From the Hematology Division, Department of Medicine,
Brigham & Women's Hospital, and Harvard Medical School, Boston,
Massachusetts 02115 and ** The Nelson Biological Laboratory, Bureau of
Biological Research, Department of Cell Biology and Neuroscience,
Rutgers University, Busch Campus, Piscataway, New Jersey 08854
Received for publication, October 18, 2000, and in revised form, January 17, 2001
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ABSTRACT |
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An organism's ability to respond to
changes in oxygen tension depends in large part on alterations in gene
expression. The oxygen sensing and signaling mechanisms in eukaryotic
cells are not fully understood. To further define these processes, we
have studied the Regardless of an organism's complexity, its ability to adapt to
changes in environmental oxygen tension is critical to its survival. In many circumstances, the adaptive response to low oxygen tension requires alterations in the expression of specific genes. In mammals, genes encoding proteins such as
erythropoietin and vascular endothelial growth factor, critical
molecules in system-wide functions such as erythropoiesis and blood
vessel formation, respectively, were among the first shown to be
regulated by hypoxia (reviewed in Refs. 1 and 2). The regulation
of these genes by hypoxia has served as a paradigm for the subsequent identification of hypoxia-regulated genes involved in basic
intracellular metabolic and biochemical pathways. For example, the
hypoxia responsiveness of genes involved in sugar transport
(e.g. GLUT1 and GLUT3) and energy production
(e.g. genes encoding certain glycolytic enzymes) is mediated
by hypoxia inducible factor-1
(HIF-1),1 the same
transcription factor that plays a critical role in erythropoietin and
vascular endothelial growth factor activation in response to low oxygen
tension (3, 4). HIF-1 is a heterodimeric protein comprised of an Although a substantial understanding of the structure and function of
HIF-1 Iron chelation also mimics hypoxia. Just as certain metals may
interfere with intracellular Fenton chemistry in some manner, decreased
intracellular iron might also decrease reactive oxygen species within
the cell, leading to altered gene expression (reviewed in Ref. 2).
HIF-1 The yeast, Saccharomyces cerevisiae, a single-celled
eukaryote, is a facultative anaerobe which respires in the
presence of oxygen but ferments under anaerobic conditions. Therefore,
it is not surprising that yeast have evolved sophisticated molecular mechanisms involving oxygen-dependent gene regulation.
Several yeast genes, exemplified by ANB1, have been shown to
be up-regulated by complete anaerobiosis, mediated in large part
through the ROX1p protein, a DNA-binding protein which functions as a
repressor (reviewed in Ref. 14). There is no evidence that any factor that mimics hypoxia in mammalian cell lines induces ANB1
expression in yeast.2
However, recently it has been shown that other genes in S. cerevisiae exhibit increased expression above baseline at low
oxygen tensions, before complete anaerobic conditions are reached (15).
One gene in particular, OLE1, encodes the Media, Chemicals, and Enzymes--
Yeast strains were grown in
YPD medium (Bio 101, Inc., Carlsload, CA) or SC dropout medium,
depending on the plasmid selectable markers. LB was used for bacteria
growth purposes. Ampicillin (U.S. Biochemical Corp.) was used as
necessary at 50 µg/ml unless indicated otherwise.
o-Nitrophenyl- Oligonucleotide Synthesis--
Oligonucleotides were synthesized
by Integrated DNA Inc. When necessary restriction sites for cloning
were added at the 5' ends of primers and were preceded by 3 to 6 nucleotides for efficient digestion. Paired oligonucleotides
used for direct cloning possess a phosphate group at the 5' end. Table
I shows the nucleotide sequences used for
polymerase chain reaction (PCR), Northern blot assay, EMSA, cloning,
and site-directed mutagenesis.
Plasmid and Plasmid Construction--
Plasmids used in this
study are shown in Table II. The
construction of several of the OLE1 promoter-lacZ
fusion deletion series was described previously (16). Construction of
pAM6, pAM7, pAM10, and pAM16 vectors containing the OLE1
LORE sequences ( Strains and Growth Conditions--
Table II contains the yeast
strains used in these studies. Yeast cells containing lacZ
fusion plasmids were grown at 30 °C on uracil dropout medium
containing dextrose (17). For unsaturated fatty acid repression
analysis, yeast were grown in medium supplemented with 1% Tergitol as
described previously (16). Cells were grown in the presence of UFAs for
6 h prior to the DNA Sequencing--
Plasmid templates for sequencing were
isolated using a QIAprep spin purification kit (Qiagen). The
fmol DNA sequencing system (Promega Corp.) was used for
sequencing according to its technical manual. Reactions were run on 6%
sequencing gels, which were dried and exposed to X-Omat AR film (Kodak)
to visualize the sequence.
Yeast Extract Preparation--
Haploid yeast (S. cerevisiae, strain RZ53-6) were cultured in 1-liter flasks
containing 200 ml of YPD (1% yeast extract, 2% peptone, 2% dextrose)
either under normoxic or hypoxic conditions, harvested at midlog phase
(A600 = 0.8), and lysed by vortexing with glass
beads according to published protocols (20). Following addition of
ammonium sulfate to 40% and incubation on a rocker table at 4 °C
for 30 min, the precipitate was collected by centrifugation at 14,000 rpm in a microcentrifuge at 4 °C for 10 min. The pellet was
resuspended in storage buffer (20 mM HEPES, pH 8.0, 5 mM EDTA, 20% (v/v) glycerol, mM
phenylmethylsulfonyl fluoride, 7 mM EMSA--
EMSA were performed essentially as described by Carey
(21) utilizing synthetic paired oligonucleotides (e.g. 10-5'
and 10-3') as a probe or a probe containing the LORE sequence made by
PCR using p62::934 as the template with
32P-labeled oligonucleotides 1-5' and yd-10 ( RNA Isolation and Northern Blot Analysis--
Total yeast RNA
was isolated as described previously (23). Equal amounts (15 µg) of
total RNA were analyzed by Northern blots according to standard
procedures for separation of RNA using 1% formaldehyde gels (15). RNA
from the gels was transferred to Nytran Plus membranes (Schleicher & Schuell Inc.) in 10 × SSC overnight. Prehybridization,
hybridization, and washing of membranes were performed as described
(24). Northern blots were quantified using a PhosphorImager (Molecular
Dynamics) and autoradiographs were also prepared on X-Omat AR film (Kodak).
To make radiolabeled cDNA probes for other genes of interest
(including ACT1 as a control), yeast genomic DNA prepared by the rapid isolation of yeast chromosomal DNA protocol (25) was subjected to PCR with appropriate pairs of primers for the particular genes of interest. The PCR products were first purified using a
QIAquick spin PCR purification kit (Qiagen), separated by agarose gel
electrophoresis in 1 × TAE, then purified by the Qiagen gel extraction kit (Qiagen) according to the manufacturer's recommendations.
For the detection of OLE1 mRNA, a radiolabeled DNA probe
was made using a 0.5-kilobase EcoRI fragment from the
OLE1 protein coding sequence. For an internal control of
cellular mRNA levels, a 1-kilobase
HindIII-KpnI fragment of the S. cerevisiae phosphoglycerate kinase gene (PGK1)
was isolated from plasmid pRIP1PGK obtained from Dr. S. Peltz (Robert
Wood Johnson Medical School). All DNA fragments were separated by
agarose gel electrophoresis in 1 × TAE and purified using the
Qiagen gel extraction kit (Qiagen) according to the manufacturer's
recommendations. The purified DNA fragments were labeled to high
specific activity with [32P]dCTP (PerkinElmer Life
Sciences) by the random primer extension method using Ready to Go DNA
labeling beads (Amersham Pharmaceutical Biotech) reaction kit.
Unincorporated nucleotides were removed from the sample using a
Sephadex G-50 spin column (Roche Molecular Biochemicals). Specific
activity of labeled probes was determined by liquid scintillation counting.
Hypoxia, the Transition Metals Cobalt and Nickel, and the Iron
Chelator 1,10-phenanthroline Increase OLE1 Expression by Northern
Analysis and
Several mammalian hypoxia and transition metal-inducible genes are also
up-regulated by the iron chelator, desferrioxamine (reviewed in Ref.
1). We, therefore, examined the effect of iron chelation on
OLE1 expression. 1,10-Phenanthroline is an iron chelator
that is routinely employed in studies with S. cerevisiae (27). Its affinity constant K for iron is 1021.
As shown in Fig. 1, exposure to
1,10-phenanthroline for 6 h resulted in a
dose-dependent increase in expression of OLE1,
as assessed by Northern blot and OLE1
promoter-lacZ reporter data (data not shown). Cell growth
was unaffected by any of the concentrations tested.
OLE1 Promoter Deletion Hypoxia-induced Activation Complex Formation with LORE--
The
analysis of the OLE1 promoter-lacZ fusion
deletion series strongly suggests that the
To further investigate the role of oligonucleotide number 10 in
hypoxia- and CoCl2-induced complex formation, EMSAs were
performed using end-labeled oligonucleotide number 10 as a probe. Fig.
4 demonstrates that crude extracts from
both hypoxia- and cobalt-treated yeast form a specific complex with
oligonucleotide number 10 in vitro. Of note, when this
shorter radiolabeled probe is used, band B2 is prominent whereas B3 is
no longer present. This is consistent with the hypothesis that B3
complex formation requires additional element(s) outside of
oligonucleotide 10. Furthermore, when mutations were introduced in
oligonucleotide 10 (m#10), the intensity of the nonspecific
B2 was further enhanced. Shortened exposure time, however, confirmed
that the specific hypoxia-induced B1 complex formation was not
observed. No specific hypoxia- or CoCl2-induced complex
formation was observed using probe number 8, confirming the previous
EMSA competition assay.
LORE Is Required for Hypoxia-induced OLE1 Expression in
Vivo--
A lacZ fusion reporter pAM4 was constructed such
that the full-length OLE1 promoter possessed three mutations
in the LORE region ( LORE Is Sufficient for Hypoxia-induced Gene Expression under the
Control of a Heterologous Promoter--
The LORE was fused to the
basal CYC1 promoter-lacZ fusion plasmid pTBA30.
As shown in Fig. 5, pAM6, carrying two
copies of the LORE in tandem, possesses robust transcriptional
activation under both hypoxic and cobalt-treated conditions with about
44- and 10-fold increases, respectively. The plasmid carrying one copy
of the LORE in both orientations also substantially stimulated the
reporter gene expression under both hypoxic and cobalt-treated conditions. In contrast, the plasmid pAM10 containing the
CYC1 heterologous promoter with three mutations in LORE did
not show induction by either hypoxia or cobalt. Considerable
variability in the basal expression of these constructs may be due to
differences in sequences related to orientation and copy number of
insert.
LORE Is Involved in OLE1 Repression under Hypoxic Conditions by
Unsaturated Fatty Acids (UFA)--
Previous experiments demonstrated
FAR elements ( Role of ROX1 in OLE1 Expression under Hypoxic
Conditions--
ROX1 plays a significant role in the
regulation of many anoxia-inducible yeast genes (reviewed in Refs. 1
and 14). Several studies have provided evidence that ROX1p functions as
a repressor of anoxia-inducible gene expression under normoxic
conditions (Ref. 28, reviewed in Refs. 29 and 30). Previous studies (28, 30, 31) had postulated that ROX1 may contribute to OLE1 induction under anoxic conditions based on predicted
potential ROX1p-binding sites in the OLE1 promoter region. A
ROX1 deletion mutant strain of RZ53-6 (RZ53-6 Sequence Specificity of the LORE Binding Activity--
To further
define the sequence requirements in LORE, we made a series of single
base pair substitutions in the site (Fig. 7A) and assayed the effects of
the mutations on DNA binding in vitro (Fig.
8, A and B). Many
of the mutant LOREs had altered DNA binding ability; in particular,
those shown in Fig. 7B are representative of the observed
range of EMSA responses. The effects on DNA binding varied from the
absence of a detectable specific complex formation B1 for mutant C337A,
to a reduction for mutant A346C, to about the same as the wild type
LORE for mutant G347T, to an increase in DNA binding for mutant T331G.
Another group of mutant LOREs (e.g. A336C and A335C)
demonstrated altered complex formation with reduction of B1 but also
creation of new shifted bands, suggesting that a new protein-DNA
complex may have been created. The DNA binding ability of all the
mutant LOREs is summarized in Fig. 8A. Substitutions that
show large decreases in specific binding complex formation are
concentrated in the center of the LORE. DNA binding was sensitive to
single nucleotide substitutions examined between Identification of a Family of Genes under Similar LORE
Control--
A search of the S. cerevisiae genome for LORE
core sequences present in the promoter regions of other genes was
carried out using DNA Pattern (32) and
PatMatch3 web-based tools.
Fig. 8A illustrates alignment of putative LOREs in the
promoter region of several genes. Some of the potential LOREs (from the
promoter sequences of TRX2, FKH1,
FTR1, RPL35A, and MET22) have exactly
the same nine core nucleotides. The potential LOREs from the
ATF1 and TIR1 promoter regions each have one
nucleotide mismatch in the core region. Another potential LORE from
SUT1 possesses two mismatches in this region. It is worth
noting that the expression of ATF1, TIR1, and
SUT1 is increased at low oxygen tensions (34-37). The
hypoxia-induced complex formation of all potential LOREs from the
listed genes was tested in vitro using EMSAs as shown in
Fig. 8B. The results demonstrate that, like the wild type
LORE from the OLE1 promoter, a clear hypoxia-induced complex
is formed with the potential LOREs from the promoters of the genes
indicated. The fact that potential LOREs from RPL35A, TRX2, and MET22 share the same core sequence but
exhibit varied degrees of complex formation ability, based on the
intensity of the B1 band shift in Fig. 8B, implies that the
nucleotides outside the core also play an important role in DNA
binding. On the other hand, the potential LOREs from ATF1,
TIR1, FTR1, and SUT1 with one or two
nucleotide mismatches still showed hypoxia-induced DNA binding (even
increased DNA binding in the case of ATF1), suggesting that
certain positions in the core sequence may be varied yet still function
as a LORE in vitro.
To test the possibility that these genes may be hypoxia-inducible,
Northern blot analyses of certain genes (ATF1, TRX2, SUT1, FTR1, and RPL35A) were performed as shown in Fig.
8C. Consistent with previous data (36), ATF1
expression was significantly induced under the hypoxic conditions
employed. This study shows for the first time that ATF1 and
TRX2 are induced by cobalt treatment as well, similar to
OLE1. About a 3-fold hypoxia and 2.5-fold cobalt induction
of TRX2 mRNA was observed. The hypoxia-induced SUT1 expression was confirmed; however, there was no
significant induction by cobalt. FTR1 mRNA was also
examined and it was confirmed that its level was decreased under
hypoxic conditions, consistent with previous studies (data not shown)
(38). The RPL35A mRNA level was not changed under
hypoxic conditions (data not shown).
RAP1p and LORE-binding Protein(s) (LBP)--
RAP1p is a yeast
multifunctional protein involved in transcriptional
activation/repression, and telomere function (Ref. 39, reviewed in
Refs. 40 and 41). Previous studies on the regulation of the
ATF1 gene identified an 18-bp element essential for
transcriptional activation in vivo (36). This element also
contains a putative LORE. A purified glutathione
S-transferase-RAP1p fusion was utilized for in
vitro EMSA using a probe from the ATF1 promoter
containing the 18-bp element. The results showed that RAP1p could form
a complex with the ATF1 promoter DNA sequence. To test the
possibility of RAP1p involvement in binding to the LORE, EMSAs were
performed. The results demonstrate that the putative LORE from
ATF1 forms a complex (B1) with the crude extracts from
hypoxia-treated yeast cells analogous to the LORE from OLE1
(data not shown). On the other hand, a constitutive, strong binding
band (B3) was observed using the RAP1p binding sequence from the
PGK1 promoter as a probe. Additional EMSAs were performed
using a very well characterized RAP1p binding sequence and mutants from
the TPI promoter as probes (42). These results confirm that
the B3 complex binds RAP1p (data not shown). A series of EMSAs were
done to investigate the relationship between B3 and B1 (data not
shown). Unlabeled LORE from either ATF1 or OLE1
could not compete out the B3 and vice versa, suggesting that the
constitutive B3 complex involved with RAP1p binding is different from
the hypoxia-induced B1 complex involving the LORE from ATF1
and OLE1. Unlabeled LORE of ATF1 could
effectively compete out the complex formed with radiolabeled OLE1 LORE and vice versa (data not shown), which together
with the previous B3 competition EMSA results suggest that the complex formed with the OLE1 LORE and the ATF1 LORE are
similar. Because there is only 1-bp difference in the core region of
the LORE between OLE1 and ATF1, a mutated LORE
T341C from OLE1 which corresponds to the putative
ATF1 LORE was examined. This single base change caused a
constitutive complex formation in crude extracts from normoxia and
hypoxia. The subsequent cold probe competition EMSAs suggested that
that complex was the same as the B3 complex formed by the RAP1p binding
sequence from PGK1 (data not shown). The B1 complex could
still be observed using hypoxic crude extract, and could be displaced
by unlabeled OLE1 LORE probe. In summary, these in
vitro results support the hypothesis that the putative LORE of
ATF1 functions like the LORE of OLE1 in the
regulation of gene expression by hypoxia. Moreover, these data do not
support a role for RAP1p binding to the LORE sequence during hypoxic induction.
OLE1 encodes the The experiments presented in this paper have confirmed previous data
showing that the expression of OLE1 mRNA is increased in
hypoxia and in the presence of the transition metals cobalt and nickel
under aerobic conditions (15). Data presented here have shown that
these stimuli induce an OLE1 promoter-lacZ
reporter gene as well. Aerobic incubation with the iron chelator
1,10-phenanthroline also leads to increased OLE1 expression
as evidenced by Northern blot and reporter assays. Subsequently, using
reporter gene assays and EMSAs, a LORE, which functions as a
transcriptional activation cis element, was identified. The
LORE, about 20 bp in length, is necessary and sufficient for
OLE1 hypoxia-induced gene expression and is also sufficient
for hypoxia-induced gene expression when placed upstream of a
heterologous promoter. Further studies demonstrated that the same LORE
sequence is involved in OLE1 repression by UFA in normoxic,
hypoxic, and cobalt containing conditions. A family of genes containing
a similar LORE in their promoter regions was identified by searching
the S. cerevisiae genome using the 9-nucleotide DNA core
binding sequence (ACTCAACAA), which was determined by performing EMSAs
using a series of single nucleotide substitutions in the
OLE1 LORE in vitro. Among them, ATF1,
TRX2, SUT1, and TIR1 may be under similar LORE
control for hypoxia-inducible gene expression.
Extensive studies over the past decade have defined the transcriptional
repression mechanism for the regulation of anoxia-inducible genes
(reviewed in Refs. 14 and 26) in S. cerevisiae. This mechanism is illustrated by the regulation of ANB1, a
prototypic anaerobic-induced yeast gene. Both genetic and biochemical
evidence have demonstrated that the normoxic repression of this gene is mediated by the ROX1p repressor through its binding to the ANB1 operator site. The full repression by ROX1p requires two general transcription mediators, SSN6p and TUP1p (43-45). In an anaerobic environment, ROX1 expression is decreased, ROX1p levels
decline, and eventually the repression of anoxia-regulated genes is
released. Heme and Hap1 are involved in ROX1 expression in
normoxic conditions (44-47). Based on the consensus binding sequence
of ROX1p, putative ROX1p binding sequences in the promoter regions of
many other genes in S. cerevisiae, including
OLE1, have been identified. The OLE1 promoter
region contains three putative ROX1p-binding sites at Multiple pathways involved in regulating hypoxic and anoxic gene
expression in yeast may exist (49). Studies of several other
hypoxic/anaerobic genes including SUT1 (34), GPD2
(50), PAU (51), and DNA1 (52) have demonstrated
ROX1p-independent hypoxic/anaerobic induction. Another
hypoxic/anaerobic gene, TIR/SRP1, has variably been reported
to be ROX1p-independent and ROX1p-dependent (35, 37). The
identification of a LORE in this study indicates that transcription
activation is crucial to the increased expression of certain yeast
genes in response to extremely low oxygen tension (versus
complete anaerobiosis). The identification of the putative transcriptional activator(s), i.e. the LBP, is under active investigation.
It is also demonstrated in this study that the LORE is involved not
only in the hypoxic induction of OLE1 and ATF1,
but also in cobalt (Fig. 5) induction. Mutations in the LORE
dramatically decrease the reporter expression in both hypoxic and
cobalt-treated conditions. However, some cobalt-induced reporter
expression is still observed (~4.5-fold induction), suggesting that
other as yet undefined elements may be involved. This result is
consistent with observations utilizing the OLE1 promoter
deletion-lacZ fusion assays (Fig. 2). In the shortest
construct (p62::255) there is still a 2.4-fold induction for
cobalt-treated yeast, despite no hypoxic induction.
The S. cerevisiae genome-wide search for LOREs in putative
promoter regions identified many genes which are potentially regulated by low oxygen tension. One of these genes, the anaerobically-induced gene, TIR1, encodes a stress-induced cell wall mannoprotein,
and is also a member of the seripauperine (PAU) family (35, 37, 53).
The in vivo function of a putative LORE in its promoter region ( Interestingly, transcription of the FTR1 gene, which
functions as an iron permease mediating high affinity iron uptake in yeast, was reported to be induced by oxygen (38). We have confirmed this result, demonstrating that the FTR1 mRNA level was
decreased under hypoxic conditions (data not shown). This suggests that the potential LORE in its promoter region is either nonfunctional or
serves as a repression cis element under hypoxic conditions. It is likely that additional factors such as adjacent transcription factor-binding sites, spacing of the binding site, or a lower factor
binding affinity in vivo may be critical in the regulation of genes possessing LOREs in their promoter regions.
OLE1 appears to be induced maximally by hypoxia as opposed
to anoxia; a genome-wide transcriptional analysis of aerobic and anaerobic chemostat cultures of S. cerevisiae demonstrated
that OLE1 has only a marginal (1.3-fold) increase under
stringent anaerobic conditions (57). Thus, OLE1 represents a
yeast gene that is regulated by hypoxia, certain transition metals
(15), and iron chelation, strikingly similar to several
hypoxia-inducible mammalian genes, such as erythropoietin, vascular
endothelial growth factor, the glucose transporters GLUT1 and GLUT3,
and several glycolytic enzymes. Given the importance of the ability to
adapt to hypoxic stress throughout evolution, it is not surprising to
find yeast and mammalian genes which are similarly regulated. Studies
of several hypoxia-inducible mammalian genes have led to the
identification of a hypoxia responsive element (58-61) and the
heterodimeric hypoxia-inducible factor HIF-1 which binds to it (62).
Although functionally the LORE and hypoxia responsive element are
similar, sequence analysis does not reveal any similarity. Differences
with respect to CoCl2-mediated induction of LORE-containing
yeast genes provide additional support to the notion that additional
factors most likely exist in S. cerevisiae oxygen-sensing
pathways which may not exist in mammalian systems. Likewise, HIF-19 fatty acid desaturase gene OLE1 in
Saccharomyces cerevisiae. We have confirmed previous data
showing that the expression of OLE1 mRNA is increased
in hypoxia and in the presence of certain transition metals.
OLE1 expression was also increased in the presence of the
iron chelator 1,10-phenanthroline. A 142-base pair (bp) region 3' to
the previously identified fatty acid response element was identified as
critical for the induction of OLE1 in response to these
stimuli using OLE1 promoter-lacZ reporter
constructs. Electromobility shift assays confirmed the presence of an
inducible band shift in response to hypoxia and cobalt. Mutational
analysis defined the nonameric sequence ACTCAACAA as necessary for
transactivation. A 20-base pair oligonucleotide containing this nonamer
confers up-regulation by hypoxia and inhibition by unsaturated fatty
acids when placed upstream of a heterologous promoter in a
lacZ reporter construct. Additional yeast genes were
identified which respond to hypoxia and cobalt in a manner similar to
OLE1. A number of mammalian genes are also up-regulated by
hypoxia, cobalt, nickel, and iron chelators. Hence, the identification
of a family of yeast genes regulated in a similar manner has
implications for understanding oxygen sensing and signaling in eukaryotes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and
subunit, both of which are basic helix-loop-helix proteins in
the PAS family of transcription factors. The previously identified
subunit is ARNT, the aryl hydrocarbon receptor nuclear translocator.
ARNT mRNA and protein levels are not significantly affected by
ambient oxygen tension. In contrast, although HIF-1
mRNA levels
are not appreciably affected by oxygen tension, HIF-1
protein is
only minimally present in normoxia, being rapidly degraded by the
ubiquitin-proteasome pathway in this condition. However, HIF-1
accumulates in response to hypoxia, certain transition metals
(e.g. cobalt and nickel), and iron chelators such as
desferrioxamine. Once the heterodimer is formed under these conditions,
it can interact with other DNA-binding proteins (such as HNF4) which function in part to provide tissue and developmental specificity (reviewed in Refs. 1 and 2). HIF-1 has also been shown to associate
with scaffold proteins such as p300/CBP (5), which have been shown to
interact with the basal transcription machinery (6, 7). The
interactions between the proteins in this complex have been the focus
of several investigators, and have been reviewed previously (reviewed
in Ref. 2).
exists, the mechanism by which mammalian cells sense hypoxia
remains poorly defined. In certain prokaryotes, a heme protein has been
shown to serve as an oxygen sensor (reviewed in Refs. 1 and 8).
Evidence supports the role for a heme protein in mammalian oxygen
sensing as well (9). Certain transition metals such as cobalt and
nickel appear to mimic hypoxia and induce several hypoxia-regulated
genes. It has been proposed that these metals may replace iron in the
heme moiety of such a protein, thereby altering its conformation to a
"deoxy" state (9). Alternatively, these transition metals may
disrupt the production of reactive oxygen species which may serve as
important signaling molecules in the oxygen response pathway (reviewed
in Refs. 1 and 10). A recent study provided evidence against metal
substitution in heme (11).
has been shown to be sensitive to these perturbations in
intracellular oxygen-free radicals (12). Finally, carbon monoxide
inhibits the expression of hypoxia-induced genes and HIF-1
activation. Carbon monoxide can bind reversibly to heme proteins, and
may lead to a change in conformation to the "oxy" state (12). A
novel human cytosolic flavohemoprotein with cytochrome
b5 and b5 reductase
domains and functional NAD(P)H oxidoreductase activity has recently
been cloned (13). This protein fulfills all of the criteria for an
oxygen sensor, although definitive data in this regard remains lacking.
9 fatty acid
desaturase and is critical for unsaturated fatty acid biosynthesis.
Incubation in the presence of cobalt or nickel leads to increased
expression of OLE1, mimicking mammalian hypoxia-regulated
genes (15). In this study, we have further defined the regulation of
OLE1 by hypoxia, certain transition metals, and iron
chelation. The cis element responsible for hypoxia induction
of OLE1 expression was identified and characterized using
reporter gene studies and electromobility shift assays. This low oxygen
response element, or LORE, was used to search the S. cerevisiae genome. This search led to the identification of a
family of yeast genes regulated in a similar manner. Taken together,
the evidence presented supports the hypothesis that critical aspects of
the oxygen sensing mechanism are highly conserved among all eukaryotes.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-galactopyranoside was obtained from ICN
Biochemicals Inc. or Sigma. Radiolabeled compounds were purchased from
PerkinElmer Life Sciences. Formamide, dextran sulfate, and Denhardt's
solution were bought from American Bioanalytical. Acrylamide,
bisacrylamide, TEMED, and protein molecular mass markers were from
Bio-Rad. Ammonium sulfate, phenylmethylsulfonyl fluoride, CoCl2, NiCl2, 1,10-phenanthroline, and Nonidet
P-40 were obtained from Sigma. SeaKem ME-agarose was from FMC
Bioproducts. T4 polynucleotide kinase and dNTPs were purchased from
Promega Corp. Shrimp alkaline phosphatase and Taq polymerase
were purchased from Roche Molecular Biochemicals; other restriction
enzymes were from New England BioLabs. All enzymes were used according
to the manufacturer's instructions.
Oligonucleotides used in this study
347 to
328 relative to the ATG translational
start codon with the A of the codon designated as +1) was performed by
inserting the synthetic paired oligonucleotides (10-5' and 10-3') into
the XhoI restriction site of pTBA30, the CYC1
basal promoter-lacZ fusion vector obtained from Dr. A. Vershon. pAM16 contains one LORE copy in the
347 to
328 forward or
(+) orientation 5' to the basal CYC1
promoter-lacZ fusion. pAM7 has one LORE copy in the
328 to
347 reverse or (
)-orientation. pAM6 contains a tandem repeat of the
LORE in the (+)-orientation. pAM10 was generated by inserting the
synthetic paired oligonucleotides (yd-19 and yd-20) into the XhoI restriction site of pTBA30. The LORE in this plasmid
has three mutations. Plasmid pAM4 is the p62::934 derivative
with three nucleotide substitutions in the LORE region (-C342T, -T341A, and -A339G) prepared utilizing three-step PCR with oligonucleotides containing site-directed mutations. Two PCR reactions with appropriate pairs of mutant primers (PCR1: yd-8 and yd-20 and PCR2: yd-19 and
lacZ-3') and Taq DNA polymerase were carried out as
recommended by Roche Molecular Biochemicals in 100-µl reactions,
using 1 ng of p62::934 as a template in a PTC-100 thermal
cycler (MJ Research, Inc., Watertown, MA) for 30 cycles (1 min at
94 °C, 1 min at 55 °C, and 1 min at 72 °C), followed by 7 min
at 72 °C. The PCR products were purified from a 1.0% agarose gel by
using a Qiaex DNA extraction kit (Qiagen, Chatsworth, CA). Then, PCR
products 1 and 2 were annealed, and amplified as above with primer yd-8
and lacZ-3'. The resulting PCR products were purified as above and
digested with restriction enzyme HindIII and
SalI. Finally this PCR product was cloned into
HindIII and SalI pretreated p62::934
plasmid to generate pAM4. All the constructs were verified by DNA
sequencing. The promoter deletion constructs were transformed into
wild-type strain RZ53-6 for subsequent
-galactosidase assays.
Yeast (S. cerevisiae) strains and plasmids
-galactosidase assays. Plasmid amplifications and bacterial transformations were performed using Escherichia coli strain DH5 (Invitrogene Corp.). Yeast
transformations were performed by the method of Elble (18). Preparative
cultures were grown aerobically in a shaker at 200 rpm (Innova 4000 incubator shaker, New Brunswick Scientific) at 30 °C to
mid-logarithmic phase. For experiments assessing yeast under hypoxic
conditions, mid-logarithmic phase preparative cultures were used to
inoculate special air-tight flasks with inlet and outlet ports to allow for equilibration with the appropriate gas mixtures. Cultures were
exposed to a continuous flow of hydrated medical grade nitrogen (BOC
Gases, Murray Hill, NJ), unless otherwise specified, for 6 h after inoculating the medium via the inlet port. Of note, medical
grade nitrogen is contaminated with trace amounts of O2 (less than 1%). The percentage of saturated O2 in each
flask was confirmed by using an oxygen monitor (G. C. Industries,
Inc.) attached to the outlet port of each culture. For experiments
assessing yeast exposed to cobalt, cobalt chloride was added to the
cultures at a concentration of 400 µM unless otherwise
noted. Cells were exposed to cobalt for a period of 6 h prior to
harvesting. The flasks contained a volume of medium that was ~1/5 the
flask volume. All experiments were performed with yeast in logarithmic
growth phase in a shaker (200 rpm) at 30 °C. Growth was monitored by measuring the yeast A600 at the completion of
each experiment.
-Galactosidase Assays--
Assays of cells containing
plasmids derived from the OLE1 promoter-lacZ
fusion p62 constructs were performed as described previously (19). Cell
densities for these assays were determined by measurement at
A600. Transformants were assayed for each of the
plasmid constructs listed in Table II.
-Galactosidase activities reported here are the results of at least two independent experiments. Each experimental assay was performed in quadruplicate.
-mercaptoethanol) and stored frozen at
80 °C. The soluble protein concentration was
determined using a Bradford dye binding assay (Bio-Rad).
397 to
234) as the primers. Synthetic paired oligonucleotides were end
labeled using polynucleotide kinase and purified using a Sephadex G-25
spin column (Roche Molecular Biochemicals) to remove unincorporated nucleotide. Probes made by PCR were purified away from labeled primers,
[
-32P]ATP and Taq polymerase using a
QIAquick spin PCR purification kit (Qiagen). The concentration of each
probe was determined on an ethidium bromide-stained agarose-mini gel
(22). In each reaction 10-20 ng of probe was used. Binding reactions
were 40 µl of buffer H (25 mM HEPES, pH 7.5, at room
temperature, 0.5 mM EDTA, 0.5 mM
dithiothreitol, 0.5 mM MgCl2, 1 mM
CaCl2, 50 mM NaCl, 7% glycerol, 1% Nonidet
P-40, 15 ng/µl poly(dA-dT)) for 20 min at room temperature. Proteins
were diluted into binding buffer on ice immediately before use.
Reactions were loaded on 5% acrylamide gels (29:1), 0.5 × TBE,
and run for 3 h at 4 °C at 15 V/cm. Gels were dried and exposed to X-Omat AR film (Kodak) to visualize the shifted bands.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Galactosidase Reporter Assay--
Previous studies
have shown that OLE1 mRNA transcript levels are
increased at low oxygen (O2) tensions, below 0.5 µmol.
Maximal expression, approaching a 4-fold increase over baseline
normoxia, was observed following 8-10 h of anoxia (15, 26). First, we confirmed that OLE1 mRNA is maximally expressed in the
presence of trace O2 (<1%) concentrations (data not
shown). Subsequently, we extended this finding utilizing a plasmid
(p62::934) in which 934 bp of the OLE1 promoter is
fused in-frame with the lacZ gene. Less than a 2-fold
induction over baseline levels in normoxia was observed in 1%
O2 although a 6-fold induction occurred at an extremely low
O2 tension (data not shown). In a similar fashion, we have
verified previous studies demonstrating increased levels of
OLE1 mRNA following incubation of S. cerevisiae with increasing concentrations of cobalt chloride
(CoCl2) and nickel chloride (NiCl2) in normoxia
(15) (data not shown). The concentrations were similar to those used in
the study of hypoxia-regulated mammalian genes such as erythropoietin
(9). These data were confirmed with experiments utilizing the
OLE1 promoter-lacZ reporter assay. The degree of
induction approached that which has been previously reported (15), even
though the metal concentrations were almost 10-fold lower. At 800 µM CoCl2 and 450 µM
NiCl2, significant differences in the growth rate of the
yeast compared with control cultures were observed in our experiments,
presumably due to direct toxicity of the culture by the metal on
S. cerevisiae. It is not immediately apparent why the
significantly lower metal concentrations used in the present studies
appear to exert the same effect on OLE1 expression as the
much higher concentrations employed in previous reports (15);
differences in exposure time may be relevant.
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Fig. 1.
OLE1 expression can be induced by
1,10-phenanthroline. A, Northern blot analysis under
1,10-phenanthroline (1,10-Ph)-treated conditions. Total RNA
was extracted from yeast cells treated with increasing concentrations
of 1,10-phenanthroline (4, 6, and 8 µg/ml) for 6 h. The RNA blot
was first hybridized with a specific OLE1 probe, stripped,
and rehybridized with a control PGK1 probe. N
indicates normoxic conditions. In the diagram OLE1 and
PGK1 indicate OLE1 and PGK1 mRNAs,
respectively. B, quantitation of OLE1 mRNA of
the Northern blot in A. Signal intensity was quantitated
with a Molecular Dynamics PhosphorImager. Transcript levels were
normalized to the level of PGK1 mRNA and are presented
as 1 relative units for the OLE1 mRNA extracted from the
yeast cell grown without 1,10-phenanthroline.
lacZ Constructs Define a 142-bp
Region (
255 to
396 Relative to the Transcription Start Site)
Critical for Induction by Hypoxia and CoCl2--
A series
of OLE1 promoter-lacZ fusion reporter constructs
were transformed into the RZ53-6 strain and incubated in hypoxia, CoCl2, or 1,10-phenanthroline. The
-galactosidase
activities of these reporter constructs are shown in Fig.
2. The removal of bases
567 through
488 resulted in an 80-fold drop in enzyme activity under normoxia,
suggesting the presence of an activating sequence in this region.
Previous work has identified the fatty acid-regulated element in this
region (16). In contrast, the deletion of sequences from
934 to
567
produced a small reduction in reporter gene expression under normoxia,
hypoxia, and cobalt-treated conditions (within 2.7-fold). Deletions 3'
to base
488 resulted in low basal reporter gene activities under
normoxic conditions. However, removal of bases
488 through
396 did
not dramatically affect the hypoxia- and cobalt-induced reporter gene
expression. In contrast, the 142-bp region between
396 and
255
proved to be critical. Its removal essentially abolished the hypoxia
induction and caused a significant reduction of
CoCl2-induced reporter gene expression. The observation
that the deletion of sequence between
567 and
488 also resulted in
about a 2-fold reduction of CoCl2-induced reporter gene
expression implies that additional regulatory elements necessary for
complete CoCl2-induced OLE1 gene expression may reside in this region.
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Fig. 2.
Deletions of the OLE1
promoter-lacZ fusion constructs and their
activities in different conditions. Deletion constructs are shown
on the left. The narrow line represents the
OLE1 promoter sequence. The numbers above each
narrow line indicate the position of the deletion end point
with respect to the ATG start codon of the wild-type base sequence with
the A of the codon designated as +1. The solid black bar
represents the amino-terminal 27 amino acids of the OLE1
coding sequence fused to E. coli lacZ (open bar).
The sequence shadowed vertically indicates the region important for
hypoxia-induced gene expression. N, H, and
CoCl2 to the right of the diagram represent the
-galactosidase activities in normoxic, hypoxic, and cobalt-treated
conditions, respectively. The
-galactosidase activity of
p62::934 in normoxia is designated as 100. Units of activity
indicated are the average of at least three independent experiments
that were performed in quadruplicate. Numbers in parentheses
are the fold induction in either H or CoCl2 compared with
N.
255 to
396 region of the
OLE1 promoter contains a cis element responsible
for hypoxic induction. The possibility of an activation complex formed
in hypoxic conditions was tested in EMSA using crude cell extracts from
normoxia, hypoxia-, and cobalt-treated yeast. A wild type
OLE1 promoter DNA fragment containing base pairs
234 to
396 was generated using PCR as a probe for the assay. Fig.
3 shows that two shifted bands (B1 and
B3) were present in hypoxia and cobalt, although the
CoCl2-treated extracts were of somewhat lower intensity.
Band B2 was constitutive and could be displaced by nonspecific DNA
(data not shown). To further define the DNA region responsible for the
hypoxia-inducible complex formation, a series of double stranded
nucleotides 20 base pairs in length covering the entire
255 to
396
region of OLE1 DNA were synthesized and used as cold
competitors for the hypoxia-inducible shifted bands (B1 and B3) in
EMSA. A double-stranded oligonucleotide 10-5'/10-3' (Table I, number
10) could effectively compete out the shifted bands as shown in Fig. 3,
whereas the remainder of the double-stranded oligonucleotides from
1-5'/1-3' (number 1) to 14-5'/14-3' (number 14) could not. Fig. 3 is a
representative gel containing paired oligonucleotides numbers 8, 9, 10, and 11. Therefore, oligonucleotide 10 appears to contain a site(s) for hypoxia- and CoCl2-induced protein(s) binding and was
designated as the LORE.
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Fig. 3.
A representative EMSA gel for
hypoxia-inducible complex formation in the OLE1
promoter. 10 µg of crude extract (CE) from
normoxia (N)-, hypoxia (H)-, and cobalt
(C)-treated yeast as described in the legend to Fig. 2 were
incubated for 20 min at 25 °C with a 32P-labeled PCR
fragment comprising the OLE1 promoter sequence from 396 to
234 and then subjected to electrophoresis at 4 °C for ~3 h. Free
probe (F) and bound complexes (B1, B2, and
B3) were detected by autoradiography. Lanes marked
indicate no CE or cold DNA competitor was added; + indicates CE added;
P represents the DNA probe; the increased amount of cold
competitor (10-, 20-, and 50-fold excess of the probe) is shown.
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Fig. 4.
Hypoxia inducible complex formation in
347 to
328 region of the OLE1 promoter. 10 µg of crude extract (CE) from normoxia (N)-,
hypoxia (H)-, and cobalt (C)-treated yeast were
incubated with 32P-end-labeled paired oligonucleotides
#10, m#10, and #8, and subjected to
electrophoresis as described in Fig. 3. Lanes marked
indicate
no CE was added; + indicates CE added; P represents DNA
probe.
328 to
347). The requirement of an intact LORE
for hypoxia-induced OLE1 expression was tested using the
-galactosidase assay in yeast containing the pAM4 reporter. In this
reporter assay, an 8-fold decrease in the basal level of expression in
normoxia compared with the nonmutated LORE reporter was observed. The
mutated LORE sequence eliminated the 6-fold hypoxic induction seen with
the wild type reporter. This result is consistent with EMSA data, which
reveal no hypoxia-induced band shift when the same mutated fragment was
utilized as a probe (data not shown). However, the CoCl2-dependent induction by reporter assay was
not affected (data not shown).
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Fig. 5.
Expression of reporter activity from the
heterologous pTBA vector containing the LORE. The diagram on the
left indicates the LORE (vertical solid bar)
inserted into the heterologous constructions employing the yeast
CYC1::lacZ gene fusion plasmids. The small
arrow in the vertical bar indicates the orientation of
the inserted LORE. The horizontal blank and solid
bars represent the CYC1::lacZ gene fusion.
The diagram on the right indicates the normalized
-galactosidase activity under normoxic (N)-, hypoxic
(H)-, and cobalt (C)-treated conditions. The
number in parentheses indicates the fold of
induction relative to N. The actual
-galactosidase units for
normoxia samples are: pTBA30, 0.028; pAM6, 1.06; pAM10, 0.045; pAM16,
0.041; pAM7, 0.813, which are the average of two independent assays
performed in quadruplicate.
466 to
576) within the OLE1 promoter
contribute to OLE1 repression by UFA in normoxic conditions
(16). However, when an OLE1-lacZ fusion containing a deleted
FAR element was tested under hypoxia, the transactivation repression by
UFA was still observed (data not shown). That observation led us to
examine whether the LORE plays a role in UFA induced OLE1
gene repression. Fig. 6A shows
that the UFA linoleic acid can strongly repress normoxia-, hypoxia-, and cobalt-induced expression of the lacZ reporter plasmid
pAM6 which contains two copies of the LORE in tandem. Inhibition showed a dose-response with an IC50 ~20 µM.
Similar dose-response inhibition was obtained using the unsaturated
fatty acids
-linolenic acid, oleic acid, and arachidonic acid but
not with the saturated fatty acid steric acid (data not shown).
Consistent with the
-galactosidase assay, the OLE1
mRNA level was also dramatically repressed by linoleic acid as
shown in Fig. 6B by Northern blot analysis. This repression
in normoxia is consistent with previous studies (16). Again, UFA
repression could not be overcome by incubation in hypoxic or
cobalt-containing conditions. Crude extracts from linoleic acid-treated
yeast were utilized for EMSA. As shown in Fig. 6C, the
hypoxia-induced LORE complex formation was significantly suppressed with disappearance of binding complex B1. The intensity of the basal
expression of the B1 complex under normoxic condition was also
repressed, implying that LORE may be involved in the basal expression
of OLE1 as well. The nonspecific band B2 was not
affected.
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Fig. 6.
The effects of unsaturated fatty acid on
OLE1 gene expression under hypoxic conditions. In
all three panels, N, H, and C indicate yeast
grown under normoxic-, hypoxic-, and cobalt-treated conditions as
described under "Materials and Methods." E and EtOH
represent the growth medium containing 1% ethanol; L.A.
indicates the growth medium containing 1 mM linoleic acid
dissolved in 1% ethanol. Panel A, histogram of
-galactosidase activity of reporter pAM6 under varying conditions.
The units of activity are the average of two independent assays.
Panel B, Northern blot assay of OLE1 gene
expression under varying conditions. ACT1 cDNA probe was
prepared using PCR (see "Materials and Methods"). In the diagram
OLE1 and ACT1 indicate OLE1 and ACT1 mRNAs,
respectively. Panel C, EMSA for the crude extracts from
yeast grown under different conditions. + indicates CE added;
indicates no CE or EtOH or linoleic acid added. P represents
DNA probe. On the right side of this panel, B1
and B2 indicate the specific and nonspecific shifted bands,
respectively; F represents the free probe.
rox1) was,
therefore, utilized to investigate the ROX1 effects on
OLE1 expression under hypoxic conditions. The results of
in vivo
-galactosidase assays of reporter p62::934 in strains RZ53-6 and RZ53-6
rox1 show that the
basal expression of the reporter gene under normoxic conditions is
essentially the same in the ROX1 deletion strain as in its
parental strain (data not shown). This suggests that ROX1 does not play
a role as a repressor in the low basal level expression of
OLE1 under normoxic conditions. Moreover, significant
induction was still observed under hypoxic conditions (4.2-fold
increase) and cobalt-treated conditions (5.1-fold increase) in the
ROX1 deletion strain. In the Northern blot analysis the
OLE1 mRNA level was induced in both strains under
hypoxia- and cobalt-treated conditions. Consistent with the in
vivo transactivation data, it was also shown that the relative
mRNA levels of hypoxia- and cobalt-treated ROX1-deleted yeast were similar to that of wild-type. The basal OLE1
mRNA expression under normoxia was similar in both strains as well
(data not shown). Another hypoxic gene ATF1 (see later
"Results") showed similar results.
343 and
335. We
have, therefore, designated this nonameric sequence (ACTCAACAA) as the
DNA-binding core of the LORE.
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Fig. 7.
A, summary of the DNA binding ability of
mutant LOREs. The first row shows the wild type (WT) LORE
sequence. G347T to T328G are examples of single nucleotide mutations in
the LORE at various positions. Values for DNA binding represent binding
activities of the LORE-binding factor(s) to the various mutant sites.
Values represent percentages relative to the wild type LORE in an EMSA,
quantitated with a PhosphorImager; * indicates altered EMSA pattern.
The nucleotides shadowed (vertically) indicate the positions important
for hypoxia induced complex formation in vitro.
B, representative EMSA gel for mutant LOREs altered in DNA
binding. Increasing amounts of crude extract (5, 10, and 50 µg)
(CE) from normoxic (N)- and hypoxic
(H)-treated yeast were incubated for 20 min at 25 °C with
a 32P-labeled wild type and mutant LOREs containing the
OLE1 promoter sequence from 347 to
328. EMSA assays and
autoradiography were performed as described in the legend to Fig.
3.
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Fig. 8.
A, representative genes containing
potential LOREs in their promoter region. Names of genes are listed on
the left. The core sequences are shadowed in the center of
the sequence. B, a representative EMSA for potential LOREs.
10 µg of crude extract (CE) from normoxic (N)-
and hypoxic (H)-treated yeast were incubated for 20 min at
25 °C with 32P-labeled wild type (OLE1) and
potential LOREs from promoter of the genes indicated, then subjected to
electrophoresis in the cold for ~3 h. Pairs of oligonucleotides for
putative LORE sequences were used as indicated in A. The
lane marked indicate no CE was added; the increased
amount of CE (5, 10, and 50 µg) was shown by the triangle.
Free probe (F), specific bound complex B1, and nonspecific
bound complex B2 were detected by autoradiography. C,
Northern blot analysis of several hypoxia- and cobalt-inducible yeast
genes. Certain genes containing a putative LORE in their promoter
region were tested for hypoxia- and cobalt-induced gene expression by
Northern blot analysis. Cells were grown to midlogarithmic phase and
total RNA was extracted as described under "Materials and Methods."
The RNA blot was first probed with one specific probe and then after
the blot was stripped and reprobed with another. The SUT1,
TRX2 cDNA probes were prepared using PCR (see
"Materials and Methods"). In the figure OLE1, ATF1, TRX2, SUT1,
ACT1, and PGK1 indicate OLE1, ATF1, TRX2, SUT1, ACT1, and PGK1
mRNAs, respectively. PhosphorImages of the resulting blots are
shown in the figure.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-9 fatty acid desaturase, an enzyme
involved in the formation of unsaturated fatty acids. This enzyme
introduces a double bond between carbons 9 and 10 of substrate
palmitoyl (16:0) or stearoyl (18:0)-CoA with molecular O2
serving as an electron acceptor to form palmitoleic (16:1) or oleic
(18:1) acid, respectively. Previous studies have demonstrated that
OLE1 is up-regulated under hypoxic conditions. Its induction
under hypoxic conditions may be in response to the limitation of
O2 as a substrate.
130,
260, and
272 relative to the first nucleotide of the translation start codon
(28, 30, 31). Previous studies suggested that OLE1 is only
slightly derepressed in a ROX1 disruptant under aerobic
conditions (31, 48). The results from the current study confirm that
ROX1 does not play a significant role in the basal aerobic
expression of the OLE1 gene. There were no significant changes in either reporter gene or OLE1 mRNA expression
in a S. cerevisiae strain in which the ROX1 gene
had been disrupted. Moreover, both hypoxia- and cobalt-induced
OLE1 gene expression was observed in this strain; the
expression of the ROX1-mediated anaerobic yeast gene
ANB1 was not up-regulated by cobalt. Of note, the hypoxic induction in the ROX1 deletion strain was not as dramatic as
its parental strain (4.2-fold versus 9.1-fold increase) in
the reporter assay although the cobalt induction in the same strain was
similar (4.2-fold versus 5.1-fold increase). This suggests
that ROX1 may be involved in the OLE1 hypoxic
induction pathway in some capacity; however, our data provide no
compelling evidence that ROX1p serves as a repressor of OLE1
expression in normoxia.
344 to
325) is not known. Another gene, SUT1,
encodes a small molecule transporter, and is up-regulated in anaerobic conditions to facilitate the uptake of sterols when sterol biosynthesis is blocked by the absence of oxygen (34). The in vivo
significance of a putative LORE found in the SUT1 promoter
region (
377 to
358 relative to the translational start codon), is
also unclear at this point. Yet another gene identified in the genomic
search for potential LOREs in promoter regions is the TRX2
gene, which encodes a small sulfydryl-rich protein thioredoxin. About a
3-fold hypoxic and 2.5-fold cobalt induction of TRX2
mRNA was observed in Northern blot analysis (Fig. 8C).
Confirming these data, in vitro EMSA demonstrated that the
potential LORE obtained from the TRX2 promoter sequence
could indeed form a hypoxia-specific complex. Nonetheless, the role of
hypoxic induction of TRX2 in vivo still requires further
investigation. The precise physiological function of thioredoxin is not
clearly understood; it appears to be part of a cellular nonenzymatic
defense system in response to oxidative stress (reviewed in Ref. 54).
Evidence for the involvement of reactive oxygen species in mammalian
oxygen sensing and signaling exists (reviewed in Refs. 1 and 2). HIF-1, the mediator of physiological and pathophysiological responses to
hypoxia, is subject to complex redox control mechanisms (Refs. 12 and
55, reviewed in Ref. 56). Therefore, it would not be surprising to
identify reactive oxygen species involved in oxygen sensing and
signaling in yeast.
homologues have been identified in several organisms, from mouse and
rat (63, 64) to Drosophila melanogaster (65-68), although
none has been found in S. cerevisiae. Further work to
identify and characterize the LBP and investigate its regulation and
relationship to the mammalian O2 sensing pathway remains of
high interest. HIF-1
is primarily regulated by the ubiquitin-proteasome degradation pathway (13, 69, 70) and studies
examining the role of this pathway in the regulation of the LBP are
ongoing. Understanding the nature of the LBP(s) will likely provide
significant insights into the molecular mechanisms which regulate
cellular responses to hypoxic stress in lower and higher eukaryotic species.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank H. Franklin Bunn and Fred Winston
for invaluable support through all phases of this project. We also
thank Steve Hanes and Robert O. Poyton for helpful discussions. We
thank Richard S. Zitomer for the gifts of RZ53-6 and RZ53-6 rox1 and
Andrew K. Vershon for the pTBA30 plasmid. We thank Andrew Mitchell for preparation of a series of OLE1 promoter-lacZ
fusion reporter constructs.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institues of Health Grants DK45098 (to M. A. G.), GM45768 (to C. E. M.), and K08 HL03599 (to M. J. V.).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.
§ Contributed equally to the results of this work.
¶ Present address: Dept. of Adult Oncology, Dana-Farber Cancer Institute, Dept. of Medicine, Brigham and Women's Hospital, and Harvard Medical School, Boston, MA 02115.
Present address: Hematology-Oncology Div., University of
Pennsylvania Medical Center, Philadelphia, PA 19104.
To whom correspondence should be addressed: Hematology Div.,
Dept. of Medicine, Brigham & Women's Hospital, Harvard Medical School,
221 Longwood Ave., Boston, MA 02115. Tel.: 617-732-5841; Fax:
617-739-0748; E-mail: Mark.Goldberg@genzyme.com.
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M009546200
2 M. Vasconcelles, unpublished data.
3 J. M. Cherry, C. Ball, K. Dolinski, S. Dwight, M. Harris, J. C. Matese, G. Sherlock, G. Binkley, H. Jin, S. Weng, and D. Botstein, unpublished info.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: HIF-1, hypoxia inducible factor-1; LORE, low oxygen response element; PCR, polymerase chain reaction; EMSA, electrophoretic mobility shift assay(s); FAR element, fatty acid-regulated element; UFA, unsaturated fatty acid; LBP, low oxygen response element-binding protein; bp, base pair(s); TEMED, N,N,N',N'-tetramethylethylenediamine.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Bunn, H. F.,
and Poyton, R. O.
(1996)
Physiol. Rev.
76,
839-885 |
2. |
Ebert, B. L.,
and Bunn, H. F.
(1999)
Blood
94,
1864-1877 |
3. |
Levy, A. P.,
Levy, N. S.,
Wegner, S.,
and Goldberg, M. A.
(1995)
J. Biol. Chem.
270,
13333-13340 |
4. | Semenza, G. L., and Wang, G. L. (1992) Mol. Cell. Biol. 12, 5447-5454[Abstract] |
5. |
Arany, Z.,
Huang, L. E.,
Eckner, R.,
Bhattacharya, S.,
Jiang, C.,
Goldberg, M. A.,
Bunn, H. F.,
and Livingston, D. M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
12969-12973 |
6. |
Yuan, W.,
Condorelli, G.,
Caruso, M.,
Felsani, A.,
and Giordano, A.
(1996)
J. Biol. Chem.
271,
9009-9013 |
7. | Kwok, R. P., Lundblad, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., Brennan, R. G., Roberts, S. G., Green, M. R., and Goodman, R. H. (1994) Nature 370, 223-226[CrossRef][Medline] [Order article via Infotrieve] |
8. | Gilles-Gonzalez, M. A., Ditta, G. S., and Helinski, D. R. (1991) Nature 350, 170-172[CrossRef][Medline] [Order article via Infotrieve] |
9. | Goldberg, M. A., Dunning, S. P., and Bunn, H. F. (1988) Science 242, 1412-1415[Medline] [Order article via Infotrieve] |
10. |
Huang, L. E.,
Arany, Z.,
Livingston, D. M.,
and Bunn, H. F.
(1996)
J. Biol. Chem.
271,
32253-32259 |
11. | Horiguchi, H., and Bunn, H. F. (2000) Biochim. Biophys. Acta 1495, 231-236[Medline] [Order article via Infotrieve] |
12. |
Huang, L. E.,
Willmore, W. G.,
Gu, J.,
Goldberg, M. A.,
and Bunn, H. F.
(1999)
J. Biol. Chem.
274,
9038-9044 |
13. |
Zhu, H.,
Qiu, H.,
Yoon, H. W.,
Huang, S.,
and Bunn, H. F.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14742-14747 |
14. | Zitomer, R. S., and Lowry, C. V. (1992) Microbiol. Rev. 5, 1-11 |
15. |
Kwast, K. E.,
Burke, P. V.,
Staahl, B. T.,
and Poyton, R. O.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5446-5451 |
16. |
Choi, J. Y.,
Stukey, J.,
Hwang, S. Y.,
and Martin, C. E.
(1996)
J. Biol. Chem.
271,
3581-3589 |
17. | Treco, D. A., and Winston, F. (1997) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struhl, K., eds) , pp. 13.2.1-13.2.12, John Wiley & Sons, New York |
18. | Elble, R. (1992) BioTechniques 13, 18-20[Medline] [Order article via Infotrieve] |
19. | Reynolds, A., Lundblad, V., Dorris, D., and Keaveney, M. (1997) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struhl, K., eds) , pp. 13.6.2-13.6.3, John Wiley & Sons, New York |
20. | Pfeifer, K., Arcangioli, B., and Guarente, L. (1987) Cell 49, 9-18[Medline] [Order article via Infotrieve] |
21. | Carey, J. (1991) Methods Enzymol. 208, 103-117[Medline] [Order article via Infotrieve] |
22. | Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual , Cold Sping Harbor Laboratory Press, Cold Spring Harbor, NY |
23. | Collart, M. A., and Oliviero, S. (1997) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struhl, K., eds) , pp. 13.12.1-13.12.5, John Wiley & Sons, New York |
24. | Brown, T., and Mackey, K. (1997) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struhl, K., eds) , pp. 4.9.1-4.9.14, John Wiley & Sons, New York |
25. | Hoffman, C. S. (1997) in Current Protocols in Molecular Biology (Ausubel, F. M. , Brent, R. , Kingston, R. E. , Moore, D. D. , Seidman, J. G. , Smith, J. A. , and Struhl, K., eds) , pp. 13.11.2-13.11.4, John Wiley & Sons, New York |
26. |
Kwast, K. E.,
Burke, P. V.,
and Poyton, R. O.
(1998)
J. Exp. Biol.
201,
1177-1195 |
27. |
Bossier, P.,
Fernandes, L.,
Rocha, D.,
and Rodrigues-Pousada, C.
(1993)
J. Biol. Chem.
268,
23640-23645 |
28. |
Deckert, J.,
Torres, A. M.,
Hwang, S. M.,
Kastaniotis, A. J.,
and Zitomer, R. S.
(1998)
Genetics
150,
1429-1441 |
29. | Zitomer, R. S., Limbach, M. P., Rodriguez-Torres, A. M., Balasubramanian, B., Deckert, J., and Snow, P. M. (1997) Methods 11, 279-288[CrossRef][Medline] [Order article via Infotrieve] |
30. | Zitomer, R. S., Carrico, P., and Deckert, J. (1997) Kidney Int. 51, 507-513[Medline] [Order article via Infotrieve] |
31. |
Stukey, J. E.,
McDonough, V. M.,
and Martin, C. E.
(1990)
J. Biol. Chem.
265,
20144-20149 |
32. | van Helden, J., André, B., and Collado-Vides, J. (1998) J. Mol. Biol. 281, 827-842[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Salceda, S.,
and Caro, J.
(1997)
J. Biol. Chem.
272,
22642-22647 |
34. | Bourot, S., and Karst, F. (1995) Gene (Amst.) 165, 97-102[CrossRef][Medline] [Order article via Infotrieve] |
35. | Donzeau, M., Bourdineaud, J. P., and Lauquin, J. (1996) Mol. Microbiol. 20, 449-459[Medline] [Order article via Infotrieve] |
36. | Fujiwara, D., Kobayashi, O., Yoshimoto, H., Harashima, S., and Tamai, Y. (1999) Yeast 15, 1183-1197[CrossRef][Medline] [Order article via Infotrieve] |
37. | Kitagaki, H., Shimoi, H., and Itoh, K. (1997) Eur. J. Biochem. 249, 343-349[Abstract] |
38. |
Hassett, R. F.,
Romeo, A. M.,
and Kosman, D. J.
(1998)
J. Biol. Chem.
273,
7628-7636 |
39. | Conrad, M. N., Wright, J. H., Wolf, A. J., and Zakian, V. A. (1990) Cell 63, 739-750[Medline] [Order article via Infotrieve] |
40. | Shore, D. (1994) Trends Genet. 10, 408-412[CrossRef][Medline] [Order article via Infotrieve] |
41. | Shore, D., and Nasmyth, K. (1987) Cell 51, 721-732[Medline] [Order article via Infotrieve] |
42. | Scott, E. W., and Baker, H. V. (1993) Mol. Cell. Biol. 13, 543-550[Abstract] |
43. | Balasubramanian, B., Lowry, C. V., and Zitomer, R. S. (1993) Mol. Cell. Biol. 13, 6071-6078[Abstract] |
44. |
Deckert, J.,
Perini, R.,
Balasubramanian, B.,
and Zitomer, R. S.
(1995)
Genetics
139,
1149-1158 |
45. | Williams, F. E., Varanasi, U., and Trumbly, R. J. (1991) Mol. Cell. Biol. 11, 3307-3316[Medline] [Order article via Infotrieve] |
46. | Lowry, C. V., and Lieber, R. H. (1986) Mol. Cell. Biol. 6, 4145-4148[Medline] [Order article via Infotrieve] |
47. | Zhang, M., Rosenblum-Vos, L. S., Lowry, C. V., Boakye, K. A., and Zitomer, R. S. (1991) Gene (Amst.) 97, 153-161[CrossRef][Medline] [Order article via Infotrieve] |
48. | Fujimori, K., Anamnart, S., Nakagawa, Y., Sugioka, S., Ohta, D., Oshima, Y., Yamada, Y., and Harashima, S. (1997) FEBS Lett. 413, 226-230[CrossRef][Medline] [Order article via Infotrieve] |
49. | Poyton, R. O. (1999) Respir. Physiol. 115, 119-133[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Ansell, R.,
Granath, K.,
Hohmann, S.,
Thevelein, J. M.,
and Adler, L.
(1997)
EMBO J.
16,
2179-2187 |
51. | Rachidi, N., Martinez, M. J., Barre, P., and Blondin, B. (2000) Mol. Microbiol. 35, 1421-1430[CrossRef][Medline] [Order article via Infotrieve] |
52. | Sertil, O., Cohen, B. D., Davies, K. J., and Lowry, C. V. (1997) Gene (Amst.) 192, 199-205[CrossRef][Medline] [Order article via Infotrieve] |
53. | Bourdineaud, J. P. (2000) Res. Microbiol. 151, 43-52[CrossRef][Medline] [Order article via Infotrieve] |
54. | Jamieson, D. J. (1998) Yeast 14, 1511-1527[CrossRef][Medline] [Order article via Infotrieve] |
55. |
Lando, D.,
Pongratz, I.,
Poellinger, L.,
and Whitelaw, M. L.
(2000)
J. Biol. Chem.
275,
4618-4627 |
56. |
Semenza, G. L.
(2000)
J. Appl. Physiol.
88,
1474-1480 |
57. |
ter Linde, J. J.,
Liang, H.,
Davis, R. W.,
Steensma, H. Y.,
van Dijken, J. P.,
and Pronk, J. T.
(1999)
J. Bacteriol
181,
7409-7413 |
58. |
Beck, I.,
Ramirez, S.,
Weinmann, R.,
and Caro, J.
(1991)
J. Biol. Chem.
266,
15563-15566 |
59. | Blanchard, K. L., Acquaviva, A. M., Galson, D. L., and Bunn, H. F. (1992) Mol. Cell. Biol. 12, 5373-5385[Abstract] |
60. | Pugh, C. W., Tan, C. C., Jones, R. W., and Ratcliffe, P. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10553-10557[Abstract] |
61. | Semenza, G. L., Nejfelt, M. K., Chi, S. M., and Antonarakis, S. E. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5680-5684[Abstract] |
62. |
Wang, G. L.,
and Semenza, G. L.
(1995)
J. Biol. Chem.
270,
1230-1237 |
63. | Ladoux, A., and Frelin, C. (1997) Biochem. Biophys. Res. Commun. 240, 552-556[CrossRef][Medline] [Order article via Infotrieve] |
64. | Luo, G., Gu, Y. Z., Jain, S., Chan, W. K., Carr, K. M., Hogenesch, J. B., and Bradfield, C. A. (1997) Gene Expr. 6, 287-299[Medline] [Order article via Infotrieve] |
65. | Bacon, N. C., Wappner, P., O'Rourke, J. F., Bartlett, S. M., Shilo, B., Pugh, C. W., and Ratcliffe, P. J. (1998) Biochem. Biophys. Res. Commun. 249, 811-816[CrossRef][Medline] [Order article via Infotrieve] |
66. | Nagao, M., Ebert, B. L., Ratcliffe, P. J., and Pugh, C. W. (1996) FEBS Lett. 387, 161-166[CrossRef][Medline] [Order article via Infotrieve] |
67. | Nambu, J. R., Chen, W., Hu, S., and Crews, S. T. (1996) Gene (Amst.) 172, 249-254[CrossRef][Medline] [Order article via Infotrieve] |
68. | Wilk, R., Weizman, I., and Shilo, B. Z. (1996) Genes Dev. 10, 93-102[Abstract] |
69. |
Huang, L. E.,
Gu, J.,
Schau, M.,
and Bunn, H. F.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7987-7992 |