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Mol Cell Biol, July 1998, p. 4252-4261, Vol. 18, No. 7
Department of Viral Oncology, Institute for
Virus Research, Kyoto University, Kyoto 606, Japan
Received 4 March 1998/Returned for modification 7 April
1998/Accepted 21 April 1998
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cytoplasmic Sequestration of the Polyomavirus Enhancer Binding
Protein 2 (PEBP2)/Core Binding Factor
(CBF) Subunit by
the Leukemia-Related PEBP2/CBF
-SMMHC Fusion Protein
Inhibits PEBP2/CBF-Mediated Transactivation
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
REFERENCES
SUMMARY
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The polyomavirus enhancer binding protein 2 (PEBP2)/core binding
factor (CBF) is a transcription factor composed of two subunits, 
and 
. The gene encoding the subunit is disrupted by inv(16), resulting in the formation of a chimeric protein, -SMMHC, which is
associated with acute myelogenous leukemia. To understand the effect of -SMMHC on PEBP2-mediated transactivation, we used a luciferase assay system in which contribution of both the and subunits was absolutely required to activate transcription. Using this
system, we found that the minimal region of the subunit required
for transactivation resides between amino acid 1 and 135, which is
known to dimerize with the subunit. In contrast, -SMMHC, despite
having this minimal region for dimerization and transactivation, failed
to support transcription with the subunit. Furthermore -SMMHC
blocked the synergistic transcription achieved by PEBP2 and
CCAAT/enhancer binding protein . By using a construct in which the
PEBP2 subunit was fused to the glucocorticoid receptor ligand
binding domain, we demonstrated that coexpressed -SMMHC tightly
sequestered the subunit in the cytoplasm and blocked dexamethasone-dependent nuclear translocation of the subunit. Thus,
the result suggess that -SMMHC inhibits PEBP2-mediated transcription via cytoplasmic sequestration of the subunit. Lastly proliferation of ME-1 cells that harbor inv(16) was blocked by an antisense oligonucleotide complementary to the junction of the
chimeric mRNA, suggesting that -SMMHC contributes to leukemogenesis by blocking the differentiation of myeloid cells.
INTRODUCTION
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A member of the transcription factor
family of proteins called polyomavirus enhancer binding protein 2 (PEBP2) or core binding factor (CBF) is composed of dimers of and
subunits. The subunit possesses regions responsible for direct
DNA binding and transactivation, while the subunit facilitates DNA
binding by the subunit (12, 32). Gene cloning
studies revealed three subunit encoding genes,
PEBP2A/ CBFA1/AML3, PEBP2B/CBFA2/AML1, and PEBP2C/ CBFA3/AML2,
in mammals, and two genes, runt and lozenge,
in Drosophila. The subunit is encoded by a single
gene, PEBP2/CBFB, in mammals, and by two genes,
brother and big brother, in
Drosophila.
Both the and subunit-encoding genes are independently
rearranged in the chromosomal abnormalities associated with leukemia. Chromosomal translocations involving AML1 include t(8;21) in
the French-American-British (FAB) M2 subtype of acute myelogenous leukemia (AML) and t(12;21) in childhood acute lymphoblastic leukemia (ALL). The t(8;21) and t(12;21) translocations produce the
chimeric proteins AML1/ETO(MTG8) and TEL-AML1, respectively
(5, 9, 23). The subunit is rearranged in inv(16) of the
FAB M4Eo subtype of AML, producing a chimeric protein,
CBF/PEBP2-SMMHC (18) (referred to here as
-SMMHC). The incidences of these fusion proteins involving
PEBP2 subunits in leukemias are as follows: AML1/ETO, 12% of AML;
-SMMHC, 12% of AML; and TEL-AML1, 20% of ALL. These
values represent a significant contribution to human leukemia in
general (19).
Recently, both the and subunits were shown to be essential for
hematopoiesis in mice. Targeted disruption of either the PEBP2B/CBFA2/AML1 or PEBP2/CBFB gene
resulted in nearly identical phenotypes of embryonic lethality with
accompanying hemorrhage of the central nervous system and defects in
fetal liver definitive hematopoiesis (25, 28, 30, 37, 38).
These studies thus proved that both subunits of PEBP2 are indispensable
for its in vivo function. Interestingly, heterozygous mice having
targeted insertion (knockin) of genes complementary to
AML1/ETO and CBF/PEBP2-MYH11 (coding for the
-SMMHC protein) displayed phenotypes similar to those of the
corresponding targeted disruptions (4, 41). This finding
indicated that both chimeric proteins acted as dominant negative
effectors.
The subunit has been shown by electrophoretic mobility shift assays
(EMSA) to act as a cofactor for DNA binding. This subunit is derived
from the single PEBP2/CBFB gene by alternative splicing to give at least three isoforms, termed 1, 2, and 3 (26, 36). None of the isoforms exhibited DNA binding by
themselves. 1 and 2 intensified DNA binding of the subunit
and gave a characteristic band supershift. In contrast, 3 only
mildly intensified DNA binding of the subunit, and the band
supershift was not readily apparent. Therefore, it was tentatively
concluded that 1 and 2 were functional but 3 was not (13,
26, 38). The only structural difference between 1 and 3 is
the absence of the exon 5-encoded region from 3 (26)
(Fig. 1A). Accordingly, two of the three
independent -gene knockout studies adapted a strategy to target exon
5 of the gene, leaving only the 3 isoform to be expressed (30,
38). These mice exhibited phenotypes identical to those observed
when exon 1 was targeted to achieve the authentic null condition
(25), a result that may simply imply that 3 is not
functional in vivo either. However, well-controlled analysis of
transcriptional potential of 1, 2, and 3 will be required
before any firm conclusions can be drawn.
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The inv(16) chromosome rearrangement produces fusion proteins
collectively called -SMMHC (18). The protein is composed of the amino-terminal portion of the PEBP2 subunit and the
carboxy-terminal portion of the smooth muscle myosin heavy chain
(SMMHC). Major chromosomal breakpoints occur in intron 5 of the
PEBP2 gene, which result in the conservation of the first
165 amino acids (aa) of the subunit in the -SMMHC protein. The
function of -SMMHC has been studied by EMSA using nuclear extracts
from -MYH11 gene-transfected cells (2).
The study showed a reduction in DNA binding of the normal -
dimers and simultaneous appearance of --SMMHC complexes.
However, the function of -SMMHC with respect to transactivation
activity of PEBP2 was not addressed.
Analysis of the function of the subunit as well as that of
-SMMHC in transactivation requires experimental systems that meet
two criteria: little or no transactivation by either subunit alone and
strong transactivation when both are present together. It is frequently
observed that the subunit alone apparently induces PEBP2
site-dependent transactivation (1, 6, 17, 22, 27, 33, 42).
The nature of -subunit-independent transactivation is unknown,
especially in terms of the extent to which this activity depends on the
endogenous subunit. This latter point needs to be emphasized, as it
is possible that other transcription factors can also facilitate DNA
binding of the subunit. Also, an earlier study on the effect of the
subunit was undermined by the relatively high activity obtained
with transfection of the subunit alone (42). Recently,
we have found that luciferase assays performed in Jurkat T cells by
using a reporter containing a regulatory element from the macrophage
colony-stimulating factor (M-CSF) receptor promoter meet the above
criteria (14). In the present study, we have analyzed the
transcriptional properties of the subunit as well as those of
-SMMHC. We show that -SMMHC fails to support PEBP2-dependent
transactivation despite the fact that it contains the region essential
for -subunit function. Subsequently we show that -SMMHC
sequesters the subunit in the cytoplasm, thereby precluding it from
acting in the nucleus.
MATERIALS AND METHODS
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Plasmid construction.
Mammalian expression vector pEF-BOS
(24) was used to make a series of expression plasmids:
pEF-B1, pEF-B1(1-243), and pEF-B1(1-183) (1);
pEF-B1(1-446), pEF-1, pEF-2, pEF-3, and
pEF--SMMHC (previously referred to as pEF-b/MYH11)
(20); pEF-AML1(453) (previously referred to as AML1b)
(45); and pEF-B1(1-411), pEF-B1(1-371),
pEF-B1(1-331), and pEF-B1(1-291) (14). For the
-deletion constructs [pEF-165 (human), pEF-135, and
pEF-117], corresponding regions of the subunit were PCR
amplified and ligated into the XbaI site of pEF-BOS. The
authenticity of the PCR-amplified sequences was confirmed by
sequencing. pMSV-C/EBP (3) and the luciferase reporter
pM-CSF-R-luc (43) have already been described.
pME-B1-GRLBD was constructed as follows. The glucocorticoid receptor
ligand binding domain (GRLBD) sequence was cut out from
pRShGRNX (7) by XhoI/BamHI
digestion and ligated to the carboxy-terminal sequence of B1 by
using the internal BssHII site and a synthetic linker to
adjust the reading frame. The entire B1-GRLBD sequence was then
cloned into pME18S, an SR promoter-driven expression plasmid
(34).
Transfection and luciferase assays. Jurkat human T cells were maintained in RPMI 1640 supplemented with 10% fetal calf serum (FCS) and antibiotics. The cells were transfected with a reporter plus expression plasmids via electroporation, and relative luciferase activities were assayed as described previously (14). The compositions of the transfected plasmids are described in the figure legends. The total amount of transfected DNA was always kept at 10 µg, using the empty vector pEF-BOS.
Indirect immunofluorescence staining.
REF52 rat fibroblasts
were cultured in Dulbecco modified Eagle medium supplemented with 10%
FCS and antibiotics. Cells were trypsinized and electroporated with 15 µg of each expression plasmid. The transfected cells were seeded onto
chamber slides (Nalge Nunc, Naperville, Ill.). After 48 h, cells
were either untreated or treated with 1 µM dexamethasone (DEX) for
1 h, fixed, and stained with a rabbit antiserum raised against the
Escherichia coli-expressed B1 and/or a hamster antiserum
raised against the E. coli-expressed 2 as described
previously (20, 45). Jurkat T cells were electroporated with
15 µg of each expression plasmid and after 48 h either untreated or treated with 1 µM DEX for 1 h. The cells were then cytospun, fixed, and stained.
Proliferation assay of ME-1 cells.
ME-1 cells
(40) were grown in RPMI 1640 supplemented with 10% FCS and
antibiotics. Proliferation assays were performed with synthesized sense
or antisense oligonucleotides as previously described (29).
The sequence of the sense oligonucleotide corresponding to the
junctional region of -MYH11 (18) is
GAAATGGAGGTCCATGAG. The sequence of the corresponding
antisense oligonucleotide is CTCATGGACCTCCATTTC.
RESULTS
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-SMMHC is required for maintenance of the proliferative state of
ME-1 leukemic cells with inv(16).
The leukemic cell line ME-1
(40) was established from a leukemic patient with inv(16),
and it was from this cell line that -MYH11 (encoding
-SMMHC) was originally cloned (18). We examined whether
the expression of -SMMHC is essential for the maintenance of the
proliferative state of this cell line. We incubated ME-1 cells with an
antisense oligonucleotide complementary to the junctional sequence of
the fusion transcript. As shown in Fig.
2, the antisense oligonucleotide, but not
the corresponding sense oligonucleotide, blocked the proliferation of
ME-1 cells. In contrast, neither the antisense nor the sense
oligonucleotide blocked proliferation of HL-60 leukemic cells, which do
not express -MYH11. After the antisense oligonucleotide
treatment, ME-1 cells showed an increase in the population of
differentiated CD13-positive cells (data not shown). The results
suggest that the fusion product, -SMMHC, may act dominantly over the
normal subunit and prevent myeloid cells from entering into
terminal differentiation, thereby supporting their continued
proliferation.
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The naturally occurring three isoforms of the subunit function
as transactivational cofactors of PEBP2.
The result described
above motivated us to investigate the function of -SMMHC at the
molecular level. We first tried to address the function of -SMMHC
and its normal counterpart, the subunit, in terms of PEBP2-mediated
transactivation. Using the M-CSF receptor promoter linked to the
luciferase gene, we previously established a reporter assay system in
Jurkat T cells, in which the function of the subunit can be
analyzed. This system requires both the and the subunits for
significant transactivation, as we have shown by using 2 as the subunit (14). Here we performed similar function assays for
the 1 and 3 isoforms. Neither 1 nor 3 alone activated the
promoter to any extent (Fig. 3A and B,
lanes 5 to 8). Increasing amounts of AML1(453) [herein, we refer to full-length human and mouse proteins encoded by the
PEBP2B/CBFA2/AML1 gene as AML1(453) and B1,
respectively] only marginally transactivated the reporter activity
when expressed alone (Fig. 3A and B, lanes 1 to 4). However, in the
presence of 1 or 3, AML1(453) transactivated the promoter in a
dose-dependent manner (Fig. 3A and B, lanes 9 to 12). Conversely, in
the presence of a fixed amount of AML1(453), a wide range of 1 or
3 concentrations enhanced AML1(453)-dependent transactivation,
suggesting that the effect of 1 or 3 was saturating above a
certain concentration (Fig. 3A and B, lanes 13 to 17). Therefore, the
naturally occurring isoforms of the subunit, 1, 2, and 3,
are all efficient for cooperative transactivation with the subunit
in vivo. Cooperation between the subunit and the different subunits was not confined to AML1(453) alone but was a general property
of other members of the subunit family, since similar results were
also obtained using PEBP2A or PEBP2C as the subunit (data not
shown).
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-SMMHC does not support cooperative transactivation with
full-length B1 or its deletion derivatives.
When
-SMMHC was used instead of the naturally occurring subunit
isoforms, -SMMHC did not cooperate with the subunit (Fig.
4). To compare the effects of 2 and
-SMMHC, we examined a series of deletion constructs of B1 (Fig.
1B) in the presence of 2 or -SMMHC. With 2, peak activities
were obtained with B1(1-371) and B1(1-331) (Fig. 4, lanes 5 and
6), confirming that the transactivation domain lies between aa 291 and
371 (14). In contrast, -SMMHC did not lead to strong
transactivation with any of the B1 deletion constructs,
demonstrating a clear difference from 2. All of the data
suggest that -SMMHC interferes with the subunit function.
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Minimal region of the subunit required for - cooperative
transactivation.
-SMMHC consists of the amino-terminal 165 aa
of the subunit and the coiled-coil tail structure of SMMHC
(18, 21) (Fig. 1A). To examine whether the inability of
-SMMHC to cooperate with the subunit is due to the lack of the
carboxy-terminal sequences present in the normal isoforms of the subunit, we constructed a series of carboxy-terminal deletion
constructs of the subunit as shown in Fig. 1A in expression
plasmids and tested their activities. As shown in Fig. 5A and
B, 135 enhanced AML1(453)-dependent transactivation, in contrast to 117, which did not exhibit any significant effect. Figure 5C shows a direct comparison of the transactivation abilities of 2, 165, 3, and 135. The
results show that these constructs are equally functional, indicating that the amino-terminal 135-aa region in the subunit is the minimal
region required for - cooperation. Therefore, the inability of
-SMMHC to cooperate with the subunit cannot be due to the absence of the cooperation domain with the subunit. Rather, the
results seem to suggest that the presence of the SMMHC region prevents
the -subunit region from properly cooperating with the subunit.
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Coexpression of -SMMHC inhibits normal - cooperative
transactivation.
There exist at least two possible mechanisms to
explain the observed negative effect of the SMMHC region. One is that
-SMMHC may not be able to interact with the subunit because of
steric inhibition by the SMMHC region. Another possibility is that
complexes of the subunit and -SMMHC, if formed, may not be able
to transactivate, which seems more likely based on already published
observations (2, 4). In the former case, -SMMHC should
not interfere with the normal - interaction, predicting that
coexpression of the -SMMHC should not affect - cooperative
transactivation. In contrast, in the latter case, -SMMHC would
compete with the normal subunit for heterodimerization with the subunit, thus inhibiting transactivation. To test these possibilities,
we examined the effect of -SMMHC on cooperative transactivation by
B1(1-331) and 2. B1(1-331) was used as the subunit
because it exhibited higher activity than full-length B1 in the
presence of 2 (Fig. 4), but similar results were obtained with
full-length B1. As shown in Fig. 6A,
-SMMHC inhibited transactivation by B1(1-331) and 2,
suggesting that -SMMHC competed with 2 for interaction with
B1(1-331), a result that favors the latter possibility. In
contrast, 165, which contains the exact -subunit region of -SMMHC and is capable of dimerizing with the subunit, did not inhibit transactivation (lane 6). This is because the subunit can
cooperate with the functional subunit regardless of whether it is
2 or 165 (Fig. 5C). Furthermore, we examined the effect of
-SMMHC on another mode of transactivation. The M-CSF receptor promoter contains a binding site for CCAAT/enhancer binding protein (C/EBP) in close vicinity to the PEBP2 binding site, and C/EBP and PEBP2 synergistically activate the promoter (44). We
have found that the transactivation domain of B1 (aa 291 to 371) is dispensable for synergistic transactivation and that the region of
B1 from aa 1 to 291 suffices for synergy with C/EBP
(15). B1(1-291) plus 2, which showed only moderate
transactivation (Fig. 4, lane 7), exhibited a remarkable increase in
transactivation activity in the presence of C/EBP (Fig. 6B, lane 3).
In this transactivation mediated by synergistic cooperation between
B1(1-291) and C/EBP, the presence of the subunit is
absolutely required. -SMMHC not only failed to substitute for the
subunit for the function (lane 4) but also inhibited the effect of
2 when coexpressed (lane 5). In contrast, 165 exhibited
cooperativity similar to that of 2 and did not inhibit the effect of
2 (lanes 8 and 9). Again, this is probably because of cooperation of
the subunit with the functional subunit regardless of whether
it is 2 or 165. On the other hand, 117 neither exhibited the
same cooperativity as 2 nor inhibited the effect of 2 (lanes 6 and 7), consistent with the fact that 117 does not dimerize with the
subunit. This makes a clear distinction with -SMMHC, which
dimerizes with the subunit and inhibits transcription.
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-SMMHC sequesters the subunit in the cytoplasm.
The
next question was why dimers composed of the subunit and -SMMHC
cannot transactivate. We previously observed that the subunit and
-SMMHC localized both in the cytoplasm and in the nucleus when
expressed together (20). When expressed separately, the subunit was exclusively nuclear whereas
-SMMHC was cytoplasmic. Thus, it seems that coexpression of the
-SMMHC affected subcellular localization of the subunit, but no
direct experimental evidence for this hypothesis was provided
(20). To examine this hypothesis more clearly, we
established a system in which translocation of one factor can be
controlled by an addition of a ligand. We constructed an expression
plasmid coding for the fusion protein B1-GRLBD. The intracellular
localization of B1-GRLBD can be controlled by DEX treatment (Fig.
7A). While parental B1 by itself is
localized exclusively to the nucleus (Fig. 7A, ii),
B1-GRLBD was localized to the cytoplasm in the absence of DEX
treatment, because it was tethered there by GRLBD (Fig. 7A, iii). With
DEX treatment, B1-GRLBD translocated into the nucleus (Fig. 7A, iv).
When B1-GRLBD and 2 were coexpressed, both were localized to the
cytoplasm in the absence of DEX treatment (Fig. 7B, left panels). With
DEX treatment, B1-GRLBD was mostly translocated into the nucleus
along with a small fraction of 2 (Fig. 7B, right panels). It is of
note that most of the subunit remained in the cytoplasm.
When B1-GRLBD and -SMMHC were coexpressed, both
were localized to the cytoplasm without DEX treatment, as
expected (Fig. 7C, left panels). Interestingly, with DEX
treatment, the majority of B1-GRLBD still remained in the cytoplasm
together with -SMMHC (Fig. 7C, right panels; see the cell in which
both were expressed). In this experiment, DEX treatment was sufficient
to induce ligand-dependent nuclear translocation, because in other
cells where only B1-GRLBD was expressed, B1-GRLBD completely translocated it into the nucleus upon DEX
treatment. These results show that nuclear translocation of
B1-GRLBD upon DEX treatment was blocked by -SMMHC but not by
wild-type 2. The same was found for Jurkat T cells, where
DEX-dependent nuclear translocation of B1-GRLBD was blocked by
-SMMHC (Fig. 7D).
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DISCUSSION
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A system for in vivo functional analysis of the subunit
has revealed that 1, 2, and 3 isoforms are all
transcriptionally functional.
Previously the function of the subunit was studied mostly by in vitro EMSA (13, 26, 38). In
the present study, the M-CSF receptor promoter-luciferase reporter was
used to assay -subunit function in Jurkat T cells, and the results
showed that the three naturally occurring isoforms of the subunit,
1, 2, and 3, are all effective in supporting -
cooperative transactivation. Previously we and others observed
prominent band supershifts of -subunit-DNA complexes with 1 and
2 but not with 3 in EMSA (13, 26, 38). However, 3
did have an effect of intensifying DNA binding of the subunit.
Thus, - dimers may be stable enough for enhanced transactivation
in vivo but not stable enough to produce a supershift in EMSA.
subunit (135) is sufficient for heterodimerization with the subunit. By
transactivation assays performed in the present study, we found
that the same 135-aa region was sufficient for cooperation with the subunit to transactivate. In these assays, it seemed that variable
regions of the subunit more carboxy terminal to the first 135-aa
region did not contribute to the transactivation function. Further
deletion of 18 aa (117) from 135 completely abolished -subunit
function in both assays. Shurtleff et al. reported that the
amino-terminal 133-aa region of the subunit exhibited weaker DNA
binding activity than 165 when mixed with the subunit
(31). Also, deletion analysis of Drosophila
Brother revealed that a construct corresponding to the amino-terminal
132 aa of the mouse subunit exhibited weaker DNA binding activity
in the presence of Runt than the corresponding amino-terminal 137 aa of
the subunit (8). It will be necessary to reevaluate
these findings by transactivation assays.
Properties of -SMMHC.
We have found that -SMMHC does not
cooperate with the subunit for transactivation despite the presence
of the minimal region for -subunit function. Rather, -SMMHC seems
to inhibit normal - cooperative transactivation. The results
suggest that -SMMHC can efficiently dimerize with the subunit,
but that SMMHC region actively participates in undermining the proper
functioning of these complexes.
-subunit region (identical to 165)? One
possible mechanism is the sequestration of the otherwise nucleus-located subunit in the cytoplasm. We showed that -SMMHC blocked nuclear translocation of B1-GRLBD upon DEX treatment, whereas 2 did not. With this GRLBD fusion system, the majority of
the --SMMHC complexes were retained in the cytoplasm. As such,
the --SMMHC complexes can no longer participate in DNA binding
and transactivation. This constitutes a form of dominant negative
inhibition. It is important to note that colocalization of -SMMHC
and the subunit was observed both in this and the previous
(20) study. However, in conventional cotransfection assays
using -SMMHC and wild-type B1, complexes of both proteins were
found to be localized in the nucleus as well as in the cytoplasm (20). We explain this as follows. When expressed separately, the subunit is localized in the nucleus because of the presence of
nuclear localization signals (NLSs) (14, 20), while
-SMMHC is localized in the cytoplasm (20), probably
because it lacks an NLS. By analogy to normal myosin structure, two
molecules of -SMMHC would be packed into a monomer by forming an
-helical coiled-coil rod. When both the subunit and -SMMHC
are coexpressed at a low level, --SMMHC complexes with a
monomeric SMMHC tail structure would form and enter into the
nucleus, utilizing the NLS of the subunit, and would bind to DNA
(2). However, with increasing levels of expression over a
"critical monomer concentration" (16), the SMMHC
portion would begin to form filaments via the carboxy-terminal
nonhelical tailpiece (11). These --SMMHC complexes
polymerized through SMMHC tails would be precluded from entering
into the nucleus. With the GRLBD fusion system, in contrast, most of
the complexes would form polymers being retained in the cytoplasm via
the GRLBD portion before DEX treatment. Thus, polymerized --SMMHC complexes would not be able to enter the nucleus upon DEX treatment.
There is a precedent for a leukemogenic fusion protein that
causes abnormal localization of transcription factors. Acute
promyelocytic leukemia retinoic acid receptor alpha (PML-RAR) is
localized to discrete subnuclear compartments called nuclear
bodies, and it subsequently causes disruption of nuclear bodies
(39). As a result of this aberrant localization, retinoid X
receptors, which are the heterodimeric partners of RAR, are
sequestered to these compartments, causing attenuated responses to
retinoids and vitamin D3 (10, 35, 39). The
situation is quite analogous to the sequestration of the subunit by
-SMMHC to the cytoplasm described in the present study. In the case
of PML-RAR, retinoic acid can restore the structure of the nuclear
bodies and differentiation responses to vitamin D3
(35, 39). By analogy, it would be intriguing to find
the molecular mechanisms of cytoplasmic retention by
-SMMHC with a view of developing specific therapeutic
agents that induce the release of the subunit.
Effect of -SMMHC on proliferation.
The present study
suggests that -SMMHC plays a key role in the maintenance of the
proliferative state of ME-1 cells which have inv(16). In contrast, Cao
et al. recently reported that -SMMHC blocks proliferation of 32D
myeloid cells at the G1/S transition (2).
Further studies will be needed to address this apparent discrepancy
between the two different systems. Nonetheless, it is interesting
that proliferation of Kasumi-1 cells, in which AML1/ETO is produced as
a result of the t(8;21) translocation, was specifically blocked by an
antisense oligonucleotide complementary to the junction region of the
AML1/ETO mRNA (29). Thus, -SMMHC and AML1/ETO
are analogous in some respects: both are dominant negative inhibitors
in developing embryos (4, 41), and both are essential for
the proliferative properties of leukemic cells that have the
corresponding chromosomal abnormalities.
Alternative interpretation of the knockout and the
-SMMHC knockin studies.
Previously, the 3 isoform was
thought to be inactive as discussed above. Accordingly, the knockout mice were produced via targeting of exon 5, leaving the 3
isoform to be expressed normally or increased in a compensatory fashion
(30, 38). These mice exhibited nearly the same phenotypes as
the mice that were targeted in exon 1, which represents the authentic
knockout (25). Together, these observations suggest that
3 is inactive in vivo. In fact, however, alternative splicing with
the skipping of exon 5 in the former group of mice did not occur so as
to quantitatively compensate for the loss of 1 and 2 (reference
38; our unpublished observation [15] on mice generated by Sasaki et al.
[30]). Thus the observed phenotypes may be attributed
to a dosage effect but not to the disabled function of 3 per se.
3 is effective in transactivation
assays. Therefore, if impaired transcriptional activity is responsible
for the knockout phenotypes, the impaired development of the exon
5-targeted mice is more likely to be due to the lack of a compensatory
increase in 3 levels. Low levels of 3, such as those naturally
present, may be insufficient, and we speculate that there might exist a
threshold level of the subunit that allows fetal liver
definitive hematopoiesis to emerge. Such a threshold level would be
less than 50% of the normal level, because heterozygous mice targeted
in exon 1 were phenotypically normal. The results of the present study
suggest that one of the mechanisms underlying the phenotypes of the
-SMMHC knockin mice may be a decrease in the available amount of the
subunit due to its aberrant sequestration. The amount of the subunit has been reported to be limiting in fetal hematopoietic
tissues, because heterozygous knockout mice for the subunit
exhibited a minor but still significant abnormality in in vitro colony
assays (37).
In conclusion, -SMMHC inhibits PEBP2-mediated transactivation via
cytoplasmic sequestration of the subunit, which is limiting in
cells. Whereas the subunit is relatively abundant, a level as low
as that of the natural amount of 3 may not be sufficient to support
the function(s) of PEBP2 in vivo. An alteration in the available
amounts of PEBP2 subunits at sites of transcription could have a
significant effect on hematopoiesis and eventually lead to leukemia.
ACKNOWLEDGMENTS
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We thank K. Umesono for pRShGRNX, D.-E. Zhang for
pM-CSF-R-luc, A. D. Friedman for pMSV-C/EBP, and K. Yanagisawa
for ME-1 cells. We also thank T. Komori and K. Sasaki for making
PEBP2 knockout mice available to us. We thank M. Osato for helpful
discussion.
The work was supported in part by a grant (FY1995, B-333) from the New Energy and Industrial Technology Development Organization and by Grant-in-Aid 0925322 for Priority Area on Cancer Research from the Minister of Education, Science and Culture, Japan.
FOOTNOTES
* Corresponding author. Mailing address: Institute for Virus Research, Kyoto University, Shogo-in, Sakyo-ku, Kyoto 606, Japan. Phone: 81-75-751-4028. Fax: 81-75-752-3232. E-mail: yito{at}virus.kyoto-u.ac.jp.
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