From the Center for Neuronal Survival, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada
Received for publication, August 21, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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Hairy/Enhancer of split 1 (Hes1) is a mammalian
transcriptional repressor that plays crucial roles in the regulation of
several developmental processes, including neuronal differentiation.
The aim of this study was to elucidate the molecular mechanisms that regulate the transcription repression activity of Hes1. It is shown
here that Hes1 associates with the nuclear matrix, the
ribonucleoprotein network of the nucleus that plays important roles in
transcriptional regulation. Nuclear matrix binding is mediated by the
same Hes1 C-terminal domain that is also required for transcriptional
repression. This domain contains the WRPW motif that acts as a binding
site for the transcriptional corepressor Groucho, which also localizes to the nuclear matrix. Both the nuclear matrix association and transcription repression activity of Hes1 are inhibited by deletion of
the WRPW motif, indicating that Groucho acts as a transcriptional corepressor for Hes1. This corepressor role is not modulated by the
Groucho-related gene product Grg5. In contrast, the Runt-related protein RUNX2, which localizes to the nuclear matrix and interacts with
Groucho and Hes1, can inhibit both the Groucho·Hes1
interaction and the transcription repression ability of Hes1. Together,
these observations suggest that transcriptional repression by Hes1
requires interactions with Groucho at the nuclear matrix and that RUNX proteins act as negative regulators of the repressive activity of
Groucho·Hes1 complexes.
In the mammalian central nervous system, progenitor cells located
in the ventricular zone of the neural tube undergo proliferation and
ultimately differentiate into neurons in response to intrinsic and
extrinsic cues. The mechanisms regulating the commitment of these
progenitor cells to the neuronal fate are controlled by either positive
or negative regulators belonging to separate families of transcription
factors containing the basic helix loop helix (bHLH)1 motif. Proteins that
promote neuronal differentiation include a number of evolutionarily
conserved transcriptional activators, generally referred to as the
proneural proteins (reviewed in Refs. 1 and 2). These include several
related molecules belonging to the Neurogenin, Ash, Ath, and NeuroD
families (3-8). Ectopic expression of proneural proteins results in
the differentiation of supernumerary neurons, whereas disruption of
their functions causes neuronal loss (4, 5, 8). Factors that antagonize neuronal differentiation include bHLH proteins which mediate
transcriptional repression rather than transactivation. These molecules
are homologous to the Drosophila Hairy/Enhancer of split
(Hes) proteins, which control insect neuronal development by negatively
regulating the functions of the neurogenic genes in response to
activation of the cell surface receptor, Notch (9-13).
The best characterized member of the mammalian Hes family is
the Hes1 gene (14). In the nervous system, Hes1
is highly expressed during the progenitor-to-neuron transition, and its
expression then decreases during the developmental maturation of
postmitotic neurons (14). Persistent expression of Hes1
inhibits neuronal differentiation in the developing telencephalon (15).
Conversely, targeted disruption of Hes1 causes premature
neuronal differentiation and up-regulation of proneural genes, in
addition to other developmental defects (16-18). Similar to
Drosophila Hes proteins, Hes1 is a transcriptional repressor
that acts as a nuclear effector of the Notch signaling pathway during
mammalian neurogenesis (14, 18, 19). Together, these findings
demonstrate that Hes1 is a crucial negative regulator of neuronal
differentiation in mammals and underscore the importance of
understanding how its cellular functions are regulated.
Although little is presently known about the molecular mechanisms that
underlie transcriptional repression by Hes1, studies in
Drosophila show that invertebrate Hes proteins interact with the general transcriptional corepressor, Groucho (11, 20, 21).
Mutations that inhibit the Groucho·Hes interaction interfere with the ability of Drosophila Hes proteins to repress
transcription, suggesting that Groucho acts a transcriptional
corepressor for these factors (11, 20, 21). These findings first raised the possibility that mammalian homologs of Drosophila
Groucho, designated as transducin-like Enhancer of split (TLE) or
Groucho-related genes (Grg) 1 through 4 (hereafter referred to as
TLE1-4) (22, 23), may be involved in transcriptional repression by
Hes1. Consistent with this notion, Hes1 and TLE
genes are coexpressed in the nervous system (14, 24-26), and
perturbations that force a persistent expression of Hes1 or
TLE1 in the mammalian forebrain result in similar phenotypes
characterized by neuronal loss (15, 27). In addition, Hes1 physically
interacts with TLE1 and TLE2 in vitro (28, 29). Taken
together with the demonstration that TLE proteins provide a
transcriptional corepressor function to a number of different
DNA-binding factors (30-33), these observations suggest that
transcriptional repression by Hes1 is regulated by interactions with
TLE family members. It remains to be determined, however, whether Hes1
and TLE proteins functionally interact in vivo and, if so,
how their transcriptional functions are regulated.
Here we describe experiments that demonstrate that Hes1 interacts with
TLEs in mammalian cells and localizes to the nuclear matrix, where TLEs
are also found. Both the nuclear matrix association and transcription
repression activity of Hes1 are inhibited by deletion of the same
C-terminal domain, indicating a correlation between nuclear matrix
binding and transcriptional repression. This observation is in
agreement with previous studies showing that the nuclear matrix is
functionally involved in the regulation of gene expression by
concentrating and localizing transcription factors and/or facilitating
the formation of appropriate chromatin structures (reviewed in Ref.
34). Our results also show that the ability of Hes1 to interact with
TLEs is required for both nuclear matrix association and
transcriptional repression, indicating that TLEs act as transcriptional
corepressors for Hes1. The involvement of TLEs in Hes1 function is not
regulated by the AES1/Grg5 (hereafter referred to as Grg5) protein.
Grg5 is a naturally occurring factor related to the first 200 amino
acids of Groucho/TLEs and has been regarded as a dominant-negative
regulator of at least certain TLE transcriptional corepressor functions
(33, 35-38). In contrast, our findings suggest that the corepressor
role of TLEs for Hes1 is negatively regulated by members of the RUNX
family of transcription factors, which are coexpressed, and interact,
with both Hes1 and TLE proteins (14, 24-26, 30, 31, 39-41). Taken
together, these findings suggest that interactions among Hes1, TLE, and
RUNX proteins at the nuclear matrix regulate the transcriptional
activity of Hes1.
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. Constructs
pEBG-Hes1, pCMV2-FLAG-Hes1, pCMV2-FLAG-Hes1-(1-275), pcDNA3-TLE1,
pCMV5-RUNX2, pGEX3-TLE3(WDR), pRc/CMV-Hes1, pBluescript-RUNX2, and
p6N- Interaction Assays in Transfected Cells and Western Blotting
Analysis--
Human 293 embryonic kidney or rat ROS17/2.8 osteoblastic
cells were grown and transfected using the SuperFect reagent (Qiagen) as described previously (31). Coprecipitation assays using plasmids pEBG-Hes1 and pEBG-Hes1-(1-275) (or
pEBG as control) and immunoprecipitation experiments with
anti-FLAG-epitope antibodies (Sigma) were performed exactly as
described previously (31, 43). Western blotting studies were performed
with either panTLE (22, 44), anti-GST (Santa Cruz Biotechnology),
anti-FLAG, anti-histone H3 (a kind gift of Dr. C. D. Allis,
University of Rochester), anti-RUNX2 (31), anti-GAL4bd (Santa Cruz
Biotechnology), or anti-lamin B1 (Chemicon) antibodies as described
previously (22, 31, 43, 44).
Isolation of Nuclear Matrix Proteins and Preparation of Nuclear
Extracts--
ROS17/2.8 cells were subjected to sequential extraction
and solubilization steps to isolate nuclear matrix proteins exactly as
described by Merriman et al. (45). High salt-resistant and -soluble nuclear fractions were prepared by subjecting isolated nuclei
to incubation in the presence of 0.5 M NaCl as described previously (43).
Transient Transfection/Transcription Assays--
Human 293 cells
were transfected using the SuperFect reagent. 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). Twenty-four hours later, transcription assays were performed as
described (29, 31), using reporter plasmid p6N- Hes1 Interacts with TLE in a WRPW Motif-dependent
Way--
Hes1 is coexpressed with TLE proteins in a number of
mammalian cell types (14, 24-26, 29) and physically interacts with TLE1 and TLE2 in vitro (20, 28, 29). These findings suggest that TLEs may form transcription repression complexes with Hes1 in vivo. To begin to examine this possibility, we first
asked whether Hes1 and TLE proteins would interact in cultured
mammalian cells. Rat ROS17/2.8 osteoblastic cells, which express high
levels of endogenous TLEs (31), were transfected with an expression construct for a fusion protein of GST and Hes1. After isolation of
GST-Hes1 on glutathione-Sepharose beads, Western blotting analysis with
panTLE monoclonal antibodies (22, 43) revealed that endogenous TLE
proteins coprecipitated with GST-Hes1 (Fig.
1A, lane 2). In contrast, TLEs did not coprecipitate with a fusion protein of GST and
Hes1-(1-275), which lacks the last six amino acids containing the WRPW
motif (Fig. 1A, lane 4). GST-Hes1 and
GST-Hes1-(1-275) were expressed at equivalent levels (Fig.
1B, lanes 2 and 4). These results show
that TLEs and Hes1 interact with each other in mammalian cells and that
this interaction requires the WRPW motif of Hes1.
Hes1 Associates with the Nuclear Matrix--
To determine whether
Hes1 and TLE proteins might colocalize to a common nuclear compartment,
it was then asked whether Hes1 could associate with the nuclear matrix,
where TLE proteins are known to localize (46). Other Hes1-binding
proteins, like RUNX family members, also associate with the nuclear
matrix (45, 47, 48). This filamentous ribonucleoprotein network of the nucleus is thought to play important roles in the regulation of gene
expression by mediating the colocalization and interaction of
transcription factors and/or facilitating the establishment of
appropriate chromatin structures. ROS17/2.8 cells were transfected with
FLAG epitope-tagged Hes1 and then subjected to two different kinds of
biochemical fractionation to determine the subnuclear localization of
Hes1. In one procedure, nuclei were isolated and high salt-soluble and
-insoluble nuclear fractions were obtained as described previously
(43). Hes1 was poorly extracted from nuclei by high ionic strength
conditions (Fig. 2A,
cf. lanes 1 and 2). This behavior is
similar to that of other nuclear matrix proteins, including RUNX family
members (45, 47), and suggests that Hes1 is tightly associated with
internal nuclear compartments. To confirm this possibility, the nuclear
matrix isolation procedure of Merriman et al. (45) was
utilized to test the possibility that Hes1 might associate with this
compartment. Cytoplasmic proteins were extracted, and the remaining
insoluble fraction was treated with DNase I and RNase A, followed by
ammonium sulfate to release chromatin components and nuclear proteins
that are not bound to the nuclear matrix. The remaining pellet was then
extracted in disassembly buffer to yield nuclear matrix proteins (45).
The effectiveness of this fractionation procedure was confirmed by Western blotting analysis of cytoplasmic, nuclease-treated, and nuclear
matrix fractions. The chromatin component, histone H3 was present only
in the nuclease-treated fraction (Fig. 2B, lane 2), whereas the nuclear matrix protein, lamin B1 was only
recovered in the nuclear matrix fraction (Fig. 2C,
lane 3). A significant amount of Hes1 immunoreactivity was
found in both the nuclease-treated and nuclear matrix fractions (Fig.
2D, lanes 2 and 3), in agreement with
the tight association of Hes1 with the high salt-resistant nuclear
fraction described above. Because TLE proteins have been shown
previously to be associated with both chromatin and the nuclear matrix
(44, 46), these findings show that Hes1 has a subnuclear distribution
similar to that of TLEs.
Elimination of the TLE binding ability of Hes1 by removal of the last
six amino acids containing the WRPW motif (resulting in the truncated
protein Hes1-(1-275)) almost completely abolished the nuclear matrix
association of Hes1 (Fig. 2E, lane 3). This situation was correlated with an increase in the amount of Hes1 found
in the cytoplasmic fraction (Fig. 2E, lane 1). A
smaller form of Hes1-(1-275), retaining the amino-terminal FLAG
epitope, was also observed under the experimental conditions used to
prepare non-nuclear and nuclear fractions (Fig. 2E,
lane 1). This faster-migrating species, likely representing
the product of a C-terminal proteolytic degradation, was predominantly
found in the cytoplasmic fraction. These findings demonstrate a
correlation between the ability of Hes1 to associate with the nuclear
matrix and its ability to interact with TLEs, suggesting that the
latter mediate the nuclear matrix association of Hes1. A larger
C-terminal deletion that removed the last 54 amino acids of Hes1,
resulting in the truncated protein Hes1-(1-227), completely abolished
the association of Hes1 with the nuclear matrix (Fig. 2F,
lane 3) and greatly reduced its association with the
nuclease-treated fraction (Fig. 2F, lane 2). This
was correlated with the accumulation of Hes1-(1-227) in the cytosolic fraction (Fig. 2F, lane 1). Together, these
results show that Hes1 can associate with the nuclear matrix and that
its C-terminal domain is necessary for this association.
To examine these possibilities further, we asked whether the C-terminal
region of Hes1 was sufficient to mediate the nuclear matrix binding of
a heterologous protein. ROS17/2.8 cells were transfected with either
the DNA-binding portion of the yeast nuclear protein GAL4 (which does
not associate with the nuclear matrix (48)) or a fusion protein of
GAL4bd and the last 88 amino acids of Hes1. GAL4bd-Hes1-(193-281) was
able to associate with the nuclear matrix (Fig.
3G, lane 3),
whereas GAL4bd alone did not bind to this compartment (Fig.
3H, lane 3), indicating that the last 88 amino
acids of Hes1 can mediate nuclear matrix binding. These results are
consistent with previous studies showing that GAL4bd-Hes1-(193-281)
interacts with TLEs, whereas GAL4bd alone does not (28). Taken
together, these findings demonstrate that the C-terminal domain of Hes1
mediates the association of this protein with the nuclear matrix.
TLE Proteins Act as Transcriptional Corepressors for
Hes1--
Based on the physical interaction and subnuclear
colocalization of Hes1 and TLEs, we next asked whether TLEs are
required for the transcription repression function of Hes1. Human 293 cells, which express endogenous TLEs (31), were transfected with a previously described (14) reporter construct containing the luciferase gene under the control of a basally active
Grg5 Is Not Involved in the Regulation of Transcriptional
Repression by Hes1--
To elucidate the molecular mechanisms that
regulate the corepressor activity of TLEs for Hes1, we asked whether
Grg5 might act as a negative regulator of this function. This
hypothesis was suggested by previous studies showing that Grg5 can
heterodimerize with TLE proteins (29, 33, 37) and, in some cases, have a dominant-inhibitory effect on the corepressor activity of TLEs (33,
38). To examine whether Hes1 might interact with Grg5 and/or TLE-Grg5
multimers, ROS17/2.8 cells were transfected with GST-Hes1 in the
presence or absence of a FLAG epitope-tagged Grg5 protein. After cell
lysis and immunoprecipitation of Grg5 with anti-FLAG antibodies (Fig.
4A, lane 2,
arrow), GST-Hes1 did not coimmunoprecipitate with Grg5 (Fig.
4B, cf. lanes 1 and 2). In contrast, endogenous TLE proteins coimmunoprecipitated with Grg5 (Fig.
4C, cf. lanes 1 and 2).
TLEs were not coimmunoprecipitated from cells transfected with the
empty FLAG vector (Fig. 4C, lane 4). In converse
experiments in which GST-Hes1 was isolated on glutathione-Sepharose
beads, Grg5 did not co-isolate with GST-Hes1 (Fig. 4D,
cf. lanes 1 and 2), whereas endogenous
TLE proteins coprecipitated with GST-Hes1 (Fig. 4E,
lane 2). These results suggest that Hes1 interacts with
neither Grg5-Grg5 oligomers nor TLE-Grg5 oligomers. To examine this
possibility further, transient transfection assays were performed to
determine whether Grg5 might negatively regulate transcriptional
repression by Hes1 in cells expressing endogenous TLEs. Transfection of
increasing amounts of a Grg5 expression plasmid had no effect on the
repressor activity of Hes1 (Fig. 4F). Expression of the Grg5
protein was confirmed by Western blotting analysis (Fig.
4G). Taken together, these studies demonstrate that Grg5
does not associate with Hes1 and does not act as a negative regulator
of the corepressor effect of TLE on Hes1.
Hes1-mediated Transcriptional Repression Is Inhibited by
RUNX2--
To examine further the mechanisms involved in the
regulation of Hes1 activity, we asked whether the Runt-related protein
RUNX2 might modulate the transcription repression ability of Hes1. This possibility was suggested by the demonstration that both Hes1 and TLE
bind to RUNX1 and RUNX2 (30, 31). Moreover, Hes1 can inhibit the
TLE·RUNX interaction and potentiate transactivation by RUNX2 (31).
Our studies showed that the ability of Hes1 to mediate transcriptional
repression was completely inhibited when it was coexpressed with RUNX2
(Fig. 5A, cf.
lanes 2 and 3). RUNX2 alone had no detectable
effect on reporter gene expression (Fig. 5A, lane
4).
The inhibition of Hes1-mediated repression did not appear to be due to
an effect of RUNX2 on the stability of this protein, because Hes1 was
equally stable in the presence of RUNX2 (Fig. 5B). We
therefore asked whether the inhibitory effect of RUNX2 might be due to
an inhibition of the interaction of Hes1 with TLEs, which are required
for Hes1-mediated repression. Based on the previous observation that
the C-terminal WDR domain of Groucho/TLEs is involved in Hes protein
binding (21), interaction assays were set up to determine whether
increasing amounts of RUNX2 would reduce binding of a fixed amount of
Hes1 to the TLE WDR domain. Binding of in vitro translated
Hes1 (shown in Fig. 5C, lane 1) to a GST-TLE(WDR)
fusion protein was compared in the absence or presence of in
vitro translated RUNX2 (shown in Fig. 5C, lane 5). Because RUNX2 also interacts with the TLE WDR domain (31), these assays were performed using very small, limiting amounts of
GST-TLE(WDR) substrate to prevent having an excess of binding sites for
both Hes1 and RUNX2. Hes1 bound to the WDR domain (Fig. 5C,
lane 2) and this interaction was gradually decreased by the addition of increasing amounts of RUNX2 (Fig. 5C,
lanes 3 and 4). At lower RUNX2 concentrations,
this decrease was not correlated with detectable binding of RUNX2 to
the TLE WDR domain (Fig. 5C, lane 3). This result
suggests that reduced Hes1 binding was due to the formation of
RUNX2·Hes1 complexes at the expense of TLE·Hes1 complexes and is in
agreement with the previous observation that RUNX2 has a higher
affinity for Hes1 than for TLE (31). At higher RUNX2 concentrations,
binding of RUNX2 to the TLE WDR domain was also observed, as described
previously (31) (Fig. 5C, lane 4). The addition
of equal amounts of unprogrammed rabbit reticulocyte lysate had no
effect on Hes1 binding to GST-TLE(WDR) (not shown). Taken together with
the observation that RUNX2 does not inhibit the DNA binding ability of
Hes1 in electrophoretic mobility shift assays,2 these findings
suggest that RUNX2 may act as a negative regulator of the transcription
repression ability of Hes1 by interfering with the interaction of Hes1
with the TLE corepressors.
Nuclear Matrix Association of Hes1 and Transcriptional
Repression--
The present studies have provided the first
demonstration that Hes1 associates with the nuclear matrix, a nuclear
compartment where TLE and RUNX proteins, both of which interact with
Hes1, are also found (45-48). The ability of Hes1 to associate with
the nuclear matrix is mediated by the same C-terminal domain that is
also required for transcriptional repression, showing a correlation between transcription repression ability and nuclear matrix targeting. No sequences resembling consensus nuclear matrix targeting signals found in other proteins (47) were identified within the C-terminal domain of Hes1. However, this region contains binding sites for TLEs,
suggesting that these proteins are involved in the nuclear matrix
association of Hes1. Consistent with this possibility, the nuclear
matrix binding of Hes1 is almost completely abolished by deletion of
the WRPW motif that mediates TLE binding. Because the cells used in our
nuclear matrix preparations express high levels of endogenous TLEs,
these findings strongly suggest that TLEs mediate the nuclear matrix
localization of Hes1. The residual nuclear matrix association observed
after deletion of the WRPW motif alone may be the result of the ability
of Hes1 to interact with RUNX proteins, which are also expressed in
ROS17/2.8 cells and localize to the nuclear matrix. This possibility is
consistent with the observation that the nuclear matrix binding of Hes1
is blocked by removal of the last 54 amino acids, which are part of the
C-terminal domain previously shown to mediate interaction with RUNX
factors (31).
We also found that TLE proteins promote transcriptional repression by
Hes1 and that inhibition of the TLE·Hes1 interaction, either by
deleting the TLE binding domain of Hes1 or overexpressing the RUNX2
protein, impairs the ability of Hes1 to mediate transcriptional repression. These findings support a model in which Hes1 and TLEs form
transcription repression complexes whose gene regulatory functions may
be regulated by association with the nuclear matrix. It must be
emphasized, however, that the transient nature of the transfection/transcription assays that were performed does not allow
the determination of whether the reporter gene targeted by Hes1 was
associated with the nuclear matrix. Future studies using stably
integrated reporter genes or in vivo targets of Hes1 will be
needed to investigate further the role played by the nuclear matrix in
the transcription repression activity of Hes1. Although much remains to
be learned about the properties of the nonchromatin elements of the
internal nuclear structure, there is increasing evidence that the
nuclear matrix may be involved in the regulation of gene expression by
supporting the formation of transcriptionally competent nuclear domains
and/or facilitating the assembly of active transcription complexes.
Thus, it is possible that nuclear architecture may play a role in
regulating the transcription functions of complexes containing Hes1 and
TLE proteins.
In the future, it will also be important to elucidate the molecular
mechanisms that underlie the repressor activity of TLE·Hes1 complexes. Previous studies have shown that Groucho/TLEs can interact with histone proteins and histone deacetylases (32, 44). Moreover, transcriptional corepression by Groucho/TLEs can be inhibited by the
histone deacetylase inhibitor, trichostatin A (32). These observations
suggest that at least one of the mechanisms utilized by Groucho/TLEs to
mediate transcriptional repression is to recruit histone deacetylases
to specific DNA sites through interaction with selected DNA-binding
proteins. Our preliminary studies suggest that Hes1-mediated
transcriptional repression also involves the activity of histone
deacetylases.2 However, it is also possible that
Groucho/TLE proteins interact with components of the basal
transcriptional machinery. This is suggested by the finding that the
yeast protein, TUP1, a general transcriptional corepressor that is
often regarded as a functional analog of Groucho/TLEs, interacts with
subunits of the yeast RNA polymerase II holoenzyme and can repress
transcription even in the absence of chromatin components (49, 50).
Thus, both histone deacetylase-dependent and -independent
mechanisms may underlie transcriptional repression by TLE·Hes1 complexes.
Grg5 Is Not Involved in the Regulation of the Transcriptional
Repression Ability of TLE·Hes1 Complexes--
Our investigations
have also shown that the naturally occurring Grg5 protein, which can
heterodimerize with full-length TLEs and has been regarded as a
possible dominant-inhibitory regulator of TLE functions, does not
associate with Hes1 and does not regulate transcriptional repression by
Hes1. This is consistent with the demonstration that the C-terminal WDR
domain of Groucho/TLEs, which is missing in Grg5, is involved in Hes
protein binding. These findings suggest that Hes1 only recruits
full-length TLE proteins to repress transcription and that neither Grg5
nor Grg5·TLE complexes are involved in the functions of Hes1.
Importantly, because Grg5 does not inhibit transcriptional repression
by TLE·Hes1, our observations also suggest that Grg5 does not act as
a dominant-negative regulator of the corepressor function that TLE
proteins provide to Hes1. In turn, this suggests that Grg5 is not a
general negative regulator of TLE activity. It is possible that Grg5
can exert a dominant-inhibitory effect on TLEs only when it shares with the latter the ability to interact directly with specific DNA-binding proteins, like Tcf/LEF family members (38) or the PRDI-BF1/Blimp1 factor (33). In this case, Grg5 may compete with full-length TLE
proteins for binding to Tcf or PRDI-BF1/Blimp1 without being able to
provide a transcription repression function, thereby inhibiting the
repressive effect of TLEs. Such a dominant-inhibitory function is
unlikely to be observed when the DNA-binding protein that requires the
TLE corepressor activity binds to neither Grg5 itself nor to TLE·Grg5
complexes, as appears to be the case for Hes1.
Involvement of RUNX2 in Transcriptional Repression by Hes1--
In
both invertebrates and vertebrates, Hes- and Runt-related proteins
contribute to the regulation of common genes (40, 46, 51) and interact
with Groucho/TLEs (30, 31, 52). Moreover, Hes1 binds to both RUNX1 and
RUNX2 and promotes transactivation by the latter (31). These
observations prompted us to determine whether RUNX family members may
be involved in regulating transcriptional repression by Hes1. Our
results show that RUNX2 can inhibit the TLE·Hes1 interaction in
vitro and Hes1-mediated transcriptional repression in transfected
cells. Taken together with the observation that the DNA-binding ability
of Hes1 does not appear to be inhibited by RUNX2, these findings
suggest that RUNX2 acts as a negative regulator of Hes1-mediated
repression by antagonizing the corepressor function provided by TLEs.
This model is consistent with the observation that a number of genes
whose promoters contain binding sites for both RUNX and Hes family
members are antagonistically regulated by these proteins. For instance,
Hes1 is a negative regulator of the expression of the
osteopontin gene in osteoblasts (40), whereas RUNX2
positively regulates this gene (46). It is possible that TLE·Hes1
complexes repress the osteopontin gene and that RUNX2
contributes to its activation both indirectly, by interfering with the
TLE·Hes1 interaction, and directly, by providing a transactivating function. A similar situation may occur in the case of the
Drosophila gene, Sex-lethal, which is positively
regulated by the Runt protein (53) and negatively regulated by the Hes
family member Deadpan in combination with Groucho (11). The further
elucidation of the molecular mechanisms that regulate the interactions
of Hes1 with TLE and RUNX proteins will be the subject of future
studies and will help clarify the cell differentiation functions of
these factors.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Act-luc have been described previously
(14, 28, 29, 31). Plasmid pEBG-Hes-(1-275) was obtained by
subcloning the appropriate PCR product into the filled-in
BamHI site of pEBG.
pCMV2-FLAG-Hes-(1-227) was generated by subcloning the
appropriate PCR product into the filled-in BamHI site of
pCMV2-FLAG. Plasmid pcDNA3-GAL4bd-Hes1-(193-281)
was obtained by digesting the pRc/CMV-Hes1 DNA with
SmaI and then subcloning the fragment encoding the last 88 amino acids of Hes1 (plus ~1.2 kb of 3'-untranslated region) into the
EcoRV site of pcDNA3-GAL4bd. Construct
pCMV-Grg5 (42) was kindly provided by Dr. T. Okamoto (Nagoya University).
Act-luc in the absence or presence of
pCMV2-FLAG-Hes1, pCMV2-FLAG-Hes1-(1-227), pcDNA3-TLE1, pCMV-Grg5, or pCMV5-RUNX2
as indicated in the figure legends. In each case, 0.25 µg of
pCMV-
-galactosidase plasmid DNA was
cotransfected to provide a means of normalizing the assays for
transfection efficiency. Results were expressed as mean ± S.D.
and were tested for statistical significance by the one-tailed Student's t test for paired differences.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Interaction of Hes1 and TLE in mammalian
cells. ROS17/2.8 cells were transfected with plasmids encoding the
indicated fusion proteins (50 ng/transfection). Cell lysates 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, 4, and 6).
One-twentieth of each input homogenate collected prior to incubation
with glutathione-Sepharose beads was also subjected to gel
electrophoresis (lanes 1, 3, and 5).
After transfer to nitrocellulose, Western blotting was performed with
either panTLE (A) or anti-GST (B) monoclonal
antibodies. The film was exposed for a shorter time in B
than in A. The positions of migration of
Mr standards are indicated.
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[in a new window]
Fig. 2.
Association of Hes1 with the nuclear
matrix. A, Western blotting analysis of soluble and
insoluble nuclear fractions. ROS17/2.8 cells were transfected with a
FLAG-Hes1 expression construct (20 ng/transfection). Nuclei were
isolated and subjected to incubation in high ionic strength conditions.
The resulting high-salt-extractable fraction (E, lane
1, ~30 µg of protein/lane) and high-salt-resistant pellet
(P, lane 2, ~20 µg of protein/lane) were
subjected to SDS-PAGE, transfer to nitrocellulose, and Western blotting
with anti-FLAG antibodies. B--H, Western blotting
analysis of cytoplasmic and nuclear fractions. Cytoplasmic
(Cyt., lane 1, ~30 µg of protein/lane),
nuclease-treated (Nuc., lane 2, ~10 µg of
protein/lane), and nuclear matrix (N.M., lane 3, ~8 µg
of protein/lane in B-F; 5 µg of protein/lane in
G and H) fractions were subjected to Western
blotting analysis with either anti-histone H3 (B),
anti-lamin B1 (C), anti-FLAG (D-F),
or anti-GAL4bd (G, H) antibodies.
D-F, cells were transfected (20 ng/transfection) with
either FLAG-Hes1 (D), FLAG-Hes1-(1-275) (E), or
FLAG-Hes1-(1-227) (F) expression constructs. G
and H, cells were transfected (20 ng/transfection) with
either GAL4bd-Hes1-(193-281) (G), or GAL4bd (H)
expression constructs. H, no GAL4bd immunoreactivity was
observed in the nuclear matrix fraction even after more prolonged
exposure of the film.
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Fig. 3.
Involvement of TLE in transcriptional
repression by Hes1. 293 cells were transfected with the
p6N- Act-luc reporter construct (2.0 µg) with
or without the following expression vectors (ng/transfection):
pFLAG-Hes1 (100 ng), pFLAG-Hes-(1-227) (100 ng),
or pcDNA3-TLE1 (75 ng). The activity of the reporter
constructs in the absence of any expression plasmid was considered as
100%. Luciferase activities were expressed as the average ± S.D.
of four independent experiments performed in duplicates. TLE1 enhanced
transcriptional repression by Hes1 (lane 3;
*p = 0.021) but had no effect on Hes1-(1-227)
(lane 5).
-actin promoter linked to six tandem copies of
a Hes1 binding site (N box). Cotransfection of Hes1 resulted in a
repression of reporter gene expression (Fig. 3, cf.
lanes 1 and 2). Importantly, the repressor
ability of Hes1 was significantly enhanced by overexpression of TLE1
(Fig. 3, lane 3), showing that Hes1-mediated repression is
promoted by TLE1. Conversely, removal of the C-terminal region of Hes1
that mediates TLE binding and nuclear matrix targeting abolished both
the repression ability of Hes1 (Fig. 3, lane 4) and the
corepressor effect of TLE1 (Fig. 3, lane 5). TLE1 alone had
no effect on reporter gene expression (Fig. 3, lane 6).
Taken together with the demonstration that Hes1 and Hes1-(1-227) were both stably expressed (see Fig. 2 above), these findings show that TLEs
act as corepressors for Hes1.
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Fig. 4.
Interaction of Grg5 with TLE but not
Hes1. A-C, coimmunoprecipitation studies.
293 cells were cotransfected with plasmids encoding FLAG-Grg5 (or FLAG
alone) and GST-Hes1 as indicated. Cell lysates were collected and
subjected to immunoprecipitation (IP) with anti-FLAG
antibodies. The immunoprecipitated material was subjected to SDS-PAGE
(lanes 2 and 4) together with one-twentieth of
each input lysate (L, lanes 1 and 3),
followed by Western blotting (WB) with anti-FLAG
(A), anti-GST (B), or panTLE (C)
antibodies. A, FLAG-Grg5 (see arrow) comigrates
with the immunoglobulin light chain (IgG LC); IgG
HC, immunoglobulin heavy chain. D and E,
coprecipitation studies. 293 cells were cotransfected with plasmids
encoding FLAG-Grg5 and GST-Hes1. Cell lysates were collected and
incubated with glutathione-Sepharose beads to isolate GST-Hes1. After
pull-down (PD), bound material was subjected to SDS-PAGE
(lane 2) together with one-tenth of the input lysate
collected prior to incubation with glutathione-Sepharose beads
(L, lane 1). After transfer to nitrocellulose,
Western blotting was performed with either anti-FLAG (D) or
panTLE (E) antibodies. The positions of migration of
Mr standards are indicated. F,
transient transfection/transcription assays. 293 cells were
cotransfected with the p6N- Act-luc reporter
construct (2.0 µg) and a Hes1 expression plasmid (20 ng) in the
absence (lane 2) or presence (lanes 3-6) of the
indicated amounts of a FLAG-Grg5 expression construct. The activity of
the reporter gene in the absence of any expression plasmid was
considered as 100%. Luciferase activities were expressed as the
average ± S.D. of at least three independent experiments
performed in duplicates. Grg5 has no effect on transcriptional
repression by Hes1. G, Western blotting analysis of Grg5.
Lysates from 293 cells transfected in the absence (lane 1)
or presence of increasing amounts of a FLAG-Grg5 expression construct
(lane 2, 100 ng; lane 3, 150 ng; lane
4, 300 ng) were subjected to Western blotting with anti-FLAG
antibodies.
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[in a new window]
Fig. 5.
Inhibition of Hes1-mediated transcriptional
repression by RUNX2. A, transient
transfection/transcription assays. 293 cells were cotransfected with
the p6N- Act-luc reporter construct (2.0 µg)
and a Hes1 expression plasmid (20 ng) in the absence (lane
2) or presence (lane 3) of a RUNX2 expression construct
(400 ng). In lane 4, the reporter plasmid was cotransfected
with the RUNX2 construct alone. Luciferase activity in the absence of
any expression plasmid was considered as 100%. RUNX2 inhibited
transcriptional repression by Hes1 but had no effect on the basal
expression of the reporter gene. B, Western blotting
analysis. 293 cells were transfected with a fixed amount (20 ng) of a
FLAG-Hes1 expression plasmid in the absence (lane 1) or
presence (lanes 2-4) of 100, 200, or 400 ng, respectively,
of a RUNX2 expression construct. Cell lysates were collected and
subjected to Western blotting with either anti-FLAG or anti-RUNX2
antibodies. C, competition binding assays. In
vitro translated 35S-labeled Hes1 (lane 1,
one-fourth the amount used in each binding assay) was incubated with 50 ng of GST-TLE(WDR) fusion protein in the absence (lane 2) or
presence of either a 5-fold (lane 3) or a 25-fold
(lane 4) volume excess of in vitro translated
35S-labeled RUNX2 (lane 5, one-tenth of the
amount used in lane 4). Due to the low amount of fusion
protein used, the pull-down efficiency was low and lanes
2-4 were exposed longer than lanes 1 and 5.
The positions of migration of Mr standards are
indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. G. Karsenty for the gift of several constructs, Dr. T. Okamoto for providing the FLAG-Grg5 expression plasmid, and Dr. C. D. Allis for anti-H3 antibodies. We also thank Dr. G. Karpati for providing access to a luminometer, and R. Lo for excellent technical assistance.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Cancer Research Society Inc. (70%) and the Medical Research Council of Canada (GR-14971) (30%) (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.
Supported by a Studentship from the Medical Research Council of Canada.
§ Supported by a Studentship from the Fonds pour la Formation de Chercheurs et l'Aide a la Recherche.
¶ 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.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007629200
2 K. McLarren, F. Theriault, and S. Stifani, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: bHLH, basic helix loop helix; AES, amino-terminal Enhancer of split; Ash, achaete-scute homolog; Ath, atonal homolog; GAL4bd, DNA binding domain of GAL4; Grg, Groucho-related gene; GST, glutathione S-transferase; Hes, Hairy and Enhancer of split; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; TLE, transducin-like Enhancer of split; WDR, WD40 repeat; kb, kilobase(s).
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