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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 9 (May 1), 1998:
pp. 3471-3480
Mouse Hypoxia-Inducible Factor-1 Is Encoded by Two Different mRNA
Isoforms: Expression From a Tissue-Specific and a Housekeeping-Type
Promoter
By
Roland H. Wenger,
Andreas Rolfs,
Patrick Spielmann,
Dieter R. Zimmermann, and
Max Gassmann
From the Institute of Physiology, University of Zürich-Irchel,
Zürich; and the Department of Pathology, University of
Zürich, Zürich, Switzerland.
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ABSTRACT |
Hypoxic induction of erythropoietin (Epo) and other oxygen-dependent
genes is mediated by the hypoxia-inducible factor-1 (HIF-1), a
heterodimeric transactivator consisting of an  and a  subunit. We
previously found that the mouse gene encoding HIF-1 harbors two
alternative first exons (I.1 and I.2), giving rise to two different
HIF-1 mRNA isoforms. Here, we show by RNase
protection analysis that the exon I.1-derived mRNA isoform is
differentially expressed in mouse tissues, being highest in kidney,
tongue, stomach, and testis, but undetectable in liver, whereas the
exon I.2 mRNA isoform is ubiquitously expressed. Sequence and
methylation analysis showed that, in contrast to exon I.1, exon I.2
resides within a region showing typical features of a CpG island, known
to be associated with the 5 end of housekeeping genes. We
identified a 232-bp minimal exon I.2 promoter that strongly induced
reporter gene expression in mouse L929 fibroblasts and Hepa1 hepatoma
cells. In contrast to L929 cells, the exon I.1 promoter was inactive in
Hepa1 cells and hypoxic exposure (1% O2) markedly reduced
exon I.2 promoter activity in Hepa1 cells. Prolonged exposure of mice to hypoxia (7.5% O2 for up to 72 hours) also caused a
decrease in liver HIF-1 mRNA, whereas aldolase mRNA levels
increased. These findings might be related to the relatively low Epo
levels in the adult liver.
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INTRODUCTION |
STIMULATION OF ERYTHROPOIESIS during
periods of limited oxygen supply improves the oxygen transport capacity
of the blood. This physiologic adaptation to hypoxia is regulated by
the glycoprotein hormone erythropoietin (Epo) that is mainly produced
in the fetal liver and the adult kidney (reviewed in
Jelkmann1). Identification of a hypoxia-responsive element
in the 3 flanking region of the gene encoding Epo led to the
discovery of the hypoxia-inducible factor-1 (HIF-1).2
HIF-1 is a basic-helix-loop-helix (bHLH) transcription factor that
is activated by exposure of cells to physiologically relevant
reductions in oxygen partial pressure (reviewed in Bunn and
Poyton,3 Wenger and Gassmann4). This activation
involves hypoxic stabilization of the protein that is otherwise
ubiquitinated and rapidly degraded in proteasomes under normoxic
conditions.5-8 Moreover, the activity of the
transactivation domain(s) can also be hypoxically
induced.6,9,10 So far, the mechanisms leading to these
hypoxic effects are not clearly understood, but there is evidence that
redox processes,5,8,11 as well as
phosphorylation,12,13 might be involved. After hypoxic exposure, HIF-1 forms a heterodimeric complex with the aryl
hydrocarbon receptor nuclear translocator (ARNT), also termed HIF-1,
which activates expression of oxygen-dependent genes.3,4
These oxy-genes4 include Epo, transferrin, vascular
endothelial growth factor, glycolytic enzymes, inducible nitric oxide
synthase, heme oxygenase, as well as other genes involved in the
adaptation of an organism to reduced oxygenation at the cellular,
local, and systemic level. ARNT also serves as a heterodimerization
partner for the aryl hydrocarbon receptor (AhR), also called dioxin
receptor, which activates genes involved in xenobiotic metabolism such
as the gene encoding cytochrome P450IA1. Besides the common bHLH DNA
binding and heterodimerization domain, all of these factors contain a
region of amino acid similarity termed PAS (PAS is an acronym for the
first described members of this family, namely Per,
ARNT, and Sim).4
We previously cloned and characterized the gene encoding mouse HIF-1
(Hif1a) and showed that it contains two different first exons
(termed I.1 and I.2).14 Expression of these two mouse HIF-1 mRNA transcripts is regulated by distinct promoters rather than being the product of differential splicing. The two alternative first exons give rise to two distinct mRNA isoforms differing in the
composition of their 5 untranslated regions (UTRs). Moreover, the predicted translation product derived from the exon I.1-containing mRNA isoform is 12 amino acids shorter than the exon I.2-containing isoform. These findings raised the possibilities of differential translational regulation and/or different protein
functions.14 So far, no human HIF-1 mRNA isoform has
been detected that corresponds to mouse exon I.1. We also analyzed the
Hif1a exon I.1 promoter and found that it is only moderately
active in the cell lines tested, suggesting that additional
cis-regulatory elements and/or so far unknown activators (eg,
developmental-stage, tissue-specific, or conditional signals) are
required for efficient exon I.1 promoter activity.14
Here, we present the sequence analysis, as well as the structural and
functional characterization, of Hif1a exon I.2 and flanking regions. In addition, we examined the expression profile of both mRNA
isoforms in various adult mouse tissues. Our results suggest that
Hif1a exon I.1 regulation exhibits tissue-specific features, whereas the exon I.2 promoter resembles a housekeeping-type promoter.
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MATERIALS AND METHODS |
Reverse transcription-polymerase chain reaction (RT-PCR) analysis.
Total RNA was isolated from mouse tissues according to the method
described by Chomczynski and Sacchi.15 Expression of the two mouse HIF-1 mRNA isoforms was examined as
described.14 Briefly, each 2.5 µg of total RNA was
reverse transcribed, and equal aliquots of the cDNA were amplified by
PCR using specific forward primers for either exon I.1 or exon I.2 and
an exon III reverse primer. Equal fractions of the reaction mixes were
electrophoresed through 1% agarose gels. Ethidium bromide-stained gels
were recorded using a video imaging system (Vilber Lourmat, Inotech,
Dottikon, Switzerland).
RNase protection assay.
Hif1a exon I.1 and exon I.2 DNA fragments were subcloned in
pBluescript (Stratagene, La Jolla, CA) and antisense cRNA probes were
obtained by in vitro transcription with T7 RNA polymerase (MBI
Fermentas, Vilinius, Lithuania) in the presence of a
32P-uridine triphosphate (UTP). After
purification by denaturing urea/polyacrylamide gel electrophoresis, the
probes were hybridized with 50 µg total RNA and digested with RNase A
and RNase T1 according to the manufacturer's directions
(RPA-II Kit; Ambion, Austin, TX). Protected products were resolved by
nondenaturing 5% polyacrylamide gel electrophoresis. Radioactive
signals were recorded and quantified by phosphorimaging (Molecular
Dynamics, Sunnyvale, CA), and the images were displayed
using a linear relationship between signal and image intensity. A mouse
-actin probe (Ambion) was used to control for equal RNA amounts
between the different samples. These reactions were performed using 2 µg total RNA only.
Cloning and sequencing.
The exon I.2-containing  phage clone H30 was cloned from a
LambdaGEM-11 (Promega, Madison, WI) genomic library as
described previously.14 A 270-bp EcoRI-NcoI
(all restriction enzymes were purchased from Fermentas) cDNA fragment
containing mouse exon I.2 sequences (kindly provided by A. Damert, Bad
Nauheim, Germany) served as hybridization probe. A 2.9-kb XbaI
fragment from H30, containing exon I.2 and flanking sequences, was
subcloned into pBluescript SK+ yielding the plasmid pH30X.
The insert of this plasmid was sequenced on both strands using a
combination of automated and manual sequencing procedures with
fluorescently labeled dideoxynucleotides or a 35S-deoxy
adenosine triphosphate (dATP) incorporation in cycle
sequencing reactions and T7 sequencing reactions, respectively,
according to the instructions provided by the manufacturers (Applied
Biosystems, Foster City, CA and Pharmacia, Uppsala,
Sweden). Sequence analysis was performed using the GCG program
package16 or the DNASIS for Windows program (Hitachi,
Tokyo, Japan).
Cell culture.
The mouse hepatoma cell line Hepa1 (also termed
Hepa1c1c7)17 was a kind gift of L. Poellinger (Huddinge,
Sweden). The mouse fibroblast cell line L929 (American Type Culture
Collection, Rockville, MD, CCL-1 NCTC clone 929) was a
kind gift of V. O'Donnall (Bern, Switzerland). Both cell lines were
cultured in Dulbecco's modified Eagle's medium (DMEM, high glucose;
Life Technologies, Basel, Switzerland) supplemented with 10%
heat-inactivated fetal calf serum (Boehringer, Mannheim, Germany), 100 U/mL penicillin, 100 µg/mL streptomycin, 1 × nonessential amino
acids, 2 mmol/L L-glutamine, and 1 mmol/L Na-pyruvate (all purchased
from Life Technologies) in a humidified atmosphere containing 5%
CO2 at 37°C. Oxygen tensions in the incubator (Forma
Scientific, model 3319; Bioblock, Frenkendorf, Switzerland) were either 140 mm Hg (20% O2 vol/vol,
normoxia) or 7 mm Hg (1% O2 vol/vol, hypoxia).
Mapping of the exon I.2 transcription initiation site.
The cap site of the mouse HIF-1 exon I.2-containing mRNA was
determined by primer extension and mung bean nuclease protection. For
primer extension, the oligonucleotide mHIFpex2 (see Fig 2) was
radioactively labeled by phosphorylation of the 5 end with [ -32P]ATP (Hartmann Analytik, Braunschweig, Germany)
and T4 polynucleotide kinase (Fermentas) as described
previously.18 Total RNA was extracted from mouse Hepa1
hepatoma cells according to Chomczynski and Sacchi.15
Poly(A)+ RNA was prepared using oligo dT cellulose spin
columns according to the manufacturer's instructions (Pharmacia).
Endlabeled mHIFpex2 (0.4 pmol) was coprecipitated with 5 µg
poly(A)+ RNA and resuspended in 10 µL 0.4 mol/L NaCl, 10 mmol/L Pipes [pH 6.4] at 96°C for 2 minutes. After annealing at
56°C for 10 minutes and at room temperature for 15 minutes, primer
extension was performed in 50 µL RT buffer (Stratagene), 1 U/µL
ribonuclease inhibitor (RNasin, Promega), 0.5 mmol/L deoxynucleotide
triphosphates (dNTPs; Promega) containing 250 U reverse transcriptase
(Stratascript, Stratagene) at 42°C for 2 hours. The reaction
products were ethanol precipitated, resuspended in 50% formamide,
electrophoresed through a 6% polyacrylamide/urea sequencing gel, and
visualized by autoradiography. A T7 polymerase sequencing reaction,
performed with the primer mHIFpex2 and pH30X single-stranded DNA
according to the manufacturer's instructions (Pharmacia), served as
position marker.
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| Fig 2.
Relative expression levels of Hif1a exon I.1
and exon I.2-containing mRNA isoforms in various mouse tissues. RNase
protection assays using each 50 µg total RNA and exon I.1 and
I.2-specific antisense probes. A total of 2 µg total RNA was used for
the -actin control reactions. Besides the sole protected 113-bp
fragment in Hepa1 cells, a second mRNA species appeared in mouse
tissues probably exceeding the 5 end of the exon I.1 probe (134 nt).
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Mung bean nuclease protection was performed essentially as described
before.14 Briefly, single-stranded DNA from the plasmid pH30X was prepared using M13K07 helper phages. The 5 endlabeled mHIFpex2 oligonucleotide was annealed to the template DNA and extended
using Klenow fragment of DNA polymerase I (Fermentas). After cleaveage
with PvuII, the single-stranded antisense probe was prepared by
alkaline agarose gel electrophoresis. This probe (100 kcpm) was
hybridized to 50 µg total RNA isolated from Hepa1 cells and digested
with 0, 10, 30, or 90 U mung bean nuclease (Life Technologies) for 30 minutes at 30°C. The products were analyzed on a sequencing
gel as described above using an unrelated sequencing reaction as length
marker.
In vivo methylation analysis.
To isolate genomic DNA, cell lines and mouse tissues were homogenized
in 0.2 mol/L NaCl, 50 mmol/L Tris-HCl [pH 8.0], 10 mmol/L EDTA, 1%
sodium dodecyl sulfate (SDS), and digested with 0.5 mg/mL Proteinase K
(Boehringer Mannheim) at 60°C for 12 hours. After addition of 0.2 mL saturated NaCl and microcentrifugation, the DNA was precipitated
from the supernatant by adding 0.45 mL isopropanol. The DNA was
resuspended in 10 mmol/L Tris-HCl [pH 7.6], 1 mmol/L EDTA, and
cleaved with XbaI, either alone or in combination with Cfr42I, SmaI, NotI, HhaI,
HpaII, or MspI. After gel electrophoresis through a
0.7% agarose gel, the DNA was transferred to uncharged Biodyne A
membranes (Pall Filtron, Northborough, UK) and cross-linked by
ultraviolet irradiation (Stratalinker; Stratagene). The membrane was
hybridized to a 561-bp Bsp1407I-BstEII fragment derived
from pH30X as described previously.14 The signals were
recorded by phosphorimaging.
Reporter gene assays.
A firefly luciferase reporter gene construct (pHXN1aluc) containing
exon I.2 upstream regulatory regions was obtained by inserting a 1.5-kb
KpnI  NheI fragment (KpnI cuts in the
polylinker 5 to the XbaI site shown in Fig 2) into the
promoterless luciferase vector pGL3Basic (Promega). Three deletion
constructs were made using BspTI, Eco91I, and
PvuII, which cleaved 801, 613, and 232 bp, respectively,
upstream of the first transcription initiation site. A similar reporter
gene construct containing 499 bp of exon I.1 upstream regulatory
sequences (pGL499Luc) was described previously.14 Tissue
culture cells (1 × 107 in 350 µL medium without
fetal calf serum) were coelectroporated with each 25 µg luciferase
reporter gene construct and a -galactosidase reference vector as
described previously.14 After incubation for 24 to 38 hours, the cells were lysed in reporter lysis buffer (Promega) and
luciferase and -galactosidase activities were determined according
to the manufacturer's instructions (Promega) using a Lumat LB9501
luminometer (Berthold, Bad Wildbad, Germany) and a DigiScan 96-well
plate photometer (ASYS, Eugendorf, Austria), respectively. Differences
in the transfection efficiency and extract preparation were corrected
by normalization to the corresponding -galactosidase activities.
Hypoxic exposure of mice.
Animals were exposed to a gas mixture containing 35% air and 65%
N2 (7.5% O2 final concentration) in an
airtight chamber containing water and nutrients ad libidum. After
exposure, the mice were killed by cervical dislocation and the organs
were withdrawn and frozen in liquid N2. Liver RNA was
extracted and analyzed by Northern blotting as described
previously.19 The filters were subsequently hybridized with
probes for mouse HIF-1,20 aldolase A,19 and ribosomal protein L28,19 the latter used to normalize for
differences in loading and blotting efficiency.
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RESULTS |
Differential tissue expression of the two mouse HIF-1 mRNA isoforms.
We previously reported that the two mRNA isoforms coding for HIF-1
are coexpressed in L929 and Hepa1 mouse cell lines.14 Here,
we examined the expression pattern of the two HIF-1 mRNAs in several
mouse tissues. To distinguish between the two isoforms, RT-PCR
reactions were performed using primers specific for either exon I.1 or
exon I.2 in combination with an exon III primer. As shown in
Fig 1, this yielded specific PCR products
of 371 bp and 472 bp for exon I.1 and exon I.2, respectively. The
specificity of these products was further confirmed by restriction
digestions and Southern blotting (data not shown). Interestingly, while
the exon I.2 mRNA isoform was ubiquitously expressed with less than fourfold variation in signal intensity between the different tissues, the exon I.1 mRNA isoform showed a distinct expression pattern. Exon
I.1 mRNA levels were highest in kidney, spleen, thymus, tongue, and the
reproductive organs (testis, ovary, and uterus); moderate in brain,
heart, lung, bone marrow, and skeletal muscle; and no exon I.1 mRNA
isoform could be detected in any liver of five mice analyzed. Exposure
of the mice to 0.1% carbon monoxide for 4 hours, causing functional
anemia,21 did not significantly alter the expression
pattern of the two HIF-1 mRNA isoforms (data not shown), whereas Epo
mRNA in kidney and liver was increased under the same conditions.21 In agreement with our previous observations
using various cell lines,14 the present RT-PCR analysis of
mouse tissues did not yield any evidence for the existence of an mRNA
isoform containing exon I.2 as an alternative splice variant, further supporting the notion that two independent promoters drive expression of the two alternative first exons.
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| Fig 1.
Hif1a exon I.1 and exon I.2-containing mRNA
isoforms are detectable in most mouse tissues. RT-PCR analysis of total
RNA isolated from the indicated mouse tissue. PCR was performed using
forward primers unique to either exon I.1 or exon I.2 and a common
reverse primer specific for exon III. Single PCR products were
indicative for the exon I.1 (371 bp) or the exon I.2 (472 bp) mRNA
isoform. Representative results of two to five independent experiments are shown. Note that the exon I.1 mRNA isoform could not be detected in
liver.
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Due to the distinct base compositions and lengths of exons I.1 and I.2,
this RT-PCR approach did not allow to determine the relative expression
levels of the two mouse HIF-1 mRNA isoforms. To circumvent this
problem, an RNase protection assay was established using labeled
antisense probes specific for exons I.1 and I.2, respectively
(Fig 2). RNase protection showed that the
exon I.1 mRNA isoform was detectable only in kidney, tongue, testis,
stomach, and embryonic tissue (embryonic day E18.5). Interestingly,
whereas the previously mapped14 113-bp protected fragment
represented the sole exon I.1 mRNA species in Hepa1 cells, an
additional mRNA species might exist in mouse tissues whose 5 end
probably extends the length of the 134 nt cRNA probe (Fig 2),
indicating that a second transcriptional start site might exist in
mouse tissues upstream to that mapped in Hepa1 cells. In contrast to
the exon I.1-derived mRNA isoform, the I.2 isoform was ubiquitously
expressed. Taking into consideration the differences in probe length,
the exon I.2 mRNA isoform was estimated to be at least sevenfold more abundant (testis) than the exon I.1 mRNA isoform.
DNA sequence of mouse Hif1a exon I.2 and flanking regions.
The ubiquitous expression of the exon I.2-derived HIF-1 mRNA isoform
implied that, unlike exon I.1, exon I.2 is expressed from a
housekeeping-type promoter. To test this hypothesis directly, we
subcloned and sequenced exon I.2 and flanking regions
(Fig 3) using the previously isolated phage H30 that bridged the gap between exon I.1 and exon
III-containing phage clones.14 Exon I.2 was 96.8%
identical to the corresponding sequence of the mouse HIF-1 cDNA
5 end reported by Li et al,9 containing three
mismatches and seven insertions. In contrast to exon I.1, exon I.2 had
an ATG translation initiation codon in frame with the ATG on exon II,
leading to a predicted translation product that was 12 amino acids
longer than the one derived from the exon I.1 mRNA isoform (Fig 3).
These 12 N-terminal amino acids were identical to those predicted from
the cDNA reported by Li et al.9 The observed translation
initiation site (TTCGCCATGG) matched the consensus reported
by Kozak (GCCRCCATGG).22 Furthermore, the
exon-intron splice junction conformed to the consensus
sequence.23
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| Fig 3.
Hif1a exon I.2 and flanking regions. The sequence
of exon I.2 is in bold, repetitive elements and restriction enzyme
recognition sites are underlined, and putative transcription factor
consensus binding sites are double underlined. The transcription
initiation sites mapped by primer extension and mung bean nuclease
protection (see Fig 4) are indicated by filled arrows and the start of
intron 1 by an open arrow. The predicted translation initiation codon and the first 12 amino acids of the exon I.2 isoform are indicated. The
location of the oligonucleotide mHIFpex2 is depicted with a line over
the sequence.
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Exon I.2 was 75% identical to the 5 UTR of the human HIF-1
cDNA and the predicted amino acid sequence differed in two of the 12 positions (Glu7 to Ala and Glu9 to Asp). Thus, exon I.2 encoded the
mouse homologue of the human HIF-1 5 end. On the other hand,
no human homologue for exon I.1 has been reported so far. Because the
in-frame ATG codon on the second exon of mouse Hif1a is not
present in the human gene, we consider that a putative human homologue
would contain the ATG initiation codon on the alternative first exon
rather than on the second exon. Alternatively, a human homologue of
mouse exon I.1 might not exist.
Mapping of the transcription initiation site of
Hif1a exon I.2.
To determine the cap site of the HIF-1 exon I.2 mRNA isoform, primer
extension analysis was performed using poly(A)+ RNA
isolated from mouse Hepa1 hepatoma cells primed with the exon
I.2-specific oligonucleotide mHIFpex2 (see Fig 3). As depicted in
Fig 4, two major bands were obtained,
corresponding to C1390 and C1397 of the sequence shown in Fig 3. To
confirm this result, we also applied a nuclease protection assay using
an endlabeled antisense DNA probe and total Hepa1 RNA. After
hybridization of the probe to the RNA, increasing amounts of mung bean
nuclease were added to digest protruding ends of the DNA-RNA hybrids,
as well as excess single-stranded antisense probe. In this case, two
major bands were identified corresponding to C1390 and C1395 (Fig 3).
Thus, the longest Hif1a exon I.2-derived transcription product
was 325 bp (corresponding to a 5 UTR of 290 bp), which was 16 bp
longer than the cDNA reported by Li et al.9 All three start
sites (indicated by arrowheads in Fig 3) conformed to the CA rule for
eukaryotic transcription initiation sites.24,25 However, as
reported by these investigators, the preferred cap site corresponds to
an adenosine rather than to a cytosine. The reason for this discrepancy
is currently unknown, but might be related to the lack of a canonical
TATA box, known to be associated with multiple transcriptional start
sites.
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| Fig 4.
Mapping of the Hif1a exon I.2 transcription
initiation sites. (A) Primer extension. Poly(A)+ mRNA was
isolated from mouse Hepa1 cells and annealed to the endlabeled,
complementary oligonucleotide mHIFpex2 (see Fig 3). The primer was
extended with reverse transcriptase and the products were resolved on a
6% denaturing polyacrylamide gel together with a sequencing reaction
performed with the same oligonucleotide as primer and a plasmid
containing the sequence shown in Fig 3 as template. (B) Nuclease
protection. Total RNA derived from Hepa1 cells was hybridized to an
endlabeled single-stranded antisense probe prepared as described in
Materials and Methods. The DNA-RNA hybrids were treated with the
indicated amounts of mung bean nuclease and separated on a sequencing
gel along with an unrelated sequencing ladder that served as length
marker. Numbers indicate the lengths of reaction products including the
mHIFpex2 primer.
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Hif1a exon I.2 is located within an essentially
methylation-free CpG island.
One intriguing feature of mouse Hif1a exon I.2 was the finding
that it is located within a G+C rich region.
Figure 5 shows a comparison of the G+C
content of the exon I.1 and I.2 loci. Whereas the exon I.1 locus
displayed an average G+C content of approximately 43%, comparable to
that of the bulk genome, exon I.2 and upstream regions had a G+C
content of 76%. After a short oligo T repeat at the beginning of
intron 1 (Fig 3), a second G+C rich (81%) stretch was found. Because
G+C rich regions associated with 5 ends of genes are indicative
for the presence of methylation-free CpG islands (reviewed in Cross and
Bird,26 Gardiner-Garden and Frommer27), we
analyzed the frequency of CpG dinucleotides, as well as the in vivo CpG
methylation pattern.
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| Fig 5.
Hif1a exon I.2 is located within a CpG island.
G+C content across the exon I.1 (A) and exon I.2 (B) regions. A
window of 100 bp shifted in steps of 1 bp was used in the
computer-assisted analysis. The positions of exon I.1 and exon I.2 are
indicated. The CpG/GpC ratio is given for each DNA segment that could
be distinguished from adjacent segments by the difference in the G+C
content.
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CpG dinucleotide frequencies are usually estimated as the ratio of CpG
(ie, observed) versus GpC (ie, expected) over a certain nucleotide
region. As shown in Fig 5A, the occurrence of the CpG dinucleotide was
suppressed in the exon I.1 locus (CpG to GpC ratio of 0.3). By
contrast, the frequency of the CpG dinucleotide in the G+C rich exon
I.2 locus was similar to the GpC frequency (Fig 5B).
CpG methylation was assessed in several mouse cell lines and tissues by
Southern blotting using CpG methylation-sensitive restriction enzymes.
As shown in Fig 6A, genomic DNA was cleaved with XbaI, either alone or in combination with a second enzyme recognizing selected sites in exon I.2 or in the 5 and 3
flanking regions. A 561-bp 5 fragment derived from a region
outside of the G+C rich CpG island served as hybridization probe. As
shown in Fig 6B, the SmaI site in exon I.2 and the NotI
site in the downstream region were entirely methylation-free.
Interestingly, methylation of the Cfr42I site in the upstream
region was about 50% in all cell lines and tissues tested. To date, we
have no conclusive explanation for this result, but it might be related to the observation of Matsuo et al,28 who showed that the
mouse experiences more accidental CpG island methylation than man,
resulting in erosion of mouse CpG islands during evolution. Because
this feature was rather unexpected for a CpG island-type promoter, we
further analyzed the 5 region using the CpG
methylation-sensitive restriction enzymes HhaI and
HpaII together with the methylation-insensitive HpaII-isoschizomer MspI. As shown in Fig 6C, the
upstream CpG dinucleotides of the G+C rich region were
methylation-free, whereas one CpG outside of the CpG island was mostly
methylated. Of note, an HpaII/MspI restriction fragment
length polymorphism was detected in these experiments (Fig 6C). Taken
together, the exon I.2 locus fulfills the criteria for a CpG island, as
it is G+C rich, shows unsuppressed CpG frequency, and is essentially
CpG methylation-free.
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| Fig 6.
Methylation pattern of the Hif1a exon I.2 region.
(A) Map of the CpG methylation-sensitive restriction enzyme recognition sites selected to assess the methylation pattern of distinct CpG dinucleotides in the exon I.2 region. The methylation status of a
particular restriction enzyme site is indicated by open and partially
filled circles. (B) Southern blot analysis of genomic DNA isolated from
various mouse cell lines and tissues. The DNA was cleaved either with
XbaI alone, or in combination with CpG methylation-sensitive
restriction enzymes cutting 5 (Cfr42I, C), within
(SmaI, S), or 3 (NotI, N) of exon I.2. (C)
Detailed exon I.2 upstream Southern blot analysis using the CpG
methylation-sensitive restriction enzymes Cfr42I (C),
HhaI (H) and HpaII (P), or the methylation-insensitive
HpaII-isoschizomer MspI (M). Note that an
HpaII/MspI restriction fragment length polymorphism
(RFLP) was detected.
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As often observed in CpG islands, we noted the lack of a canonical TATA
box and a high number of putative Sp1 binding sites (Fig 3). It has
been reported that Sp1 binding is implicated in maintaining CpG islands
methylation-free.26 Furthermore, two putative HIF-1
binding sites were identified, which perfectly matched the consensus
sequence we reported previously.4 However, because HIF-1
mRNA levels in cell lines29 and mice20 were not
upregulated by hypoxia, these putative sites are probably not
functional. Notably, also a putative HIF-1 binding site in the exon I.1
promoter was found to be nonfunctional.14
Comparison of the Hif1a exon I.1 and I.2 promoter
activities.
To analyze the presumed exon I.2 promoter activity, a 1.4-kb fragment
of the exon I.2 upstream region was inserted into a promoterless
firefly luciferase reporter gene vector. To directly compare the
respective promoter activities of exon I.1 and exon I.2 upstream
sequences, a 499-bp exon I.1 promoter-containing luciferase construct
was also analyzed. As reported previously,14 the exon I.1
promoter activity using either a 0.5-kb or a 1.0-kb fragment was about
equal. Mouse L929 fibroblasts and Hepa1 hepatoma cells were transiently
transfected with both constructs and incubated for 24 to 28 hours at
normoxic conditions. A cotransfected -galactosidase expression
vector served as a reference to correct for differences in transfection
efficiency and extract preparation. As shown in Fig 7, the exon I.2 promoter construct was
10-fold and fivefold more active than the exon I.1 promoter construct
in L929 and Hepa1 cells, respectively. Interestingly, while in these
experiments the exon I.1 promoter stimulated basal luciferase
expression ninefold in L929 fibroblasts, only very low promoter
activity could be detected in Hepa1 hepatoma cells, suggesting that the
exon I.1 promoter was not active in hepatoma cells. This finding is in line with the lack of detectable exon I.1 mRNA isoform expression in
mouse liver (see above).
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| Fig 7.
Comparison of the Hif1a exon I.1 and exon I.2
promoter activities. The empty parental vector pGL3Basic or the same
vector containing either the exon I.1 promoter or the exon I.2 promoter were transiently transfected into mouse L929 fibroblast and Hepa1 hepatoma cells. Luciferase activity was determined after 24 to 28 hours
of normoxic incubation. A cotransfected -galactosidase expression
vector served as internal control for transfection efficiency and
extract preparation. All values were normalized to the respective
luciferase activity obtained with the empty vector pGL3Basic, which was
arbitrarily defined as 1. Mean ± standard deviation (SD) of three
independent experiments are shown. Note the different scales.
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Hypoxia decreases Hif1a exon I.2 promoter activity in
mouse hepatoma cells.
To further analyze the exon I.2 promoter, several deletion constructs
were prepared containing 1389, 801, 613, or 232 bp of exon I.2 upstream
regulatory sequences. After transient transfection, L929 and Hepa1
cells were split and exposed to normoxia (20% O2) or
hypoxia (1% O2) for 36 to 38 hours. As shown in
Fig 8, deletion of most of the 1389-bp
fragment only marginally reduced exon I.2 promoter activity in normoxic
cells, and the 232-bp fragment was almost equally active as the 1389-bp
fragment. Interestingly, exposure of the cells to 1% oxygen reduced
luciferase expression about fivefold in Hepa1 cells, but only
marginally in L929 cells. Conversely, parallel transfections with an
SV40 promoter-driven luciferase vector containing three concatamerized
Epo-derived HIF-1 binding sites17,18,30 showed about
twofold hypoxic induction (data not shown). These results imply that
the previously observed17,30 time-dependent hypoxic
decrease in HIF-1 steady-state mRNA levels in Hepa1 cells is due to
a concomitant reduction in exon I.2 promoter activity.
View larger version (25K):
[in this window]
[in a new window]
| Fig 8.
Hypoxic downregulation of Hif1a exon I.2 promoter
activity. Exon I.2 upstream sequences of various length as indicated
were placed in front of the firefly luciferase reporter gene. These constructs were transiently transfected into mouse L929 fibroblasts or
Hepa1 hepatoma cells, followed by exposure to normoxia or hypoxia for
36 to 38 hours. Subsequently, luciferase expression was determined as
described in Fig 7. All values were normalized to the respective normoxic luciferase activity obtained with the construct containing the
longest 5 region, which was arbitrarily defined as 100. Mean ± SD of three independent experiments are shown.
|
|
Hypoxic reduction of HIF-1 mRNA in mouse liver.
The hypoxic decrease of exon I.2 mRNA levels and promoter activity in
mouse hepatoma cells and the lack of the exon I.1 mRNA expression in
mouse liver implied that chronic hypoxia might result in reduced
hepatic HIF-1 mRNA levels. To test this hypothesis, mice were
exposed in triplicates to hypoxic hypoxia (7.5% O2) for up
to 3 days. After exposure, liver RNA was isolated and analyzed by
Northern blotting. Figure 9 depicts the
ratio between HIF-1 mRNA and the ribosomal protein L28 mRNA, which
was used to normalize for differences in loading and blotting
efficiency. Interestingly, the HIF- to L28 ratio slightly decreased
in hypoxic mouse liver by 20% to 30%, whereas the hypoxia-inducible
glycolytic enzyme aldolase A mRNA to L28 ration transiently increased
2.2-fold after 24 hours and decreased again to 1.6-fold over normoxic
controls after 48 to 72 hours.
View larger version (17K):
[in this window]
[in a new window]
| Fig 9.
Hypoxic reduction of HIF-1 mRNA levels in mouse liver.
Three mice each were exposed to 7.5% O2 for 0 to 72 hours
and liver RNA was analyzed by Northern blotting. Shown are the ratios
of HIF-1 mRNA and aldolase A mRNA, respectively, and the ribosomal protein L28 mRNA (mean ± SD).
|
|
| |
DISCUSSION |
In this work, we showed that the mouse HIF-1 exon I.1-containing
mRNA isoform is tissue-specifically expressed, whereas the exon I.2
isoform displays an ubiquitous expression pattern in all mouse tissues
examined. Ubiquitous HIF-1 expression correlates with the previous
observation that HIF-1 DNA binding activity and HIF-1-mediated
transactivation of reporter genes is widespread in mammalian
cells.31,32 We sequenced and functionally characterized the
alternative mouse Hif1a exon I.2 and 5 flanking regions
that we previously mapped approximately 6 kb downstream of the
Hif1a exon I.1.14 As predicted from the G+C rich
5 UTR of human and mouse HIF-1 mRNA,14 exon I.2
is located within a 1.3-kb G+C rich, mostly methylation-free CpG
island, known to be associated with the promoters of housekeeping
genes. This observation is in agreement with the ubiquitous expression
of the exon I.2 mRNA isoform. However, because about 40% of all
tissue-specifically expressed genes also contain CpG island-type
promoters,26,27 we cannot exclude the possibility that the
Hif1a exon I.2 promoter works in a cell type-specific manner in
vivo. The RT-PCR and RNase protection approaches used in this work were
not suitable to identify the cell type expressing a particular mRNA.
Even if the exon I.2 promoter is ubiquitously active in cell culture,
in situ hybridization experiments of mouse tissues will be
necessary to unambigously determine the HIF-1 mRNA levels in a
particular cell type. In view of the fact that adaptation to different
oxygenation is likely to be mandatory for every single cell, a
tissue-specific HIF-1 expression seems to be rather improbable.
However, tissue-specific expression of the alternative mouse HIF-1
mRNA isoforms, as well as the recently discovered hypoxia-inducible
HIF-24 (also termed EPAS1,33
HLF,34 or HRF35) might complement each other to
ensure ubiquitous HIF-1-like activity. Of note, also two tissue- and
developmental stage-specifically expressed ARNT relatives
(ARNT236,37 and BMAL138), as well as several
other bHLH-PAS proteins, have recently been detected, implying a
complex network of spatial and temporal formation of heterodimeric
bHLH-PAS transcription factors.
A remarkable finding of our HIF-1 mRNA expression analysis in
various mouse tissues is the lack of detectable exon I.1 mRNA isoform
and a slight hypoxic reduction of the exon I.2 mRNA isoform in mouse
liver. In mouse Hepa1 hepatoma cells in culture, reporter gene
experiments showed that the Hif1a exon I.1 promoter is inactive and hypoxic exposure reduced exon I.2 promoter activity. These data are
consistent with our previous finding that hypoxia time-dependently decreases HIF-1 (but not ARNT) mRNA levels in Hepa1
cells,17,30 and they might also explain why reporter gene
experiments using hypoxia-responsive luciferase constructs consistently
gave lower induction levels in Hepa1 cells than in other cell
lines.18,30,39 Both promoters were much more active in L929
cells compared with Hepa1 cells and the hypoxic downregulation could
neither be found in L929 nor in any other human hepatoma (Hep3B and
HepG2) or nonhepatoma cell line examined (unpublished observation, June
1997).
Overall, our in vitro data provide an explanation for the in vivo
findings and it is tempting to speculate that altered HIF-1 mRNA
levels might also influence target gene expression. For example, endogenous transferrin mRNA was less induced by hypoxia in Hepa1 compared with the human hepatoma cell lines Hep3B and
HepG2,40 and oxygen-regulated Epo expression is also
exclusively found in Hep3B and HepG2,3 but was undetectable
in Hepa1 cells (our unpublished observations, August 1995). One
hallmark of Epo expression is the switch during development from the
fetal liver to the adult kidney as the main source of Epo synthesis. In
mammals, this switch takes place in the third trimester of
gestation.1 However, the molecular mechanism(s) underlying
this phenomenon are currently unknown. At least in the adult mouse, our
results on HIF-1 mRNA expression might provide a (partial)
explanation of why hypoxia-dependent Epo production is lowered in the
adult liver. Clearly, examination of HIF-1 mRNA isoform expression
in murine liver and kidney during development will be required to
elucidate a possible role of HIF-1 in tissue-specific and developmental
stage-specific Epo expression.
The kinetics of hypoxic HIF-1 protein activation appears different from
that of HIF-1 mRNA downregulation in Hepa1 cells. Four hours of 1%
oxygen are sufficient to induce HIF-1 DNA binding activity in Hepa1
cells.17,29,30 Four hours of 0.1% carbon monoxide
treatment are also typically used to elicit a hypoxic response in
mice.21 However, 4 hours of hypoxia does not alter expression of the two HIF-1 mRNA isoforms in Hepa1
cells17,29,30 or in mice,20 but rather, at
least 8 hours hypoxia are required to downregulate HIF-1 mRNA in
Hepa1 cells.17,30 For transient expression experiments, we
typically induced the cells for 36 to 38 hours, and 24 hours of hypoxic
exposure was also necessary to see a change in mouse liver HIF-1
mRNA. Thus, hypoxic HIF-1 protein activation precedes its mRNA
downregulation. The presence of a putative HIF-1 binding site in the
Hif1a exon I.2 5 flanking region, which perfectly
matched the tentative consensus sequence,4 opens the
possibility that HIF-1 could downregulate HIF-1 expression in an
autoregulatory loop. Indeed, in the ARNT-deficient subline Hepa1C4,
known to be incapable of activating hypoxia-responsive reporter
genes,17,30 we observed no hypoxic reduction in HIF-1 mRNA levels,30 supporting the hypothesis of a feedback
inhibition. However, deletion of this putative HIF-1 binding site in
the 232-bp exon I.2 upstream reporter gene construct did not alter the
hypoxic downregulation of luciferase expression, suggesting that this putative HIF-1 binding site is not functional. Of note, a nonfunctional HIF-1 binding site is also present in the exon I.1
promoter,14 confirming that one such element in isolation
is not sufficient to convey hypoxic activation of gene
expression.4 However, this result cannot exclude that HIF-1
might be involved in the regulation of its own expression.
In summary, the present report completes our work on the two
alternative mouse Hif1a promoters, establishes a
housekeeping-type major HIF-1 mRNA isoform corresponding to the one
known in humans, and opens intriguing questions on the biological
function(s) of the tissue-specific alternative mRNA isoform.
| |
FOOTNOTES |
Submitted July 21, 1997;
accepted December 29, 1997.
Supported by the Swiss National Science Foundation (Grant No.
31-47111.96). R.H.W. is a recipient of the Sondermassnahmen des Bundes
zur Förderung des akademischen Nachwuchses.
The novel nucleotide sequence reported in this paper has been deposited
with the EMBL/GenBank/DDBJ data bases and is available under accession
number Y13656.
Address reprint requests to Roland H. Wenger, PhD, Physiologisches
Institut der Universität Zürich-Irchel, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We are grateful to A. Damert, V. O'Donnall, S. Kozlov, U. Müller, and L. Poellinger for the gifts of material; H. Marti and A. Görlach for helpful discussions and critically reading the manuscript; R. Städeli for technical help; C. Gasser for the artwork; and C. Bauer for support.
| |
REFERENCES |
1.
Jelkmann W:
Erythropoietin: Structure, control of production, and function.
Physiol Rev
72:449,
1992[Free Full Text]
2.
Wang GL,
Jiang B-H,
Rue EA,
Semenza GL:
Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension.
Proc Natl Acad Sci USA
92:5510,
1995[Abstract/Free Full Text]
3.
Bunn HF,
Poyton RO:
Oxygen sensing and molecular adaptation to hypoxia.
Physiol Rev
76:839,
1996[Abstract/Free Full Text]
4.
Wenger RH,
Gassmann M:
Oxygen(es) and the hypoxia-inducible factor-1.
Biol Chem
378:609,
1997
5.
Huang LE,
Arany Z,
Livingston DM,
Bunn HF:
Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its subunit.
J Biol Chem
271:32253,
1996[Abstract/Free Full Text]
6.
Pugh CW,
O'Rourke JF,
Nagao M,
Gleadle JM,
Ratcliffe PJ:
Activation of hypoxia-inducible factor-1; definition of regulatory domains within the subunit.
J Biol Chem
272:11205,
1997[Abstract/Free Full Text]
7.
Kallio PJ,
Pongratz I,
Gradin K,
McGuire J,
Poellinger L:
Activation of hypoxia-inducible factor 1: Posttranscriptional regulation and conformational change by recruitment of the Arnt transcription factor.
Proc Natl Acad Sci USA
94:5667,
1997[Abstract/Free Full Text]
8.
Salceda S,
Caro J:
Hypoxia-inducible factor 1 (HIF-1) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes.
J Biol Chem
272:22642,
1997[Abstract/Free Full Text]
9.
Li H,
Ko HP,
Whitlock JP Jr:
Induction of phosphoglycerate kinase 1 gene expression by hypoxia. Roles of ARNT and HIF1.
J Biol Chem
271:21262,
1996[Abstract/Free Full Text]
10.
Jiang B-H,
Zheng JZ,
Leung SW,
Roe R,
Semenza GL:
Transactivation and inhibitory domains of hypoxia-inducible factor 1. Modulation of transcriptional activity by oxygen tension.
J Biol Chem
272:19253,
1997[Abstract/Free Full Text]
11.
Wang GL,
Jiang B-H,
Semenza GL:
Effect of altered redox states on expression and DNA-binding activity of hypoxia-inducible factor 1.
Biochem Biophys Res Commun
212:550,
1995[Medline]
[Order article via Infotrieve]
12.
Wang GL,
Jiang B-H,
Semenza GL:
Effect of protein kinase and phosphatase inhibitors on expression of hypoxia-inducible factor 1.
Biochem Biophys Res Commun
216:669,
1995[Medline]
[Order article via Infotrieve]
13.
Salceda S,
Beck I,
Srinivas V,
Caro J:
Complex role of protein phosphorylation in gene activation by hypoxia.
Kidney Int
51:556,
1997[Medline]
[Order article via Infotrieve]
14.
Wenger RH,
Rolfs A,
Kvietikova I,
Spielmann P,
Zimmermann DR,
Gassmann M:
The mouse gene for hypoxia-inducible factor-1. Genomic organization, expression, and characterization of an alternative first exon and 5 flanking sequence.
Eur J Biochem
246:155,
1997[Medline]
[Order article via Infotrieve]
15.
Chomczynski P,
Sacchi N:
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:156,
1987[Medline]
[Order article via Infotrieve]
16.
Devereux J,
Haeberli P,
Smithies O:
A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Res
12:387,
1984
17.
Gradin K,
McGuire J,
Wenger RH,
Kvietikova I,
Whitelaw ML,
Toftgård R,
Tora L,
Gassmann M,
Poellinger L:
Functional interference between hypoxia and dioxin signal transduction pathways: Competition for recruitment of the Arnt transcription factor.
Mol Cell Biol
16:5221,
1996[Abstract]
18.
Kvietikova I,
Wenger RH,
Marti HH,
Gassmann M:
The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site.
Nucleic Acids Res
23:4542,
1995[Abstract/Free Full Text]
19.
Wenger RH,
Marti HH,
Schuerer-Maly CC,
Kvietikova I,
Bauer C,
Gassmann M,
Maly FE:
Hypoxic induction of gene expression in chronic granulomatous disease (CGD)-derived B cell lines: Oxygen sensing is independent of the cytochrome b558-containing nicotinamide adenine dinucleotide phosphate oxidase.
Blood
87:756,
1996[Abstract/Free Full Text]
20.
Wenger RH,
Rolfs A,
Marti HH,
Guénet J-L,
Gassmann M:
Nucleotide sequence, chromosomal assignment and mRNA expression of mouse hypoxia-inducible factor 1.
Biochem Biophys Res Commun
223:54,
1996[Medline]
[Order article via Infotrieve]
21.
Marti HH,
Wenger RH,
Rivas LA,
Straumann U,
Digicaylioglu M,
Henn V,
Yonekawa Y,
Bauer C,
Gassmann M:
Erythropoietin expression in human monkey and murine brain.
Eur J Neurosci
8:666,
1996[Medline]
[Order article via Infotrieve]
22.
Kozak M:
An analysis of vertebrate mRNA sequences: Intimations of translational control.
J Cell Biol
115:887,
1991[Abstract/Free Full Text]
23.
Mount SM:
A catalogue of splice junction sequences.
Nucleic Acids Res
10:459,
1982[Abstract/Free Full Text]
24.
Bucher P:
Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences.
J Mol Biol
212:563,
1990[Medline]
[Order article via Infotrieve]
25.
Penotti FE:
Human DNA TATA boxes and transcription initiation sites. A statistical study.
J Mol Biol
213:37,
1990[Medline]
[Order article via Infotrieve]
26.
Cross SH,
Bird AP:
CpG islands and genes.
Curr Opin Genet Dev
5:309,
1995[Medline]
[Order article via Infotrieve]
27.
Gardiner-Garden M,
Frommer M:
CpG islands in vertebrate genomes.
J Mol Biol
196:261,
1987[Medline]
[Order article via Infotrieve]
28.
Matsuo K,
Clay O,
Takahashi T,
Silke J,
Schaffner W:
Evidence for erosion of mouse CpG islands during mammalian evolution.
Somat Cell Mol Genet
19:543,
1993[Medline]
[Order article via Infotrieve]
29.
Wenger RH,
Kvietikova I,
Rolfs A,
Gassmann M,
Marti HH:
The hypoxia-inducible factor-1 is regulated at the post-mRNA level.
Kidney Int
51:560,
1997[Medline]
[Order article via Infotrieve]
30.
Gassmann M,
Kvietikova I,
Rolfs A,
Wenger RH:
Oxygen- and dioxin-regulated gene expression in mouse hepatoma cells.
Kidney Int
51:567,
1997[Medline]
[Order article via Infotrieve]
31.
Maxwell PH,
Pugh CW,
Ratcliffe PJ:
Inducible operation of the erythropoietin 3' enhancer in multiple cell lines: Evidence for a widespread oxygen-sensing mechanism.
Proc Natl Acad Sci USA
90:2423,
1993[Abstract/Free Full Text]
32.
Wang GL,
Semenza GL:
General involvement of hypoxia-inducible factor 1 in transcriptional response to hypoxia.
Proc Natl Acad Sci USA
90:4304,
1993[Abstract/Free Full Text]
33.
Tian H,
McKnight SL,
Russel DW:
Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells.
Genes Dev
11:72,
1997[Abstract/Free Full Text]
34.
Ema M,
Taya S,
Yokotani N,
Sogawa K,
Matsuda Y,
Fujii-Kuriyama Y:
A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1 regulates the VEGF expression and is potentially involved in lung and vascular development.
Proc Natl Acad Sci USA
94:4273,
1997[Abstract/Free Full Text]
35.
Flamme I,
Fröhlich T,
von Reutern M,
Kappel A,
Damert A,
Risau W:
HRF, a putative basic helix-loop-helix-PAS domain transcription factor is closely related to hypoxia-inducible factor-1 and developmentally expressed in blood vessels.
Mech Dev
63:51,
1997[Medline]
[Order article via Infotrieve]
36.
Hirose K,
Morita M,
Ema M,
Mimura J,
Hamada H,
Fujii H,
Saijo Y,
Gotoh O,
Sogawa K,
Fujii-Kuriyama Y:
cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS factor (Arnt2) with close sequence similarity to the aryl hydrocarbon receptor nuclear translocator (Arnt).
Mol Cell Biol
16:1706,
1996[Abstract]
37.
Drutel G,
Kathmann M,
Heron A,
Schwartz J-C,
Arrang J-M:
Cloning and selective expression in brain and kidney of ARNT2 homologous to the Ah receptor nuclear translocator (ARNT).
Biochem Biophys Res Commun
225:333,
1996[Medline]
[Order article via Infotrieve]
38.
Ikeda M,
Nomura M:
cDNA cloning and tissue-specific expression of a novel basic helix-loop-helix/PAS protein (BMAL1) and identification of alternatively spliced variants with alternative translation initiation site usage.
Biochem Biophys Res Commun
233:258,
1997[Medline]
[Order article via Infotrieve]
39.
Kvietikova I,
Wenger RH,
Marti HH,
Gassmann M:
The hypoxia-inducible factor-1 DNA recognition site is cAMP-responsive.
Kidney Int
51:564,
1997[Medline]
[Order article via Infotrieve]
40.
Rolfs A,
Kvietikova I,
Gassmann M,
Wenger RH:
Oxygen-regulated transferrin expression is mediated by hypoxia-inducible factor-1.
J Biol Chem
272:20055,
1997[Abstract/Free Full Text]
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|
|
Y. Makino, H. Nakamura, E. Ikeda, K. Ohnuma, K. Yamauchi, Y. Yabe, L. Poellinger, Y. Okada, C. Morimoto, and H. Tanaka
Hypoxia-Inducible Factor Regulates Survival of Antigen Receptor-Driven T Cells
J. Immunol.,
December 15, 2003;
171(12):
6534 - 6540.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
W. Ma, L. Tessarollo, S.-B. Hong, M. Baba, E. Southon, T. C. Back, S. Spence, C. G. Lobe, N. Sharma, G. W. Maher, et al.
Hepatic Vascular Tumors, Angiectasis in Multiple Organs, and Impaired Spermatogenesis in Mice with Conditional Inactivation of the VHL Gene
Cancer Res.,
September 1, 2003;
63(17):
5320 - 5328.
[Abstract]
[Full Text]
[PDF]
|
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|
|
|
|
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P. B. Freeburg, B. Robert, P. L. St. John, and D. R. Abrahamson
Podocyte Expression of Hypoxia-Inducible Factor (HIF)-1 and HIF-2 during Glomerular Development
J. Am. Soc. Nephrol.,
April 1, 2003;
14(4):
927 - 938.
[Abstract]
[Full Text]
[PDF]
|
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|
|
|
|
|
T. Daikoku, H. Matsumoto, R. A. Gupta, S. K. Das, M. Gassmann, R. N. DuBois, and S. K. Dey
Expression of Hypoxia-inducible Factors in the Peri-implantation Mouse Uterus Is Regulated in a Cell-specific and Ovarian Steroid Hormone-dependent Manner. EVIDENCE FOR DIFFERENTIAL FUNCTION OF HIFs DURING EARLY PREGNANCY
J. Biol. Chem.,
February 21, 2003;
278(9):
7683 - 7691.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. D. Powell, R. Elshtein, D. J. Forest, and M. A. Palladino
Stimulation of Hypoxia-Inducible Factor-1 Alpha (HIF-1{alpha}) Protein in the Adult Rat Testis Following Ischemic Injury Occurs Without an Increase in HIF-1{alpha} Messenger RNA Expression
Biol Reprod,
September 1, 2002;
67(3):
995 - 1002.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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R. H. WENGER
Cellular adaptation to hypoxia: O2-sensing protein hydroxylases, hypoxia-inducible transcription factors, and O2-regulated gene expression
FASEB J,
August 1, 2002;
16(10):
1151 - 1162.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. H. Marti, D. M. Katschinski, K. F. Wagner, L. Schaffer, B. Stier, and R. H. Wenger
Isoform-Specific Expression of Hypoxia-Inducible Factor-1{alpha} During the Late Stages of Mouse Spermiogenesis
Mol. Endocrinol.,
February 1, 2002;
16(2):
234 - 243.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
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D. M. STROKA, T. BURKHARDT, I. DESBAILLETS, R. H. WENGER, D. A. H. NEIL, C. BAUER, M. GASSMANN, and D. CANDINAS
HIF-1 is expressed in normoxic tissue and displays an organ-specific regulation under systemic hypoxia
FASEB J,
November 1, 2001;
15(13):
2445 - 2453.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Wenger
Mammalian oxygen sensing, signalling and gene regulation
J. Exp. Biol.,
January 4, 2000;
203(8):
1253 - 1263.
[Abstract]
[PDF]
|
|
|
|
|
|
|
S. BICHET, R. H. WENGER, G. CAMENISCH, A. ROLFS, W. EHLEBEN, T. PORWOL, H. ACKER, J. FANDREY, C. BAUER, and M. GASSMANN
Oxygen tension modulates ß-globin switching in embryoid bodies
FASEB J,
February 1, 1999;
13(2):
285 - 295.
[Abstract]
[Full Text]
|
|
|
|
|
|
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L. M. Neckers
aHIF: the Missing Link Between HIF-1 and VHL?
J Natl Cancer Inst,
January 20, 1999;
91(2):
106 - 107.
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Lukashev, C. Caldwell, A. Ohta, P. Chen, and M. Sitkovsky
Differential Regulation of Two Alternatively Spliced Isoforms of Hypoxia-inducible Factor-1alpha in Activated T Lymphocytes
J. Biol. Chem.,
December 21, 2001;
276(52):
48754 - 48763.
[Abstract]
[Full Text]
[PDF]
|
|
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Abstract
Introduction
Abstract