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Erythropoietin mRNA Levels Are Governed by Both the Rate of Gene
Transcription and Posttranscriptional Events
By Mark A. Goldberg, Christopher C. Gaut, and H. Franklin Bunn
The human hepatoma cell line, Hep3B. synthesizes large
quantities of erythropoietin (Epo) mRNA and protein in a
regulated manner in response to hypoxia and cobaltous
chloride (CoCI,). To further understand the regulation of Epo
gene expression, we studied the effects of hypoxia and CoCI,
on the rate of Epo gene transcription. While Northern blot
analyses showed that steady-state Epo mRNA levels increase more than 50-fold in response to hypoxia or CoCI,,
nuclear run-off experiments demonstrated only a 10-fold
increase in Epo gene transcription in response to these
stimuli. In the presence of either actinomycin D (Act D) or
cycloheximide, the stability of biologically functional Epo
mRNA was much greater than that observed in the absence
of these agents and much greater than that which has been
reported in vivo. These findings suggest that the stability of
Epo mRNA is modulated by the transcription and translation
of rapidly turning over gene product(s). Thus, Epo mRNA
levels are determined by both the rate of gene transcription
and posttranscriptionalevents. These experiments demonstrate a potential pitfall in estimating mRNA half-lives based
on Act D chase experiments alone.
o 1991by The American Society of Hematology.
R
Hep3B cell line, a homogeneous population of Epoproducing cells.
EGULATION OF the expression of highly inducible
genes, such as growth factors and oncogenes, has
been the subject of much in~estigation.'.~The protein
products of these genes are frequently quite labile and their
in vivo levels may fluctuate by several orders of magnitude
depending on particular physiologic conditions. The production of these proteins is dependent on precise regulation of
mRNA levels. In turn, the steady-state levels of mRNA are
determined by both the rate of gene transcription and the
rate of mRNA decay.2.8-'o
Erythropoietin (Epo) is a glycoprotein hormone essential
for the regulation of red blood cell production. It is
produced in the fetal liver" and the adult kidney" in
response to hypoxia and circulates in the plasma with a
~
levels may increase as
half-life of 4 to 7 h 0 ~ r s . lSerum
much as 1,000-fold in severe anemia. Conversely, inappropriate overproduction of Epo may cause life-threatening
polycythemia due to markedly increased blood viscosity and
decreased blood f l o ~ . ' ~Thus,
, ' ~ the Epo-synthesizing cells
must possess the ability to regulate the rate of Epo
production over a wide range. One of the most puzzling
aspects of erythropoiesis is the mechanism by which hypoxia triggers increased expression of the Epo gene.
We have previously demonstrated that the human hepatoma cell line. Hep3B, synthesizes large quantities of Epo
in a regulated manner in response to hypoxia and cobaltous
chloride (CoCl,), and that this regulation occurs at the Epo
mRNA le~e1.'~~''
In addition, we have previously shown that
ongoing protein synthesis is required for both the hypoxia
and cobalt induction of increased levels of Epo mRNA in
Hep3B cells." Northern blot analyses of RNA from murine
kidneys show greater than a 100-fold increase in the level of
Epo mRNA in the presence of hypoxia or cobaltous
To assess changes in the rate of Epo gene
~hloride.'~,'~
transcription that might account for this marked increase in
steady-state Epo mRNA levels, Schuster et alZoperformed
nuclear run-off experiments on nuclei isolated from the
whole kidneys of hypoxic and nonhypoxic mice. They could
detect Epo gene transcription only in nuclei from hypoxic
kidneys. A very small percentage of this mixed population
of cells were potential Epo-producing cells.21To gain a
better understanding of the level of regulation of the Epo
gene, we have examined the influence of hypoxia and CoCl,
on Epo gene transcription and Epo mRNA stability in the
Blood, Vol77, No 2 (January 15), 1991: pp 271-277
MATERIALS AND METHODS
Cell culture. Hep3B cells were obtained through American
Type Culture Collection. They were cultured in MEM Alpha
medium (GIBCO, Grand Island, NY) supplemented with penicillin (100 U/mL), streptomycin (100 pg/mL), and 10% defined
supplemented bovine calf serum (Hyclone, Logan, UT) and were
maintained in a humidified 5% CO, incubator at 37°C. The cells
were grown hypoxically either as described previously'6 or in a
controlled atmosphere chamber (Plas Labs, Lansing, MI) supplied
with a constant flow of a hydrated 1% 0,, 5% CO, balance N, gas
mixture. All experiments were begun when the Hep3B cells
approached confluence.
Radioimmunoassay (RIA). The RIA for Epo was performed
using a high-titer polyclonal rabbit antiserum raised in our laboratory against human recombinant Epo. lZsIrecombinant human Epo
was obtained from Amersham, Inc (Arlington Heights, IL). Standards were prepared using recombinant human Epo from Amgen,
Inc (Thousand Oaks, CA) diluted in MEM Alpha medium containing 10% defined supplemented calf serum (Hyclone) and 0.05%
sodium azide, pH 7.4. Aliquots of 0.2 mL of standard or sample
were placed in 5 mL conical polypropylene tubes. To this was
added 0.1 mL of rabbit antiserum diluted 1:15,000 in phosphatebuffered saline (PBS) containing 0.5% bovine serum albumin. The
mixture was diluted to 0.7 mL using the same diluent used for the
recombinant Epo standards and was incubated at room temperature for 2 hours. '''I Epo, 0.1 mL (approximately 10,000 cpm),
prepared in the same diluent was then added, the mixture was
briefly vortexed and incubated overnight at 4°C. One milliliter of
Amerlex-M (Donkey antirabbit globulin, Amersham, UK) was
From the Department of Medicine, Hematology Divhion, Brigham
and Women's Hospital and Harvard Medical School, Boston, MA.
Submitted April 30, 1990; accepted September 4, 1990.
Supported in part by National Institutes of Health (NIH) Grant No.
DK41234. M.A.G. is the recipient of an NIH Physician Scientist
Award (Grant No. DK01401).
Address reprint requests to H. Franklin Bunn, MD, Brigham and
Women's Hospital, Hematology Division, 75 Francis St, Boston, M A
02115.
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 I734 solely to
indicate this fact.
0 I991 by TheAmerican Society of Hematology.
0006-4971/91/7702-01I9$3.00/0
27 1
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GOLDBERG, GAUT, AND BUNN
272
then added to each tube and the tubes were placed at 4°C with
constant shaking for 2 hours. The Amerlex was pelleted by
centrifugation for 15 minutes at 1,500gat 4”C,washed once with 1.0
mL PBS, and counted in an LKB model 1282gamma counter.
Northern blot anaiysis. Total RNA was prepared from cultured
cells as described by Chirgwin et al.’, The RNA was denatured in
formaldehyde, electrophoresed on a 1%agarose gel containing 2.2
m o m formaldehyde and a trace amount of ethidium bromide, and
transferred to a Genescreen Plus filter (New England Nuclear,
Boston, MA) using 1OX standard saline citrate (1.5 mol/L NaCI,
0.15 mol/L sodium itr rate)?^ Epo cDNA in an SP65 plasmid was
digested with the restriction enzyme EcoR1, the Epo insert isolated
by agarose gel electrophoresis, followed by electr~elution~~
and
”P-labeled to a specific activity of between 3 x 108 and 1.2 x lo9
cpm/kg of c D N A . ~The radiolabeled cDNA was then mixed with
carrier salmon sperm DNA, denatured by boiling for 10 minutes,
and hybridized to the filter at 5 x lo5 cpm/mL of hybridization
solution (50% formamide, 1 m o m NaCl, 1% sodium dodecyl
sulfate, 10% dextran). Hybridization was performed at 42°C for 20
hours. The final washing was done in 0.5X standard saline citrate
(0.075 mol/L NaCl, 0.0075 m o m sodium citrate) at 65°C. Autoradiography was performed with intensifying screens at -80°C using
Kodak X-Omat AR film (Eastman Kodak, Rochester, NY).
Mouse p-actin cDNA was also radiolabeled and hybridized to
the same filters to provide an internal control for the efficiency of
RNA transfer to the filters.
Nuclear run-off experiments. The transcriptional component of
Epo gene regulation was assessed by nuclear run-off experiments,
as described by Greenberg.= Hep3B cells were exposed to 75
pmol/L CoCI, or 1% 0, for varying lengths of time. After isolation
of nuclei by lysis of cells in NP-40 buffer, in vitro transcription was
completed in the presence of aZ2P-UTP.Radiolabeled RNA was
then isolated and the amount of trichloroacetic acid precepitable
radioactivity was quantitated. At each time point an equal amount
of ”P-RNA was hybridized to nitrocellulose filters containing a
linearized plasmid possessing either a nearly full-length Epo
cDNA clone or a full-length, human Epo genomic clone (4.0 kb
Epo gene extending from 0.4 kb upstream from the putative start
site to 0.7 kb downstream from the polyadenylation signal). A
plasmid containing a linearized mouse p-actin cDNA fragment
served as a positive control while a linearized plasmid containing a
human P-globin genomic clone provided a negative control. Densitometric scanning was performed using a Molecular Dynamics
Model 300A computing densitometer. Relative levels of Epo
mRNA transcription represent arbitrary units normalized to p-actin mRNA transcription. Normalization to p-actin mRNA was
used because this is an abundant mRNA and its transcriptional
rate has been shown to be minimally influenced by hypoxia?6
Epo mRNA stubiliv. Hep3B cells were placed in either a 1% or
21% 0, containing atmosphere for 18 to 24 hours at 37°C. At “0
time” the 0, content of the atmosphere was manipulated as
described below and either actinomycin D (Act D) or cycloheximide was added to final concentrations of 5 @mL or 20 pg/mL,
respectively. After varying periods of incubation, total cellular
RNA was isolated from the cells and Northern blot analyses were
performed. Densitometric scanning was performed with a Molecular Dynamics Model 300A computing densitometer. Relative Epo
mRNA levels represent arbitrary units normalized relative to
p-actin mRNA levels.
RESULTS
Northern blot analyses were performed on RNA obtained from Hep3B cells grown for 18to 24 hours in either a
1% or 21% oxygen atmosphere. The RNA blots were
hybridized with Epo and actin radiolabeled probes and the
levels of each of these mRNAs was quantitated by densitometry as described above. Analysis of 14 such experiments demonstrated a greater than 50-fold mean increase
in steady-state Epo mRNA levels (representative blots are
shown in lanes 1 and 2 of Figs 2B and 3B). This stated
increase in steady-state Epo mRNA levels represents a
minimal estimate because densitometry loses linearity in
the very low and very high ranges. In contrast, actin mRNA
levels were not significantly influenced by hypoxia as
estimated by concordance with the levels of 18s and 28s
ribosomal RNA levels observed on the ethidium stained
gels.
To assess the relative contribution of changes in the rate
of Epo gene transcription to this marked increase in Epo
mRNA steady-state levels, nuclear run-off experiments
were performed. Initial experiments were performed with a
nearly full-length Epo cDNA probe as detailed in the
Materials and Methods section. However, with this probe it
was difficult to detect a hybridizing signal in experiments
performed on the nuclei of either hypoxic or nonhypoxic
cells. In an attempt to increase sensitivity we used a human
Epo full-length genomic clone that has greater homology
with the unspliced primary RNA generated in the in vitro
transcription reaction. With this probe we were able to
detect low-level transcription even in the absence of hypoxia. In contrast to the more than 50-fold increase in
steady-state Epo mRNA levels observed in RNA blot
analyses, three nuclear run-off experiments performed on
Hep3B cells (Fig 1) demonstrated a 5-to 10-fold increase in
Epo mRNA transcription in response to hypoxia or CoCI,.
Important to the interpretation of the experiments shown
in Fig 1 is the finding that the total counts per minute of
32P-radiolabeledRNA recovered from the in vitro transcription reactions on nuclei of cells grown for 8 hours in 21%
oxygen, 1% oxygen, or 75 p”l/L CoCI,, were within 10%
of one another, suggesting that 8 hours of hypoxia or
exposure to 75 kmol/L CoCI, had no significant change on
overall gene transcription.
Assuming a zero-order rate of formation of Epo mRNA
and a first-order rate of mRNA decay:”
a mere 10-fold
increase in the rate of Epo mRNA synthesis cannot by itself
explain the greater than 50-fold increase in steady-state
Epo mRNA levels observed after stimulation by hypoxia or
cobalt. Therefore, experiments were undertaken to investigate posttranscriptional influences on steady-state Epo
mRNA levels. Initially, Hep3B cells were grown in 1% 0, to
increase Epo mRNA levels, and subsequently were switched
to 21% 0,. Epo mRNA levels were then determined after
varying periods of time by Northern blot analyses. As
demonstrated in Figs 2 and 3, in 21% 0, Epo mRNA has a
rapid rate of decay with steady-state levels decreasing by
50% within 1.5 to 2 hours and with almost undetectable
levels by 6 hours. This finding is similar to reported changes
in kidney Epo mRNA levels in mice switched from a
hypoxic to a nonhypoxic environment:’ and represents a
maximal estimate of the in vivo half-life of the Epo mRNA
(because new transcription was not blocked). To distinguish between transcriptional and posttranscriptional con-
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REGULATION OF ERYTHROPOIETIN MRNA LEVELS
A
273
EPO
Actin
Globin
hours hypoxia
0
2
4
6
8
12001
a
z
U
E
0
n
W
-Q
.->
Fig 1. Transcriptional component of Epo gene regulation. Nuclear
run-off experiments, as described by Greenberg,” were performed on
near confluent Hep3E cells exposed to 75 pmol/L CoCI, or 1% 0, for
varying lengths of time as indicated. (A) Autoradiogram of a fitter
demonstratinghypoxia-induced increase in Epo gene transtription. (E)
Results of densitometry normalized to mouse p-actin as described in
Materials and Methods. [-E-),
1% oxygen; (-m-),
cobalt.
tributions to this observed decrease in steady-state Epo
mRNA levels, Act D chase experiments were performed.
Initial experiments demonstrated that Act D at a concentration of 5 pg/mL blocked greater than 98% of the incorporation of ’[HI-uridine into total cellular Hep3B RNA under
both hypoxic and nonhypoxic conditions. Hep3B cells were
made hypoxic to allow an increase in Epo mRNA levels.
After the addition of Act D the cells were either kept
hypoxic or rapidly equilibrated with a 21% 0, atmosphere
for various periods of time. The stability of the Epo mRNA
in the presence of Act D at both 0, tensions was much
greater than that observed in the absence of Act D (Fig 2)
and much greater than that reported in vivo. Thus, the
addition of Act D resulted in an increased stability of Epo
mRNA (estimated T,, = 7 to 8 hours). Of note, when
Hep3B cells were grown continually in 1% 0, in the
absence of Act D, Epo mRNA levels continued to remain
elevated (ie, did not peak and then decline) throughout the
entire period of study.
If Act D was preventing the transcription of rapidly
turning over mRNA whose protein product or products
participate in the degradation of Epo mRNA, one would
expect cycloheximide to have a similar effect on the stability
of Epo mRNA. Hence, parallel experiments were performed using 20 pg/mL cycloheximide instead of Act D.
These experiments demonstrated a reproducible increase
in the stability of the Epo mRNA in the presence of
cycloheximide as well (Fig 3). Evidence that the cycloheximide enhances Epo mRNA stability rather than de novo
transcription is provided by the fact that cycloheximide
blocks hypoxia and CoCI,-induced increases in Epo mRNA”
and cycloheximide by itself does not cause an increase in
m
a
U
0
1
0
.
I
2
.
I
.
I
6
l i m e (hours)
4
.
,
8
.
1
10
steady-state Epo mRNA levels.” However, as seen in Fig 3,
the effect of cycloheximide on increasing Epo mRNA
stability is not quite as marked as the effect of Act D. This
may be due to the inability of cycloheximide to block
translation for a sustained period of time” as completely as
Act D blocks transcription.
To assess the possible biologic relevance of the prolonged stability of Epo mRNA in the presence of Act D,
experiments were performed to determine if this stabilized
mRNA was available for translation into Epo protein.
Hep3B cells were grown hypoxically for 20 to 24 hours. The
culture medium was removed and frozen for subsequent
determination of Epo concentration by RIA (mean 2 1
standard deviation = 91 2 8 mU/mL, n = 32), the cells
were washed thoroughly, and fresh medium was added. The
cells were then grown in 1% or 21% 0, in the presence or
absence of Act D. After 14 hours the medium was removed
and assayed for the presence of Epo by RIA. As shown in
Fig 4, Hep3B cells switched to 21% 0, in the presence of
Act D were fully capable of sustaining Epo production and,
in fact, secreted significantly more Epo protein into the
culture medium than cells switched to 21% 0, in the
absence of Act D (mean 2 1 standard deviation = 52 2 9
mU/mL v 33 2 8 mU/mL, P < .OS). This finding strongly
supports the conclusion that the increased Epo mRNA
levels observed in the presence of Act D are not due to
nonspecific cytotoxic effects. Furthermore, the observation
that Hep3B cells grown continually in 1% 0, in the absence
of Act D secreted even more Epo than cells grown in 1% 0,
in the presence of Act D suggests that both the rate of Epo
gene transcription and posttranscriptional events influence
steady-state Epo mRNA levels.
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GOLDBERG, GAUT, AND BUNN
274
a
z
U
E
0
P
W
Q)
.->
c
Q
Q)
U
A
0
4
8
12
16
20
time (hours)
28s -
18S-
AcfinomycinD
kb/rSOfZf%02
B
- - - t - + - t
6 6
24 0 2 2 4 4
ACTIN
DISCUSSION
These experiments suggest that the hypoxia and cobalt
induced increase in Epo mRNA steady-state levels cannot
be accounted for by increased Epo gene transcription
alone. As noted previously, in the nuclear run-off experiments described here, a human Epo full-length genomic
clone was used to probe the in vitro transcription products.
While the genomic probe allowed for increased sensitivity
and enabled detection of low-level transcription even in the
absence of hypoxia, it is not without potential drawbacks. If
the intervening sequences include repetitive sequences that
are transcribed elsewhere as well, the run-off assay may not
be totally specific for Epo mRNA. In fact, in the intervening
sequence between exons I11 and IV is a member of the Alu
family of repeated sequences.%This region is 70% homologous to the consensus Alu sequence?" Because the 100%
homologous Epo cDNA gave a relatively weak signal, it is
unlikely that under the hybridization conditions used this
relatively short, imperfectly conserved, repetitive sequence
would give a significant hybridization signal. Furthermore,
Fig 2. Epo mRNA stabiiity. Experiments were
performed as described in the Materials and Methoda
section. Relative Epo mRNA levels represent arbiWary units normalized relative to p-actin mRNA levels. (A) (-A+,
21% oxygen; (-A+,
21% oxygen Act D; (U
1%
)
oxygen
,
+ Act D. Each data
point in (A) is the average (+1 standard deviation) of
at least two experimentswith most points representing the average of three or more experiments. (E) A
representativeRNA blot demonstratingthe effects of
Act Don Epo mRNA stability.
+
when the Epo cDNA probe was used, a weak hybridization
signal was seen even from nuclei of cells grown in 21% 0,
(data not shown). This result argues strongly that some
baseline Epo gene transcription is ongoing in 21% 0,.
Hence, the hybridization signal seen at 21% 0, using the
genomic Epo probe is unlikely to simply represent nonspecific, background hybridization.
Like Schuster et al,m we also note an increase in Epo
gene transcription in response to hypoxia, although the
degree of increase appears to be less. Our nuclear run-off
experiments used nuclei from a homogeneous population
of Epo-producing human hepatoma cells grown in continuous culture. On the other hand, Schuster et almused nuclei
from a heterogeneous population of primary murine kidney
cells, only a very small proportion of which were actually
Epo-producing cells. These differences may account for the
difference in the degree of increase in Epo gene transcription.
The data presented in this report suggest that Epo
mRNA levels are governed by a complex mechanism
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REGULATION OF ERYTHROPOIETIN MRNA LEVELS
275
1
2
0
6
4
8
(hours)
time
28S-
18S-
1
+
Ben; (-A+,
21% oxygen
cyclohex. Each data
point In (A) is the average ( 2 1 standard deviation) of
at least two experiments, with most points representing three or more experiments. (B) A representative
RNA blot demonstrating the effect of cycloheximide
on Epo mRNA stability.
B
involving both the rate of gene transcription and posttranscriptionalevents. The fact that steady-state Epo mRNA
levels increase more than 50-fold in response to hypoxia
and CoCI, while Epo gene transcription increases only
about 10-fold in Hep3B cells suggests that there is a
significant posttranscriptional component to Epo gene
regulation in this cell line. Numerous examples of posttranscriptional regulation of mRNA have been well documented, particularly at the level of mRNA
Many
examples of regulated mRNA stability involve genes whose
protein products play a crucial role in cell growth regulation
and s ~ r v i v a l . ~Although
~ . ~ ~ ~ ”still
~ ~poorly
~
understood, multiple mechanisms for this regulation of the rate of mRNA
degradation have been demonstrated2; however, many of
these mechanisms seem to share certain common features.
In many cases mRNA degradation requires ongoing protein
synthesis because stabilization occurs in the presence of
various protein synthesis inhibitor^.""^'^^ Two explanations for the requirement of ongoing protein synthesis have
been proposed: (1) certain mRNAs may be degraded by
rapidly turning over specific ribonucleases, and (2) the
degradation of some mRNAs may be directly coupled to
their translation. Cotranslational degradation has been
+
+- +
3 3 5 5
1
L
ACT1 N
21% 0 2 -> 21% 0 2
1% 0 2
* 21% 0 2 + Act
D
1% 0 2 -w 1% 0 2 + Act D
1%
o 2 * 1% 0
*
.
0
.
20
.
40
.
.
.
60
80 100
Epo mU/ml
.
120
.
140
Fig 4. Biologic activity of Epo mRNA. After approaching confluence, the media was changed and the cells were grown in an
atmosphere containing either 1% or 21% 0, for 20 to 24 hours. The
media was then removed, tha calls were washed thoroughfy with
PBS. and incubated with 5 mL of fresh medium with or without Act D
(5 pg/mL) in either a 1% or 21% 0, environment, as indicated In the
figure, for an additional 14 hours. The media was then removed and an
Epo RIA was performed in duplicate on an aliquot. Each data bar
represents the average ( 2 1 standard deviation) of eight separate
experiments.
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GOLDBERG. GAUT, AND BUNN
276
elegantly demonstrated for tubulin and histone mRNAs.”.”
On the other hand, although indirect evidence suggests the
presence of rapidly turning over specific ribonucleases:
thus far none of these nucleases have been well characterized.
If the expression of these putative nucleases is regulated
at the level of transcription, one would expect that inhibitors of transcription as well as inhibitors of protein
synthesis would stabilize the mRNA targets of these ribonucleases. This is not the case for the protein synthesisrequiring degradation of the c-myc, c-fos, 32,34 and granulocyte-macrophage colony-stimulating factor7 mRNAs.
However, the transcription inhibitor Act D does stabilize
poly(1) poly((=)-induced fibroblast interferon
as
well as prevent the decay of human transferrin receptor
mRNA.” Furthermore, recent studies by Shyu et a1%
demonstrate two independent determinants of c-fos mRNA
instability, one of which appears to be dependent on
ongoing transcription. Epo is another case in which mRNA
instability requires both ongoing transcription and protein
synthesis. However, unlike many of the other known examples, Epo’s biogenesis”r3’ and its biologic action^'^ are
extremely tissue specific.The data presented here support a
mechanism in which Epo mRNA degradation requires both
the transcription and translation of a rapidly turning over
mRNA (or mRNAs) whose protein product(s) participates
in the degradation of Epo mRNA. In addition, this phenomenon clearly demonstrates a potential pitfall in relying on
Act D chase experiments alone to estimate mRNA halflives.
The evidence presented here suggesting the existence of
a rapidly turning over Epo mRNA degrading protein raises
the possibility that the concentration and/or the activity of
this protein may be decreased during hypoxia or CoCl,
exposure. However, direct demonstration of PO,-dependent changes in Epo mRNA stability in intact Hep3B cells
is impossible using Act D because the Act D itself alters the
mRNA stability by blocking synthesis of the presumptive
Epo mRNA destabilizing protein. One alternative might be
to perform ’[HI-uridine pulse-chase experiments, but this
approach requires medium to high abundance mRNA
Another more direct approach, currently underway, is to characterize this Epo mRNA destabilizing protein, and the sequences to which it binds, in an in vitro
system.@
In the case of Epo gene regulation, the finding that
steady-state Epo mRNA levels may be significantly influenced by a rapidly turning over Epo mRNA degrading
protein is particularly intriguing because it may have
biologic significance. The combination of the hypoxiainduced increased Epo gene transcription coupled with
increased Epo mRNA stability permits a marked amplification of Epo mRNA levels. A modest increase in the rate of
Epo gene transcription and a modest increase in Epo
mRNA stability acting in synergy can explain how the cell is
able to regulate Epo mRNA levels such that during
hypoxia, circulating levels of Epo protein can increase by
three orders of magnitude.
ACKNOWLEDGMENT
The full-length human genomic Epo clone and the Epo cDNA
clone were kindly provided by Genetics Institute, Inc (Cambridge,
MA). Mouse p-actin cDNA was a gift from Dr Bruce Spiegelman
of the Dana-Farber Cancer Institute (Boston, MA). We thank
Thomas Schneider for his excellent technical assistance.
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REGULATION OF ERYTHROPOIETINMRNA LEVELS
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1991 77: 271-277
Erythropoietin mRNA levels are governed by both the rate of gene
transcription and posttranscriptional events
MA Goldberg, CC Gaut and HF Bunn
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