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J. Biol. Chem., Vol. 277, Issue 11, 9118-9126, March 15, 2002
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From the Department of Laboratory Medicine, Division of Molecular
Medicine, Lund University, University Hospital MAS,
S-205 02 Malmö, Sweden
Received for publication, August 13, 2001, and in revised form, November 30, 2001
It is assumed that the Id helix-loop-helix (HLH)
proteins act by associating with ubiquitously expressed basic HLH
(bHLH) transcription factors, such as E47 and E2-2, which prevents
these factors from forming functional hetero- or homodimeric DNA
binding complexes. Several tissue-specific bHLH proteins, including
HASH-1, dHAND, and HES-1, are important for development of the nervous system. Neuroblastoma tumors are derived from the sympathetic nervous
system and exhibit neural crest features. In differentiating neuroblastoma cells, HASH-1 is down-regulated, and there is
coincident up-regulation of the transcriptional repressor HES-1, which
is known to bind the HASH-1 promoter. We found that the
three Id proteins expressed in neuroblastoma cells (Id1, Id2, and Id3) were down-regulated during induced differentiation, indicating that Id
proteins help keep the tumor cells in an undifferentiated state.
Studying interactions, we noted that all four Id proteins could
dimerize with E47 or E2-2, but not with HASH-1 or dHAND. However, the
Id proteins did complex with HES-1, and increased levels of Id2 reduced
the DNA binding activity of HES-1. Furthermore, HES-1 interfered with
Id2/E2-2 complex formation. The ability of Id proteins to affect HES-1
activity is of particular interest in neuronal cells, where regulation
of HES-1 is essential for the timing of neuronal differentiation.
Neuroblastoma is a childhood tumor derived from sympathetic
neuroblasts of the peripheral nervous system (1). This part of the
nervous system originates from the neural crest, which, in addition to
the cells of the peripheral nervous system, gives rise to cartilage and
smooth muscle cells, as well as melanocytes (2). In recent years,
several transcription factors that control differentiation of the
developing nervous system have been identified, and many of these
belong to the basic helix-loop-helix
(bHLH)1 family of proteins
(3). This family can be divided into different subgroups on the basis
of structure, expression pattern, and DNA binding activity (4, 5). The
E proteins (also designated class A), have four mammalian members:
E2-2, HEB, and the two E2a gene products E12 and E47. These
proteins are ubiquitously expressed, and they can bind DNA as
homodimers or as heterodimers with the class B or tissue-specific bHLH
transcription factors. The homo- or heterodimeric DNA binding complexes
activate transcription of target genes that contain the E-box motif
CANNTG in their promoter (4). There is also a subgroup of
tissue-specific bHLH proteins that repress transcription, typified by
hairy/enhancer of split homologue-1 (HES-1) (6). The basic DNA-binding
domains of these proteins contain a proline that alters the DNA binding
specificity, as compared with other bHLH proteins (6). The HES-related
proteins can achieve their repressive functions in two ways: by binding DNA and recruiting the corepressor transducin-like enhancer of split;
or, in a non-DNA-binding manner, by interfering with complex formation
between E and bHLH proteins (6, 7). From a mechanistic point of view,
the latter function resembles the effect of the Id (inhibitors of
differentiation or inhibitors of DNA binding) proteins, which are a
group of regulatory proteins in the helix-loop-helix (HLH) network. The
Id proteins are dominant-negative inhibitors since they lack the basic
DNA-binding domain (8). The four mammalian Id proteins (Id1-Id4) form
transcriptionally inactive heterodimers, primarily with the E proteins,
and thereby prevent the E proteins from forming functional heterodimers
with tissue-specific bHLH proteins (9).
Mammalian achaete-scute homologue-1 (MASH-1 in the mouse and HASH-1 in
humans) is a vital bHLH protein in the developing sympathetic nervous
system. MASH-1 is expressed in restricted regions of
the embryonic brain and in sympathetic and enteric precursor cells (10). Gene-targeting experiments have shown that MASH-1 is
needed for proper development of autonomic and olfactory neuroblasts, neuroendocrine cells of the lung, and certain regions of the
telencephalon (11-13). Another tissue-specific bHLH protein is dHAND,
which is expressed in the embryonic sympathetic and enteric nervous
systems (14-16).
Neuroblastoma cells show characteristics of developing sympathetic
neuroblasts, and we have previously studied the expression of
dHAND and HASH-1, in both primary tumors and
neuroblastoma cell lines. At the mRNA level, we detected expression
of these two genes in all neuroblastoma cell lines and a majority of
the primary tumors we analyzed (16-18). Furthermore, during induced differentiation of neuroblastoma cells, we observed rapid
down-regulation of HASH-1 that was accompanied by a
transient up-regulation of HES-1 (17). HES-1 is an important
protein in the Notch-1 signaling cascade that has been shown to bind
the HASH-1 promoter and thereby inhibit expression of
HASH-1 (19). Accordingly, down-regulation of
HASH-1 induced by increased expression of HES-1 may be a
prerequisite for neuronal differentiation of neuroblastoma cells
(17).
In the present study, we analyzed expression of the Id genes in
relation to some tissue-specific bHLH factors that are expressed in
differentiating neuroblastoma cells. We found varying levels of Id1,
Id2, and Id3, but not Id4, in neuroblastoma cell lines, and the
expression of these proteins was down-regulated when differentiation was induced. To further elucidate the dynamics of the bHLH network, we
also performed experiments to ascertain the capacity of Id proteins to
interact with bHLH proteins involved in neurogenesis. The results show
that the Id proteins form complexes with HES-1, both in
vitro and in vivo, which demonstrates novel functions of HES-1 and the Id proteins and provides evidence of an additional level of regulation within the bHLH network. This could have
implications for understanding of the formation of the neural crest and
the lineage determination of neuronal cells, as well as the function of
these proteins in general.
Cell Culture--
The neuroblastoma cell lines SH-SY5Y and
SK-N-BE (2) and the neuroepithelioma cell line SK-N-MC were grown in
Eagle's minimum essential medium (Invitrogen) supplemented with 10%
fetal calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml) at 37 °C in 5% CO2. The SH-SY5Y cells were
induced to differentiate by exposure to 16 nM
12-O-tetradecanoylphorbol-13-acetate (TPA), and the SK-N-BE
(2) cells were differentiated with 10 µM
all-trans-retinoic acid (RA, Sigma) for 0, 2, 8, 24, and
96 h. The neuroblastoma cell lines (IMR-32, LA-N-1, LA-N-2, and
LA-N-5) and the Chinese hamster ovary cell line (CHO) were maintained
in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf
serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). The
rat pheochromocytoma cell line PC12 was grown in RPMI 1640 medium
supplemented with 10% horse serum, 5% fetal calf serum,
penicillin (100 units/ml), and streptomycin (100 µg/ml).
Western Blot Analysis--
Total cell homogenates were prepared
in Nonidet P-40 lysis buffer (1% Nonidet P-40, 10% glycerol, 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, and 4%
complete protease inhibitor mixture (Roche Molecular Biochemicals).
Using 40 µg of protein per lane, we performed SDS-PAGE and then
blotted to PVDF filters (Immobilon). The filters were probed with
polyclonal anti-Id1 or anti-Id3 antisera (Santa Cruz) diluted 1:1000 or
polyclonal anti-Id2 antiserum (Santa Cruz) diluted 1:200. HES-1 and
HASH-1 were detected with polyclonal anti-HES-1 antiserum (kindly
provided by Dr. Tetsuo Sudo (20)) diluted 1:8000 and a monoclonal
anti-MASH-1 antibody (Pharmingen) diluted 1:125. Fusion proteins
containing VP16, GAL4, and EGFP domains were, respectively, detected
with a monoclonal anti-VP16 antibody (Santa Cruz), a monoclonal
anti-GAL4-DNA-binding domain antibody (Santa Cruz), and a polyclonal
anti-GFP antiserum (CLONTECH). Immunoreactivity was
visualized by enhanced chemiluminescence (Pierce).
Mammalian Two-hybrid Analysis--
Mammalian two-hybrid analyses
were performed using the Checkmate system (Promega). Constructs
encoding VP16 transactivating (pACT) and GAL4-DNA-binding (pBIND)
proteins were created by cloning PCR-generated fragments into
BamHI/SalI-digested vectors. The fragments
comprised the following amino acids: dHAND, 1-217; E2-2, 1-668; E47,
508-654 (spanning the bHLH region); HASH-1, 1-180; HES-1, 90-281;
Id1, 1-154; Id2, 1-134; Id3, 1-119; Id4, 1-162. The pBIND-MyoD
vector was provided in the Checkmate kit (Promega). The
pG5luc reporter plasmid contains five GAL4-binding sites
upstream of the coding sequence for firefly luciferase. The pMACT
vector also encodes renilla luciferase downstream of a
constitutively active cytomegalovirus promoter, hence it was
possible to use a Dual Luciferase kit (Promega) for both luminometric
measurement of the interaction and determination of transfection
efficiency. The analyses were done on CHO cells (1.5 × 105 per 35-mm dish), which were transfected with the
appropriate vectors and the pG5luc reporter (0.6 µg of
each plasmid per dish) using LipofectAMINE (Invitrogen). The
experiments were performed in triplicate, and the results were recorded
as the relative luciferase activity (i.e. the ratio of
firefly luciferase activity to renilla luciferase activity).
The expression plasmids were sequenced, and expression of all proteins
was verified by Western blot analysis. The mammalian two-hybrid system
was also employed to assess the ability of HES-1 and Id2 to interfere
with the formation of bHLH dimers in a dominant-negative manner. CHO
cells were co-transfected with pACT-E2-2 and pBIND-HASH-1 or pBIND-Id2
(0.2 µg of each plasmid per dish), along with increasing amounts of
pMYC-HES-1 or pEGFP-Id2 (0.2-0.8 µg of plasmid per dish). The total
amount of DNA was equalized with either pMYC or pEGFP. The pMYC-HES-1
and pEGFP-Id2 were made as described above, but, for HES-1, the
full-length sequence (amino acids 1-281) was used. The results are
given as percentage of the initial activity.
Transient Transfections and Co-immunoprecipitations--
The
following expression plasmids were used: pBIND-HASH-1, pBIND-HES-1,
pBIND-Id2, pBIND-E2-2, pBIND-E47, pEGFP-HASH-1, and pEGFP-HES-1. To
make pEGFP-HASH-1 (amino acids 1-238) and pEGFP-HES-1 (amino acids
1-281), PCR-generated fragments were inserted into a
BamHI/XhoI-digested pEGFP-C2 vector
(CLONTECH). All transfections were performed using
LipofectAMINE according to the recommendations of the manufacturer
(Invitrogen). CHO cells were plated (1 × 106
cells/100-mm dish) and transfected with 5 µg of total DNA. Sixteen hours after transfections, total cell lysates were prepared in modified
RIPA buffer (1% Nonidet P-40, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 4% complete protease
inhibitor mixture (Roche Molecular Biochemicals)). The lysates were
centrifuged at 14,000 × g for 15 min to remove debris
and then precleared with protein G-Sepharose beads (Amersham
Biosciences, Inc.) for 10 min at 4 °C. Immunoprecipitations were
performed with a monoclonal anti-GAL4-DNA-binding domain antibody. The
immune complexes were recovered on protein G-Sepharose beads for 1 h at 4 °C and then washed four times with phosphate-buffered saline.
The precipitated proteins were resuspended in sample buffer, boiled for
5 min, fractionated by SDS-PAGE on a 12.5% gel, and thereafter blotted to a PVDF filter. The filter was incubated with polyclonal anti-GFP antiserum diluted 1:100. Immunoreactivity was detected by enhanced chemiluminescence (Pierce).
For co-immunoprecipitations of endogenous proteins, PC12 cells were
plated (10 × 106 cells/100-mm dish), and cell lysates
were prepared in modified RIPA buffer (as above). Immunoprecipitations
were performed with 1 µg of polyclonal anti-Id1 antiserum (Santa
Cruz) or 1 µg of rabbit immunoglobulin (negative control, Dako) for
1.5 h at 4 °C. The immune complexes were precipitated with
protein G-Sepharose for 1 h at 4 °C and then washed twice with
modified RIPA buffer and twice with phosphate-buffered saline. The
samples were separated by electrophoresis on a 12.5% acrylamide gel
and the proteins were transferred to a PVDF filter. HES-1 was detected
with polyclonal anti-HES-1 antiserum diluted 1:16,000, and Id1 with
polyclonal anti-Id1 antiserum diluted 1:1000. Immunoreactivity was
detected as described above.
Electrophoretic Mobility Shift Assay--
Using LipofectAMINE,
pACT-HES-1 or pACT-Id2 were transfected into CHO cells seeded at a
density of 1 × 106 cells/100-mm plate. Sixteen hours
after transfection, the cells were harvested in lysis buffer (10 mM Tris-HCl, 50 mM NaCl, 30 mM
Na4P2O7, 50 mM NaF, and
5 µM ZnCl2 (pH 7.1), supplemented with 1%
Triton X-100, 5% protease inhibitor mixture (Sigma), and 6.7 mM dithiothreitol). Transcription and translation of Id2
in vitro were carried out using a TNT-coupled reticulocyte
lysate kit (Promega) and the pACT-Id2 plasmid. DNA-binding reactions
were done in a total volume of 35 µl containing 20 mM
HEPES (pH 7.3), 50 mM KCl, 3 mM
MgCl2, 1 mM EDTA, 8% glycerol, 5 µg/ml
aprotinin, 1 mM Expression of Id Proteins in Neuroblastoma Cell Lines--
The
expression patterns of Id1, Id2, Id3, Id4, HASH-1, and HES-1 were
analyzed at the protein level in neuroblastoma cell lines and in two
other cell lines derived from related tumors, the rat pheochromocytoma
cell line PC12 and the neuroepithelioma cell line SK-N-MC (Fig.
1). The PC12 cells and all but one of the
neuroblastoma cell lines (i.e. SH-SY5Y, IMR-32, LA-N-2,
LA-N-1, and SK-N-BE (2), but not LA-N-5) expressed detectable levels of
HASH-1. HES-1 was clearly detected in PC12 and SK-N-MC cells, and low
levels of this protein was also found in four neuroblastoma cell lines
(SH-SY5Y, LA-N-5, LA-N-1, and SK-N-BE (2)). These results corroborated
our previous mRNA data showing that there is an inverse correlation
between HASH-1 and HES-1 expression in
neuroblastoma cells and that HES-1, but not
HASH-1, is expressed in SK-N-MC cells (17). Varying levels
of Id1 and Id2 were expressed in all neuroblastoma cell lines tested:
high levels of Id1 in SH-SY5Y, IMR-32, LA-N-1, and SK-N-BE (2); and
substantial expression of Id2 in LA-N-2 and SH-SY5Y (Fig. 1). All the
neuroblastoma lines except IMR-32 and LA-N-1 expressed Id3 (Fig. 1),
whereas none of the cell lines investigated expressed Id4 (data not
shown).
Expression of Id Proteins in Differentiating Neuroblastoma
Cells--
In many cell systems, Id expression decreases during
differentiation (9). To study the expression pattern of HASH-1, HES-1, and the Id proteins in differentiating neuroblastoma cells, we used two
different cell systems: SH-SY5Y, which lacks N-myc
amplification; and SK-N-BE (2), in which each cell contains ~85
copies of the N-myc gene (21). The SH-SY5Y cells were
induced to undergo robust and well characterized sympathetic neuronal
differentiation by exposure to 16 nM TPA (22, 23) and the
SK-N-BE (2) cells were induced to differentiate with 10 µM RA, which led to a less distinct neuronal
differentiation (17, 18). Within 2 h of TPA treatment, the level
of HES-1 in the SH-SY5Y cells reached a maximum, and there was a
concurrent down-regulation of HASH-1 expression (Fig.
2); neither of these proteins was
detected after 96 h of differentiation. In accordance with these
findings, we have previously studied the corresponding genes and
observed a similar rapid down-regulation of HASH-1 at the
mRNA level, with a concomitant transient up-regulation of
HES-1 (17). In differentiating SH-SY5Y cells, the Id1 level
remained constant up to 24 h, but had decreased markedly after
96 h (Fig. 2). By comparison, Id2 and Id3 were down-regulated more
quickly: both showed a distinct decrease after only 8 h, and,
after 96 h, Id3 was undetectable, and the level of Id2 was low
(Fig. 2).
After 2 h of differentiation induced by 10 µM RA,
the SK-N-BE (2) cells exhibited a decrease in the level of HASH-1, and this was accompanied by an increase in HES-1 (Fig.
3). After 96 h, the former protein
was barely detectable, and the level of HES-1 had decreased to
baseline, which reflects our previously reported mRNA data (17).
The changes in Id levels that occurred in this cell line with
progressing differentiation were not as marked as those observed in
SH-SY5Y cells undergoing TPA-induced differentiation. Moreover, in the
SK-N-BE (2) cells, the level of Id1 was virtually constant, while the
level of Id2 decreased to some extent, and there was a slight transient
up-regulation of Id3 expression after 8 h (Fig. 3). Thus, the
SK-N-BE (2) and SH-SY5Y cells were similar in regard to regulation of
expression of HASH-1 and HES-1, but they exhibited slightly different
Id expression patterns.
Mammalian Two-hybrid Analysis of Interactions between Id Proteins
and Neuronal bHLH Transcription Factors--
We used the mammalian
two-hybrid system to investigate the ability of the Id proteins to form
complexes with bHLH transcription factors (24). Expression plasmids
encoding GAL4 fusion proteins of Id1 to Id4 and VP16 fusion proteins of
a panel of bHLH transcription factors were constructed (Fig.
4A). Western blot analyses
revealed that the levels of expression of the different fusion proteins were similar (Fig. 4, B and C). The modest
variations that were noticeable were not correlated with the degree of
reporter gene activation in the mammalian two-hybrid assays, as
exemplified by HES-1 and dHAND (Fig. 4B), as well as dHAND
and Id1 (Fig. 4C). As expected, there was a clear
interaction between Id1 and the ubiquitously expressed E proteins E47
and E2-2 (Fig. 4B). Comparable results were obtained with
the Id2, Id3, and Id4 constructs (data not
shown), and, in the subsequent mammalian two-hybrid experiments, all
four Id proteins displayed similar capacities to interact with bHLH
proteins. In agreement with previous reports (25, 26), we found that
all four Id proteins interacted with MyoD (Fig. 4B and data
not shown). Moreover, although the four Id proteins did not form
complexes with the tissue-specific proneuronal transcription factors
dHAND and HASH-1, they did dimerize with HES-1 (Fig. 4B and
data not shown).
The ability of HES-1 to dimerize with both the Id and the E proteins
distinguished it from the other tissue-specific bHLH transcription
factors we investigated. The difference between dHAND and HES-1 in
regard to interaction capacity was further illustrated by coexpression
of VP16-dHAND or VP16-HES-1 with GAL4 fusion proteins of a panel of
HLH/bHLH factors. We thereby generated a "fingerprint" of the
abilities of dHAND and HES-1 to interact with other proteins of the HLH
network (Fig. 4, C and D). The background
activity of each GAL4 fusion protein was assessed in combination with
the empty pACT vector. These analyses highlighted the following
characteristic difference in the interaction patterns of these two
proteins: HES-1 formed complexes with the E and the Id proteins,
whereas dHAND dimerized only with the E proteins.
Mammalian Two-hybrid Analysis of Dominant-negative Effects of HES-1
and Id2--
We extended the mammalian two-hybrid analyses to
ascertain whether HES-1 and Id2 interfere with the formation of HLH
dimers in a dominant-negative manner. Increasing amounts of HES-1 had no effect on dimerization between HASH-1 and E2-2 (Fig.
5A). In contrast, Id2
decreased the reporter gene activity even at the initial concentration
we tested (Fig. 5B). At this level, the EGFP-Id2 fusion
protein was hardly detectable by Western blot analysis. This effect was
most likely due to the formation of E2-2/Id2 dimers, since the Id
proteins do not interact with HASH-1 (Fig. 4B and data not
shown). Accordingly, we investigated the effect of HES-1 on formation
of the E2-2/Id2 dimer and discovered that reporter gene activation
decreased with increasing amounts of HES-1 (Fig. 5C).
Theoretically, this may have been due to binding of either Id2 or E2-2
to HES-1. However, HES-1 did not affect the interaction between E2-2
and HASH-1 (Fig. 5A), hence we suggest that this inhibitory
effect of HES-1 was mainly the result of complex formation with Id2.
Thus, in these experiments in vivo, HES-1 sequestered Id2
and in that way prevented it from dimerizing with the E protein
E2-2.
Co-immunprecipitation of Id2 and HES-1--
We performed
co-immunoprecipitation assays to prove that HES-1 and Id proteins can
interact in mammalian cells. CHO cells were transfected with expression
plasmids for EGFP-tagged HES-1 or HASH-1 and GAL4-tagged Id2 (Fig.
6A), and Western blotting revealed that EGFP-HES-1 and EGFP-HASH-1 were expressed at similar levels (data not shown). Sixteen hours after transfection, the cells
were lysed, and Id2 with associated proteins were immunoprecipitated with an anti-GAL4 antibody conjugated to protein G-Sepharose beads. The
precipitates were subjected to Western blot analysis using a polyclonal
anti-EGFP antiserum, which showed that HES-1, but not HASH-1, was
co-immunoprecipitated with Id2 (Fig. 6A). Use of an empty
GAL4-expressing vector showed that the Id2 part of the fusion protein
was necessary for co-immunoprecipitation to occur. We included the E
proteins E2-2 and E47 as positive controls in these experiments. Our
findings confirmed the results obtained using the mammalian two-hybrid
system, that is, both E2-2 and E47 formed complexes with HES-1, whereas
there was no detectable association between HASH-1 and HES-1 or between
HASH-1 and Id2 (Fig. 6A). Apparently, the amounts of HES-1
immunoprecipitated by Id2, E47, and E2-2, respectively, were to some
extent correlated with the level of reporter gene activation observed
in the mammalian two-hybrid system, because we found that more HES-1
was co-immunoprecipitated with Id2 than with the E-proteins.
Furthermore, E47 was less efficient in precipitating HES-1 than E2-2
(Figs. 4B and 6A). It will be necessary to use
other experimental techniques to determine whether these observations
reflect different affinities between the various HLH proteins.
We continued our work by examining the ability of HES-1 to form
complexes with the Id proteins in cells derived from the sympathetic nervous system. We chose to analyze Id1 instead of Id2 due to the
superior immunoprecipitating capacity of the anti-Id1 compared with the
anti-Id2 antiserum. The rat pheochromocytoma cell line PC12 was used,
because these cells express high levels of HES-1 and Id1. We found that
the anti-Id1 antiserum did immunoprecipitate HES-1 from PC12 extracts
(Fig. 6B), which confirmed the results of the
co-immunoprecipitations using extracts from transfected cells.
Therefore, we conclude that Id1 can complex with HES-1 in sympathetic
neuronal cells.
Id2 Inhibits Binding of HES-1 to an N-box
Oligonucleotide--
HES-1 and other hairy-related bHLH proteins bind
to a distinct hexameric sequence called the N- or C-box (CACNAG) (6). For electrophoretic mobility shift assays, we transfected CHO cells
with a VP16-tagged HES-1 expression construct and then
prepared cell lysates. A prominent complex appeared when
32P-labeled N-box-containing oligonucleotide was mixed with
cell extract from HES-1-transfected CHO cells, whereas no
such complex was detected when this oligonucleotide was mixed with
extract from untransfected cells (Fig.
7A, lanes 2 and
4). The complex was supershifted when we mixed extract from
HES-1-transfected cells with an anti-HES-1 serum, which
confirms that this complex arose due to HES-1 DNA binding activity
(Fig. 7A, lane 3). To verify the specificity of
this binding, we performed competition experiments, in which an excess
of unlabeled wt or N-box-mutated oligonucleotide was added to the
reaction mixtures, and found that only the wt oligonucleotide could
diminish the DNA binding of HES-1 (Fig. 7A, lanes
5 and 6). To study the effect of Id2 on DNA binding by
HES-1, we preincubated extract from HES-1-transfected cells
with Id2 translated in vitro or, as a control, with an equal amount of a lysate of mock-translated rabbit reticulocyte lysate. The
results show that binding of HES-1 to the N-box oligonucleotide was
decreased by the in vitro translated Id2, but not by the
control mock-translated lysate (Fig. 7B, lanes 2 and 3). Extending these experiments, we mixed increasing
amounts of cell extract from Id2-transfected CHO cells with
a constant amount of cell extract from HES-1-transfected CHO
cells, and found that binding of HES-1 to the N-box-containing
oligonucleotide decreased with increasing Id2 concentration (Fig.
7C). Together, these results show that HES-1 can bind an
N-box-containing oligonucleotide, and that addition of Id2 translated
in vitro or extract from Id2-transfected cells decreases this DNA binding activity of HES-1.
To gain a better understanding of the biological activity and
function of the different Id proteins, we analyzed these proteins in
regard to their expression patterns and their ability to form complexes
with bHLH factors expressed in differentiating neuroblastoma cells. All
the neuroblastoma cell lines we investigated expressed Id1 and Id2, and
most of them also expressed Id3.
It has been reported that, in a number of model systems, Id levels are
down-regulated during induced differentiation, and there is concomitant
cell cycle withdrawal (27). Consistent with those findings, we have
previously noted that all Id genes expressed in our experiments
(Id1, Id2, and Id3) were
down-regulated during TPA-induced differentiation and growth inhibition
in SH-SY5Y cells (28), but it is not clear whether the down-regulation of Id proteins is a prerequisite for the decreased proliferation. By
comparison, in the present study, we detected only modest changes in Id
levels in SK-N-BE (2) cells that had been induced to differentiate. The
differences in Id levels may reflect the varying extent of neuronal
differentiation in these two cell lines, with a more robust neuronal
phenotype in the SH-SY5Y cells than in the SK-N-BE (2) cells.
Considering the decreased proliferation during induced differentiation,
it should be mentioned that Id proteins also have several effects on
cell cycle regulatory proteins. One such effect is the direct
interaction between Id2 and proteins of the retinoblastoma family (pRB,
p107, and p130) (29). It is assumed that Id2 interferes with the
association between pRB and E2F-DP1 by binding hypophosphorylated pRB,
which results in E2F-DP1-driven transcription of genes required for
S-phase progression. Thus, a possible explanation for the reduced
proliferation seen in neuroblastoma cells undergoing induced
differentiation is that a lower level of Id2 increases the pool of free
hypophosphorylated pRB. In support of this concept, ectopic expression
of the Id proteins leads to a blocked differentiation and increased
proliferation in several model systems (9). However, we could not
determine whether this is also the case in neuroblastoma cells, because they died upon overexpression of Id proteins (data not shown). Of
specific interest for the genesis of neuroblastoma is a study indicating that Id2 is a transcriptional target of N-myc
(30). Since N-myc amplification, and thereby dysregulated
expression, is a cardinal feature of high stage neuroblastoma tumors
these findings could shed light on some puzzling features of
neuroblastomas, such as the lack of mutations in the pRB pathway
(31).
Within the bHLH network, the Id proteins exert their effect primarily
by binding to E proteins. This was readily revealed by the mammalian
two-hybrid analyses, which showed that all four Id proteins interacted
with E47 and E2-2 (Fig. 4B and data not shown). We
observed that the Id proteins interacted with MyoD, as also reported by
other investigators (8, 26), but not with the proneuronal bHLH proteins
HASH-1 and dHAND. These data corroborate the concept that the Id
proteins act by sequestering the E proteins, thereby preventing complex
formation between proneuronal bHLH proteins and their obligatory
protein dimerization partners (4). Furthermore, we found that HES-1
interacted with all four Id proteins, and the difference in interaction
pattern between HES-1 and the proneuronal bHLH protein dHAND was
clearly demonstrated by generating mammalian two-hybrid interaction
fingerprints (Fig. 4, C and D). Whereas
dHAND interacted solely and specifically with the E proteins, HES-1
formed complexes with both the E and Id proteins. Although the level of
reporter gene activation in the mammalian two-hybrid system does not
necessarily reflect the exact strength of the interaction, our
co-immunoprecipitation data support the suggestion that the association
between HES-1 and Id2 is of considerable strength (Fig. 6A).
Moreover, the co-immunoprecipitation experiments of endogenous proteins
showed that a considerable fraction of HES-1 in PC12 cells is complexed
with Id1 (Fig. 6B), which is the predominant Id protein in
this sympathetic nervous system-derived cell line. This clearly
suggests that the interaction influences the activity of both proteins
in vivo.
We extended the mammalian two-hybrid analyses to include a third
factor that was expressed together with the GAL4 and VP16 fusion
proteins. We reasoned that if this additional protein bound either of
the two fusion proteins, we would detect decreased reporter gene
activation. Indeed, introduction of increasing amounts of Id2 to cells
with a transactivating HASH-1·E2-2 complex showed that Id2 had a
dominant-negative effect on the HASH-1/E2-2 reporter gene activation
(Fig. 5B). We also found that HES-1 can interfere with the
dimerization between Id2 and E2-2, but not that between HASH-1 and
E2-2, which suggests that HES-1 in vivo can modulate the
dominant-negative effect that Id2 has on an E protein without directly
interfering with dimerization between HASH-1 and E2-2 (Fig. 5). This
reveals a novel level of regulation within the HLH network: in an
in vivo situation in which the four proteins compared in
this experiment are present, Id2 may act as a dominant-negative regulator of the HASH-1/E2-2 complex, and HES-1 will, in turn, act as a
negative regulator of Id2, without affecting the association between
HASH-1 and E2-2. Our results may also have implications beyond the bHLH
network, since HES-1 might interfere with other functions of the Id
proteins, such as the interaction between Id and pRB (29), and in that
way directly affect cell proliferation. Furthermore, Id2 decreased the
binding of HES-1 to an N-box-containing oligonucleotide (Fig.
7C). Thus, Id2 can sequester HES-1 and prevent it from
binding DNA in a manner similar to the dominant-negative effect that
the Id proteins exert on the E proteins.
Low levels of HES-1 are expressed in many types of cells, and
this expression is at least partly controlled by Notch-1 signaling (32). Moreover, there is a negative HES-1 autoregulation component at
N-boxes in the HES-1 promoter (33), and Id protein levels may therefore
influence this autoregulatory loop. Interestingly, studies of
Drosophila have provided genetic evidence linking the function of the Notch signaling cascade with extramacrochaetae (emc),
the Drosophila orthologue of the Id proteins. In
Drosophila, the function of Notch-1 is important for proper
wing formation, and, during that event, there is an association between
expression of Notch and emc. This suggests that,
in certain developmental stages, emc collaborates with
downstream genes that are targets of Notch-1, such as the hairy-related
Enhancer of split-mb gene (34). However, biochemical studies
of the Drosophila proteins have excluded a direct
interaction between emc and the hairy-related proteins, in contrast to
our results which are based on the mammalian homologues and show a
relatively strong interaction between HES-1 and the Id proteins (Fig.
4, B and C, and 6A). Both HES-1 and Id2 play important roles in development of the neural crest.
Gene-targeting experiments in mice have shown that HES-1 controls the
proper timing of neurogenesis and regulates neural tube morphogenesis by repressing expression of proneuronal genes such as MASH-1 (35-37). In the chicken, it has been demonstrated that overexpression of Id2,
which is normally expressed in the cranial neural folds, converts
ectodermal precursor cells into a neural instead of an epidermal
lineage (38), and the authors suggested that epidermalization may
be promoted by a bHLH partner of Id2. This bHLH partner should have
properties that are compatible with the known functions of HES-1 as an
inhibitor of neuronal differentiation.
Mutual regulation of HES-1 and Id proteins may also be important in
other tissues and cell types in which involvement of HES-1 has been
implicated, for instance, the developing central nervous system (39),
We thank Dr. Karim Dib for helpful advice on
the co-immunoprecipitation experiments.
*
This work was supported by grants from the Swedish Cancer
Society, the Children's Cancer Foundation of Sweden, Inga and John Hain's Foundation, Åke Wiberg's Foundation, the Crafoord Foundation, HKH Kronprinsessans Lovisas förening för
barnasjukvård, Hans von Kantzow's Foundation, and
Malmö University Hospital Research Funds.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: Dept. of Medical Biochemistry and Microbiology,
Uppsala University, BMC, P.O. Box 582, S-751 23 Uppsala, Sweden.
¶
To whom correspondence should be addressed: Dept. of
Laboratory Medicine, Div. of Molecular Medicine, Lund University,
University Hospital MAS, Entrance 78, S-205 02 Malmö, Sweden.
Tel.: 46-40337621; Fax: 46-40337322; E-mail:
hakan.axelson@molmed.mas.lu.se.
Published, JBC Papers in Press, December 27, 2001, DOI 10.1074/jbc.M107713200
The abbreviations used are:
bHLH, basic
helix-loop-helix;
HES-1, hairy/enhancer of split homologue-1;
TPA, 12-O-tetradecanoylphorbol-13-acetate;
RA, retinoic acid;
CHO, Chinese hamster ovary;
PVDF, polyvinylidene difluoride;
EGFP, enhanced green fluorescent protein.
Modulation of Basic Helix-Loop-Helix Transcription Complex
Formation by Id Proteins during Neuronal Differentiation*
,,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
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-mercaptoethanol, 10 mM
dithiothreitol, 0.1 mg/ml salmon sperm DNA, and 0.2 ng of 32P-labeled wild type (wt) oligonucleotide
(5'-CCGCCAGGCGCACGCACTGCAACAA-3'). Competition experiments were done
with 175 ng of unlabeled wt oligonucleotide or 175 ng of N-box-mutated
oligonucleotide (5'-CCGCCAGGCGACTAAGCTGCAACAA-3'). For supershift
experiments, the cell extracts were preincubated with polyclonal
anti-HES-1 antiserum (CeMines) for 10 min at room temperature.
Thereafter, the labeled probe was added, and the samples were further
incubated for 30 min at 30 °C. Id2 translated in vitro or
an equal amount of mock-translated rabbit reticulocyte lysate was mixed
with protein extract from HES-1-transfected CHO cells, and the mixture
was preincubated for 10 min at room temperature, after which the
labeled probe was added. In the Id2 competition experiments, increasing
amounts of cell extract from Id2-transfected CHO cells were mixed with
constant amounts of cell extract from HES-1-transfected CHO cells and
then preincubated for 10 min at room temperature. Cell extract from
untransfected CHO cells was used as a negative control. The binding
reactions were then subjected to electrophoresis on a 4%
polyacrylamide gel (30:1 acrylamide:bisacrylamide).
RESULTS
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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View larger version (88K):
[in a new window]
Fig. 1.
Western blot analysis of neuroblastoma and
related cell lines, showing the expression patterns of HASH-1, HES-1,
Id1, Id2, and Id3. Cell extracts (each containing 40 µg of
protein) from the neuroblastoma cell lines SH-SY5Y, IMR-32, LA-N-5,
LA-N-2, LA-N-1, and SK-N-BE(2), as well as the rat pheochromocytoma
cell line PC12 and the human neuroepithelioma cell line SK-N-MC, were
subjected to 12.5% SDS-PAGE. The proteins were transferred to a PVDF
filter and detected using antibodies directed toward HASH-1, HES-1,
Id1, Id2, and Id3.
View larger version (58K):
[in a new window]
Fig. 2.
Expression patterns of HASH-1, HES-1, Id1,
Id2, and Id3 in differentiating SH-SY5Y cells. Cell extracts (40 µg) from SH-SY5Y cells treated with 16 nM TPA for 2 to
96 h was subjected to Western blotting. The samples were
fractionated by SDS-PAGE in a 12.5% gel and then transferred to a PVDF
filter. The proteins were detected using antibodies directed toward
HASH-1, HES-1, Id1, Id2, and Id3.
View larger version (70K):
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Fig. 3.
Western blot analysis of HASH-1, HES-1, Id1,
Id2, and Id3 expressed in RA-treated SK-N-BE(2) cells. The cells
were treated with 10 µM RA for 2 to 96 h. Cell
extracts containing 40 µg of protein were subjected to 12.5%
SDS-PAGE and then transferred to a PVDF filter. Proteins were detected
using antibodies directed toward HASH-1, HES-1, Id1, Id2, and
Id3.
View larger version (31K):
[in a new window]
Fig. 4.
Mammalian two-hybrid analysis of the ability
of Id proteins to dimerize with bHLH transcription factors in
vivo. A, schematic representation of the GAL4 or VP16
fusion proteins tested in the mammalian two-hybrid system. The proteins
were cloned in-frame with the GAL4 domain in the pBIND vector or the
VP16 domain in the pACT vector. The amino acids of the proteins
included in the individual constructs are indicated. The shaded
boxes represent the GAL4 or VP16 domains, and the striped
boxes indicate the approximate locations of the HLH domains. All
constructs were sequenced and found to express proteins of the expected
molecular weights. B, a mammalian two-hybrid system was
employed to study the ability of Id1 to form heterodimers with a
collection of bHLH transcription factors expressed in human
neuroblastoma cells, using MyoD as a positive control. Plasmids
encoding the DNA-binding GAL4 domain fused to Id proteins were
co-transfected into CHO cells, together with plasmids encoding the VP16
transactivating domain fused to bHLH factors. The degree of interaction
is given as the "relative luciferase activity," which was
calculated as the ratio of firefly luciferase activity to
renilla luciferase activity. The interaction patterns
obtained in experiments with Id2, Id3, and Id4 (data not shown) were
identical to the pattern for Id1. The Western blot panel shows the
expression levels of the VP16 fusion proteins. The mammalian two-hybrid
system was used to analyze VP16 fusion proteins of either HES-1
(C) or dHAND (D) regarding their ability to form
complexes with a panel of GAL4-fused HLH proteins. The shaded
columns represent the relative luciferase activity recorded when
pACT-HES-1 or pACT-dHAND was co-transfected with different pBIND-HLH
plasmids. The unshaded columns represent the background
relative luciferase activity detected when the various pBIND-HLH
vectors were co-transfected with empty pACT vector. Expression of GAL4
fusion protein was analyzed by Western blotting (C).
View larger version (26K):
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Fig. 5.
Analysis of the ability of Id2 and HES-1 to
interfere with formation of bHLH dimers in vivo. The
expression vectors of the mammalian two-hybrid system were used to
assess the capacity of HES-1 and Id2 to interfere with the formation of
transcriptionally active bHLH dimers in a dominant-negative manner.
A, effect of HES-1 on the interaction between HASH-1 and
E2-2. CHO cells grown in 35-mm culture dishes were co-transfected with
pBIND-HASH-1 and pACT-E2-2 (0.2 µg each) and increasing amounts
(0.2-0.8 µg) of pMyc-HES-1. The total amount of DNA was balanced
with empty pMyc-vector. B, effect of Id2 on the interaction
between HASH-1 and E2-2. The cells were co-transfected with HASH-1 and
E2-2 as described above, as well as increasing amounts (0.2-0.8 µg)
of pEGFP-Id2. C, effect of HES-1 on the interaction between
Id2 and E2-2 interaction. The CHO cells were co-transfected with
constant amounts of pBIND-Id2 and pACT-E2-2, and increasing levels of
pMyc-HES-1, as in A. The interaction between the GAL4 and
the VP16 fusion proteins is recorded as the relative luciferase
activity, representing the ratio of firefly luciferase activity to
renilla luciferase activity. The value representing the
activity of the GAL4 and VP16 fusion proteins alone was considered to
be 100%. Western blot analysis using anti-HES-1 (A) or
anti-GFP (B) antiserum was performed to confirm that the
protein levels did increase with increasing amounts of expression
vector.
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Fig. 6.
Co-immunoprecipitation assays confirming
interactions between the Id proteins and HES-1. A, CHO
cells were transfected with the indicated plasmids, using pEGFP-HASH-1,
pBIND-HASH-1, and pBIND as negative controls. Cell extracts were
immunoprecipitated with a monoclonal anti-GAL4 DNA-binding domain
antibody, and the immunoprecipitated protein complexes were subjected
to 10% SDS-PAGE and blotted to a PVDF filter. Crude cell extract from
CHO cells transfected with pEGFP-HES-1 and pBIND-Id2 was run in a
parallel lane. Co-immunoprecipitated EGFP-HES-1 fusion protein was
detected by Western blotting with a polyclonal anti-GFP antiserum.
B, co-immunoprecipitation of endogenous HES-1 and Id1
proteins from extracts of PC12 pheochromocytoma cells. The proteins
were immunoprecipitated with a polyclonal anti-Id1 antiserum or with
normal rabbit immunoglobulin (Ig) as a negative control. The
immunoprecipitate was subjected to 12.5% SDS-PAGE, and proteins were
transferred to a PVDF filter. Crude cell extract from PC12 cells was
run in a parallel lane. Immunodetection was done with polyclonal
anti-HES-1 or anti-Id1 antiserum, as indicated.
View larger version (63K):
[in a new window]
Fig. 7.
Electrophoretic mobility shift assay of N-box
binding activity in cell extracts from HES-1-transfected CHO
cells. A, a 32P-labeled oligonucleotide
containing a HASH-1 promoter sequence, including the N-box
CACGCA, was incubated with extract from HES-1-transfected CHO cells
(lanes 2, 3, 5, and 6). In
lane 3, a polyclonal anti-HES-1 antiserum was added to the
reaction mixture. Lane 4 shows untransfected CHO cell
extract; the complex that appeared in this lane was of unknown origin.
As competitors, an excess of the unlabeled wt oligonucleotide probe was
added (lane 5), or an unlabeled mutant version of the
oligonucleotide (lane 6). B, cell extract from
HES-1-transfected CHO cells was mixed with a 32P-labeled
N-box oligonucleotide. Before adding the labeled probe, aliquots of the
extract were preincubated with rabbit reticulocyte lysate (lane
2) or an equal amount of Id2 translated in vitro
(lane 3). C, increasing amounts (3, 5, 7, and 9 µg, lanes 3-6) of cell extract from Id2-transfected CHO
cells were mixed with a constant amount of cell extract from
HES-1-transfected CHO cells, after which the labeled N-box
oligonucleotide was added. The same amounts of cell extract from
untransfected CHO cells (i.e. 3, 5, 7 and 9 µg,
lanes 7-10) were used as a negative control.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells of the pancreas (40), and tumors such as small cell lung
cancer (19). During differentiation in SH-SY5Y neuroblastoma cells,
HES-1 is transiently up-regulated before the Id proteins are
down-regulated (Fig. 2). Consequently, it is possible that
differentiation is initiated by the short-lived up-regulation of HES-1
and that the subsequent down-regulation of Id proteins is necessary
during later stages of differentiation and for cessation of
proliferation. In this scenario, the interaction between HES-1 and the
Id proteins could have several effects, one of which might be to
balance the level of functional HES-1, and, conversely, HES-1 might
affect the activity of the Id proteins. Furthermore, the induction of
HES-1 during neuroblastoma cell differentiation may result in decreased
levels of free Id2 and an increased pool of hypophosphorylated pRB,
which would impede cell cycle progression. Concomitantly, the HES-1/Id2
interaction may relieve the dominant-negative effect of Id2 on the E
proteins, allowing them to dimerize with the proneuronal bHLH proteins
and permitting the differentiation program to proceed. HES-1 that is
not associated with Id proteins may have a negative autoregulatory effect and also repress HASH-1 transcription. Our finding
that interaction can occur between HES-1 and Id protein may be of
importance not only in further studies of neuroblastoma cell
differentiation, but also to elucidate the role of these proteins in general.
ACKNOWLEDGEMENT
FOOTNOTES
These authors contributed equally to this work.
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INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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 A. Fischer and M. Gessler Delta Notch and then? Protein interactions and proposed modes of repression by Hes and Hey bHLH factors Nucleic Acids Res., July 14, 2007; 35(14): 4583 - 4596. [Abstract] [Full Text] [PDF]
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 Y. Li, J. Yang, J.-H. Luo, S. Dedhar, and Y. Liu Tubular Epithelial Cell Dedifferentiation Is Driven by the Helix-Loop-Helix Transcriptional Inhibitor Id1 J. Am. Soc. Nephrol., February 1, 2007; 18(2): 449 - 460. [Abstract] [Full Text] [PDF]
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 S. M. Nemetski and L. B. Gardner Hypoxic Regulation of Id-1 and Activation of the Unfolded Protein Response Are Aberrant in Neuroblastoma J. Biol. Chem., January 5, 2007; 282(1): 240 - 248. [Abstract] [Full Text] [PDF]
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J. M. Jones, M. Montcouquiol, A. Dabdoub, C. Woods, and M. W. Kelley Inhibitors of Differentiation and DNA Binding (Ids) Regulate Math1 and Hair Cell Formation during the Development of the Organ of Corti J. Neurosci., January 11, 2006; 26(2): 550 - 558. [Abstract] [Full Text] [PDF]
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Q. Shen and S. Christakos The Vitamin D Receptor, Runx2, and the Notch Signaling Pathway Cooperate in the Transcriptional Regulation of Osteopontin J. Biol. Chem., December 9, 2005; 280(49): 40589 - 40598. [Abstract] [Full Text] [PDF]
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T. Lofstedt, A. Jogi, M. Sigvardsson, K. Gradin, L. Poellinger, S. Pahlman, and H. Axelson Induction of ID2 Expression by Hypoxia-inducible Factor-1: A ROLE IN DEDIFFERENTIATION OF HYPOXIC NEUROBLASTOMA CELLS J. Biol. Chem., September 17, 2004; 279(38): 39223 - 39231. [Abstract] [Full Text] [PDF]
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 J. Samanta and J. A. Kessler Interactions between ID and OLIG proteins mediate the inhibitory effects of BMP4 on oligodendroglial differentiation Development, September 1, 2004; 131(17): 4131 - 4142. [Abstract] [Full Text] [PDF]
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