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INTRODUCTION |
Opioid receptors are the molecular targets for analgesic
compounds. From studying genetically altered animal models, we have begun to understand the roles of opioid receptors in animals, including
a direct role in mediating the pharmacological action and certain side
effects of morphine, as well as some behavior changes (1-6).
Three opioid receptor genes have been cloned, and their genomic
structures have been determined (7-9). The expression of opioid
receptor genes in animals has been examined mostly by in
situ hybridization, immunohistochemistry, and ligand binding
assays (10-16). The genetic basis underlying the ontogenesis of opioid
receptors in animals has been disclosed for the kappa opioid receptor
(KOR)1 from studying
transgenic animal models in our laboratory (17).
Opioid receptors are expressed specifically in the central nervous
system in the adult, primarily in the areas associated with pain
sensation and behavior changes, but how they are expressed in
these tissues/cells is not understood. Recently, we have demonstrated the expression of opioid receptors in early developing animals (18). In
a KOR-lacZ transgenic reporter mouse model (17), we
have demonstrated that an ~4-kilobase pair sequence, which spans 3 kilobase pairs of the 5' untranscribed region, the first exon,
the first intron, and the translational control located in exon 2 of
the mouse KOR gene, is able to direct lacZ reporter expression, recapitulating most of the endogenous KOR expression patterns during developmental stages as revealed by in situ
hybridization and immunohistochemistry (16). Most significantly,
KOR-lacZ is expressed widely in early developing embryos and
is restricted more to the nervous system and sensory organ primordia in
mid- to late-gestation stages (17).
To understand how the KOR gene is regulated for specific expression in
developing animals, we have tested various hormones and opioid
compounds in developing animals as well as an embryonal carcinoma cell
model, P19, which constitutively expresses the KOR gene (18). We found
retinoic acid (RA) to be a potent suppressor of the KOR gene in
both animals and P19 cells. This study aims at understanding the
mechanism of RA suppression of KOR gene expression. We now report the
identification of an intronic silencer element that contains a binding
site for the Ikaros 1 (Ik-1) transcription factor. Ik-1 is
induced by RA in P19, and its overexpression in P19 results in
suppressed KOR mRNA expression, accompanied by chromatin histone
deacetylation on the KOR promoters. This study proposes a novel
mechanism of targeted suppression of the opioid receptor gene by RA
through Ik-1 binding to an intronic silencer, which renders histone
deacetylation of the promoters.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs and Determination of Reporter Gene Activity in
COS-1 and P19 Cells--
The homologous reporters were constructed by
inserting different KOR genomic DNA fragments (19) into a luciferase
cassette lacking the promoter pGL3B (Promega, Madison, WI). K45
contained a KOR genomic fragment of 1320 bp from the BamHI site to the
ATG codon. K18 and K19 were truncated from K45, generating reporters starting from the end of exon I (retaining intact intron 1) or deleted
in intron 1, respectively. K20 was generated by removing intron I from
its original position in K45 and inserting it in front of the BamHI
site. K27 was made by deleting 76 bp (SphI site) from K20. The
heterologous reporters were constructed by fusing various KOR genomic
fragments to either the tK (K38, K50, K49, and K48) or SV40
(K85) promoter. K38, K50, K49, and K48 each contained a KOR sequence of
387 bp (
401 to
15), 310 bp (
324 to
15), 257 bp (
271 to
15),
and 149 bp (
163 to
15), respectively. K85 contained a 128-bp KOR
sequence (
269 to
142). All of the nucleotide positions were
numbered in relationship to the initiating codon, ATG. The expression
vector of Ik-1 was a gift from Dr. S. T. Smale (20).
Mutated reporters K97 (m1) and K99 (m2) were made by introducing these
mutated sequences into the background of K45 by polymerase chain
reaction (PCR), and K98 and K100 were made in the background of K85.
COS-1 cells were transfected using the calcium phosphate precipitation
method as described (21). P19 cell were transfected using Superfect
lipofection (Qiagen, Santa Clarita, CA). A specific reporter
(0.5 µg) and 0.05 µg of cytomegalovirus-lacZ
internal control were used in each transfection. Cells were harvested
48 h after transfection, and specific reporter gene activity,
after normalizing to internal control and represented as relative
luciferase units, was determined as described previously (21). All of
the transfection experiments were carried out at least three times with
duplicate or triplicate cultures to obtain the means and the standard
errors of the means.
Analysis of Endogenous mRNA in P19 Cells--
Two methods
were used to examine endogenous Ik-1 expression in P19 cells, including
reverse transcription-PCR (RT-PCR) and Northern blot analyses. P19
cells were transfected with either an Ik-1 expression vector (20) or an
empty vector, pcDNA3 (Invitrogen, Carlsbad, CA) by Superfect
lipofection. Twenty hours after transfection, total RNA was isolated,
and KOR expression was detected with an established RT-PCR protocol
(22). For RA treatment, P19 cells were treated with 1 µM
all-trans-RA for 18, 24, or 48 h or for 3 days. The Ik-1 specific
primers are 5'-GGTGAACGGCCTTTC3-' (exon 4) and 5'-TTCTTCCTTAATGAC-3'
(exon 6) (20). Amplified KOR (730, 760, and 800 bp for isoforms a, b,
and c, respectively) and Ik-1 (304 bp) fragments were detected on
Southern blots and quantified using a PhosphorImager and ImageQuant
software (Molecular Dynamics, Sunnyvale, CA). Actin-specific primers
were included for internal control in each RT-PCR. For Northern blot
analysis, RNA was isolated from P19 and separated on a denaturing
agarose gel followed by blotting onto a nylon membrane and detection
with Ik-1 cDNA probes.
Gel Shift Assay--
The sequence of the wild type Ik element on
intron 1 of the KOR gene is 5'-GGGTGGGAAGGGGATTT-3', and two mutants
with mutations (underlined) introduced into either the first or the
second repeat are 5'-GGGTGTTAAGGGGATTT-3' (m1) and
5'-GGGTGGGAAGGTTATTT-3' (m2), respectively. The annealed
oligos were end-labeled with -[
-32P]dCTP by Klenow
fragment. Ik-1 protein was synthesized in TNT reactions (Promega) from
a T7 expression vector (pSP73, Promega) that contained Ik-1 cDNA.
DNA-protein interactions were detected as described (23). Briefly, 2 µl of TNT product was incubated with 2 ng of labeled DNA fragments at
4 °C for 30 min, and the reaction mixture was resolved on 5%
polyacrylamide gels followed by PhosphorImager analyses. For antibody
reactions, 2 µl of antibody (20-fold dilution) and TNT product were
allowed to react at 4 °C for 15 min followed by the addition of DNA
probes for 30 min at 4 °C.
Chromatin Immunoprecipitation (ChIP) Assay--
COS-1 cells
(10-cm plate) were transfected with a KOR reporter (K45) containing
both promoters 1 and 2 together with either the Ik-1 expression vector
or an empty vector. The ChIP assay was performed according to the
manufacturer's recommendation (Upstate Biotechnology, Lake Placid,
NY). Briefly, histone was cross-linked to DNA by the addition of
formaldehyde to a final concentration of 1%, and cells were sonicated
in 200 µl of lysis buffer. One-quarter of the total lysate was used
for monitoring total DNA input, which was diluted 20-fold in PCR. The
rest of the lysate was cleared with 80 µl of salmon sperm DNA/protein
G-agarose slurry. One-half of the cleared lysate was incubated with an
anti-acetylated Histone 3 antibody (Upstate Biotechnology) overnight at
4 °C, and the other half was used for a negative control
(nonspecific antibody). After reversing the cross-link, the eluted
immunocomplex was digested with proteinase K, and DNA was purified by
phenol extraction. DNA was precipitated for detection by PCR with
primers specific to the KOR promoter regions, i.e.
5'-GATGCACAGTAGCTTTCC-3' and 5'-GCAAGGAAGCAAGTGGTAGC-3' for promoter 1 and 5'-GAGGGTGAAGCA-3' and 5'-CTGGAAAGCGAGAAGGTG-3' for promoter 2. Amplified fragments were analyzed on a 1.2% agarose gel. For the ChIP
assay to determine Ik-1 binding to the KOR sequence in P19, an anti-Ik
antibody was used to precipitate chromatin. To amplify sequence
flanking the Ik-binding site, primers 5'-CCCGAGGGTGAAGCAA-3' and
5'-TGGCCGCCCATCCCAA-3' were used to amply a fragment of 126 bp flanking
the Ik-binding site.
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RESULTS |
RA Suppresses KOR Gene Expression, Mediated by a Putative
Ik-binding Sequence in Intron 1--
Previously, we showed that three
mouse KOR isoform mRNA species could be generated as a result of
using dual promoters and alternative splicing (19, 22). Recently, we
detected constitutive expression of the KOR gene in mouse embryonal
carcinoma P19 stem cells (18). In an attempt to examine the KOR gene
expression pattern in RA-induced P19 cell differentiation model, we
found that RA suppressed total KOR gene expression in P19. To examine the suppressive nature of RA on the expression of KOR mRNA
isoforms, we used an established RT-PCR procedure with the help of
specific primers to detect the expression of specific KOR mRNA
isoforms. As shown in Fig. 1, it appeared
that all three isoforms, a, b, and c, were suppressed by RA in P19
cells.

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Fig. 1.
RA suppresses KOR mRNA isoforms a, b, and
c expression in P19. A, maps of KOR mRNA isoforms a, b,
and c. Specific primer positions for amplifying each isoform are
indicated under the maps. B, expression of KOR
mRNA in P19 was detected by RT-PCR as described under
"Experimental Procedures." Lanes: ctrl, control culture;
RA 3 days, RA treatment for 3 days.
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An Ik-binding Sequence in Intron 1 of the KOR Gene Is a Functional
Silencer--
Because the potential regulatory sequence common to the
three isoform mRNA species is intron 1, we then determined whether intron 1 was involved in KOR suppression by using reporter assays conducted in P19 cells, as shown in Fig.
2A. Three KOR reporters, K45, K19, and
K20, each contained or deleted in intron 1, were generated and
tested in P19 cells. The two constructs, which contained intron 1 in
either its natural position (K45) or a relocated upstream position
(K20), were both suppressed as compared with the construct deleted in
intron 1 (K19), suggesting that intron 1 contained a sequence
responsible for suppression of the KOR gene in P19 background. An
Ik-binding site (5'-GGGAAGGGGAT-3') located at
278 to
288, ~185
bp upstream of the promoter 2 initiation site, was found by sequence
alignment. Intron 1 sequence was then further dissected and fused to a
heterologous promoter, thymidine kinase (tK) promoter, and
tested in P19 cells as shown in Fig. 2B. All three of the reporters
(K38, K50, and K49) that retained this putative Ik-binding site were
suppressed, whereas the reporter deleted in this putative Ik-binding
site (K48) was not suppressed as compared with the control
tk reporter. These results suggested that the Ik-binding
sequence of intron 1 in the KOR gene could be a negative element that
might be responsible for its suppression by RA in P19.

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Fig. 2.
KOR intron 1 encodes a negative Ik-binding
element. A, KOR-luc reporter activities
determined in P19. K45 is driven by a contiguous KOR genomic segment
containing KOR promoters, K19 is driven by the same KOR genomic segment
deleted in intron 1, and K20 is derived from K19 with the intron 1 sequence placed at the upstream region. RLU,
relative luciferase units. B, heterologous reporter
activities determined in P19. The intact KOR intron 1 (K38)
and its partial sequences (K50, K49, and K48)
were placed upstream of a tk-luc reporter
(Control), and the specific reporter activities were
determined in P19.
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We then determined whether Ik-1 had a suppressive effect on KOR
reporter gene expression and whether the putative Ik-binding site was a
functional regulatory sequence. For a functional assay of Ik-1 activity
on KOR gene expression, we first examined a panel of KOR reporters,
including K45, K19, K20, generated previously (Fig. 2A), and two more
deletions, K18 (retaining only intron 1 at its natural position) and
K27 (a shorter intron 1 placed at the upstream of promoter 1), in COS-1
cells cotransfected with an Ik-1 expression vector as shown in Fig.
3A. All of the KOR reporters that
retained the Ik-binding sequence were suppressed by Ik-1 expression in
COS-1 cells, whereas the reporter (K19) deleted in this sequence was
not suppressed. This Ik-binding site was further confirmed to be
effective in the context of heterologous promoters, because the SV40
promoter driven by this sequence was also suppressed in COS-1 cells
cotransfected with an Ik-1 expression vector, as shown in Fig. 3B.
Moreover, the reporter containing this sequence was suppressed by Ik-1
in a dose-dependent manner, as shown in Fig. 3C. Therefore,
it was concluded that the Ik-binding element on KOR intron 1 was a
functionally negative element responsive to Ik-1-mediated gene
suppression, as determined in the context of both homologous and
heterologous promoters.

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Fig. 3.
The Ik-binding element of KOR intron 1 is a
functional, repressive element. A, biological activities of
Ik-binding sequence in the context of natural KOR promoters determined
in COS-1 cells. Different KOR-luc reporter activities were
determined in COS-1 cells cotransfected with a control (open
bars) or an Ik-1 expression vector (filled bars). K45,
K19, and K20 were the same as that shown in Fig. 2. K18 was deleted
from K45 by truncating the 5' upstream region that contains promoter 1 and leaving intron 1 intact. K27 was deleted from K20 by truncating the
5' flanking sequence of the Ik-binding site. RLU,
relative luciferase units. B, biological activities of the
Ik-binding sequence in the context of heterologous promoters determined
in COS-1 cells. The Ik-binding sequence was fused upstream to a
reporter driven by SV40 promoter (Control), generating K85.
Cells were cotransfected with the specific reporter and a control
expression vector (open bars) or the Ik expression vector
(filled bars). C, Ik-1 suppresses K85 reporter
activities in a dose-dependent manner in COS-1 cells.
Different amounts of Ik expression vector and a fixed amount of K85
reporter were cointroduced into COS-1 cells and specific reporter
activities were determined as described for panel B.
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Ik-1 Suppresses Endogenous KOR Expression in P19 Cells--
The
data presented above showed that Ik-1 was a negative factor for KOR
expression as determined in reporter assays. To further determine
whether Ik-1 could suppress endogenous KOR expression in P19 stem
cells, we then introduced Ik-1 expression vectors into P19 stem cells
and analyzed the expression of endogenous KOR mRNAs 20 h
following transfection, as shown in Fig.
4. The results revealed that all three of
the KOR mRNA isoforms were suppressed, although to different
extents, confirming that elevated Ik-1 expression down-regulated
endogenous KOR gene expression for all three mRNA isoforms.

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Fig. 4.
Suppression of endogenous KOR mRNA
isoforms by Ik-1 expression in P19. Expression of KOR mRNA
isoforms a, b, and c in P19 following transfection with a control
vector (ctrl) or the Ik expression vector
(Ikaros) for 20 h was detected by RT-PCR as described
under "Experimental Procedures."
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RA Induces Ik-1 Expression in P19 Cells--
RA suppressed
endogenous KOR gene expression in P19, following treatment for ~2-3
days (Fig. 1), suggesting an indirect nature of the suppressive effect
of RA. It was then predicted that RA treatment might induce regulatory
components, such as Ik-1, responsible for KOR suppression. To
examine such a possibility, we then determined whether Ik-1 could be
induced by RA treatment in P19 cells using both RT-PCR and Northern
blot analyses. Using specific primers in PCR that allowed Ik-1 to be
amplified to a 304-bp fragment, it was found that Ik-1 mRNA
expression was indeed elevated in RA treated cultures. As shown in Fig.
5, A and B, Ik-1 was induced by RA after
18 h of treatment, and the induction peaked at 24 h and then
leveled off. Fig. 5C shows the Northern blot analysis of Ik-1 mRNA
expression in untreated P19 cells (ctrl) and RA-induced P19 (RA,
24 h). The bottom panel shows the amount and integrity of these
RNA preparations. Ik-1 mRNA was clearly induced by RA as
demonstrated in both RT-PCR and Northern blot.

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Fig. 5.
RA induction of Ik-1 in P19 cells.
A, P19 cells were induced with RA for a duration as
indicated, and analyzed for Ik-1 mRNA expression by RT-PCR as
described under "Experimental Procedures." ctrl,
control. B, a statistical analysis of three independent
experiments as shown in panel A. Ik-specific signals were
normalized to actin-specific signals to obtain the relative intensity
of the Ik signal in each culture. C, Northern blot analysis
of Ik-1 expression in P19 cells. Thirty µg of total RNA from
untreated P19 (ctrl) or cells induced with RA for 24 h
(RA) was analyzed on a Northern blot, probed with Ik-1
cDNA. The bottom panel shows 28 S and 18 S rRNA.
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The experiments described above demonstrated that, in P19 cells, either
RA or Ik-1 expression could suppress the expression of all three KOR
mRNA isoforms. In addition, RA induced Ik-1 expression, preceding the suppression of KOR gene for ~24 h. Therefore,
Ik-1 could be a physiological mediator responsible for the suppression of the KOR gene by RA.
Ik-1 Binds to the Putative Ik-binding Site on Intron 1 of the KOR
Gene--
To examine whether the Ik response element was indeed an
Ik-binding element, gel mobility shift experiments were performed as
shown in Fig. 6A. The wild type Ik
element was bound specifically by Ik-1 protein as evidenced by the
competition of cold probes (lanes 2-6). We then mutated either one of
the two repeats and tested these mutated sequences for Ik-1 binding in
gel shift assays. Interestingly, mutation at the first repeat (m1, lane
7) did not affect Ik-1 binding, whereas mutation at the second repeat
(m2, lane 8) abolished the specific binding. Furthermore, when an
anti-Ik antibody was added into the reaction mixture, the shifted band diminished, indicating a disruption of Ik-DNA interaction by this antibody. In the presence of preimmune serum, the specifically retarded
band remained the same (lane 10), supporting the specificity of the
effect of antibody observed on lane 9. The gel shift patterns remained
the same when nuclear extracts isolated from P19 transfected with the
Ik-1 expression vector were used (data not shown).

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Fig. 6.
Specificity of Ik-1 binding site.
A, gel mobility shift demonstrating specific binding of
Ik-1. Gel mobility shift experiments were conducted as described under
"Experimental Procedures." Lanes 1-6 show competition
experiments. A specifically retarded band is indicated by an
arrow. Lanes 7 and 8 show the results of mutant 1 (m1) and mutant 2 (m2) Ik-binding sequences.
Lane 9 shows antibody interference of Ik-1 binding to the
wild type (wt) sequence, and lane 10 shows a
preimmune serum control. B, biological activities of mutated
Ik-1 binding sequence. Mutants 1 and 2 were introduced into
both the KOR reporter (K45) as K97 and K99 and the
heterologous reporter (K85) as K98 and K100, respectively.
The biological activities of these mutant reporters were compared with
the wild type reporters K45 and K85 in COS-1 cells as described in Fig.
3. RLU, relative luciferase units.
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To further confirm the biological activity of this
Ikaros-binding sequence, the two mutations (m1 and m2) were
introduced into KOR reporter K45 and the heterologous reporter K85, and
Ikaros-mediated repression of these reporters was examined
in COS-1 cells. Mutations on the background of K45 are designated as
K97 and K99 for mutants 1 and 2, respectively. Mutations on the
background of K85 are designated as K98 and K100 for mutants 1 and 2, respectively. As shown in Fig. 6B, mutant 1 had no effect on
Ikaros-mediated repression of these reporters (K97 and K99),
whereas mutant 2 abolished Ikaros-mediated repression (K98
and K100). This result confirmed that repression of reporters
containing the Ikaros-binding sequence was indeed mediated
by Ikaros binding to this sequence. Therefore, it was
concluded that the putative Ik-binding sequence on intron 1 of the KOR
gene was a functional Ik-1 binding site, which could mediate the
suppression of KOR gene expression.
Ik-1 Expression Renders Histone Deacetylation on KOR
Promoters--
Ik-1 has been shown to be associated with histone
deacetylase (HDAC) complexes (24), which would silence target genes by deacetylating specific gene promoters. To determine whether Ik-1 binding to the KOR sequence in P19 could be enhanced by RA and whether
Ik-1 expression would affect the acetylation status of histone proteins
on the KOR promoters, ChIP assays were performed as shown in Fig.
7. The principle was based upon
immunoprecipitation of acetylated chromatin, but not deacetylated
chromatin, by a specific anti-acetylated histone antibody followed by
PCR detection of the immunoprecipitated DNA sequences. If, as
predicted, Ik-1 could bring the HDAC complexes to KOR intron I, which
was in close proximity to promoters 1 and 2, KOR promoter regions would
be deacetylated upon the overexpression of Ik-1. Thus, chromatin in KOR promoters would not be immunoprecipitated with the
anti-acetylated histone antibody, and no KOR promoter DNA sequences
could be detected by PCR. To examine this scenario, we first tested the
histone acetylation status of KOR promoters in the presence or absence of Ik-1 in COS-1 cells, as shown in Fig. 7A. COS-1 cells were transfected with the KOR reporter containing both promoters 1 and 2, K45, in the presence or absence of Ik-1 expression vector. An
anti-acetylated histone antibody was used to precipitate chromatin. Immunoprecipitated chromatin DNA was then detected by PCR with primers
flanking either KOR promoter 1 (top panel) or promoter 2 (middle
panel). It is obvious that only in the absence of Ik-1 were KOR
promoter histones acetylated, and thereby immunoprecipitated, and the
DNA sequences of these regions detected (lane 2). On the contrary, in the presence of Ik-1, these KOR sequences could not be
detected (lane 1), indicating histone deacetylation on these KOR
promoter regions presumably brought about by Ik-1 binding. For a
control of antibody specificity, a nonspecific antibody (anti-TR2) was
used in parallel reactions as shown in lanes 3 and 4. With this
nonspecific antibody, no PCR products were detected, confirming the
specificity of immunoprecipitation of acetylated chromatin detected in
lane 2. Lanes 5 and 6 are positive controls of input, lane 7 shows a
negative control reaction of water, and lane 8 shows a positive control
using plasmid DNA for PCR. For the control of equal loading of
reactions in the presence (lane 1) or absence (lane 2) of Ik-1, a
nonspecific promoter, the cytomegalovirus promoter of the
expression vector, was also examined, as shown on the bottom panel of
this figure. On this nonspecific promoter, chromatin acetylation was
the same for reactions in the presence (lane 1) or absence (lane 2) of
the Ik-1 expression vector, confirming equal loading of these two
reactions. These results support the effect of Ik-1 expression on the
acetylation status of KOR promoters, i.e. Ik-1 expression
brings about histone deacetylation of KOR promoters.

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Fig. 7.
ChIP assays for histone deacetylation on KOR
promoters and Ik-binding to KOR promoter region. A, dChIP
assays were conducted as described under "Experimental Procedures"
to detect histone acetylation on KOR promoters. The top
panel shows the results of promoter 1, the middle panel
shows the result of promoter 2, and the bottom panel shows
the result of a nonspecific promoter. Input DNA (1 µl from a 20×
dilution) from each lysate before immunoprecipitation
(lanes 5 and 6) and K45 plasmid DNA
(lane 8) were used as positive controls in PCR. Negative
controls are shown in lanes 3 and 4 for reactions
with a nonspecific antibody and in lane 7 for water control.
Ik-1 expression renders histone deacetylated on KOR promoters, and
therefore no KOR-specific PCR products (140 bp for promoter 2 and 240 bp for promoter 1) are detected (lane 2). On the contrary,
these promoter regions are acetylated in cultures expressing an empty
expression vector, and therefore DNA is precipitated and detected
(lane 1, bottom panel). The equal loading of lanes 1 and 2 is demonstrated in parallel reactions examining a nonspecific
promoter as shown in the bottom panel. B, ChIP assay
demonstrating Ik-1 binding to KOR DNA. Anti-Ik antibody was used in
ChIP assay as described above, and the sequence flanking the Ik-1
binding site on the KOR gene was examined. Lanes 1 and
2 show the result of positive reaction using anti-Ik
antibody. Lanes 3 and 4 show the negative results
using preimmune sera to precipitate, lanes 5 and
6 are control of input, lane 7 shows a negative
control of water, and lane 8 shows a positive control using
plasmid DNA in PCR.
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To provide evidence for changes in Ik-1 binding to the Ik-binding site
of KOR in P19 cells following RA induction, a ChIP assay was conducted
using an anti-Ik antibody to precipitate chromatin, as shown in Fig.
7B. P19 cells were transfected with K45 and treated with RA or vehicle
for 24 h. Cells were lysed for the ChIP assay as described
under "Experimental Procedures." Anti-Ik antibody was used
to precipitate DNA, and the precipitate was subjected to PCR with
specific primers flanking the Ik-binding site. As shown in lanes 1 and
2, specific fragments were amplified only in the presence of anti-Ik
antibody. Moreover, RA enhanced Ik-1 binding (lane 2) to the Ik-1
binding site as compared with an untreated culture (lane 1). In the
absence of anti-Ik antibody, no PCR products were detected (lanes 3 and
4). Lanes 5 and 6 show input control, lane 7 shows water control, and
lane 8 shows the plasmid control.
These data provide physiologically relevant evidence for RA induction
of Ik-1 which acts as a negative regulator for KOR gene expression. The
effect of Ik-1 is mediated by its binding to the Ik-1 element of KOR
intron 1, which recruits HDAC and renders histone deacetylation of KOR
promoter regions.
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DISCUSSION |
We present the first evidence for a negative regulation of KOR
gene expression by RA and provide a molecular mechanism underlying this
suppressive phenomenon. P19 stem cells express KOR constitutively at a
basal level, and RA induces an elevation of Ik-1 expression in these
cells within 20 h. An Ik-binding element was identified in the
first intron of this gene, which can be bound specifically by Ik-1
protein. This Ik-binding element is a position-independent, functional,
negative DNA element as demonstrated in reporters driven by the KOR
promoters or heterologous promoters. Elevation of Ik-1 in P19
suppresses the endogenous KOR gene expression for all three of the
mRNA isoforms, accompanied by increased binding of Ik-1 to its
intron 1 sequence and histone deacetylation in the KOR promoter
regions. This study is the first to demonstrate hormonal regulation of
opioid receptor gene expression, which involves chromatin remodeling
brought about by a negative transcription factor that is induced by
RA.
The suppression of the KOR gene by RA does not occur until 2-3 days of
treatment (Fig. 1), suggesting the indirect nature of RA suppression
of the KOR gene. This hypothesis is further supported by the
fact that within a 4-kilobase pair regulatory region of the
mouse KOR gene, no typical RA response element can be found.
Interestingly, Ik-1 is transiently induced by RA within 20 h,
preceding the suppression of KOR by RA for ~24 h. This would leave
enough time for Ik-1 to regulate the KOR gene. Because induction of
Ik-1 expression occurs relatively early and levels off as culture is
depleted of RA (Fig. 5), it is possible that the Ik-1 gene contains an
RA response element. However, this possibility remains to be examined experimentally.
The presence of a negative element in the first intron of KOR gene is
interesting. This intron also encodes the second promoter of the KOR
gene, and therefore, it may potentially be involved in both the
transcriptional control of promoter 2 and alternative splicing of
transcripts initiated from promoter 1. The fact that the suppressive
activity of this intron, particularly the Ik-binding element, is
position-independent and is functional in the context of heterologous
promoters would suggest that this intron is a common regulatory element
for both promoters 1 and 2 of the KOR gene. Furthermore, because Ik
binding renders both promoters 1 and 2 of the KOR gene deacetylated, a
most probable hypothesis for HDAC action on this gene is a spreading
model. Upon Ik binding, HDAC is recruited and spread along the
regulatory region of KOR gene. Both promoters are thus deacetylated and
tightly packed. However, the extent of the suppressive effects of Ik-1
on RNA variants transcribed from the two KOR promoters may vary because this sequence can also affect splicing of transcripts derived from
promoter 1 (isoforms a and b). This hypothesis is supported by
the results of overexpressing Ik-1 in P19, where the three isoforms are
all suppressed but to different extents (Fig. 4). According to the DNA
sequence, the KOR gene belongs to the housekeeping gene category
because no TATA or CCAAT boxes can be found; this is in agreement with
the fact that KOR is constitutively expressed in P19 stem cells at a
basal level. In our transgenic mouse model (17), KOR-lacZ
reporter gene is also more widely expressed in early gestation stages
before the birth of neurons. However, in older embryos,
KOR-lacZ expression becomes restricted to specific areas of
the central nervous system, primarily in sensory organ primordia. It is
possible that the chromatin structure of the KOR gene is readily open
in certain stem cells, such as P19 and those of young embryos, for a
basal level of gene activity. When cells undergo differentiation
such as being induced by RA, KOR expression is shut down in
most cell lineages except specific mature KOR neurons. The silence of
the KOR gene may be brought about by specific negative regulatory
factors induced by RA, such as Ik-1 protein. However, it remains to be
determined what positive factors are required to maintain or induce the
expression of KOR gene in mature KOR neurons.
Ikaros transcription factors, also named LyF-1 (25),
are found to be relatively restricted to the early stages of lymphoid cells (26). KOR expression is apparent in P19 stem cells, which constitute a pool of precursor cells for a variety of cell lineages. It
is widely recognized that P19 is induced by RA to differentiate into
various cell types including neurons. However, neurons, especially KOR-positive neurons, do not appear until the later stages of differentiation (18). It is conceivable that KOR-expressing stem cells
will gradually change cell fate as various transcription factors are
induced by RA. Ikaros could be one of these important factors that help to shape early cell differentiation processes and
turn off certain genes such as the KOR gene. In fact, KOR expression
has been detected in lymphoid cell lineages, such as R1.1, in addition
to mature KOR neurons (19). Therefore, the suppression of the KOR gene
by a lymphoid transcription factor is physiologically relevant.
Important questions to be answered in the future would be why the KOR
gene is active in undifferentiated cells and how the KOR gene is
reactivated in mature KOR neurons after being suppressed during early
stage of differentiation.
Recently, two reports have demonstrated the recruitment of
mSin3A·HDAC complexes to a neurally restrictive silencer element that
regulates more than 20 neuron-specific genes (27, 28). Our
study demonstrates the presence of a novel gene silencer in the KOR
gene intron 1, which is a target of a specific transcription factor,
Ik-1. The repressive activity of Ikaros has first been suggested to involve its interaction with mSin3, which binds to histone
deacetylases (24). Recently, it was shown that Ikaros can
also repress gene expression by interacting with a repressor, CtBP,
that acts in a HDAC-independent manner (29). Our current studies show
increased Ik-1 expression in RA-induced P19, which renders the KOR gene
suppressed and histone deacetylated in the KOR promoter regions,
presumably brought about by recruiting HDAC complexes. Although our
studies support HDAC-dependent action of Ikaros
on KOR gene expression, it cannot be ruled out that other mechanisms
may exist for Ikaros-mediated KOR gene regulation, such as a
potential role of other repressors in KOR regulation. Our study is a
first example of negative regulation of an opioid receptor gene by RA,
mediated by a transcription regulator that can potentially cause
histone deacetylation on the promoters and interact with other gene repressors.