mRNA. In this
context, it is important to point out that a previous report
demonstrated that, in C3H 10T1/2 cells, the half-life of c-fos and c-jun mRNA was 15 and 30 min, respectively, in the
presence of subinhibitory concentrations of anisomycin, whereas their
half-lives were more than 6 h in the presence of inhibitory
concentrations of anisomycin(23) . However, not available were
the half-lives of these mRNAs in the absence of anisomycin in C3H
10T1/2 cells. Consequently, it is not clear whether these mRNAs are
more stable in the presence of subinhibitory concentrations of
anisomycin than in the absence of anisomycin. Therefore, our
observation that a subinhibitory concentration of anisomycin stabilizes
the ATF3 mRNA does not necessarily contradict their results.Regulation of the ATF3 Promoter by Anisomycin
In
this report, we demonstrate that anisomycin activates the ATF3 gene in tissue culture cells. Although anisomycin is a protein
synthesis inhibitor, this activation is independent of its ability to
inhibit protein synthesis, because anisomycin at 50 ng/ml, a
concentration that does not significantly affect protein synthesis, can
still activate the ATF3 gene. Therefore, anisomycin activates
the ATF3 gene, not by its ability to generally inhibit protein
synthesis, but by its intrinsic ability to activate certain
intracellular machinery. One candidate for such a machinery is the
JNK/SAPK signal transduction pathway, because anisomycin has been
demonstrated to activate this pathway independent of its ability to
block protein synthesis(23, 32, 33) .
Therefore, our results indicate a correlation between the activation of
the JNK/SAPK pathway and the activation of the ATF3 gene.
)-D-ribofuranosylbenzimidazole; JNK/SAPK,
C-Jun NH-terminal kinase/stress-activated protein kinase;
CREM, cAMP-responsive element modulator.))
We thank Dr. W. Herr for pCG, Dr. T. Curran for
pCMV-Jun, Drs. G. Gill and R. Tjian for pG6TI, Drs. S. Liang and M.
Ptashne for pEC, Dr. A.P. Young for pRSV-CAT and the human placenta
tissue, Dr. J. Lang for HeLa cells, and Dr. L. Johnson for NIH 3T6
cells.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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