From the
To examine the function of conserved noncoding regions in the
erythropoietin (Epo) gene, we have prepared clones and pools of Hep3B
cells stably transfected with a marked 4.1-kilobase Epo gene and
deletions thereof. The marked transcripts had single base substitutions
at three sites in the coding portion of Exon 5, enabling them to be
distinguished from endogenous Epo mRNA by ribonuclease protection and
competitive polymerase chain reaction. The basal expression and hypoxic
induction of the marked Epo gene that had no deletions were
indistinguishable from that of the endogenous Epo gene. Likewise,
deletion of conserved intervening sequence 1 had minimal effect on
hypoxic induction. In contrast, a 3`-deletion that included the
conserved 3`-enhancer element resulted in a substantial, but not
complete, suppression of hypoxic induction while a 3`-deletion
downstream of the enhancer resulted in enhancement. A 188-base pair
deletion of a conserved 3`-untranslated region in Exon 5 had minimal
effect on hypoxic induction. However, the truncated Epo mRNA had a
markedly prolonged half-life (15 h) in comparison to the endogenous Epo
mRNA (2.0 h) or the marked full-length Epo mRNA (2.1 h). Further
deletions in the 3`-UTR showed that a relatively small region of
approximately 50 bases is responsible for the relatively rapid turnover
of Epo mRNA. These experiments provide information on cis-acting
elements of the Epo gene that cannot be obtained from conventional
reporter gene transfection experiments.
Erythropoietin is a 30.4-kDa glycoprotein that regulates red
cell mass
(1) . Reduction in oxygen tension markedly increases
the production of this hormone in the kidney and liver as well as in
two human hepatoma cell lines, Hep3B and HepG2
(2, 3) .
Hypoxia causes about a 100-fold enhancement in Epo
A logical and often fruitful
approach in identifying important cis-acting elements in a gene is to
test highly conserved sites in the 5`-flank as well as in noncoding
regions downstream. As shown in Fig. 1, segments of strong
homology in the noncoding portions of the human and mouse Epo genes
include: a stretch upstream of the transcriptional start site; two
segments within the first intron; all of the untranslated region in the
5th exon (excluding a B1 repeat insertion in the mouse gene); and, 130
bp downstream of the polyadenylation site, a segment extending 371 bp
(13, 14, 15) . Transfection experiments into
Hep3B and HepG2 cells utilizing some of these conserved elements linked
to reporter genes have proven useful in determining their contribution
to hypoxic induction. For example, the region of homology downstream
from the termination of transcription contains a 40-bp enhancer which
plays a critical role in hypoxic induction
(16, 17, 18, 19, 20, 21) .
This 3`-enhancer interacts with a hypoxia inducible transcription
factor, HIF-1
(20, 22, 23) , and, 14 bp
downstream, with the nuclear receptor HNF-4
(24) . Functional
analysis of the Epo promotor indicates that 118 bp upstream of the
start site are required for both basal transcription as well as for
hypoxic induction
(19) . It is likely that the full response
requires interaction and perhaps cooperativity between the enhancer and
promotor elements.
Constructs A genomic clone of human Epo in pUC18 was kindly provided by Genetics
Institute, Cambridge, MA. The Epo gene insert is a 4100-bp
HindIII- EcoRI fragment (GenBank
The Epo
gene in this construct was ``marked'' by site-directed
mutagenesis. Three base substitutions in the coding region of Exon 5
(2691:A
To facilitate preparation of
deletion constructs the upstream HindIII site in HUMERPA was
replaced by a unique BamHI site. Since the Epo gene has a weak
promoter
(19) , it was necessary to prevent cryptic promoter
activity from site(s) upstream of the vector. Therefore a 625-bp
NdeI- BamHI fragment containing a tandem repeat
transcriptional stop sequence from pXP2 ( A
Constructs expressing wild type and marked Epo riboprobes were
prepared by inserting a PvuII- NcoI Epo fragment
(2650-2890) from wild type Epo or from the marked Epo (mE-FL)
into pBluescript KS(+). This fragment of Epo genomic DNA extended
from the 5`-end of Exon 5 into its noncoding portion of Exon 5, thereby
including the three sites in the coding region of Exon 5 that were
``marked'' by substitution. Because the construct was
linearized in the polylinker, 15 bp downstream of the Epo insert, the
full length riboprobe could be readily distinguished from protected
fragments. Transfections Stable transfections were performed by electroporation. 10 µg of
pSVNeo (expressing the neomycin resistance gene driven by the strong
promoter of SV40 large T) and 300 µg of supercoiled marked Epo
constructs were co-transfected into 3
Fig. 1
depicts the marked wild type and mutant Epo
constructs used in our experiments. Hep3B cells were transfected with
these constructs as described under ``Materials and
Methods.'' Initially, all of these constructs were transiently
transfected into Hep3B cells both as calcium phosphate precipitates and
by electroporation, under conditions which we have previously shown to
give maximum transfection efficiency
(19, 27) .
Unfortunately, analysis of Epo mRNA by reverse transcription and
competitive PCR revealed that the expression of the marked gene in both
normoxic and hypoxic cells was less than 10% that of the endogenous
gene (data not shown). This low level of expression, presumably a
reflection of the inherent weakness of the Epo promotor
(19, 27) , precluded our use of transient transfections.
To assure a higher level of expression of the marked Epo gene, we
prepared stably transfected cell lines. In most experiments, neomycin
resistant colonies were pooled in order to avoid bias owing to
variability in integration site and its possible effect on basal
expression and hypoxic induction of the marked gene. The average number
of copies of the marked gene per cell in a given pool varied
considerably from 0.2 to 13 with a mean of 3.1 ± 0.8 (S.E.,
n = 18) for all of the cell pools. We observed no
significant correlation between the copy number of the marked gene and
its expression. Subconfluent adherent cells were incubated for 12 h
either at 21% O
The validity of our experimental design required
demonstration that the expression of the transfected full-length Epo
gene (mE-FL) was comparable to that of the endogenous gene both
constitutively (21% O
The most commonly employed strategy for studying the function
of cis-acting elements in a gene is to transfect into an appropriate
cell line a construct in which a given element is attached to a
reporter gene, not normally expressed by that cell. Although this
approach is highly adaptable and informative, it suffers from obvious
drawbacks. First, gene fragments artificially inserted into reporter
gene constructs may give misleading and unreliable results with respect
to tissue and developmental specificity as well as response to specific
cellular signals. Second, such experiments provide no information about
post-transcriptional gene regulation. To circumvent these problems we
have studied the Epo gene in its most native possible context by
marking the Epo gene and using the marked transcript as the reporter.
Baseline experiments using the ``full-length'' (FL) 4.1-kb
marked Epo gene provide evidence that this approach is valid. As shown
in Figs. 3 and 5 A and , basal expression, hypoxic
induction and stability of the mEpo-FL mRNA were indistinguishable from
that of the endogenous Epo gene. These results offer satisfactory
assurance that 385 bp of upstream flank and 762 bp of downstream flank
are sufficient for the stably integrated gene to function normally in
Hep3B cells. Moreover, marking the gene by 3-bp substitutions in the
coding region of Exon 5 did not significantly affect expression,
hypoxic induction or mRNA stability. Finally, the fact that
reproducible results were obtained with replicate pools as well as
clonal lines of stable transfected cells indicates that integration
site did not impact significantly on the function of the marked gene.
Thus, our demonstration that the expression of mE-FL faithfully mimics
that of the endogenous Epo gene indicates that experiments testing
deletions of this construct can be interpreted with confidence.
As
shown in Fig. 4and , we observed a marked diminution
in hypoxic induction of the marked Epo mRNA when the transfected
construct contained a deletion that included the 3`-enhancer
(mE
During the past
decade, there has been growing appreciation of the importance of
sequences in the 3`-untranslated region as determinants of mRNA
stability. Examples of special biologic importance include the AU rich
motif that is shared among the 3`-UTRs of a number of transcription
factor and cytokine mRNAs
(29, 30, 31) and the
iron regulatory stem loop motif in the mRNA of the transferrin receptor
(32, 33) . The 3`-UTR of Epo's mRNA lacks any
known sequence homology except perhaps to the 3`-UTR of the tyrosine
hydroxylase gene
(12) . We have sought to determine whether Epo
gene expression is affected by post-transcriptional regulation. Nuclear
run-on experiments in Hep3B cells have demonstrated at least a 10-fold
increase in Epo gene transcription in response to hypoxia and cobalt
(9) . Because the level of Epo gene expression in unstimulated
cells is so low, the quantitation of the signal was imprecise.
Therefore it is possible that the induction of transcription is
considerably greater than 10-fold. In order to assess whether hypoxia
also affected Epo mRNA stability, an actinomycin D chase experiment was
performed
(9) . Surprisingly, inhibition of de novo transcription by actinomycin D increased the half-life of
Epo mRNA by a factor of four. Cycloheximide, an inhibitor of protein
synthesis, also resulted in a marked prolongation in the half-life of
Epo mRNA. Electrophoretic mobility gel shift experiments on extracts
from normoxic and hypoxic Hep3B cells demonstrated 70- and 135-kDa
cytosolic proteins which bound specifically to a portion of Epo mRNA in
the 3`-untranslated region immediately downstream of termination of
translation (approximately 2664-2884)
(10) . This region
includes a 28-base pyrimidine rich stretch that is homologous to a
region in the 3`-UTR of tyrosine hydroxylase mRNA that bound
preferentially to cytosolic extracts from hypoxic cells
(12) .
In contrast, the Epo mRNA bound equally well to protein in normoxic and
hypoxic extracts
(10) . Furthermore, there was no apparent
correlation between the tissue distribution of these proteins and the
ability of cells to express Epo
(10) . Taken together, these
results suggest that the half-life of Epo mRNA is reduced by the
specific binding of proteins to the 3`-UTR. However, there is no
evidence thus far that these proteins are either stimulus-specific or
tissue-specific. Because the stability of Epo mRNA is affected by
ongoing transcription and protein synthesis, it is difficult to design
and execute experiments that address this problem.
In this study, we
demonstrate that a conserved
The first column shows the marked
Epo construct tested. The second column shows the number of pools
tested. The next two columns show ratios of marked to endogenous
reverse transcribed Epo mRNA from cells exposed to 21% O
We appreciate helpful discussions with Kerry
Blanchard, Debby Galson, and Mark Goldberg.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
mRNA expression in both the murine kidney
(4, 5, 6) and in Hep3B cells
(7) , an
induction due primarily to enhanced transcription
(8, 9) . In addition, the Epo gene may also be regulated
at the level of mRNA stability
(9, 10) , although the
evidence for this is indirect and therefore less convincing than
recently published studies on another hypoxia inducible gene, tyrosine
hydroxylase
(11, 12) .
Figure 1:
Diagrams of the wild
type human Epo gene, the marked full-length ( mE-FL) and some
of the marked deletion constructs used in this study. The five exons
are shown in rectangles; the coding regions are
shaded. Regions of sequence homology between human and mouse
Epo genes are shown at the top. Black, >75% homology;
shaded, 50-75% homology. MP, minimal promotor;
3`UTR, = 3`-untranslated region; A, the tandem
repeat polyadenylation sequence; IVS1, first intervening
sequence. The following restriction sites are shown: AccI
( Acc) in the wild type gene was mutated to HindIII
( Hind) in the marked genes; E, EspI, S,
SacI; A, ApaI, H, HphI;
Eco, EcoRI.
Much less information is available about the
possible functional roles of other regions of homology in the Epo gene.
Segments in the first intron and in the 3`-untranslated region may
impact on mRNA processing and stability. As such, they cannot be tested
by conventional reporter gene analyses. To address this question, we
have devised a strategy that has enabled us to investigate cis elements
of the Epo gene in their native context. We have used the Epo gene as
its own reporter by marking it with innocuous point mutations in the
coding region, thereby enabling its transcript to be readily
distinguished from endogenous Epo mRNA. We prepared stable
transfectants expressing this marked Epo gene and deletions thereof.
Analyses of the expression of the marked Epo gene, in comparison to the
endogenous gene has provided useful new information on functionally
important cis elements.
file
HUMERPA (accession no. M11319))
(25) extending from 385 bp
upstream of the transcriptional start site to 762 bp downstream of the
polyadenylation signal. In this report, as in HUMERPA, the numbering of
bases begins with the 5`-A of the HindIII site.
G; 2694: C
T; 2706: C
T) obliterated an
AccI site and created a HindIII site 13 bp upstream
of it. As demonstrated below, these changes enabled the marked
transcript to be distinguished from the endogenous transcript by means
of both ribonuclease protection analysis (RPA) and competitive PCR.
These three substitutions were at third bases of codons thereby
preserving Epo's native amino acid sequence. This precaution
would be important in the unlikely event that the expression of steady
state mRNA levels is influenced by the protein product, analogous to
the regulation of tubulin gene expression
(26) . We have
designated this construct mE-FL ( marked Epo gene with
full- length sequence) in order to distinguish it from
constructs having deletions ( e.g. mE
IVS1 = marked
Epo gene with deletion of IVS1).
in Fig. 1) was inserted into the NdeI and
BamHI sites upstream of the marked Epo gene. Deletions in the
mE-FL were generally made by digestions at selected restriction sites
followed by filling in overhangs and blunt end ligation. Several of the
constructs involving non-unique restriction sites required partial
digestions. The
IVS1 construct was prepared by substituting an
EclXI- Asp718 fragment of Epo cDNA for an
EclXI- Asp718 fragment of Epo genomic DNA.
10
Hep3B
cells with a voltage of 250 and a capacitance of 1,080 µF. After
incubation for 48-72 h in standard medium, 0.4 mg/ml G418 was
added. Previous dose-response experiments indicated that this was an
appropriate dose to select neomycin-resistant cells. Approximately 3
weeks were required for the outgrowth of neomycin-resistant colonies.
For most experiments, colonies on a 10-cm plate were pooled in order to
avoid bias owing to the site of integration of the Epo gene. The number
of colonies per pool ranged from 5 to greater than 50. For a few
experiments, individual colonies were isolated and expanded by
trypsinization in a 1-cm cylindrical ring which was fixed to the
culture plate by autoclaved vacuum grease. In each transfection
procedure, a plate of cells was mock transfected and selected in the
same concentration of G418 to assure that no breakthrough colonies
occurred. Stably transfected and nontransfected Hep3B cells were split
into equal aliquots grown to approximately 70% confluence in 15 cm
plates and incubated for 12 h in either 1 or 21% O
as
described above. Following trypsin treatment to release cells from the
plates, DNA and RNA samples were prepared by standard protocols. Analysis
Ribonuclease Protection
Ribonuclease protection was
employed to provide quantitation of absolute levels of marked and
endogenous Epo mRNAs. For most of the experiments (and all the results
shown in Figs. 3-5), the marked riboprobe was used (see section
above under ``Constructs''). In the few experiments in which
wild type riboprobe was used, the results were fully consistent. The
riboprobe plasmid was linearized at a site distal to the Epo insert and
radiolabeled antisense cRNA was prepared by incubating 1 µg of DNA
in the presence of T7 RNA polymerase and [P]CTP.
An aliquot of labeled riboprobe containing 2
10
cpm
was mixed with 20 µg of RNA sample incubated overnight at 55 °C
and then digested with RNase T, and RNase A for 45 min. Samples were
then loaded onto a 6% polyacrylamide gel and electrophoresed at 1700 V
(80-100 watts) for approximately 2 h. Autoradiograms were
prepared by exposure to X-Omat film. Endogenous and marked Epo mRNA was
quantitated by scanning the dried gel with a PhosphorImager (Molecular
Dynamics Inc.), and the respective bands were quantitated by ImageQuant
software.
Competitive PCR
Competitive PCR was used to assay
both the number of copies of marked Epo constructs integrated into
stably transfected cells and the expression of marked Epo mRNA,
relative to endogenous Epo mRNA. The locations of the PCR primers are
shown in Fig. 2 A. Different upstream primers were used
for amplification of genomic DNA versus cDNA. For the former,
a 24-base sequence in Intron 4 was used (5`3`):
AGGAGCGGACACTTCTGCTTGCCC. For amplification of cDNA the upstream primer
consisted of 28 bases of sequence at the 3`-end of Exon 4
(ACTCTGCTTCGGGCTCTGGGAGCCCAG) followed by 3 bases of sequence at the
5`-end of Exon 5 (AAG). The use of this primer prevented any
amplification of genomic DNA or transfected plasmid DNA. The same
downstream primer was used for both amplifications:
AAGCAATGTTGGTGAGGGAGGTGGTGGAT. cDNA was prepared from 1-µg samples
of RNA by incubation in 1
RT buffer
(7) , 4 units
reverse transcriptase and 2 µg random oligonucleotide primers. PCR
reactions were carried out as described previously
(7, 28) . One aliquot of the PCR products was digested
with AccI, which cleaves only the endogenous genomic DNA and
cDNA fragments. Another aliquot was digested with HindIII
which cleaves only the marked fragments. In keeping with the principle
of competitive PCR, the endogenous and marked DNAs are co-amplified
with equal efficiencies
(28) . Therefore, the ratio of their
amplification products will be identical to the ratio in the sample
prior to amplification, irrespective of number of cycles or incubation
conditions. Accordingly, competitive PCR gives an accurate measurement
of the ratio of marked to endogenous genomic DNA and the ratio of
marked to endogenous mRNA. The ethidium bromide stained PCR gels (shown
in Fig. 2 B) were analyzed by a Molecular Dynamics
densitometer utilizing ImageQuant software and also by visual
inspection. The reliability of our PCR analysis was considerably
enhanced by digestion of each specimen with two enzymes ( AccI
and HindIII), thereby providing an internal check. The
fractions of fragments formed by digestion with either enzyme was
predictably less than the fraction of parent (pre-amplification) Epo
DNA having that restriction site, owing to the inability of the enzyme
to cut heteroduplexes.
(
)
Accordingly, the ratio
of marked to endogenous product was calculated using the binomial
distribution to correct for heteroduplex formation.
Figure 2:
Analysis of genomic DNA and reverse
transcribed RNA by PCR. A, PCR amplification of marked
( M) and endogenous ( E) Epo genes and cDNAs. The
dark rectangles represent exon 4 ( left) and exon 5
( right) separated by intron 4. In exon 5, the AccI
site (2702) of wild type Epo and the HindIII site (2689) of
marked Epo are shown. The upstream primer used to amplify genomic DNA
( dotted arrow) hybridizes to the 5`-end of intron 4. The
upstream primer use to amplify cDNA contains 28 bases of sequence at
the 3`-end of exon 4 and 3 bases of sequence at the 5`-end of exon 5.
The same downstream primer was used for amplification of both genomic
DNA and cDNA. B, ethidium bromide-stained gel showing
amplification products of reverse transcribed RNA from cells stably
transfected with marked Epo genes. Following digestion with
AccI ( top) and HindIII ( bottom),
fragments of expected size were obtained. Lower left-hand
lane, size markers ( HaeIII-digested X174 DNA).
Two left-hand lanes, mE-FL3, the marked
``full-length'' gene having 400 bp upstream of the promotor.
Middle two lanes, m5`E-FL1, a construct having 5 kb of Epo
flanking sequence upsteam of the promotor; this construct failed to be
incorporated into the genome and therefore produced no transcripts.
Two right-hand lanes, m
BgS-1, having a deletion in the
3`-UTR.
or at 1% O
. The expression of
the endogenous and the marked Epo genes was analyzed by RPA as well as
by competitive PCR.
) and following hypoxic challenge (1%
O
). Fig. 3shows RPA gels for RNA samples from five
independent pools transfected with mE-FL containing from 5 to greater
than 50 colonies per pool. In each of the five pools incubated at 21%
O
, there was barely detectable expression of both the
marked Epo gene and, as expected, the endogenous gene. Moreover, in
each of the five pools, the expression of the marked gene was
substantially enhanced by hypoxic challenge, as shown in the hypoxia
lanes in Fig. 3. These RPA results were generally in good
agreement with the competitive PCR data. In three of the five pools
tested, the hypoxic induction of the marked gene was the same as that
of the endogenous with ratios of M/E 1% to M/E 21% that were close to
unity (). In the other two pools (mE-FL7 and mE-FL10)
hypoxic induction of the marked gene was less than that of the
endogenous gene, probably because a few colonies in each pool
constitutively overexpressed Epo at 21% O
, thereby
increasing the M/E ratio. Nevertheless, as shown by RPA in
Fig. 3
, even in these two pools, the expression of the marked Epo
gene undergoes robust enhancement following exposure to hypoxia. Taken
together, the RPA and competitive PCR results show that the marked wild
type Epo gene underwent normal or nearly normal hypoxic induction when
stably transfected into Hep3B cells.
Figure 3:
Ribonuclease protection analysis of RNA
from pools of cells stably transfected with the 4.1-kb
``full-length'' marked Epo gene (mE-FL). The antisense
riboprobe had the sequence of the marked Epo gene (intron 4 and exon 5)
and therefore protected a larger (242-base) fragment of marked Epo mRNA
in comparison to endogenous wild type (180-base fragment).
Hep3B, nontransfected Hep3B cells expressing only the
endogenous Epo mRNA. Y, yeast RNA; N, normoxia (21%
O); H, hypoxia (1%
O
).
We then tested marked Epo genes
in which putative regulatory elements were deleted. As shown in
Fig. 4
, the marked IVS1 Epo gene, in which exons 1 and 2
were fused, thereby eliminating intron 1, appears to have nearly normal
basal expression and hypoxic induction. The competitive PCR data in
also suggests normal induction although there is more
variability among the three
IVS pools than among the other sets of
data. In contrast, deletion downstream of the ApaI site in the
3`-flank (
AE), thereby eliminating the above-mentioned 40-bp
enhancer, resulted in a marked decrease in hypoxic induction of the
marked gene in two independent cell pools. The RPA result is supported
by the competitive PCR analysis () showing that the ratio
of expression of the marked to endogenous Epo mRNA fell markedly when
the cells were challenged by hypoxia. Nevertheless, as shown in
Fig. 4
, significant hypoxic induction was retained, indicating
that the 3`-enhancer is not solely responsible for hypoxic
up-regulation of Epo gene expression. In a construct having a deletion
further downstream, thereby preserving the 3`-enhancer, hypoxic
induction was enhanced above normal, as shown both by RPA
(Fig. 4) as well as by competitive PCR (). This
result suggests the presence of cis elements in the 3`-flank between
3550 and 4100 that impact negatively on hypoxic induction.
Figure 4:
Ribonuclease protection analysis of RNA
from pools of cells stably transfected with: mE-IVS1 (deletion of
first intervening sequence); mE
AE (3`-deletion that includes the
3`-enhancer); mE
HE (3`-deletion downstream of the 3`-enhancer);
and mE
ES (deletion of highly conserved segment within the 3`-UTR).
As in Fig. 3, the marked Epo riboprobe was used. The protected fragment
of the marked Epo mRNA from mE
ES was 22 bases shorter because of
the deletion in the 3`-UTR. N, normoxia (21% O
);
H, hypoxia (1% O
).
The
function of the highly conserved 3`-untranslated region was examined by
means of a series of marked deletion mutants. RPA analysis
(Fig. 4) shows near normal induction of the mutant ES
(EspSac) having the largest deletion in the 3`-UTR (2873-3061).
However, both the RPA and the competitive PCR analyses ()
showed that at 21% O
the basal expression of the marked
ES gene was somewhat higher than that of the endogenous
gene.
(
)
This result, observed in five out of six
separate cell pools, could be due either to increased basal
transcription of the marked gene or enhanced mRNA stability. In order
to distinguish between these possibilities, the in vivo decay
of the marked Epo mRNA was compared to that of the endogenous Epo mRNA
by inducing high level expression with a 12 h hypoxic challenge,
followed by rapid replacement with 21% O
. RNA samples were
prepared at 0, 1, 2, 4, and 8 h. The radioactivity of the protected
bands in the RPA gels was measured by a PhosphorImager. As shown in
Fig. 5
, the half-life of the endogenous Epo mRNA was 2.0 ±
0.1 h ( n = 4), a value very close to those that we
previously obtained by Northern blot analysis (1.5 h)
(9) and
by competitive PCR (2.0 h)
(7) . As shown in
Fig. 5A, the marked wild type Epo mRNA (mWT) had a
half-life of 2.1 ± 0.1 h ( n = 5), a value
indistinguishable from that of the endogenous mRNA. This result was
fully anticipated since the two mRNAs differ only by 3 bases in the
coding portion of exon 5, a region not expected to affect mRNA
stability. In contrast, as shown in Fig. 5, B and
C, the large (187 bases) deletion in the conserved 3`-UTR
(
ES) resulted in the expression of a truncated mRNA with markedly
prolonged projected half-life of 15 ± 3 h ( n =
5). The large difference between the stability of the marked wild type
mRNA compared to the
ES mRNA that was observed with cell pools was
also observed in single clonal colonies of mE-FL and
ES. In order
to further delineate the site that confers relative instability on Epo
mRNA, a set of smaller deletions were tested. The half lives of these
mutant Epo mRNAs are shown in Fig. 5 C.
BsS, a
58-base deletion at the 3`-end of ES, had a normal half-life (2.1 h),
while that of
EBg, an 84-bp deletion at the 5`-end of ES, was
nearly normal (3.2 h).
(
)
These results taken
together suggests that the major contributor of Epo mRNA's
relative instability lies in the 46-base middle region
(Fig. 5 C). In support of this conclusion the mutant
BgS mRNA, which has a deletion that includes this 46-base segment,
had a prolonged half-life (5.8 h). Thus, these experiments testing
deletions in the 3`-UTR suggest that a relatively small stretch is
responsible for its relatively rapid degradation and short half-life.
Figure 5:
Stability of endogenous and marked Epo
mRNA. High levels of Epo mRNA were induced by overnight exposure to 1%
O. At time 0, the cells were rapidly exposed to 21% O
and the levels of Epo mRNA were analyzed by ribonuclease
protection and quantitated by PhosphorImaging. A, decay of
endogenous Epo mRNA ( squares), marked Epo mRNA expressed by
cells stably transfected with full-length Epo gene, mE-FL
( circles) and marked truncated Epo mRNA expressed by cells
stably transfected with mE
ES ( triangles). B,
ribonuclese protection analysis of RNA from cells stably transfected
with the marked gene having a deletion in the 3`-UTR (mE
ES1).
C, restriction sites in 3`-UTR used for preparation of
deletion constructs. The brackets depicted above show the
t
in hours for each of the four deletions that
were tested. Restriction sites: Esp, 2874; Bgl, 2957;
Bsm, 3003; Sac, 3061.
AE). This experiment demonstrates that this enhancer is
functional in the context of the intact Epo gene. Nevertheless, even in
the absence of this downstream element, the Epo gene still responded
substantially to hypoxic challenge. This observation poses a challenge
to conclusions in the recent literature that Epo's 3`-enhancer is
both necessary and sufficient for response to hypoxia in Hep3B cells
(18, 20) . The induction that we observed in the absence
of this 3`-enhancer is consistent with reporter gene experiments
demonstrating that Epo's promotor per se can confer a significant
induction by hypoxia
(19, 24) .
46 base sequence in the Epo 3`-UTR
(2957-3003) is required for the relatively short half-life of Epo
mRNA. Deletions in this region which prolonged the t
of the marked mRNA generally gave rise to enhanced basal (21%)
expression of marked Epo mRNA as assayed by PCR. (See and
Fig. 2B, bottom panel, showing increased ratios
of marked to endogenous mRNAs from cells exposed to 21% O
).
Despite the semiquantitative nature of these PCR analyses, they provide
information that could not be obtained from ribonuclease protection
assays, owing to the weakness of the RPA signals from 21% O
samples. This 46-base segment is at least 67 bases downstream of
the protein binding region described above. It is possible that the
upstream binding of proteins mediates ribonuclease cleavage at the
downstream site. It will be of considerable interest to ascertain
whether the same cytosolic proteins bind to the mRNAs of Epo and
tyrosine hydroxylase and perhaps to mRNAs of other oxygen sensitive
genes.
Table:
Stable transfections with marked Epo
gene: analysis by competitive PCR
,
and from cells exposed to 1% O
. The right hand column
(1%/21%) shows the hypoxic induction of the marked Epo gene relative to
the endogenous. 1%/21% =
[M
/E
]/[M
/E
].
Since the expression of the endogenous gene
(E
/E
) varies only slightly among the pools,
the data in this 1%/21% column reflects the induction of the marked Epo
gene relative to the endogenous; a value of 1 means that the marked
gene is equally as inducible as the endogenous. This table shows mean
values ± S.E.
+
2ab + b
). Thus, if the endogenous (E) and marked (M)
Epo transcripts were present in equivalent amounts, the final PCR
products would be 25% EE, 50% EM, and 25% MM. AccI would
digest only EE and HindIII would digest only MM.
ES construct at 21% O
was 2.1 ± 0.4 (S.E.),
compared to a mean M/E of 1.24 ± 0.31 for the mE-FL construct
( p = 0.05).
) expression of these two deletion mutants (
EBg and
BsS) relative to endogenous Epo was low (0.4 and 0.4,
respectively) compared to values of
ES and
BgS which have
long half-lives.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.