(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-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.
Transcriptional regulation plays an important role in both
differentiation and homeostasis (for reviews see (1) and (2) ). We have been studying the ATF/CREB ()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.
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. ()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.
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
ATF3Zip. Schematic representations of the mRNAs and
proteins for ATF3 and ATF3
Zip 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.
As reported
previously(20) , there is an alternatively spliced isoform of
ATF3, ATF3Zip. ATF3
Zip 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 ATF3
Zip was
shown previously(20) ; the exon organization of ATF3
Zip is
shown in Fig. 2.
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 accession number is
U37542.
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-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.
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 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.
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
[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.
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.