(Received for publication, July 13, 1995; and in revised form, November 9, 1995)
From the
Increased expression of the transcription factor Spi-1 (PU.1)
results from retroviral insertion in nearly all Friend spleen
focus-forming virus-transformed murine erythroleukemia cell lines and
exposure of these cells to MeSO, induces their
differentiation and decreases Spi-1 mRNA level by 4-5-fold. While
these results suggest that alterations in Spi-1 expression have
significant effects on erythroblast growth and differentiation, neither
the cause nor the effect of the decrease in Spi-1 expression that
follows Me
SO exposure has been established. The experiments
described here demonstrate that the effect of inducers on Spi-1
expression is regulated post-transcriptionally. Nuclear run-off
transcriptions demonstrated that Spi-1 transcription was not decreased
following Me
SO exposure. Additionally, expression of a
recombinant Spi-1 mRNA under transcriptional control of a
constitutively active Rous sarcoma virus promoter was regulated
identically to endogenous Spi-1 mRNA. The ability of Me
SO
to destabilize Spi-1 mRNA was selective, as the stability of the
erythroid transcription factors GATA-1 and NF-E2 were not similarly
effected. The effect of Me
SO on the stability of Spi-1 mRNA
provides a novel means of altering gene expression in these cells and
is likely to have significance for the differentiation of these cells.
SFFV()-transformed MEL cells are an established
system for studying erythroid growth and differentiation. These cells
are derived from mice infected with the Friend retrovirus complex,
which includes the defective SFFV and a helper Friend murine leukemia
virus(1) . Three discrete molecular events have been
identified, which contribute to the transformation of these
erythroblasts. An early, proliferative phase of the disease results
from interaction of the mutant env gene product of the SFFV
with the erythropoietin receptor, producing ligand-independent receptor
activation(2) . These infected erythroblasts are still able to
differentiate and withdraw from the cell cycle. The subsequent acute
erythroleukemic phase of the disease is associated with accumulation of
undifferentiated erythroblasts. For essentially all of these
erythroleukemias, genetic changes can be demonstrated in two additional
loci. These include mutations or deletions of p53 (3, 4) and retroviral insertion and transcriptional
activation of the Spi-1 (PU.1) gene(5, 6) , which
encodes a transcription factor(7, 8) . How these
changes interact to transform these cells remains unknown.
Spi-1 encodes a protein related to the ets family of transcription factors (9) which has a demonstrated role in regulating gene expression in monocytes and B-lymphocytes(10, 11, 12) . Additional evidence suggests this transcription factor also plays a role in erythroid differentiation. Spi-1 is normally expressed in erythroid progenitors (CFU-E)(13) , and mice lacking this gene die in utero, displaying a general defect in hematopoiesis including defective maturation of developing erythroblasts(14) . In the erythroleukemic stage of Friend SFFV disease, overexpression of Spi-1 is a universal occurrence(6) , and overexpression of Spi-1 has also been shown to immortalize erythroblasts in long-term bone marrow cultures(15) . In addition, two related ets family genes, fli-1 and v-ets, have also been implicated in erythroleukemic transformation(16, 17) . These findings strongly suggest that increased expression of Spi-1 is significant for erythroblastic transformation and for blocking the normal differentiation of these cells.
MEL cells undergo terminal
erythroid differentiation when exposed to MeSO or a number
of other inducing agents(18) , and the expression of Spi-1
decreases 4-5-fold in Me
SO-exposed
cells(13, 19) . To understand how inducers cause
differentiation of these cells, we investigated the regulation of Spi-1
expression in inducer-exposed cells. The data presented here
demonstrate that transcription of Spi-1 is unaltered by
Me
SO exposure, suggesting that the stability of this mRNA
is decreased by inducer exposure. A Spi-1 cDNA transcribed by a
constitutively active RSV promoter is regulated identically to
endogenous Spi-1, confirming that expression of this mRNA is regulated
post-transcriptionally and confining the required sequences for this
regulation to the coding region plus 10 nucleotides of 5`- and 3`-UT
sequences. In contrast, the stability of mRNAs encoding the erythroid
transcription factors, GATA-1 and NF-E2, are unaffected by inducer
exposure. These findings demonstrate that inducers can alter gene
expression via post-transcriptional mechanisms. The ability of
Me
SO to differentially affect transcription factor
stability suggests that this mechanism plays a significant role in
altering gene expression in cells exposed to this agent.
Expression of the recombinant Spi-1 transcripts were determined by
blot hybridization of RNAs separated by electrophoresis in 3.5%
polyacrylamide gels, as described by Stoeckle and Guan(23) , or
by RNase protection. For the RNase protections, a 271-base pair BamHI-XbaI fragment containing the bovine growth
hormone 3`-UT sequences and polyadenylation signal from the pRC/RSV
expression vector was used since RNA transcripts that included 3`-Spi-1
sequences were poorly digested with either RNase A and T1 or RNase One
(Promega), presumably due to stable secondary structure in this GC-rich
(62%) sequence(13) . The 3`-UT sequences were ligated between
the BamHI-XbaI sites in pBlueScript II SK and P-labeled antisense RNAs transcribed with T7 RNA
polymerase, as described by the manufacturer (Stratagene).
300,000-600,000 dpm of this radiolabeled RNA was hybridized with
15 µg of cytoplasmic RNA for 18 h at 60 °C in 0.4 M sodium chloride, 40 mM PIPES, 1 mM EDTA, 80%
formamide. The unhybridized RNA was digested to completion with RNase
One as described by the manufacturer (Promega), and the undigested
fragments were separated by electrophoresis in denaturing 6%
polyacrylamide gels and visualized by autoradiography.
The DNAs used in these experiments included
cloned DNAs encoding hsc70 (24) , -actin(25) ,
-globin(26) , Band 3(27) , GATA-1(28) ,
NF-E2 (29) , and Spi-1(13) . The cloning vectors for
these cDNAs, pUC19, pBlueScript II, and pGEMM were used as negative
controls. For determination of sense and antisense transcription,
pBlueScript II SK(+) and pBlueScript II SK(-) phagemids
containing the respective cDNAs were rescued by infection with VCS-M13
interference-resistant helper phage. Virus was isolated from the
supernatants by precipitation with polyethylene glycol as described by
Stratagene. Phage DNA was purified by phenol and chloroform extraction
and ethanol precipitation. For the experiments requiring probes
specific to the 3`-end of the Spi-1 transcript, a 250-base pair
fragment of 3`-untranslated Spi-1 sequence was excised by digestion
with ApaI and XhoI. The fragment was isolated by
agarose gel electrophoresis and ligated into the polylinker of
pBlueScript II SK(+) at the ApaI and XhoI sites.
Figure 1:
MeSO exposure causes a
selective decrease in Spi-1 mRNA abundance. MEL cells were grown in the
presence of Me
SO, and cytoplasmic mRNA was extracted at the
indicated times. Equal amounts of RNA were separated by gel
electrophoresis, and mRNA levels were determined by Northern blot
hybridization with
P-labeled cDNAs encoding Spi-1,
-actin, ribosomal protein S12, and
-globin. Results were
quantitated by densitometry and standardized for loading by normalizing
to results obtained by rehybridizing the blots with a labeled fragment
of the 18 S rRNA gene.
To determine if the decreased abundance of Spi-1 mRNA was
due to a decrease in its transcription, run-off transcriptions were
performed on isolated nuclei from cells grown under normal conditions
or following 24 h of MeSO exposure. Since previous studies
had demonstrated that antisense transcription occurs across some genes
in MEL cells (including c-myc(33) and
hsc70(32) ), the labeled run-off transcripts were hybridized to
single-stranded sense and antisense cDNA probes for Spi-1. As
demonstrated in Fig. 2A, a low level of antisense
transcription (detected by hybridization with the cDNA encoding the
positive strand) is detected across the Spi-1 gene in these cells. This
antisense transcription appears authentic, since no signal is detected
hybridizing with the phagemid vector. The sense strand (hybridizing to
the cDNA encoding the negative strand) is transcribed at approximately
10-fold the rate of the antisense transcript. However, as demonstrated
here, neither sense nor antisense transcription rate is significantly
altered following inducer exposure.
Figure 2:
MeSO exposure does not affect
transcription rate nor cause transcriptional attenuation of Spi-1. A, run-off transcriptions were performed on nuclei prepared
from control and Me
SO-exposed cells as described in the
text. Antisense Spi-1 transcripts were detected by hybridization of the
labeled transcripts with single-stranded phagemid DNA containing the
Spi-1 sense (+) strand. Sense transcripts were detected by
hybridization with single-stranded phagemid containing the Spi-1
antisense(-) strand. The remaining DNAs used in this experiment
were double-stranded. The plasmid DNAs pUC19 and pBlueScript II SK
(+) were included as negative controls. B, run-off
transcriptions were performed as above, and the labeled transcripts
were hybridized with a cloned Spi-1 cDNA, which contained only the
3`-terminal 250 nucleotides of the mature transcript. Other plasmids
included cDNAs with sequences encoding band 3,
-globin, and
-actin. The plasmid DNAs, pUC19 and pBlueScript II, were included
as negative controls.
The early decrease in expression
of c-myc mRNA that follows MeSO exposure of MEL
cells is due to transcriptional attenuation(34) . To determine
if Me
SO caused attenuation of Spi-1 transcription, the
run-off transcriptions were repeated and hybridized with a cDNA
fragment of Spi-1, which included only the 3`-UT sequences of this
mRNA. Transcripts extending to the 3`-UT of this gene were equally
represented in the run-off transcripts from both control and
Me
SO-exposed cells (Fig. 2B), demonstrating
that attenuation of transcription had not occurred on this gene. These
assays were sensitive to changes in transcription, since the
transcription of
-globin at this time had increased from 3 to 17%
of the rate actin transcription. The transcription of band 3 also
appeared to increase, although this could not be quantified due to the
lack of a detectable signal in the control cells. These results
demonstrate that the transcription of Spi-1 in MEL cells was unaffected
by Me
SO exposure, and thus the decreased accumulation of
this mRNA was mediated post-transcriptionally.
To determine the
half-life of Spi-1 mRNA following MeSO exposure, cells were
exposed to DRB, a specific inhibitor of RNA polymerase II
transcription, since actinomycin D has previously been shown to induce
MEL cell differentiation(35) . We analyzed mRNA half-life
following 24 h of inducer exposure, since the full extent of the
decrease in expression of Spi-1 was evident by this time (see Fig. 1). Cells were grown in the presence or absence of
Me
SO for 24 h, then DRB was added for an additional 4 h.
Cytoplasmic RNA was extracted at hourly intervals following the
addition of DRB, and Spi-1 mRNA levels were determined by Northern
blotting. The results shown in Fig. 3are normalized for RNA
loading as determined by rehybridizing the blots with an 18 S rRNA
fragment and represent the average of two separate experiments. For
control cells, the half-life of Spi-1 mRNA was determined to be 8.2 h,
while following 24 h of Me
SO exposure the half-life was
reduced to 3 h. The decay was noted to be biphasic for both control and
Me
SO-treated cells, with the initial decrease (at 1-2
h of DRB exposure) accounting for the entire difference in half-life.
Other investigators have also noted biphasic mRNA decay following
exposure to actinomycin D(30) , suggesting that a secondary
response to transcriptional inhibitors accounted for this latter rate.
While the measured decrease in half-life difference was slightly less
than the 3-4-fold decrease in Spi-1 mRNA abundance that followed
inducer exposure, these results supported the conclusion that the
stability of Spi-1 mRNA was decreased by inducer exposure.
Figure 3:
MeSO decreases Spi-1 mRNA
half-life. MEL cells were grown in the presence or absence of
Me
SO for 24 h; transcription was then inhibited by the
addition of DRB, and the cells were incubated for an additional 4 h.
RNA was extracted from the cells at 1-h intervals, and the Spi-1 mRNA
level was determined by Northern blot hybridization. The experiments
were performed in duplicate, and the results were quantitated by
densitometry and standardized for loading by normalizing to results
obtained by rehybridizing the blots with a cloned fragment of the 18 S
rRNA gene. Representative blots are shown in panel A, and the
results of the densitometric quantitation are shown in panel
B. The results are expressed as a percentage of Spi-1 mRNA present
at time 0, prior to the addition of DRB. The vertical bars in
panel B represent the range for each
determination.
The effect of MeSO on expression of
the recombinant transcript was determined on eight independent clones
that expressed detectable levels of the recombinant transcript. The
results of nuclease protection assays utilizing a probe derived from
the 3`-UT sequences of the vector are shown for four of these clones (Fig. 4A). A fragment of 135 nucleotides, which
corresponded to the expected length of the 3`-UT of the recombinant
transcript, was detected in all the clones. The smaller fragment
resulted from use of an alternative cleavage and polyadenylation site
present in the 3`-UT(36) . Hybridizing sequences were not
detected in control cells. Exposure to Me
SO (24 h) resulted
in decreased expression of the recombinant transcript in all analyzed
clones. To compare this decrease with that of endogenous Spi-1 mRNA,
clone pRC/Spi-1(8) was analyzed by blot hybridization of RNA separated
in 3.5% denaturing acrylamide gels. As demonstrated in Fig. 4B, following 24 h of Me
SO exposure
the extent of the decrease in expression of the recombinant mRNA was
identical to that of the endogenous mRNA. Similar behavior was observed
on two other clones similarly screened (not shown).
Figure 4:
MeSO exposure decreases the
expression of a Spi-1 mRNA transcribed by the RSV promoter. A Spi-1
cDNA containing 10 nucleotides of 5`- and 3`-untranslated sequence
under the transcriptional control of the RSV promoter was introduced
into MEL cells, and stable clones expressing the recombinant transcript
were selected in G418. A, the effect of Me
SO on
the expression of the recombinant transcript in four of these clones
was determined by RNase protection, utilizing a probe that hybridized
with the unique 3`-UT sequences derived from the vector. Each lane
represents the results of a hybridization with 20 µg of cytoplasmic
RNA from either the parental cell line (MEL) or from the cloned cells
(indicated by name above each paired set of lanes). Nuclease
protections were performed with RNA from uninduced cells (C)
and following 24 h of Me
SO exposure (D). A
protected fragment of the correct predicted size (135 nucleotides) was
detected in the selected clones but not in the parental cell line. The
smaller fragment results from the use of an alternative cleavage and
polyadenylation site in the vector. B, the effect of
Me
SO on the expression of both recombinant and endogenous
Spi-1 mRNAs was determined by electrophoresis of RNAs in 3.5%
acrylamide gels and blot hybridization as described in the text. The
results for clone pRC/Spi-1(8) are shown in the panel. Endogenous Spi-1
transcripts (wt) and the recombinant transcripts (
5`/3`) are indicated by the arrows to the right of the autoradiograph. Expression of the recombinant
transcripts in two other analyzed clones exhibited a similar
change.
The conclusion
that expression of the recombinant Spi-1 transcript was regulated
post-transcriptionally was inferred from evidence that the RSV promoter
is constitutively active under these conditions, as determined by
transient assays. ()However, to confirm this, the effect of
Me
SO on transcription of the recombinant Spi-1 gene in
clone pRC/Spi-1(8) was determined by run-off transcriptions. The vector
(pRC/RSV) without inserted cDNA sequences was used to detect the
3`-sequences uniquely present in the recombinant transcripts. Run-off
transcripts that hybridized to vector sequences were easily detected in
the transfected clone, and their transcription was unaffected by
Me
SO (Fig. 5). Similar results were found with
analysis of an additional clone. There was no significant hybridization
detected with the pRC/RSV vector sequences in the parental cell line
(see Fig. 6C). A weak signal was detected hybridizing
to the plasmid DNA, pUC19, in the electroporated cells. This was
detected in repeated experiments and was also noted for pGEMM plasmid
DNA (data not shown). Since this was not detected in the nuclear
run-off transcriptions performed on untransfected cells (see Fig. 1and Fig. 2), this may represent hybridization with
transcripts from the expression vector that extended into the plasmid
backbone, since transcription past the cleavage and polyadenylation
site would be an expected occurrence. These experiments confirmed that
transcription from the vector was constant and unaffected by
Me
SO exposure. Therefore, the decreased expression of the
recombinant Spi-1 mRNAs detected in the preceding experiments must be
mediated post-transcriptionally.
Figure 5:
Transcription of the recombinant Spi-1
transcript is unaffected by MeSO exposure. Transcriptional
activity of the RSV promoter was determined for the clone shown in Fig. 4B by nuclear run-off transcription in cells grown
with and without Me
SO (1.5%) for 30 h as described in the
text. Labeled transcripts were hybridized with 10 µg of linearized
plasmid DNAs. Plasmids containing cDNA inserts encoding
-globin
and the ribosomal protein L26 or the plasmid pUC 19, without inserted
sequences, were included as controls. Spi-1 transcripts derived from
the expression vector were detected by hybridization with 3`-UT
sequences present in the pRC/RSV vector to avoid detection of
endogenous Spi-1 transcripts. Hybridization with pRC/RSV sequences was
not detected in run-off transcriptions done on the parental cell line
MEL cells (see Fig. 6C).
Figure 6:
The stability of GATA-1 and NF-E2 mRNAs
are unaffected by MeSO exposure. A, RNA was
extracted from MEL cells at the times of Me
SO exposure
indicated, and expression of GATA-1 and NF-E2 mRNA assessed by Northern
blot hybridization is shown.
-globin mRNA expression was similarly
assessed. B, the results were quantitated by densitometry and
normalized for loading by hybridization with an 18 S rRNA gene
fragment. The results are presented graphically at the right of the figure. The increase in
-globin gene expression was
not quantified, since expression in uninduced cells was below the
limits of detection by densitometry. C, nuclear run-off
transcriptions were performed on uninduced cells and at 30 h of
Me
SO exposure. The preparation of nuclei and
P
labeling of run-off transcripts were performed as described in the
text. The labeled transcripts were hybridized with 10 µg of
slot-blotted plasmid DNAs with inserted cDNA sequences encoding
r-protein L26 and
-globin or with single-stranded phagemids with
inserted sequences corresponding to GATA-1 and NF-E2 antisense strands
to detect hybridization with the sense transcripts of these genes.
Phagemid DNA without inserted sequences (pBSSK) was included as
control. The plasmid pRC/RSV was also included as a control for the
run-off transcriptions performed on the electroporated cell lines shown
in Fig. 5.
The transient decrease in GATA-1 and NF-E2 mRNA
levels that followed MeSO exposure suggested that a
decrease in the stability of these mRNAs could have been offset by a
subsequent increase in their transcription. To determine if this was
the case, we assessed the transcription of these genes in control cells
and at 30 h of Me
SO exposure by nuclear run-off
transcription. At this time of inducer exposure, GATA-1 and NF-E2 mRNAs
were expressed at approximately 110 and 125% of their respective levels
in uninduced cells. Run-off transcriptions demonstrated that at 30 h of
Me
SO exposure, the transcription of these genes was
unchanged relative to their transcription rates in the control cells.
In contrast, an increase in transcription of
-globin was clearly
evident at this time. These results demonstrate that the stability of
these two mRNAs was not significantly altered by Me
SO
exposure. Therefore, the decrease in Spi-1 mRNA stability that occurs
following inducer exposure is not the result of a global decrease in
stability but exhibits specificity since neither GATA-1 nor NF-E2 mRNAs
are similarly affected.
Exposure of MEL cells to the inducer of differentiation,
MeSO, causes a 3-5-fold decrease in accumulation of
mRNA encoding the transcription factor Spi-1(13, 19) .
The data presented here demonstrate that post-transcriptional
regulatory mechanisms are responsible for this decrease. In contrast,
the stability of mRNAs encoding the transcription factors, GATA-1 and
NF-E2, is unaffected by inducer exposure, demonstrating specificity to
this regulation. These findings provide insight into the regulation of
Spi-1 expression and suggest that differential stability of
transcription factor mRNAs following inducer exposure may play a role
in establishing the pattern of gene expression in the differentiating
cells.
Exposure of MEL cells to MeSO results in a
70-75% decrease in accumulation of Spi-1 mRNA. Nuclear run-off
transcriptions demonstrated that this decrease was mediated
post-transcriptionally. Since increased expression of Spi-1 in Friend
virus-transformed cells is due to retroviral insertional activation of
transcription, these results suggest that the transcriptional activity
of the retroviral long terminal repeat is unaffected by
Me
SO, and results of transient transfection assays have
reached a similar conclusion. (
)Thus, the decreased
expression of Spi-1 is regulated post-transcriptionally. While the data
presented here do not exclude that Me
SO blocked
nucleocytoplasmic transport of this mRNA, this appears unlikely since
Schuetze et al.(19) detected a similar decrease in
Spi-1 expression when whole cell mRNA was analyzed.
Determination of
Spi-1 mRNA half-life by transcriptional inhibition demonstrated that
MeSO exposure decreased the stability of Spi-1 mRNA. Since
the expression of a recombinant Spi-1 cDNA transcribed by the
constitutively active RSV promoter was regulated similarly to
endogenous Spi-1, this limits the sequence required for this regulated
change in stability to the coding region plus the adjoining 10
nucleotides of 5`- and 3`-untranslated sequences. The coding region of
a number of other mRNAs, including
-tubulin, c-fos, and
c-myc, has also been shown to play a role in determining
stability(43, 44, 45, 46) . The
mechanisms by which these sequences regulate mRNA stability remain to
be established. However, the ability of inducers of differentiation to
regulate the effect of these elements provides an additional means to
investigate these mechanisms.
The degradation of mRNAs is frequently
preceded by their
deadenylation(30, 37, 38, 39, 40) ,
and this is consistent with our recent observation that the inducers
MeSO, hypoxanthine, and A23187 all cause substantial
deadenylation of mRNAs. (
)Spi-1 is one of the mRNAs so
affected. In contrast, for two ``housekeeping'' mRNAs
(specifically ribosomal protein L26 and S12), inducer exposure affected
neither abundance nor poly(A) tail length. Although the mechanisms
resulting in this selectivity of deadenylation are unknown, this likely
plays a role in the differential stabilities of these mRNAs following
inducer exposure. Current studies are assessing the effect of
Me
SO on the adenylation of GATA-1 and NF-E2 mRNAs.
Regardless of the mechanism(s) that account for the differential
stability of Spi-1 and GATA-1 and NF-E2 mRNAs in inducer-exposed MEL
cells, these differences are likely to be significant for the
regulation of gene expression in differentiating cells. While the
MeSO-induced decrease in expression of Spi-1 has not yet
been demonstrated to play a role in MEL cell differentiation, the
consistent overexpression of Spi-1 during leukemogenesis of these cells
suggests that this decrease is unlikely to be without effect. Further,
the ability of Me
SO to destabilize mRNAs is unlikely to be
limited to Spi-1. It is noteworthy that expression of a recombinant Id
transcript in MEL cells has been reported to be decreased by
Me
SO(48) , suggesting that Me
SO also
destabilizes this mRNA. The stability of GATA-1 and NF-E2 following
inducer exposure has increased significance in light of evidence
suggesting that the activity of GATA-1 containing promoters can be
regulated by competition for binding with other transcription
factors(49, 50) . Thus, the ability of
Me
SO to destabilize Spi-1 or other, yet to be identified
transcription factor mRNAs may lead to a functional increase in
activity of those mRNAs unaffected by this change. Further knowledge of
how inducers affect mRNA stability should provide additional insights
into the mechanisms by which these agents cause differentiation of
these cells.