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J Biol Chem, Vol. 275, Issue 1, 530-538, January 7, 2000
From the Drosophila Runt is the founding
member of a family of related transcription factors involved in the
regulation of a variety of cell-differentiation events in invertebrates
and vertebrates. Runt-related proteins act as both transactivators and
transcriptional repressors, suggesting that
context-dependent mechanisms modulate their transcriptional
properties. The aim of this study was to elucidate the molecular
mechanisms that contribute to the regulation of the functions of the
mammalian Runt-related protein, Cbfa1. Here we provide the first
demonstration that Cbfa1 (as well as the related protein, Cbfa2/AML1)
physically interacts with the basic helix loop helix transcription
factor, HES-1, a mammalian counterpart of the Drosophila
Hairy and Enhancer of split proteins. This interaction is mediated by
the carboxyl-terminal domains of Cbfa1 and HES-1, but does not require
their respective tetrapeptide motifs, WRPY and WRPW. Our studies also
show that HES-1 can antagonize the binding of Cbfa1 to mammalian
transcriptional corepressors of the Groucho family. Moreover, HES-1 can
potentiate Cbfa1-mediated transactivation in transfected cells. Taken
together, these findings implicate HES-1 in the transcriptional
functions of Cbfa1 and suggest that the concerted activities of Groucho
and HES proteins modulate the functions of mammalian Runt-related proteins.
Invertebrate and vertebrate Runt-related proteins are
transcription factors involved in the regulation of a variety of
cell-differentiation events and aberrant functions of members of this
protein family correlate with developmental abnormalities and
neoplastic transformations (1-5). In particular, the mouse
Runt-related protein, core-binding factor Studies in invertebrate and vertebrate species implicate Runt-related
proteins in both transactivation and transcriptional repression,
suggesting that their transcription functions may be regulated in
context-dependent ways by interactions with other proteins
(1, 9, 12-17). In this regard, Drosophila Runt has recently
(18) been shown to interact with the protein Groucho, a general
transcriptional repressor involved in a variety of gene regulatory
events (19-21). In particular, genetic studies show that repression of
certain Runt-regulated genes is dependent on interaction with Groucho
and is sensitive to Groucho dosage (18). These results implicate
Groucho in the regulation of the transcriptional functions of Runt in
Drosophila.
A number of observations have suggested that the functions of mammalian
Runt-related proteins are also modulated by Groucho homologs,
designated as the transducin-like Enhancer of split (TLE) or
Groucho-related gene products 1 through 4 (hereafter referred to as
TLE1-4) (22-24). TLE proteins are co-expressed with the Runt-related
proteins, Cbfa1 and Cbfa2/AML1 (AML1), in a variety of cell types (7,
14, 25-27). In addition, AML1 and TLE proteins can physically interact
with each other (28). Furthermore, transient transfection studies in
mammalian cells have shown that TLE proteins can inhibit the
transactivation mediated by both Cbfa1 and AML1 (14, 28, 29). Together,
these findings strongly suggest that TLE proteins are involved in the
regulation of the transcriptional functions of mammalian Runt-related proteins.
Studies in Drosophila have also implicated a second
evolutionarily conserved family of transcription factors in the
regulation of the functions of Runt-related proteins. Specifically, the
basic helix loop helix proteins of the Drosophila
Hairy/Enhancer of split (HES) family are co-expressed with Groucho and
Runt in a variety of cell types and physically interact with Groucho
(18, 20, 21, 30). Moreover, genetic studies in Drosophila
show that runt and HES genes contribute to common
gene regulatory events important for sex determination and segmentation
(30-32). Mammalian HES and runt-related genes
are also co-expressed with TLE genes in a variety of cell
types and their protein products participate in common developmental
mechanisms (14, 27, 33, 34). Together, these findings raise the
possibility that Runt-related and HES proteins may interact.
Here we describe experiments designed to test whether Cbfa1 can
functionally interact with the HES family member, HES-1, which is
co-expressed with Cbfa1 in mammalian skeletal cells (9, 33). Our
results demonstrate that Cbfa1 can physically interact with both HES-1
and TLE proteins and identify the domains of these molecules involved
in these interactions. HES-1 also interacts with AML1, in addition to
Cbfa1. Our studies also show that HES-1 can potentiate Cbfa1-mediated
transactivation in transfected cells, possibly by virtue of its ability
to antagonize the interaction of Cbfa1 with TLE proteins. Taken
together, these results strongly suggest that the transcriptional
functions of Cbfa1 are modulated by interactions with both TLE and HES
family members.
Plasmids--
The following is a summary of the names and
origins of the constructs used in these studies. Additional information
on cloning strategies and oligonucleotide primers used in PCR
experiments is available upon request. Vent DNA polymerase was used and
PCR products were routinely sequenced before subcloning into the
appropriate vectors. For expression of glutathione
S-transferase (GST) fusion proteins in bacteria, the
pGEX1-TLE1(32-770) (near full-length TLE1 lacking the first
31 amino acids), pGEX2-TLE1(1-135) (Q domain of TLE1),
pGEX3-TLE3(490-774) (WDR domain of TLE3), and
pGEX1-HES-1(3-281) (the entire HES-1 sequence except for
the first 2 amino acids) constructs were generated as described
previously (35-37). The pGEX1-TLE1(290-461) plasmid was
obtained by subcloning a PCR product encoding amino acids 290 through
461 (SP domain) of TLE1 into the BamHI and
HindIII sites of pGEX-1. The
pGEX2-HES-1 Interaction Assays in Transfected Cells and Western Blotting
Analysis--
ROS17/2.8 cells were grown as described previously (9,
14) and transfected using the SuperFect reagent (Qiagen) according to
the manufacturer's instructions. In each experiment, cells were
co-transfected with 1.0 µg of either pCMV5-Cbfa1 DNA or
pCMV2-FLAG-AML1b DNA and 1.0 µg of pEBG-HES-1
DNA (or pEBG as control). Approximately 24 h after
transfection, cells were collected, homogenized as described previously
(40), and incubated in the presence of glutathione-Sepharose beads for
60 min. The beads were then collected by centrifugation, washed five
times with buffer 1 (25 mM Tris/HCl, pH 7.8, 200 mM NaCl, 0.5% Triton X-100), and immediately incubated in
2 × electrophoresis sample buffer, followed by SDS-PAGE and transfer to nitrocellulose. Nitrocellulose replicas were subjected to
Western blotting analysis with previously described anti-Cbfa antibodies (9, 13) or anti-FLAG-epitope monoclonal antibodies (Sigma).
In Vitro Fusion Protein Interaction Assays--
Proteins were
in vitro translated and GST fusion proteins were expressed
in bacteria and purified on glutathione-Sepharose beads as described
previously (36, 37). Unless otherwise indicated, roughly 1 µg of each
fusion protein was incubated at 4 °C with glutathione-Sepharose
beads for 60 min. After this time, the appropriate in vitro
translated proteins were mixed with the beads as described previously
(36, 37) and incubations were prolonged for 3 h. Beads were then
collected by centrifugation, washed five times with buffer 1, and
incubated in 2× electrophoresis sample buffer, followed by SDS-PAGE,
and autoradiography. Bacterial preparations of GST-HES-1 fusion
proteins contained a number of lower Mr species. Western blotting analysis showed that these components were recognized by anti-GST antibodies (Santa Cruz Biotechnology), indicating that they
represented degradation products of the full-length GST-HES-1 fusion
proteins and not unrelated bacterial proteins that co-isolate with
GST-HES-1.
Transcription Assays--
Human 293 and rat ROS17/2.8 cells were
cultured as described previously (9, 14, 37), and transfected using
SuperFect. Cells were incubated in the presence of the Superfect-DNA
complexes for 24 h. The amount of DNA transfected was adjusted
using pcDNA3 plasmid so that the total amount of DNA
used in each transfection was the same (3.0 µg). Transcription
studies with the reporter plasmid p6OSE2luc (0.5 µg),
plasmids pCMV5-Cbfa1 or pCMV5Cbfa1 Other Methods--
Yeast transformations and two-hybrid
interaction assays were performed exactly as described previously (36,
37). Western blotting analysis with panTLE monoclonal antibodies was as
described elsewhere (22, 23, 25).
HES-1 Physically Interacts with Cbfa1 and AML1--
Based on the
participation of runt and HES genes in common
developmental pathways in Drosophila (30-32) and the shared
ability of both HES and Runt-related proteins to bind to
transcriptional corepressors of the Groucho/TLE family (14, 18, 21, 28, 36), we asked whether specific mammalian HES and Runt-related proteins
might associate with each other. To address this question, we first
performed coprecipitation assays using cells co-transfected with
expression plasmids for HES-1 and Cbfa1. These factors are normally
coexpressed in mammalian osteoblastic cells (9, 33, 34, 41, 42). Rat
ROS17/2.8 osteoblastic cells were transfected with either GST or a
fusion protein of GST and HES-1 that contained the entire HES-1
sequence except for the first two amino acids (GST-HES-1(3-281)).
Twenty-four hours later, cells were homogenized and the GST proteins
were isolated on glutathione-Sepharose beads. Western blotting analysis
of the fractions bound to the beads using anti-GST antibodies revealed
that GST and GST-HES-1 were both stable and expressed at equivalent
levels (Fig. 1C, lanes 2 and
4). Western blotting analysis with previously described (9,
13) anti-Cbfa antibodies revealed that full-length Cbfa1 (Cbfa1(1-528); refer to Fig. 1A for Cbfa1 structure)
co-precipitated with GST-HES-1 (Fig. 1B, lane 4) but not
with GST (Fig. 1B, lane 2). Consistent with this result, the
Cbfa1-related protein, AML1b, which represents the full-length form of
AML1 (6, 16, 28), coprecipitated with GST-HES-1 (Fig.
2A, lane 2), but not with GST
(Fig. 2A, lane 4). Together, these results show that HES-1 can associate with both Cbfa1 and AML1 in transfected cells.
The coprecipitation of Cbfa proteins with HES-1 may result from either
a direct interaction between these molecules or an indirect association
mediated by endogenous TLE proteins, which can bind to both Cbfa- and
HES family members. To distinguish between these possibilities, we
tested the Cbfa/HES-1 interaction in the absence of TLEs by performing
in vitro pull-down experiments and yeast two-hybrid
interaction assays. In the former, GST-HES-1 was purified from bacteria
and incubated with in vitro translated AML1 proteins. AML1b
bound to GST-HES-1 (Fig. 2B, lane 2), but not to GST (Fig.
2B, lane 3). As previously reported (41), in vitro translated AML1b migrated as a doublet. Interestingly, HES-1 interacted preferentially with the more slowly migrating form of this
doublet. Our studies also revealed that the carboxyl-terminal WRPY
motif shared by all Runt-related proteins was not necessary for these
interactions, since HES-1 bound to AML1(1-472) (also referred to as
AML1a; Ref. 28), which is identical to AML1b except that it contains a
different carboxyl terminus lacking the WRPY motif (Fig. 2B, lane
6). Similarly, removal of the the carboxyl-terminal WRPW motif of
HES-1 did not disrupt the AML1/HES-1 interaction (Fig. 2C, cf.
lanes 1 and 2), nor the Cbfa1/HES-1 interaction.2 The rabbit
reticulocyte system used for in vitro translation reactions
did not contain endogenous TLE proteins (Fig. 2E, lane 1),
indicating that the interaction between AML1 and HES-1 did not require TLEs.
These findings were confirmed and extended by performing two-hybrid
interaction assays in yeast cells previously shown to be devoid of TLE
proteins (40). Co-transformation of yeast cells with plasmids encoding
GAL4ad-Cbfa1(468-528), containing the last 60 amino acids of Cbfa1,
and GAL4bd-HES-1(193-281), containing the last 88 residues of HES-1,
resulted in a specific reconstitution of GAL4 transcriptional activity
(Fig. 3A). This result was
indicative of an interaction between these factors in the absence of
TLE proteins.
Taken together, these results show that HES-1 can interact with the
mammalian Runt-related proteins, Cbfa1 and AML1, in a TLE-independent
way. Moreover, they reveal that the Cbfa1/HES-1 interaction involves
sequences located within the last 60 amino acids of Cbfa1 and the last
88 residues of HES-1, respectively.
Cbfa1 Interacts with TLE Proteins--
Previous studies have shown
that the carboxyl-terminal domains of Drosophila Runt and
human AML1, including but not limited to the WRPY motif, mediate
interactions with Groucho/TLE proteins (18, 28). Based on those
results, we asked whether Cbfa1 might also physically interact with TLE
proteins and, if so, whether its carboxyl-terminal domain might be
involved in these interactions. Pull-down assays using in
vitro translated full-length Cbfa1 and a previously described (37)
fusion protein of GST and near full-length TLE1 (GST-TLE1(32-770))
showed that Cbfa1 and TLE1 can bind to each other (Fig.
4B, lane 2). Further studies
with fusion proteins of GST and individual TLE domains (see Fig.
4A for description of TLE structure) revealed that Cbfa1
interacted with the first 135 amino acids (Q domain) of TLE1 (Fig.
4C, lane 2), whereas no detectable interaction was observed
with residues 290 through 461 (SP domain) of TLE1 (Fig. 4C, lane
3). Cbfa1 also bound specifically to a fusion protein containing
the highly conserved carboxyl-terminal WDR domain shared by all
Groucho/TLEs (Fig. 4C, lane 4). Since we were unable to
obtain a stable preparation of a fusion protein containing the WDR
domain of TLE1, these studies were performed using a fusion protein,
GST-TLE3(490-774), containing the WDR domain of TLE3, which is 93%
identical to the corresponding domain of TLE1 (22).
In agreement with these findings, yeast two-hybrid interaction assays
demonstrated that co-transformation with plasmids encoding GAL4ad-Cbfa1(468-528) and GAL4bd-TLE1(1-770) (full-length TLE1) resulted in reconstitution of GAL4 transcriptional activity, indicative of an interaction between TLE1 and the carboxyl-terminal domain of
Cbfa1 (Fig. 5A). An active
GAL4 transcription complex was also reconstituted after co-transforming
GAL4ad-Cbfa1(468-528) with either GAL4bd-TLE1(1-435), which contains
the amino-terminal half of TLE1 excluding the WDR domain (Fig.
5B), or GAL4bd-TLE1(444-770), which contains only the WDR
domain (Fig. 5C).
Further investigations revealed that the carboxyl-terminal WRPY motif
conserved in all Runt-related proteins was not necessary for the
interaction of Cbfa1 with TLE proteins. In particular, pull-down assays
using in vitro translated Cbfa1(241-523), which extends
from the end of the Runt domain to the carboxyl-terminal end but lacks
the last 5 amino acids, VWRPY, showed that this protein was competent
to bind to both the Q- and the WDR domains of TLEs (Fig.
5E). In contrast, Cbfa1(241-442), which lacks not only the
WRPY motif but also most of the previously described carboxyl-terminal
repression domain of Cbfa1 (14), did not display TLE binding activity
(Fig. 5F). These results are in agreement with previous
studies showing that the WRPY motif of AML1 contributes to, but is not
necessary for, the TLE/AML1 interaction (28). Taken together, these
findings show that Cbfa1 can interact with TLE proteins and that amino
acids 468-528 of Cbfa1 contain sequences that can interact with both
amino- and carboxyl-terminal TLE domains.
HES-1 Can Potentiate Cbfa1-mediated Transactivation--
The
previous results show that the last 60 amino acids of Cbfa1 can mediate
interaction with both HES-1 and TLE proteins. We therefore asked
whether HES-1 could antagonize the interaction between Cbfa1 and TLE
proteins by performing binding assays using the GST-TLE1(1-135) fusion
protein, which mediates interaction with Cbfa1 (Fig. 4C) but
not HES-1 (see below). In vitro translated Cbfa1 was
incubated with GST-TLE1(1-135) in the absence (Fig. 6A, lane 3) or presence of an
excess of either in vitro translated HES-1 (Fig. 6A,
lane 5) or unprogrammed rabbit reticulocyte lysate (Fig. 6A,
lane 4). The addition of HES-1 resulted in a significant decrease
in the amount of Cbfa1 bound to TLE1(1-135), while the presence of the
unprogrammed lysate had no effect. The competing effect of HES-1 did
not correlate with binding of this protein to GST-TLE1(1-135) (Fig.
6A, lane 5), suggesting that neither HES-1 alone nor
Cbfa1·HES-1 complexes were able to bind to this fusion protein. We
obtained the same result when similar experiments were performed using
AML1 instead of Cbfa1. In particular, addition of an excess of in
vitro translated HES-1 significantly reduced the binding of the
more slowly migrating form of AML1 to GST-TLE1(1-135). This effect was
specific, since only a small inhibition of the binding of the faster
AML1 form to GST-TLE (1-135) was observed (Fig. 6B, cf. lanes
2 and 3). These observations are in agreement with the
finding that HES-1 interacts preferentially with the more slowly
migrating form of in vitro translated AML1 (see Fig. 2B). Together, these results suggest that by interacting
with Cbfa proteins, HES-1 may interfere with the association of the latter with TLE proteins.
To examine this possibility further, we hypothesized that if HES-1 can
interfere with the Cbfa1/TLE interaction in transfected cells, then the
transactivating ability of Cbfa1 might be potentiated as a result of
the reduction/elimination of the repressive effect that TLE proteins
can exert on Cbfa1 (14). To test this possibility, we examined the
transactivating ability of Cbfa1 in rat ROS17/2.8 cells, which express
high levels of endogenous TLE proteins (14, 36). These cells were
transfected with a reporter plasmid containing the
luciferase gene under the control of six tandem copies of a
canonical Cbfa1-binding site, the OSE2 cis-acting element found in the
promoter of the mouse osteocalcin gene (13). Co-transfection of increasing amounts of a Cbfa1 expression plasmid resulted in a
dose-dependent, but somewhat weak, activation of reporter
gene expression (Fig. 7A).
Importantly, this transactivation was significantly increased
(approximately 15-fold) when increasing amounts of Cbfa1 were
coexpressed with a fixed amount of HES-1, showing a functional cooperation between these proteins (Fig. 7A). Expression of
HES-1 alone had no effect on reporter gene expression. Neither Cbfa1 alone nor the combination of Cbfa1 and HES-1 activated the expression of the reporter gene when the latter was placed downstream from a
mutated OSE2 element (9) containing a 2-base pair mutation that
abolishes Cbfa1 binding.2 Similar experiments were
performed using human embryonic kidney 293 cells, which were shown to
express relatively low levels of endogenous TLE proteins (36, 37).
Unlike ROS17/2.8 cells, Cbfa1 mediated a strong transactivation of
reporter gene expression when expressed in 293 cells (Fig.
7B). Similar to ROS17/2.8 cells, however, coexpression of
Cbfa1 and HES-1 in 293 cells also resulted in a potentiation of the
transcriptional activity of Cbfa1 (Fig. 7B), indicating that
the interaction between these two proteins is not cell-type specific.
To exclude the possibility that the observed effect of HES-1 on the
transcriptional activity of Cbfa1 were the result of nonspecific
interactions mediated by the Cbfa1 QA domain, which consists of
contiguous long stretches of glutamine and alanine residues (14), we
tested whether HES-1 could potentiate the transcriptional activity of a
truncated form of Cbfa1 (Cbfa1
We next asked whether the ability of HES-1 to promote Cbfa1-mediated
transactivation was contingent on its ability to interact with TLE
proteins. Since previous studies have shown that HES factors are
incapable of binding to Groucho/TLEs if their carboxyl-terminal WRPW
motif is deleted (20, 21) we examined whether a truncated form of HES-1
lacking the last six amino acids, WRPWRN (referred to as
HES-1
Taken together, these results indicate that HES-1 can promote the
transactivating ability of Cbfa1 and that this positive effect does not
depend on the formation of TLE·HES protein complexes. This suggests
that HES-1 can potentiate Cbfa1 activity due to the formation of
Cbfa1·HES-1 complexes that have stronger transcriptional activity
than Cbfa1 alone, rather than by simply "titrating away" TLEs from Cbfa1.
HES-1 Interacts with Cbfa Proteins--
Our studies have provided
the first evidence that HES-1 can bind to Cbfa family members in
cultured mammalian cells. The observed interactions appear to be direct
and not mediated by TLE proteins since they can also be demonstrated in
transformed yeast cells devoid of the latter. Moreover, they were
observed in pull-down assays with bacterially expressed GST-HES-1
fusion proteins and preparations of AML1 and Cbfa1 obtained by in
vitro translation using rabbit reticulocyte lysates devoid of
detectable TLE immunoreactivity.
The finding that HES and Cbfa proteins can physically interact with
each other is consistent with a number of previous results. First,
expression studies show that, in both invertebrates and vertebrates,
HES and Runt-related proteins are coexpressed in a variety of cell
types (18, 20, 21, 33, 34). Second, both of these proteins interact
with Groucho/TLE family members (18, 20, 21, 28, 36), suggesting that
HES- and Runt-related proteins may come in contact with each other at
least during mechanisms involving Groucho/TLEs. Third, genetic studies
in Drosophila show that runt and HES
genes participate in common developmental mechanisms involved in the
control of sex determination and segmentation (30-32). For instance,
both Runt and the HES family member, Deadpan, can bind to the promoter
of the Sex-lethal gene and regulate its expression (32, 43).
Finally, Cbfa1 and HES-1 contribute to the regulation of mammalian
osteoblast-specific genes; for instance, they provide antagonistic
inputs to the control of the expression of the osteopontin
gene (14, 33, 44). Our first demonstration of a direct link between
mammalian HES and Cbfa proteins will now facilitate the study of how
these factors interact with each other and with TLE proteins. Moreover,
it will be important to determine whether invertebrate members of these
protein families also interact with each other in similar ways.
Overlapping Domains of Cbfa1 Mediate Interaction with Both HES-1
and TLE Proteins--
Our studies have also shown that the
carboxyl-terminal domains of Cbfa1 and HES-1 are involved in their
interaction. Specifically, the last 60 amino acids of Cbfa1 can
interact with the last 88 residues of HES-1. Importantly, we have found
that the same carboxyl-terminal region of Cbfa1 involved in HES-1
binding also contains binding sites for TLE proteins, raising the
possibility that HES-1 and TLE proteins may compete with each other for
Cbfa1 binding (see below).
The observation that the carboxyl-terminal region of Cbfa1 is involved
in TLE binding is consistent with the previous identification of a
transcriptional repressor function within this domain (14) and suggests
that this repressor activity is due to the recruitment of the TLE
corepressors. Interestingly, the region of Cbfa1 containing residues
443-516 is ~70% identical to amino acids 366 through 438 of mouse
Cbfa2 (14). Since this domain of Cbfa2 also harbors a transcription
repression function (17), it is possible that TLE-binding sites are
present within this carboxyl-terminal region of Cbfa2.
We have also shown that binding of TLE proteins to Cbfa1 is not
dependent on the presence of a carboxyl-terminal WRPY motif. This
result is in agreement with previous studies that also showed that
binding of TLE1 to AML1 occurs even in the absence of the WRPY motif
(28). Moreover, these findings are consistent with transcription
studies in transfected mammalian cells showing that TLE overexpression
reduces transactivation by both Cbfa1 and a truncated Cbfa1 form
lacking the WRPY motif (albeit not as effectively in the latter case)
(14). These combined results differ from the previous report that
binding of Drosophila Groucho to Runt requires the
carboxyl-terminal WRPY motif of the latter (18). However, those same
studies did show a weak Groucho/Runt interaction when a truncated form
of Runt lacking solely the WRPY motif was used. Only when additional
sequences were deleted together with the WRPY motif did Runt fail to
bind to Groucho, suggesting that other elements in addition to the WRPY
tetrapeptide may mediate this interaction. It is also possible that the
difference between the investigations in Drosophila (18) and
mammals (28, this study) may reflect differences between
Drosophila Runt and its mammalian counterparts or may derive
from the use of different experimental protocols.
Finally, our studies have also revealed that Cbfa1 can interact with
two separate TLE domains located within either the amino-terminal Q
region or the carboxyl-terminal WDR domain, both of which are highly
conserved among all Groucho/TLE family members (22). The identification
of the WDR domain of Groucho/TLEs as a protein-protein interaction
element is not surprising given the demonstrated involvement of WD40
repeats in molecular interactions (45, 46) and the previous
demonstration that the WDR domain of Drosophila Groucho is
involved in the interaction with the HES protein, Hairy (20). The
amino-terminal Q domain of TLE proteins has also been shown previously
to mediate protein-protein interactions, including those with the
PRDI-BF1/Blimp-1 (47) and UTY (37) proteins. Moreover, in agreement
with our results, Cbfa1 has recently been shown to interact with the
product of the Grg5 gene, which encodes a roughly 200-amino
acid protein homologous to the amino-terminal Q domain of Groucho/TLEs
but lacking the carboxyl-terminal SP and WDR
regions.3 Thus, it appears
that TLE proteins utilize both of their recognized protein-protein
interaction domains to interact with Cbfa family members. Although the
specific contributions of these separate TLE domains to the interaction
with Cbfa proteins remain to be determined, it is worth mentioning that
we have recently found that TLEs also utilize both the amino-terminal Q
domain and the carboxyl-terminal WDR domain to associate with specific
members of the family of winged-helix DNA-binding
proteins.4 This suggests that
the use of separate protein-protein interaction domains may be a
feature underlying the association of Groucho/TLE proteins with
distinct DNA-binding factors.
Implications for Transcriptional Regulation by Cbfa and HES
Proteins--
The present demonstration that both Cbfa1 and AML1 can
interact with HES-1 suggests that members of these two protein families can regulate each other's transcriptional functions. In agreement with
this possibility, we have observed that HES-1 can potentiate Cbfa1-mediated transactivation in transfected cells. A number of
observations suggest that HES-1 may perform this function by binding
directly to Cbfa1 and inhibiting the interaction between Cbfa1 and
endogenous TLE proteins, thereby reducing/inhibiting the repressive
effect that TLEs can exert on the transactivating function of Cbfa1
(14). First, Cbfa1 and HES-1 can directly bind to each other. Moreover,
binding sites for both HES-1 and TLE proteins are present within the
same carboxyl-terminal domain of Cbfa1. In addition, HES-1 can
interfere with the Cbfa1/TLE interaction in in vitro binding
assays. Finally, Cbfa1-mediated transactivation can be potentiated by a
truncated form of HES-1 that does not interact with TLEs due to the
loss of its carboxyl-terminal WRPW motif but is still competent to bind
to Cbfa proteins. Together, these observations suggest that the
positive effect of HES-1 on the transcriptional activity of Cbfa1 may
involve an active competition with TLEs for direct binding to Cbfa1,
rather than a situation in which HES-1 simply titrates away TLEs from
Cbfa1 but does not associate with the latter.
Alternative mechanisms can also be proposed. In particular, our results
raise the interesting possibility that HES factors may mediate
transcriptional activation, instead of repression, when they are
associated with Cbfa proteins rather than with TLEs. Although a number
of previous studies have shown that invertebrate and vertebrate HES
proteins generally act as transcriptional repressors (20, 21, 34),
recent investigations in Xenopus have implicated certain HES
family members in both negative and positive feedback loop mechanisms
that either repress or maintain the expression of genes of the Notch
signaling pathway during embryonic somitogenesis (48). Together with
our present observations, this finding suggests that, perhaps under
appropriate conditions in which they escape interactions with
Groucho/TLE proteins, HES factors may contribute to the transcriptional
activity of other transcription factors.
The possibility that HES-1 may interfere with the Cbfa1/TLE interaction
and, vice versa, that Cbfa1 may interfere with the HES-1/TLE
interaction may help to explain the finding that HES-1 can repress the
expression of the osteopontin gene in osteoblasts (33),
whereas Cbfa1 can activate osteopontin expression (9, 44).
It is possible that HES-1·TLE complexes keep the
osteopontin promoter silent and that, by becoming recruited
to the promoter, Cbfa1 may contribute to gene activation both directly,
by providing a transactivating function, and indirectly, by interfering
with the TLE/HES-1 interaction. These combined functions may mediate a
shift from transcriptional repression mediated by DNA-bound HES-1·TLE
complexes to transcriptional activation mediated by Cbfa1. In this
model, the direct interaction between Cbfa1 and HES-1 may provide a way
to prevent the interaction of Cbfa1 with TLEs. Specifically, by
interacting with HES-1, Cbfa1 may become unavailable to TLE proteins
and thus protect its transactivation ability from the repressive effect
of the TLEs. This situation may provide a molecular explanation for the
ability of Cbfa1 to promote transactivation of osteopontin
and other osteoblast-specific genes even in the presence of TLE
proteins (14, 44). This would likely not be possible if Cbfa1 were
simply titrating away TLEs from HES-1, because the resulting
Cbfa1·TLE complexes would probably not be able to promote
transactivation (14, 28, 29).
This model is also consistent with the involvement of
Drosophila Runt and Deadpan in the regulation of the
Sex-lethal gene. Deadpan mediates repression of
Sex-lethal and Groucho is required for this function (21,
32). Conversely, Runt can bind to the Sex-lethal promoter
and stimulate its activation (43). It is possible that in males, where
Runt dosage is one-half of that in females, Deadpan binds to the
Sex-lethal promoter and, together with Groucho, mediates
transcriptional repression. In females, Runt may be able to antagonize
the Deadpan/Groucho-mediated repression by interacting with Deadpan and
disrupting the repressive complexes of Deadpan and Groucho. The ensuing
Runt-Deadpan complexes may then be able to promote transcription. This
model would thus provide a way to regulate the Runt/Groucho interaction
through the formation of Runt-Deadpan complexes, a situation that might
help to explain the apparent paradox that Runt can activate
Sex-lethal expression while at the same time mediating
repression of other target genes in the same cells (43).
In summary, the present results implicate the activities of TLE and HES
proteins in the modulation of the transcriptional functions of
mammalian Cbfa proteins and suggest that the study of the interactions
between these proteins may provide important information about the
mechanisms underlying the transcriptional functions of Runt-related and
HES proteins.
We thank Yoram Groner, Dwayne Barber, and
Eseng Lai for the gift of several plasmids, George Karpati for
providing access to a luminometer, and Yanling Liu for technical assistance.
*
This work was supported by National Institutes of Health
Grants DE11290 and HD97006 and a Basic Science Award of the March of
Dimes Foundation (to G. K.) and Medical Research Council of Canada
Grants MT-13957 and GR-14971 (to S. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Present address: Endocrine Research, DC 0403, Lilly Research
Labs, Indianapolis, IN 46285.
2
K. McLarren, R. Lo, and S. Stifani, unpublished data.
3
W. F. Wang and B. Olsen, personal communication.
4
J. Yao, E. Lai, and S. Stifani, unpublished data.
The abbreviations used are:
Cbfa, core binding
factor
The Mammalian Basic Helix Loop Helix Protein HES-1 Binds to and
Modulates the Transactivating Function of the Runt-related Factor
Cbfa1*
,,,
Center for Neuronal Survival, Montreal
Neurological Institute, McGill University, Montreal, Quebec, H3A 2B4
Canada and the § Department of Human and Molecular Genetics,
Baylor College of Medicine, Houston, Texas 77030
ABSTRACT
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
INTRODUCTION
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2
(Cbfa2)1 is essential for
fetal liver hematopoiesis (6-8) and its human homolog, AML1, is
frequently targeted by chromosomal translocations that lead to acute
myeloid leukemia (4, 6). A related mouse protein, Cbfa1, plays an
essential role in osteoblast differentiation and mutations interfering
with its function correlate with defects in ossification in mice and
humans (3, 5, 9-11).
EXPERIMENTAL PROCEDURES
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REFERENCES
C (truncated HES-1 lacking the
last 6 amino acids, WRPWRN) was generated by subcloning a PCR product
encoding this portion of HES-1 into the SmaI site of
pGEX-2. The DNA construct for expression of GST-HES1 in
mammalian cells, pEBG-HES-1(3-281), was obtained by
subcloning a PvuII fragment from the rat HES-1
cDNA into the filled-in ClaI site of the pEBG
vector (38). Plasmids for in vitro translation reactions
were obtained as follows. pcDNA3-GAL4bd-Cbfa1(241-523) and pcDNA3-GAL4bd-Cbfa1(241-442) (encoding the
DNA-binding domain of GAL4 fused to either residues 241-523 or
241-442 of Cbfa1, respectively) were obtained by subcloning
XhoI/XbaI fragments from the previously described
(14) constructs pSG424-Cbfa1(241-523) and
-Cbfa1(241-442) into the pcDNA3-GAL4bd
vector (36, 37). Plasmids pcDNA3-GAL4bd-AML1(2-453)
(fusion protein of GAL4bd and AML1b) and
pcDNA3-GAL4bd-AML1(2-472) (fusion protein of GAL4bd and
AML1a) were generated by digesting pBluescript-AML1b or
-a cDNAs (obtained from Y. Groner, The Weizmann
Institute) with XhoI (followed by filling-in with Klenow DNA
polymerase) and EcoRI, and by subcloning into
pcDNA3-GAL4bd digested with EcoRV and
EcoRI. DNA constructs for yeast two-hybrid assays were
obtained as follows. pGAD424-Cbfa1(468-528) (activation
domain of GAL4 fused to residues 468 through 528 of Cbfa1) was
generated by subcloning a SmaI fragment from
pBluescript-Cbfa1 into the SmaI site of
pGAD424. The plasmids pGBT9-TLE1(1-770),
-TLE1(1-435), and -TLE1(444-770) (GAL4bd fused to full-length TLE1, the first 435 amino acids of TLE1, or residues 444 through 770 of TLE1, respectively) were obtained as described previously (36, 37). pGBT9-HES-1(193-281) (GAL4bd fused to amino acids 193 through 281 of HES-1) has been described previously (35). Plasmids pCMV5-Cbfa1 (full-length Cbfa1),
pCMV5-Cbfa1
49-96 (truncated form of Cbfa1 lacking the QA
domain), and p6OSE2luc (luciferase reporter gene
under the control of six Cbfa1-binding sites, the OSE2 element) have
been described previously (9, 14). The pRc/CMV-HES-1
construct (full-length HES-1) has been described (39). The
pCMV2-FLAG-AML1b plasmid was generated by digesting
pcDNA3- GAL4bd-AML1(2-453) with EcoRI and
XbaI, followed by filling-in with Klenow DNA polymerase and
subcloning into the SmaI site of pCMV2-FLAG. The
pCMV2-FLAG-HES1(3-281) plasmid was obtained by subcloning a
PvuII fragment from the rat HES-1 cDNA into
the SmaI site of pCMV2-FLAG. Construct
pCMV2-FLAG-HES-1C was generated by subcloning
a PCR product encoding this portion of HES-1 into the filled-in
BamHI site of pCMV2-FLAG.
49-96 (0.5-1.0 µg), and constructs pCMV2-FLAG-HES-1(3-281) or
pCMV2-FLAG-HES-1C (0.1-0.3 µg) were
performed as described previously (9, 14, 36, 37). In each case, 0.25 µg of pCMV-
-galactosidase plasmid DNA was
co-transfected to provide a means of normalizing the assays for
transfection efficiency. Results were expressed as mean value ± S.D. and were tested for statistical significance by the one-tailed Student's t test for paired differences.
RESULTS
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Fig. 1.
Interaction of Cbfa1 and HES-1 in transfected
cells. A, schematic representation of the domain
structure of Cbfa1 and truncated derivatives thereof. The location of
the different activation domains (AD1-3), the repression
domain (RD), the Runt domain, and the WRPY motif is shown.
Activation domain 2 corresponds to the QA domain (14). Also shown are
some of the deletion derivatives of Cbfa1 used in this study, named
according to the residues contained in each protein. B and
C, transfection/coprecipitation assays. Rat ROS17/2.8
osteosarcoma cells were co-transfected with the pCMV5-Cbfa1
plasmid to express full-length Cbfa1 (lanes 1-4) and either
the pEBG (lanes 1 and 2) or
pEBG-HES-1(3-281) (lanes 3 and 4)
plasmid to express GST or GST-HES-1, respectively. Cell homogenates
were then collected and incubated with glutathione-Sepharose beads. The
material that remained bound to the beads after extensive washing was
subjected to SDS-PAGE (lanes 2 and 4). One-tenth
of each input homogenate collected prior to incubation with
glutathione-Sepharose beads was also subjected to gel electrophoresis
(lanes 1 and 3). After transfer to
nitrocellulose, Western blotting was performed with either anti-Cbfa
(B) or anti-GST (C) antibodies.
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Fig. 2.
Interaction of AML1 and HES-1.
A, transfection/coprecipitation assays. Rat ROS17/2.8
osteosarcoma cells were co-transfected with the
pCMV2-FLAG-AML1b plasmid to express AML1b (lanes
1-4) and either the pEBG-HES-1(3-281) (lanes
1 and 2) or pEBG (lanes 3 and
4) plasmids to express GST-HES-1 or GST, respectively. Cell
homogenates were collected and incubated with glutathione-Sepharose
beads. The material that remained bound to the beads after extensive
washing was subjected to SDS-PAGE (lanes 2 and 4)
together with one-tenth of each input homogenate collected prior to
incubation with glutathione-Sepharose beads (lanes 1 and
3). An equivalent aliquot of homogenate from control
non-transfected cells was loaded onto lane 5. After transfer
to nitrocellulose, FLAG-AML1b (see arrow) was visualized by
Western blotting with anti-FLAG antibodies. These antibodies
cross-reacted with other components that were also present in
non-transfected cells (cf. lanes 1, 3, and
5) but did not coprecipitate with GST-HES-1 (cf.
lanes 1 and 2). B-D,
pull-down assays with GST-HES-1 fusion proteins. In each experiment,
the indicated in vitro translated 35S-labeled
protein (one-half of the amount used in each reaction; lanes
1 and 5 in B; lane 1 in
D) were incubated in the presence of roughly 1.0 µg of the
indicated fusion proteins, followed by incubation in the presence of
glutathione-Sepharose beads. The material bound to the beads was
subjected to SDS-PAGE, followed by autoradiography. The
GST-HES-1(3-281) and GST proteins used in the experiments depicted in
B and D are shown in B (lanes
7 and 8, respectively) after staining with Coomassie
Blue. E, Western blotting analysis with panTLE monoclonal
antibodies. Aliquots of either rabbit reticulocyte lysate (lanes
1 and 3) or ROS 17/2.8 cell lysate (lanes 2 and 4) were subjected to SDS-PAGE and transfer to
nitrocellulose. The replicas were first stained with Ponceau S
(lanes 3 and 4) and then incubated with panTLE
antibodies (lanes 1 and 2). Blots were developed
using ECL detection. No TLE immunoreactivity was detected in the rabbit
reticulocyte lysate, even after prolonged exposure. The positions of
migration of Mr standards are indicated.
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Fig. 3.
Interaction of Cbfa1 and HES-1 in yeast
two-hybrid interaction assays. Yeast cells were co-transformed
with plasmids encoding the indicated combinations of proteins, cultured
and subjected to a filter assay for -galactosidase activity.
A, the ability of the transformed cells to turn blue
(dark streaks) in the presence of the -galactosidase
substrate,
5-bromo-4-chloro-3-indolyl--D-galactopyranoside
indicated that a transcriptionally competent GAL4 complex was
reconstituted due to the interaction between Cbfa1 and HES-1.
B, no interaction between Cbfa1 and GAL4bd was observed.
C, HES-1 did not interact with GAL4ad.
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Fig. 4.
Interaction of Cbfa1 with amino- and
carboxyl-terminal domains of TLEs. A, schematic
representation of the domain structure of TLE proteins and truncated
derivatives thereof, using TLE1 as an example. Domains shown include
the amino-terminal Gln-rich Q domain, the Gly/Pro-rich GP domain, the
internal CcN domain containing a nuclear localization sequence and
putative phosphorylation sites for casein kinase II and
p34cdc2, the Ser/Thr/Pro-rich SP domain, and the
carboxyl-terminal domain containing tandem WDRs (22). Also shown are
deletion derivatives of TLE1 and TLE3 used in this study, named
according to the residues contained in each protein. B and
C, pull-down assays using GST-TLE fusion proteins. In
vitro translated 35S-labeled Cbfa1(1-528) (one-half
of the amount used in each binding assay was loaded onto lane
1 in B and C) was incubated in the presence
of ~1.0 µg of the indicated fusion proteins, followed by incubation
in the presence of glutathione-Sepharose beads. The material that
remained bound to the beads was subjected to SDS-PAGE, followed by
autoradiography. D, analysis of individual GST-TLE fusion
proteins by SDS-PAGE. Roughly equivalent amounts of GST-TLE1(1-135)
(lane 2), GST-TLE1(290-461) (lane 3),
GST-TLE3(490-774) (lane 4), or GST (lane 5)
proteins were visualized by staining with Coomassie Blue.
B-D, empty lanes were sometime left between
individual samples to prevent possible spill-over artifacts. In
D, the positions of migration of Mr
standards are indicated in lane 1.
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Fig. 5.
Interaction between the carboxyl-terminal
domain of Cbfa1 and separate TLE1 domains. A-D, yeast
two-hybrid interaction assays. Yeast cells were co-transformed with
plasmids encoding the indicated combinations of proteins, cultured and
subjected to a filter assay for -galactosidase activity.
A-C, the ability of the transformed cells to turn
blue (dark streaks) in the presence of the -galactosidase
substrate,
5-bromo-4-chloro-3-indolyl--D-galactopyranoside
indicated that a transcriptionally competent GAL4 complex was
reconstituted due to the interaction between Cbfa1 and TLE1 (or domains
thereof). D, no interaction between Cbfa1 and GAL4bd was
observed. E and F, pull-down assays with GST-TLE
fusion proteins. GAL4bd-Cbfa1(241-523) (lane 1 in
E, see small arrow; one-eighth of the amount used
in each binding assay) or GAL4bd-Cbfa1(241-442) (lane 1 in
F, see big arrow; one-half of the amount used in
each binding assay) were incubated in the presence of 1.0 µg of the
indicated fusion proteins, followed by incubation in the presence of
glutathione-Sepharose beads. The material that remained bound to the
beads was subjected to SDS-PAGE, followed by autoradiography. In
E, lanes 1 and 2 were originally
separated by an empty lane and a lane loaded with
Mr standards. In F, autoradiography
was intentionally prolonged to exclude the possibility of weak
interactions. GAL4bd does not interact with the GST-TLE fusion proteins
used in these experiments (36, 37). The positions of migration of
Mr standards are indicated.
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Fig. 6.
Competitive interactions between Cbfa1,
HES-1, and TLE proteins. A, in vitro
translated 35S-labeled Cbfa1(1-528) (lane 2,
see open arrow; one-half of the amount used in each binding
assay) was incubated with 1.0 µg of GST-TLE1(1-135) protein in the
absence (lane 3) or presence of a 20-fold volume excess of
either unprogrammed rabbit reticulocyte lysate (lane 4) or
in vitro translated 35S-labeled full-length
HES-1 (lane 5, see arrowhead; 20-fold the amount
shown in lane 1). Lanes 1 and 2 were
originally separated by a lane loaded with Mr
standards. B, in vitro translated
35S-labeled GAL4bd-AML1b (lane 1, one-half of
the amount used in each binding assay) was incubated with 1.0 µg of
GST-TLE1(1-135) in the absence (lane 2) or presence of a
20-fold volume excess of in vitro translated full-length
HES-1 (lane 3). The arrow points to the more
slowly migrating form of AML1b whose binding to TLE1(1-135) was
specifically reduced by HES-1. The positions of migration of
Mr standards are indicated.
49-96) that lacked the QA domain.
These studies showed that HES-1 potentiated the transactivating ability
of Cbfa149-96 and full-length Cbfa1 to similar
extents.2
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Fig. 7.
Potentiation of the transactivating ability
of Cbfa1 by HES-1. A and B, transient
transfection, transcription assays. Rat ROS17/2.8 (A) or
human 293 (B) cells were transfected with the
p6OSE2luc reporter construct in the absence or presence of
varying amounts (indicated in micrograms) of plasmids encoding the
indicated combinations of proteins. Luciferase activities were
determined in each case and values shown represent the mean ± S.D. of at least five independent transfection experiments performed in
duplicates. C, Western blotting analysis of HES-1 and
HES-1 C proteins. Lysates from ROS17/2.8 cells
transfected with plasmids encoding either FLAG-HES-1 (lane
1) or FLAG-HES-1C (lane 2) were
subjected to SDS-PAGE, transfer to nitrocellulose, and incubation with
anti-FLAG epitope antibodies.
C), could potentiate the transcriptional activity
of Cbfa1. As shown above in Fig. 2C, deletion of these amino
acids did not impair the ability of HES-1 to interact with Runt-related
proteins. More importantly, HES-1C was capable of
potentiating Cbfa1-mediated transactivation, albeit to a somewhat
lesser extent than full-length HES-1 (Fig. 7A). Both HES-1
and HES-1C were expressed at equal levels in transfected
cells (Fig. 7C).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Scholar of the Fonds de la Recherche en Sante du Quebec and
Killam Scholar of the Montreal Neurological Institute. To whom correspondence should be addressed: Tel.: 514-398-3946; Fax:
514-398-1319; E-mail: mdst@musica.mcgill.ca.
ABBREVIATIONS
;
AML, acute myeloid leukemia;
GAL4ad, activation domain of
GAL4;
GAL4bd, DNA-binding domain of GAL4;
GST, glutathione
S-transferase;
HES, Hairy and Enhancer of split;
PAGE, polyacrylamide gel electrophoresis;
TLE, transducin-like Enhancer of
split;
WDR, WD40 repeat;
PCR, polymerase chain reaction.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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