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INTRODUCTION |
The basic helix-loop-helix
(bHLH)1 proteins are
intimately associated with developmental events such as cell
differentiation and lineage commitment (1-6). The HLH domain in the
bHLH motif is responsible for dimerization, whereas the basic region
mediates DNA binding (1). Based on sequence alignment and domain
analysis, human DEC (differentially expressed
in chondrocytes), mouse STRA (stimulated with
retinoic acid), and rat SHARP
(split and hairy related
protein) constitute a new and structurally distinct
class of bHLH proteins (7-10). These proteins are distantly related to
Drosophila Hairy and E(spl) as well as the mammalian
homologues (e.g. HES) with the highest sequence identity
(~40%) in the bHLH region (1, 11, 12). Like Hairy/E(spl)/Hes,
DEC/STRA/SHARPs contain an orange domain and a proline residue in the
DNA binding domain. However, the proline is located 2 residues more
toward the NH2 terminus (1, 8). Another major structural
difference on the functional domains is that DEC/STRA/SHARPs, unlike
Hairy/E(Spl)/Hes proteins, lack the COOH-terminal WRPW tetrapeptide
motif (13). Through this sequence, Hairy/E(spl)/Hes recruit corepressor
Groucho to the transcription regulatory complex (13). Recruitment of Groucho is responsible for a vast array of biological activities of
Hairy/E(spl)/Hes proteins including cellular differentiation and
lineage commitment (14-18).
Two members of DEC/STRA/SHARP proteins are identified in each mammalian
species studied with a sequence identity of >90% in the bHLH region
and ~40% in the total proteins, respectively (8). They exhibit an
overlapping tissue distribution, and their expression is highly
elevated in response to environmental stimuli (7-10). In rats that
undergo seizure induction by kainic acid, the levels of mRNA
encoding SHARP1 or -2 are sharply increased within 1 h in the
brain (9). In cultured human cells, both DEC1 and DEC2 are markedly
induced in response to hypoxia (19). Co-transfection experiments with
promoter reporters have identified functional hypoxia response elements
in both DEC1 and DEC2 genes. These elements show high affinity toward
hypoxia-inducible factor-1
and -
, providing a molecular
explanation on the co-regulatory phenomena of DEC1 and DEC2 during
hypoxia response (19). Rapid induction of these proteins in response to
environmental stimuli suggests that DEC/STRA/SHARPs are protective
against detrimental conditions.
In addition to a potential protective role against environmental
stimuli, DEC/STRA/SHARPs have been implicated in cell differentiation (7, 10, 20), maturation of lymphocytes (21), and regulation of
molecular clock (22). In a cell culture system, mouse STRA13 promotes
neuronal but represses mesodermal and endodermal differentiation (7).
Consistent with the inductive effect on neuronal differentiation, rat
SHARP proteins are abundantly expressed in a subset of mature neurons
(9). DEC1 has recently been shown to promote chondrocyte differentiation at the early and terminal stages (20). STRA13-deficient mice, although surviving to adulthood, develop autoimmune
diseases accompanied by accumulation of spontaneously activated T and B cells (21). In addition, the mouse proteins are recently found to
regulate the expression of biological clock regulator Per
(22). Recently, we and other investigators have recently demonstrated that deregulated cell survival by DEC1 may have oncogenic significance. In paired samples, DEC1 is abundantly expressed in colon carcinomas but
not in the adjacent normal tissues (23). High levels of DEC1 transcript
are also detected in an array of cancer cell lines derived from a wide
range of organs (24). Cells that lack the functional tumor suppressor
VHL (von Hippel-Lindau) express higher levels of DEC1 (24). Forced
expression of DEC1 antagonizes serum deprivation-induced apoptosis and
selectively inhibits the activation of procaspases (23). These findings
suggest that overexpression of DEC1 provides cells with an unusual
survival mechanism and thus is oncogenic.
The present study was undertaken to extend the expression study on DEC1
and to determine whether DEC1 and DEC2 displayed similar expression
patterns among paired tumor-normal tissues from the colon, lung, and
kidney. Without exceptions, DEC1 was expressed markedly higher in the
carcinomas, whereas DEC2 was expressed markedly higher in the adjacent
normal tissues. Forced expression of DEC1 sharply decreased the
expression of DEC2 and markedly repressed the activity of a DEC2
promoter reporter. Co-transfection experiments with mutant reporters
and electrophoretic mobility shift assay (EMSA) located, in the
proximal promoter, an E-box that supports DEC1-mediated repression.
These findings provide direct evidence that DEC1 negatively regulates
the expression of DEC2, which is largely achieved through direct DNA
binding to the E-box in the proximal promoter of DEC2.
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MATERIALS AND METHODS |
Chemicals and Supplies--
Tri-reagent, FLAG-cytomegalovirus
vector, and anti-FLAG antibody were purchased from Sigma. The goat
anti-rabbit-IgG conjugated with alkaline phosphatase or horseradish
peroxidase and ECL substrate were from Pierce. Dulbecco's modified
Eagle's medium, LipofectAMINE, and the ThermoScript I reverse
transcription-coupled PCR kit were from Invitrogen. The Dual-Luciferase
reporter assay system and DNA binding buffer were from Promega. Unless
otherwise indicated, all other reagents were purchased from Fisher.
Tissue Collection and Processing--
Samples were collected
from patients who underwent surgical resection for histologically
confirmed adenocarcinoma. As paired controls, specimens from the
adjacent, grossly normal tissues were harvested. The samples (12 pairs)
were collected from the colon, kidney, and lung with four pairs from
each organ. The age of the patients was between 23 and 68 with seven
male and five female. The size of tumors was generally 2-5 cm in
diameter, and the degree of differentiation of tumors was moderate or
poor as determined by pathological examination. Samples were freshly
processed for RNA isolation and protein extraction. Total RNA was
isolated with a Tri-reagent as described previously (25). For the
preparation of protein extracts, tissues were homogenized in lysis
buffer (20 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1%
sodium deoxycholate, 0.1% SDS, 0.2 mM phenylmethylsulfonyl
fluoride, and 1 mM dithiothreitol). The homogenates
were centrifuged at 12,000 × g for 30 min to remove any insoluble precipitates. The protocol of using human pathological tissues was reviewed by the Institutional Review Board.
RT-PCR--
The expression of DEC1 and DEC2 in human tissues and
cultured cells was primarily determined by RT-PCR experiments with a ThermoScript I kit. Total RNA (2 µg) was subjected to the synthesis of the first strand cDNA with an oligo(dT) primer and a
ThermoScript reverse transcriptase. The reactions were incubated
initially at 50 °C for 30 min and then at 60 °C for 60 min after
additional reverse transcriptase was added. The cDNAs were then
subjected to PCR amplification with cycling parameters as follows:
95 °C for 30 s, 52 °C for 30 s, and 68 °C for 30 or
40 s for a total of 32 cycles. The primers for DEC1 amplification
were 5'-GTCTGTGAGTCACTCTTCAG-3' and 5'-GAGTCTAGTTCTGTTTGAAGG-3'. The
primers for DEC2 amplification were 5'-CGCCCATTCAGTCCGACTTGGAT-3' and
5'-TGGTTGATCAGCTGGACACAC-3'. The primers for
-actin amplification
were 5'-GTACCCTGGCATTGCCGACAGGATG-3' and
5'-CGCAACTAAGTCATAGTCCGCCTA-3'. The PCR-amplified products were
analyzed by agarose gel electrophoresis.
Plasmid--
A cDNA encoding the full-length DEC1 was
isolated by a cDNA-trapping method (23, 26). Several DEC1 mutant
constructs were prepared by PCR with the full-length DEC1 as the
template. These mutants had a specific sequence deleted or one or more
amino acids substituted. Some of the mutant constructs were prepared
with the SPORT vector (the NH2-terminal truncated mutants),
whereas others (the COOH-terminal truncated mutants) were prepared with the FLAG vector to facilitate immunodetection. In some cases, a Kozak
sequence was introduced for effective translation initiation. The DEC2
promoter reporter was prepared with the pGL3-basic luciferase vector
(Promega). Human genomic DNA was isolated from the placenta with a DNA
extraction kit (Qiagen) according to the manufacturer's instruction. A
genomic fragment (
1,888 to +11) was generated by PCR with
5'-AACAGATGAACTGAACGGACCG-3' and 5'-CCTCAGTGCAGTGTTGAAAGTG-3'. This PCR
fragment was ligated to the pGL3 vector. Deletion mutants of this
reporter were prepared by endonuclease digestion followed by ligation
or PCR.
Site-directed Mutagenesis--
The DEC2 promoter reporter had
two E-box motifs that probably interact with DEC1, and the studies with
deletion mutants suggested that the E-box in the proximal region
supports DEC1-mediated repression. In order to definitively establish
such a role, site-directed mutagenesis was performed to substitute two
of the six nucleotides. The mutant construct was prepared with a
QuikChange site-directed mutagenesis kit (Stratagene). Complementary
oligonucleotides
(5'-GATGGTACGTTCCGAACGGGAGCTGGGTGCTGG-3') were
synthesized to target this region. To perform the substitutions, the
primers were annealed to a DEC2 promoter reporter and subjected to a
thermocycler for a total of 15 cycles. The resultant PCR-amplified constructs were then digested with DpnI to remove the
nonmutated parent construct. The mutated PCR-amplified constructs were
used to transform XL1-Blue. The same approach was used to prepare three DEC1 mutants that had single or double residues substituted in the DNA
binding domain (P56A, R58P, or both). The general sequence for the
site-directed mutagenic oligonucleotides was
5'-GAGACCTACAAATTGGCGCACCCGCTCATCGAGAAAAAGAG-3' with the nucleotides in boldface type substituted individually or
simultaneously. All mutated constructs were subjected to sequencing analysis to confirm the desired mutation being made without secondary mutations.
Co-transfection Experiment--
Cells (293T) were plated in
24-well plates in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum at a density of 1.6 × 105
cells/well. Transfection was conducted by lipofection with
LipofectAMINE according to the manufacturer's instructions.
Transfection mixtures contained DEC1 or a mutant construct (100 ng),
reporter plasmid (100 ng), and the pRL-TK Renilla plasmid (1 ng). If a DEC1-stable line was used, DEC1 or its mutant construct was
omitted from the transfection mixture. The transfected cells were
cultured for an additional 24 h, washed once with
phosphate-buffered saline, and resuspended in passive lysis buffer
(Promega). The lysed cells were subjected to two cycles of
freezing/thawing. The reporter enzyme activities were assayed with a
Dual-Luciferase reporter assay system. This system contained two
substrates, which were used to determine the activity of two
luciferases sequentially. The firefly luciferase activity, which
represented the reporter gene activity, was initiated by mixing an
aliquot of lysates (20 µl) with Luciferase Assay Reagent II. Then the
firefly luminescence was quenched, and the Renilla
luminescence was simultaneously activated by adding Stop & Glo reagent
to the sample wells. The firefly luminescence signal was normalized
based on the Renilla luminescence signal. In cases where the
reading on the luciferase activity was too high, the lysates were
diluted, and luciferase activities were then determined to minimize the
interference on the reading of the Renilla luciferase activity.
EMSA--
Cells (293T) were transfected with DEC1 or a mutant,
and nuclear extracts were prepared with a nuclear extraction kit
(Active Motif). In some cases, DEC1-stable transfected cells were used but cultured in the presence or absence of tetracycline to modulate the
expression of transfected DEC1. Nuclear proteins (10 µg) were incubated with radiolabeled double-stranded oligonucleotides
(5'-CGTTCCGCACGTGAGCTGGG-3') in a final volume of 10 µl containing
1× DNA binding buffer. For competition experiments, nuclear extracts
were first incubated with a 10- or 50-fold molar excess of cold
probe and then mixed with the radiolabeled probe.
Oligonucleotides with a disrupted E-box were also used in the
competition assays. For supershift assays, the anti-DEC1 or an
anti-FLAG antibody was added either before or after the nuclear
extracts were incubated with the radiolabeled probe. The protein-DNA
complexes were resolved in 6% PAGE and visualized by autoradiography.
Other Analyses--
Western analyses were conducted as described
previously (27). The anti-DEC1 antibody against the COOH-terminal
peptide was described elsewhere (23). Protein concentration was
determined with BCA assay (Pierce) with bovine serum albumin as the
standard. Data are presented as mean ± S.D. of at least four
separate experiments, except where results of blots are shown, in which
case a representative experiment is depicted in the figures.
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RESULTS |
DEC1 and DEC2 Are Inversely Expressed in Paired Carcinomas and
Adjacent Normal Tissues--
We have reported that DEC1 is abundantly
expressed in colon carcinoma but not in the adjacent normal tissues
(23). The initial focus of the present study was to extend the
expression study on DEC1 and to determine whether DEC1 and DEC2 shared
similar expression patterns among paired cancer-normal tissues from the colon, kidney, and lung. RT-PCR experiments with primers specific to
DEC1 and DEC2 were performed. As shown in Fig.
1, without exceptions, the levels of DEC1
mRNA were markedly higher in the carcinomas, whereas the levels of
DEC2 mRNA were markedly higher in the adjacent normal tissues.
Between paired samples, the levels of
-actin mRNA were
comparable. The carcinoma-related increase in DEC1 expression was also
detected by Western blot (top of each depicted figure), suggesting that mRNA levels are indicative of the overall
expression of these two genes.

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Fig. 1.
Inversed expression patterns of DEC1 and DEC2
in the carcinoma and the adjacent normal tissues from the colon,
kidney, and lung. Total RNA (5 µg) of carcinoma-normal paired
samples from the colon, kidney, and lung was subjected to RT-PCR
analyses with a ThermoScript I kit. For PCR amplification, a master
tube containing all common reagents was prepared and equally
distributed to individual PCR tubes (DEC1, DEC2, and -actin). PCR
amplification was conducted with cycling parameters as follows:
95 °C for 30 s, 52 °C for 30 s, and 68 °C for 30 or
40 s for a total of 32 cycles. The PCR-amplified products were
analyzed by agarose gel electrophoresis and visualized by ethidium
bromide staining. For Western blotting analysis, homogenates (10 µg)
were subjected to SDS-PAGE. The immunoblot was incubated with the
antibody against DEC1. The primary antibody was then located by
horseradish peroxidase-conjugated goat anti-rabbit IgG and visualized
with chemiluminescent substrate.
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Forced Expression of DEC1 Proportionally Decreases the Expression
of DEC2--
The inversed expression patterns between DEC1 and DEC2
suggest that DEC1 negatively regulates the expression of DEC2 or
vice versa. In order to directly test this possibility,
DEC1-stable transfected lines were used to study the expression
relationship between DEC1 and DEC2. Two clonal stable lines were
included: one expressing DEC1 (wild type) and the other expressing
DEC1-M, which lacked the DNA binding domain. The stable lines were
prepared with 293T cells and the pcDNA6/TR-pcDNA4 expression
system; therefore, the expression of DEC1 and DEC1-M was inducibly
regulated by tetracycline as described previously (23). As expected,
the addition of tetracycline caused a
concentration-dependent increase on the levels of DEC1 as
determined by Western blots (Fig.
2A, top).
Consistent with the inducible increase in the levels of DEC1 protein,
the levels of DEC1 mRNA were proportionally increased (data not
shown). In contrast to the increased expression of DEC1, the levels of
DEC2 mRNA were proportionally decreased (Fig. 2A).
However, such inversed expression patterns were observed only in the
cells expressing wild-type DEC1 (Fig. 2A) and not the cells
expressing the DEC1 mutant, although the levels of DEC1-M were markedly
induced by tetracycline (Fig. 2B).

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Fig. 2.
Repressed expression of DEC2 by DEC1 in
stable transfected cells. Stable transfected cells by DEC1
(A) or DEC1-M (B) were seeded in six-well plate.
After reaching ~80% confluence, cells were treated with tetracycline
at various concentrations (0-1 µg/ml) for 24 h. Total RNA and
homogenates were prepared and analyzed for the expression of DEC1 by
Western blots or DEC2 by RT-PCR as described in the legend for Fig. 1.
Similarly, the expression of -actin was determined and served as
control.
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The E-box in the Proximal Promoter of DEC2 Is the Sequence Targeted
by DEC1--
The inability of DEC1-M to down-regulate the expression
of DEC2 suggests that DEC1-mediated repression is achieved through a
DNA-binding mechanism. In order to directly test this possibility, reporter experiments and EMSA were conducted. A DEC2 promoter reporter
(pLuc-1888) was constructed to contain the basal promoter and other
potential regulatory sequences of the DEC2 gene (
1,888 to +11). This
region was chosen because it contained two E-box motifs that commonly
serve as target sequences for bHLH transcription factors (1). A series
of 5' deletion mutants of this reporter was also prepared and designed
to specify the location of DNA sequence that is targeted by DEC1 (Fig.
3A, left).
Co-transfection experiments were conducted to test these reporters for
their ability to support DEC1-mediated activity. The stable transfected
line (wild-type DEC1 only) was transfected again with a reporter
construct and cultured in the presence or absence of tetracycline to
modulate the expression of DEC1. The pRL-TK Renilla plasmid
was also included in the transfection mixture to normalize transfection
efficiency. As described in Fig. 3A (right), the
addition of tetracycline decreased the activity of the pLuc-1888
reporter by as much as 90%. Similar repression was observed with the
reporters that had the sequence deleted up to nucleotide
535. In
contrast, reporter pLuc-125, which had a further deletion from
nucleotide
535 to
125, simultaneously lost the basal transcription
activity and the ability to respond to DEC1, suggesting the importance
of this region (
535 to
125) in both basal and regulatory
transcription.

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Fig. 3.
DEC1-mediated repression on the DEC2 promoter
reporter and binding to the proximal E-box. A,
DEC1-mediated repression on the DEC2 promoter reporter. Deletion and
site-directed mutants of the DEC2-promoter reporter (pLuc-1888) were
prepared by endonuclease digestion followed by ligation or by PCR with
a QuikChange site-directed mutagenesis kit. DEC1-stable transfected
cells were cultured in 24-well plates at ~80% confluence and
transfected again with a reporter construct (100 ng) and the pRL-TK
Renilla (1 ng). The retransfected cells were cultured in the
presence or absence of tetracycline (1 µg/ml) for 24 h. The
cells were collected, washed once with phosphate-buffered saline, and
resuspended in passive lysis buffer. The reporter enzyme activities
were assayed with a Dual-Luciferase reporter assay system. The firefly
luminescence signal was normalized based on the Renilla
luminescence signal. B, EMSA DEC1-stable transfected cells
were cultured in the presence or absence of tetracycline
(tet; 1 µg/ml) for 24 h, and nuclear extracts were
prepared with a nuclear extraction kit (Active Motif). Nuclear proteins
(10 µg) were incubated with radiolabeled double-stranded
oligonucleotides harboring the proximal E-box in a final volume of 10 µl containing 1× DNA binding buffer. For competition experiments,
nuclear extracts were first incubated with excess cold probe (50× in
lane 1 or 10× in lane 3)
and then mixed with the radiolabeled probe. Oligonucleotides
(M) with the E-box disrupted were also used in the
competition assays (50× in lane 2). For
supershift assays, the anti-DEC1 antibody (D) was added
either before (lane 5) or after (lane
6) the nuclear extracts were incubated with the radiolabeled
probe. As a control, the anti-DEC1 antibody was replaced by an
anti-FLAG antibody (F, lane 4). The
protein-DNA complexes were resolved in 6% polyacrylamide gel
electrophoresis and visualized by autoradiography.
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We next examined whether responsiveness to DEC1 could be separated from
the basal transcription activity in the DEC2 promoter reporter. Given
the fact that this region (
535 to
125) contains a single E-box that
is probably targeted by DEC1, a reporter with this E-box disrupted was
tested for the ability to confer basal transcription. Reporter pLuc-535
was subjected to site-directed mutagenesis to selectively disrupt the
E-box (CACGTG to AACGGG).
Similarly, co-transfection experiments were performed. As shown in Fig.
3A (bottom), disruption of this E-box
(pLuc-535-M) caused little change in the basal activity (cultured
without tetracycline), suggesting that this E-box contributes little to
basal transcription. In contrast, the reporter mutant (pLuc-535-M)
exhibited only ~35% repression in response to DEC1 (Fig.
3A, lane 8), which contrasts
strikingly with 90% repression observed with the corresponding
nonmutagenic reporter (Fig. 3A, lane
5). These findings suggest that the proximal E-box is
largely responsible for DEC1-mediated repression. It should be
emphasized that a similar observation was made with a substitution
mutant reporter prepared from the longest reporter pLuc-1888, and the expression levels of DEC1 were comparable among all cells as determined by Western blots (data not shown).
We next examined whether this E-box interacted directly with DEC1. The
DEC1-stable line was cultured in the presence or absence of
tetracycline, and nuclear extracts were prepared. Double-stranded oligonucleotides harboring this E-box were synthesized and
radiolabeled. The labeled probe was incubated with the nuclear extracts
and analyzed by EMSA. As shown in Fig. 3B, incubation with
the extracts from the cells cultured in the presence of tetracycline
yielded a shifted band (lane 8). This band was
not detected when incubation was performed with the extracts from the
cell cultured without tetracycline (lane 7). The
shifted band was competed completely by 50× (lane
1) or partially by 10× excess cold probe (lane
3). However, the oligonucleotides (50×) that harbored a
mutated E-box (E-box-M) showed no competitive activity (lane
2). In addition, the shifted band was supershifted by the
anti-DEC1 but not the anti-FLAG antibody. The supershifted band
appeared whether the antibody was added before or after the
DEC1-DNA complexes were formed (lanes 5 and
6), suggesting that the antibody binding does not interfere
with interactions between DEC1 and its element (the antibody directed
against the COOH-terminal sequence of DEC1).
DNA Binding Is Required to Effectively Repress the DEC2 Promoter
Reporter--
Disruption of the proximal E-box caused drastic but
incomplete loss of responsiveness to DEC1 (Fig. 3A),
suggesting that DNA binding is not the only mechanism involved in
DEC1-mediated repression on the DEC2 reporter or that an additional
DEC1 binding site exists in this region. We next tested whether DEC1
mutants, defective of DNA binding, had any repressive activity. These
mutants had one or more residues in the DNA binding domain substituted
or one or more structural domains deleted (Fig.
4A). A total of three deletion
mutants (DEC1-M, DEC1105-412 and DEC1237-412) were prepared, and all of them lacked the DNA binding domain. As shown
in Fig. 4A, additional sequences were also deleted in DEC1105-412 (the HLH motif) and DEC1237-412
(the HLH motif and orange domain). The HLH motif and the orange domain are shown in other bHLH proteins to mediate dimerization and protein interactions, respectively (1). Similarly, three substitution mutants
were prepared, including DEC1P56A, DEC1R58P,
and DEC1P56A/R58P. The rationale for preparing the
substitution mutants was that proline 56 was assumed to be critical in
DNA binding based on studies with other bHLH proteins (1). However,
there is a major difference regarding the location of this proline. In
other bHLH proteins, the proline is located 2 residues more carboxyl
terminal (corresponding to residue 58 in DEC1) (7, 8). Therefore, the
mutants represented substitution of proline 56 with an alanine (DEC1P56A), arginine 58 with a proline
(DEC1R58P), or both (DEC1P56A/R58P).

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Fig. 4.
Essentiality of DNA binding for DEC1 to
repress DEC2 promoter activity. A, co-transfection
experiment. Cells (293T) were cultured in 24-well plates and
transiently transfected with DEC1 or a DNA binding defective mutant
(100 ng), DEC2 promoter reporter (pLuc-1888; 100 ng) and the pRL-TK
Renilla (1 ng). After a 24-h incubation, cells were
collected and analyzed for luciferase activities. Similarly, firefly
luminescence signal was normalized based on the Renilla
luminescence signal, and the ratios from the cells transfected with the
vector were calculated as 100%. B, immunoblotting analysis.
The cell lysates (10 µg) from the cells used for reporter activity
were analyzed for the expression of DEC1 or its mutants by anti-DEC1
antibody as described in the legend to Fig. 1. C, EMSA.
Nuclear contracts were prepared from cells transiently transfected with
DEC1 or a mutant and incubated with the radiolabeled proximal E-box
probe. The DNA-protein complexes were resolved by PAGE.
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Co-transfection experiments were conducted to test these DEC1 mutants
for their ability to repress the DEC2 reporter (pLuc-1888). As shown in
Fig. 4A (top), all deletion mutants (DEC1-M,
DEC1105-412, and DEC1237-412) exhibited
little repressive activity toward this reporter. In contrast, all
substitution mutants repressed the DEC2 reporter, but the overall
repressive activity varied markedly among them. The
DEC1P56A mutant showed a similar potency as the wild-type
DEC1 (~90% repression), whereas the other two mutants
(DEC1R58P and DEC1P56A/R58P) caused only ~65
and ~50% repression, respectively. The expression of DEC1 and its
mutants was comparable with the exception of DEC1-M that was expressed to a higher extent (Fig. 4B), excluding the possibility that
lack of expression was a contributing factor to the weaker
repression by some of the mutants (e.g.
DEC1P56A/R58P). In order to determine whether these
mutants, particularly the mutants DEC1R58P and
DEC1P56A/R58P, indeed lost DNA binding ability, nuclear
extracts from the respective transfected cells were incubated with the
radiolabeled E-box oligonucleotides, and the corresponding DNA-protein
complexes were analyzed by EMSA. As predicted, all deletion mutants
(DEC1-M, DEC1105-412, and DEC1237-412) showed
no DNA binding activity (result shown for DEC1-M only) (Fig.
4C). In contrast, DNA binding activity varied among the
substitution mutants. DEC1P56A showed a similar binding
ability as DEC1, whereas DEC1R58P and
DEC1P56A/R58P had no DNA binding activity, consistent with
the fact that DEC1P56A was the only substitution mutant
that effectively repressed the promoter activity of DEC2 (Fig.
4A).
DNA Binding Is Not Sufficient to Confer Repressive
Activity--
The studies with DNA binding defective mutants clearly
demonstrated the importance of DNA binding in repressing the DEC2
promoter. We next examined whether DNA binding was sufficient to exert
repression. In order to directly test this possibility, DEC1 mutants
were prepared to keep the bHLH motif intact (DNA binding) but have sequences with various lengths deleted from the COOH terminus (Fig.
5A). These COOH-terminal
truncated mutants were subcloned in the FLAG vector to facilitate
immunodetection. Similarly, co-transfection experiments were performed
with DEC1 or a mutant along with the DEC2 reporter (pLuc-1888). As
shown in Fig. 5A, deletion of the COOH-terminal 65 residues
(FLAG- DEC11-347) caused no changes in the repressive
activity (1). In contrast, deletions of additional COOH-terminal
sequence caused a partial or a complete loss of repressive ability. As
a matter of fact, FLAG-DEC11-150 no longer had any
repressive activity. Western analyses were performed to confirm that
the mutants were actually expressed slightly higher than the wild type
DEC1 (Fig. 5B).

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Fig. 5.
DNA binding is not sufficient for DEC1 to
repress DEC2 promoter activity. A, co-transfection
experiment. Cells (293T) were cultured in 24-well plates and
transiently transfected with DEC1 or a COOH-terminal truncated mutant
(100 ng), DEC2 promoter reporter (pLuc-1888; 100 ng), and the pRL-TK
Renilla (1 ng). Determination and calculation of the
luciferase activities were described in the legend to Fig. 4.
B, immunoblotting analysis. The cell lysates (10 µg) from
the cells used for reporter activity were analyzed for the expression
of DEC1 or its mutants by an anti-FLAG antibody as described in the
legend to Fig. 1. C, EMSA. Nuclear contracts were prepared
from cells transiently transfected with DEC1 or a mutant and incubated
with radiolabeled oligonucleotides harboring the proximal E-box.
Similarly, competition experiments were performed with excess cold
probe (E) or a mutant probe (M) as described in
the legend to Fig. 3B. For supershift assays, an anti-FLAG
(F) or the anti-DEC1 (D) was added to the
incubation mixtures before being analyzed by PAGE.
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Next, we examined whether these mutants actually retained DNA binding
activity. EMSA was performed with the nuclear extracts from the cells
used for reporter assays. As shown in Fig. 5C, a shifted
band was detected with all COOH-terminal truncated mutants. The
relative electrophoretic mobility was generally associated with the
size of a mutant. For example, FLAG-DEC11-150 was the
shortest among the mutants, and the shifted band with this mutant
exhibited the fastest mobility. More importantly, the addition of an
anti-FLAG antibody into the binding reactions resulted in the
appearance of a supershifted band accompanied by the disappearance of
the original shifted band, providing direct evidence that the observed
protein-DNA interactions were highly specific. These findings also
suggest that DNA binding, although essential, is not sufficient to
confer repressive effect.
The HLH Motif Is Required for Dominant Interfering
Regulation--
The inability of FLAG-DEC11-150 to exert
repression, although it bound effectively to DNA, points to two
important possibilities: the deleted region from residue 150 to 347 has
intrinsic repressive activity, or this region is responsible for
recruiting protein(s) that causes repression. Apparently, comprehensive
experiments are required to definitively establish the involvement of
each possibility. However, we examined the second possibility by
testing mutants that contained part or the entire sequence of this
region for the ability to function as a dominant interfering regulator. Co-transfection experiments were conducted with DEC1 in the
presence and absence of a mutant. Among mutants DEC1-M,
DEC1105-412, and DEC1237-412, only DEC1-M
effectively reversed DEC1-mediated repression (Fig.
6A), although they all shared
two important features; they lacked the entire DNA binding domain and
lacked repressive activity themselves (Fig. 4A). Among the
substitution mutants, DEC1R58P and
DEC1P56A/R58P but not DEC1P58A partially but
significantly reversed DEC1-mediated repression, consistent with the
fact that DEC1P58A was a potent repressor itself (as potent
as wild type DEC1), whereas DEC1R58P and
DEC1P56A/R58P were much less repressive (Fig.
4A). It should be emphasized that the expression patterns in
the cells co-transfected with DEC1 and a mutant were consistent with
what was predicted; a band with more intensified staining was detected
if a mutant co-migrated with DEC1 (e.g.
DEC1P56A/R58P); otherwise, an additional band
(e.g. DEC1105-412) was detected if a mutant and
DEC1 were electrophoretically distinct.

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Fig. 6.
Dominant interfering regulation on
DEC1-mediated repression. A, effects of DNA
binding-defective mutants on the repressive activity by DEC1. Cells
(293T) were cultured in 24-well plates and transiently transfected with
DEC1 (50 ng) in the presence or absence of a DNA binding-defective
mutant (100 ng). Vector construct was used to equalize the amount of
plasmid in each transfection. Similarly, the pRL-TK Renilla
plasmid (1 ng) was included in the transfection mixture to normalize
transfection efficiency. Determination and calculation of the
luciferase activities were described in the legend to Fig. 4. To
determine the expression levels of transfected constructs, cell lysates
(10 µg) from the cells used for reporter activity were analyzed for
the expression of DEC1 and its mutants by the anti-DEC1 antibody
(specific to the COOH terminus of DEC1). B, effects of the
COOH-terminal truncated mutants on the repressive activity by DEC1. The
co-transfection and immunodetection were performed as described in the
legend to Fig. 6A. However, both anti-DEC1 and anti-FLAG
antibodies were simultaneously used for the immunodetection, because
the COOH-terminal truncated constructs were prepared with the
FLAG-cytomegalovirus vector.
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We also tested all COOH-terminal truncated mutants for the ability to
function as dominant interfering regulators. Generally, these mutants
either partially or completely antagonized DEC1-mediated repression,
depending on the relative potency to act as a repressor by its own
(Fig. 6B). Mutants with less intrinsic repressive activity exhibited a higher potency to reverse the repression by DEC1. For
example, FLAG-DEC11-150 itself had no repressive activity (Fig. 4A) but completely reversed DEC1-mediated repression
(Fig. 6B). Among all mutants that were less repressive than
wild type DEC1, only DEC1105-412 and
DEC1237-412 failed to reverse the repression by DEC1 (Fig.
6), and they were the only mutants that did not contain the HLH domain
(Figs. 4A and 5A), suggesting that the dominant
interfering regulatory activity is achieved through the HLH domain. The
HLH domain is known to mediate dimerization (1), and mutants with an
intact HLH domain probably form dimers with wild type DEC1, but the
resultant dimers have no DNA binding activity or are transcriptionally
inactive. In support of the first possibility, we performed EMSA and
found that DEC1R58P and DEC1P56A/R58P (DNA
binding-defective mutants) markedly abolished the DNA binding ability
of DEC1 when cells were co-transfected together with DEC1 and
DEC1R58P or DEC1P56A/R58P (data not shown).
 |
DISCUSSION |
The bHLH proteins are intimately associated with developmental
events such as cell differentiation and lineage commitment (1). Based
on sequence alignment and functional domain analyses, human DEC
proteins, along with mouse STRA and rat SHARP, constitute a new class
of bHLH transcription factors (7-10). These proteins are shown to play
important roles in cell differentiation, regulation of molecular clock,
immune response, and xenobiotic response (7, 10, 19-22, 28). Recently,
we have reported that DEC1 is abundantly expressed in colon
carcinomas, antagonizes serum deprivation-induced apoptosis, and selectively inhibits the activation of procaspases (23).
In this report, we describe inversed expression patterns between DEC1
and DEC2 among paired tumor-normal samples from the colon, lung, and
kidney. Experimentally forced induction of DEC1 causes proportional
decreases in the expression of DEC2. Given the fact that DEC/STRA/SHARP
proteins are highly identical (>90%) in the DNA binding region, but
very diverse in other areas (<40%), our findings described in
this report provide an important mechanism by which the cellular
function of target genes probably shared by these proteins can be
coordinately affected by members within the same class.
DNA binding is probably the primary mechanism responsible for
DEC1-mediated repression on the expression of DEC2, although members of
DEC/STRA/SHARP protein family have been shown to use non-DNA binding
mechanism(s) (29-31). Several lines of evidence presented in this
study support this notion. First, studies with deletion and
site-directed reporter mutants identify the proximal E-box that
supports the repression by DEC1. This E-box exhibits a high affinity
toward DEC1, and disruption of this E-box markedly reduces its
responsiveness to DEC1 (Fig. 3), suggesting that DNA binding is
involved in the DEC1-mediated repression. Second, DEC1 deletion mutants
(DEC1-M, DEC1105-412, and DEC1237-412), which
lack the entire DNA binding domain, show neither DNA binding ability
nor repressive activity (Fig. 4A), providing direct evidence that DNA binding is required for DEC1 to repress the DEC2 promoter. Finally, among the substitution mutants, DEC1P58A binds to
the E-box as much as wild type DEC1 and is equally active in
repression, whereas DEC1R58P and DEC1P56A/R58P
show no DNA binding ability and are much less repressive (Fig.
4A), further supporting the notion that DEC1-mediated
repression is largely achieved through DNA binding. It remains to
be determined whether DEC1R58P and DEC1P56A/R58P, although lacking DNA binding ability, cause
some repression. It is likely that these two mutants retain some DNA binding ability within the cells, but the conditions employed for EMSA
fail to support such interactions. Alternatively, they exert repression
through non-DNA binding mechanisms (29-31).
DNA binding, although required to exert effective repression, is not
sufficient to repress the DEC2 promoter. Mutant
FLAG-DEC11-150, for example, binds effectively to DNA but
shows no repressive activity (Fig. 5A). As a matter of fact,
mutants, with a deletion in the region from residue 150 to 347, all
bind to DNA as effectively as wild type DEC1 but are markedly less
repressive (Fig. 5A). In this region, several helical
structures and particularly an orange domain are located (7, 8). These
structures are assumed to mediate protein-protein interactions based on
studies with other bHLH proteins (1, 7, 8). It is likely that this region recruits proteins that cause repression. However, the necessity of protein recruitment to repress DEC2 is unlikely, because
mutants such as DEC1105-412 contain the entire sequence
of this region but show no dominant interfering activity against wild type DEC1 (Fig. 6A), suggesting that this region has
intrinsic repressive activity. Alternatively, proteins assumed to be
recruited are abundantly expressed in the cells employed in this study. Although we have not provided sufficient data to support protein recruitment in repressing DEC2, it cannot be excluded that such events
are involved in the regulation of other genes by DEC1, particularly
given the fact mouse STRA13, a highly identical protein to DEC1, has
been shown to interact directly with TFIIB through this region
(29).
DEC/STRA/SHARPs differ significantly from other bHLH proteins in terms
of binding to DNA. Most bHLH proteins bind to E-box (CANNTG) or N-box
(CANNAG). Binding preference is specified by the sequence in the basic
region. Generally, proline-containing basic regions have higher
affinity toward the N-box (1, 32), whereas the basic regions without a
proline preferentially recognize the E-box. DEC/STRA/SHARPs contain a
proline; however, this proline (residue 56 based on DEC1) is located 2 residues more amino-terminal (8). Instead, DEC/STRA/SHARPs have an
arginine (residue 58) that substitutes for the conserved proline among
N-box binding bHLH proteins. Although initial studies suggest that
STRA13 has no binding activity toward classic E- or N-box (7),
PCR-based site selection experiments have recently identified a class B type E-box (CACGTG) that is effectively bound by DEC1 and STRA13 (28,
33). In this study, we have demonstrated that the contribution of
Pro56 to DNA binding is insignificant because mutant
DEC1P58A is equally effective as wild type DEC1 in DNA
binding (Fig. 4C). In contrast, introduction of a proline by
substituting Arg58 completely eliminates DNA binding
activity (Fig. 4C), suggesting that residue in this location
is indeed important for E-box binding. It would be interesting to test
whether DEC1R58P and DEC1P56A/R58P show an
increase in binding to an N-box sequence. In addition, the DEC2
reporter contains two identical E-box sequences (proximal and distal)
(Fig. 3A); however, only the proximal E-box is required for
responding to the repression by DEC1. The precise mechanism for such a
difference remains to be determined. It is likely that the genomic
context rather than an E-box alone determines intracellular DNA
binding. In support of this possibility, STRA13 has been shown to
preferably bind to an E-box flanked with certain nucleotides.
DEC1 and DEC2 share the DNA binding domain with an exception of a
single residue (aspartate versus glutamate, the farthest NH2-terminal residue of this domain) (8); therefore, DEC2
probably acts as an autoregulator. This possibility is further
supported by their highly identical sequences flanking the DNA binding
domain. Immediately COOH-terminal to the DNA binding domain is the
helix-loop-helix domain that is identical between DEC1 and DEC2, and
NH2-terminal to this domain is an acidic residue-rich
stretch in both proteins (8). The DNA binding domain and its highly
identical flanking sequences suggest that DEC1 and DEC2 have
overlapping target genes, particularly those that are regulated through
direct DNA binding. In support of this notion, mouse proteins (STRA13
and DEC2) have been recently shown to repress Clock/Bmal1-induced
activation of the Per promoter (22), a gene that is involved
in the regulation of the molecular clock. Therefore, it is likely that
DEC/STRA/SHARP proteins are functionally redundant on some target
genes, and such a redundant mechanism provides a possible explanation
that STRA13 knockout mice develop to adulthood and show no discernible phenotypic differences compared with their wild-type littermates (21).
It should be emphasized, however, that DEC1 and DEC2 may not
necessarily exert the same biological activity on all target genes and
in all cell types, particularly given the fact that they have a minimal
sequence identity (<40%) in the COOH-terminal half and exhibit
several major structural differences (8). Both DEC1 and DEC2 have an
orange domain (two helical structures spanned by ~50 residues);
however, the overall sequence identity in this domain is only moderate
(~50%). In addition, an alanine/glycine-rich region is present in
DEC2 but absent in DEC1. Previous studies with STRA13 as well as the
findings described in this study have demonstrated that the region
harboring the orange domain is required to exert effective repression
by both proteins (Fig. 5A) (7). Amino acid repeats,
on the other hand, are implicated in protein folding, protein-protein
interactions, and degradation (34).
DEC1-mediated repression is probably responsible for the differences on
cell and tissue distributions between DEC1 and DEC2. Although Northern
analyses have shown that DEC1 and DEC2 have an overlapping tissue
distribution (8, 10), it remains to be determined whether they are
actually expressed in the same cell type and to a similar extent (8,
10). Some organs with high levels of DEC1 (e.g. liver)
express lower levels of DEC2 (10). Very recently, DEC1 and DEC2 were
found to regulate the mammalian molecular clock, but they exhibit
distinct and area-dependent expression patterns in the
brain (21). In this report, we have demonstrated that these two
proteins exhibit inversed expression patterns among the paired
tumor-normal tissues, and forced expression of DEC1 causes proportional
decreases in the expression of DEC2 (Figs. 1 and 2), providing direct
evidence that increased expression of DEC1 is at least in part
responsible for decreased expression of DEC2 in a given cell context.
DEC1-mediated repression, although profound, may not always dictate the
expression of DEC2. For example, DEC1 and DEC2 are both up-regulated in
response to hypoxia induction (19). Acute hypoxia is considered severe
cytotoxic stimulus, and rapid induction of both genes maximizes the
cellular survival mechanism based on our recent report that DEC1 is
antiapoptotic (23), although it remains to be determined whether DEC2
is actually antiapoptotic as well.
In summary, we have demonstrated that DEC1 is a negative regulator on
the expression of DEC2. These two proteins exhibit inversed expression
patterns among paired samples from the colon, kidney, and lung. An
inducible expression system demonstrates that increased expression of
DEC1 proportionally decreases the expression of DEC2. The DEC1-mediated
repression is primarily achieved by binding to the E-box in the
proximal promoter of DEC2. Site-directed mutagenesis studies show that
arginine 58 in the DNA binding domain is critical for DEC1 to interact
with this E-box. Given the fact that DEC/STRA/SHARP proteins are
emerging as very important regulators in a vast array of cellular
events including cell differentiation, maturation of lymphocytes,
oncogenesis, molecular clock, and xenobiotic response, our findings
described in this study provide an important mechanism by which
these proteins regulate the cellular function by not only modulating
the expression of their target genes but also the expression of the
members within the same class.