From the Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455
Received for publication, August 27, 2002, and in revised form, November 20, 2002
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ABSTRACT |
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We have identified transcription factors that
bind to specific sequences in 5'-distal promoter regulatory sequences
of the mouse µ opioid receptor (mor) promoter using the
yeast one-hybrid system. The sequence between Opioids have many pharmacological and physiological effects,
including analgesia, sedation, euphoria, and respiratory depression and
also induce tolerance and physical dependence when administered chronically. Pharmacological, physiological, receptor binding, and
molecular cloning studies (1) have revealed that opioids interact with
three major types of opioid receptors in the mammalian central nervous
system and peripheral tissues, µ, Mor is expressed mainly in the central nervous system, where
it exhibits distinct temporal and spatial patterns of distribution (1).
Recently, in situ hybridization studies and other methods (e.g. RPA (RNase protection assay) and reverse
transcriptase-PCR) indicate that expression of mor mRNA
is regulated at the transcriptional level, especially during embryonic
development (4-7). The molecular mechanisms underlying this regulation
are not completely understood, but analysis of the 5'-upstream
sequences from the translation initiation sites of the mor
gene has shown that expression of mor is driven by two
promoters, a distal and a proximal promoter (8). Both promoters exhibit
characteristics of housekeeping genes lacking a TATA box (9, 10), with
the proximal promoter regulating mor transcription from four
major initiation sites located in a region 291-268 bp upstream of the
translation initiation site. The distal promoter initiates
mor transcription from a single transcription initiation
site located 794 bp upstream of the translation initiation site (8) and
is known to be 20-fold less active than the proximal promoter, based on
quantitative reverse transcriptase-PCR using adult mouse brain mRNA
(9). Thus, in adult mouse, the proximal promoter confers the largest
part of the cell and tissue-specific expression of mor (9).
It is also important to note that the 34-bp cis-acting
element positioned between The results demonstrate the ability of the cis-acting
element to augment the mor distal promoter activity in
co-transfection analysis using deletional promoter constructs. Three
kinds of Sox genes were isolated using the yeast one-hybrid system,
which helped to identify the trans-acting factors that bind
to the positive cis-acting element of the mor
distal promoter. The Sox proteins belong to the high mobility group
(HMG) box superfamily of DNA-binding proteins and are found throughout
the animal kingdom (12). These proteins are involved in the regulation
of such diverse developmental processes as germ layer formation, organ
development, and cell-type specification. Hence, deletion or mutation
of Sox proteins often results in developmental defects and congenital
disease in humans. Sox proteins perform their function in a complex
interplay with other transcription factors in a manner highly dependent
on cell type and promoter context. They exhibit remarkable cross-talk and functional redundancy among each other (12, 13). Therefore, Sox
proteins may play an important role in regulating the expression of the
mor gene, temporally, spatially, and developmentally as well
as gender-dependently in brain. In this report, we
characterized the nature and function of these nuclear proteins, which
regulate the expression of µ opioid receptor gene.
Yeast One-hybrid Screening for cDNAs Encoding Positive
Regulatory DNA-binding Proteins--
The MATCHMAKER One-Hybrid System
(Clontech) was used according to the supplier's
protocol. Four tandem repeats of the Plasmid Construction and in Vitro Translation--
Constructs
for the recombinant luciferase reporter gene plasmids pL1.3K/687,
pL1.3K/775, pL1.3K/728, pL1.3K/721, and pL1.3K/717 have been previously
described (11). To further define the positive regulatory region of the
mor distal promoter, pL1.3K/747, pL1.3K/738, pL1.3K/731, and
pL1.3K/725 were generated from pL1.3K/687 by PCR with the
primers shown in Fig. 1; that is, forward (mor5'),
5'-GTCTGTGAGAATTCAGTTAAACTTCTACAACC-3' (EcoR1 site at nucleotide
In vitro translation was carried out with pc1-Sox18,
pc1-Sox21, and pc1-Sox6 in a reaction mixture containing
[35S]methionine (Amersham Biosciences) using a TNT
quick-coupled transcription/translation system (Promega). The labeled
proteins were then electrophoresed by 10% SDS-PAGE, and their sizes
were compared with the predicted sizes.
Cell Culture, Transfection, and Reporter Gene Assay--
Human
neuroblastoma NMB cells were grown in RPMI 1640 medium supplemented
with 10% heat-inactivated fetal bovine serum (Invitrogen) at 37 °C
in a humidified atmosphere of 5% CO2. Cells were plated in
6-well dishes at a concentration of 1 × 106
cells/well and cultured overnight before transfection. Various plasmids
at concentrations indicated in each figure were used with the SuperFect
Transfection reagent (Qiagen) as described by the manufacturer.
Briefly, for 3' deletional analysis of the distal promoter, 2 µg of
the reporter plasmids were mixed with SuperFect Transfection reagent
for 15 min before being added to NMB cells. Forty-eight hours after
transfection, cells grown to confluence were washed once with 1×
phosphate-buffered saline and lysed with lysis buffer (Promega). To
correct for differences in transfection efficiency, a one-fifth molar
ratio of a pCH110 plasmid (Amersham Biosciences) containing the
Electrophoretic Mobility Shift Assay (EMSA)--
Nuclear extract
from NMB cells was prepared using a modification of procedures of
Dignam et al. (14) and Hennighausen et al. (15).
The upper and lower strands of each probe were annealed, and the
double-stranded oligonucleotides were then end-labeled with
[ DNase I Footprint Analysis--
The mor distal
promoter region from Identification of Functional Cis-acting Elements of mor Distal
Promoter--
Our previous results have shown that the 5'-flanking
region of the mouse µ opioid receptor (mor) gene has two
major promoters, designated as the proximal and distal (9). Choe
et al. (11) from our laboratory have also reported a
positive cis-acting element between the nucleotides
To determine whether this positive cis-acting element of
NMB Nuclear Extracts Specifically Bind to a Sox-binding Motif
within the Identified Cis-acting Element--
The SOX family consists
of a group of transcription factors that recognize a specific common
SOX protein binding site (5'-WWCAAWG-3', where W = A or T) (17).
These factors are thought to bind to the minor groove of
double-stranded DNA and induce DNA bending, resulting in chromatin
structure favorable to regulation of promoter transcription (18).
Analysis of the O1 cis-acting element revealed at least four
putative Sox-binding sites within this short DNA sequence, as indicated
in Fig. 3. To further characterize Sox binding to O1, EMSAs were carried out using O1 as probe and a consensus Sox binding sequence,
5'-GATCCGCGCCTTTGTTCTCCCCA-3' (SSC, as
shown in Fig. 3B) (19) as a competitor. Efficient
competition was observed with a 40-fold excess of the SOX consensus
competitor and maximal competition with a 100-fold excess of the
competitor, confirming the probe's SOX factor binding property.
The O1 cis-acting element contains four putative Sox-binding
sites in very close proximity to each other. Similar multiple Sox-binding sites have also been previously reported from several other
genes (20, 21). To define exactly which of these Sox-binding sites is
essential to the promoter activity, five additional oligonucleotides spanning the entire region were synthesized. They were designated as O3
to O7, as indicated in Fig. 3A. These fragments were then tested for their ability to compete with the DNA-protein complexes from
the O1 element. The DNA-protein complexes were abolished by a 100-fold
excess of competitors O2, O3, and O7 but not by O4, O5, and O6. These
data suggest that the double-stranded DNA sequence shared by O2, O3,
and O7 is probably important for binding of NMB nuclear extracts to the
O1 cis-acting elements. Examination of this common
sequence revealed a putative Sox-binding site, 5'-CTTTGAA-3', as
underlined and in bold in Fig. 3A.
Furthermore, this putative Sox-binding site is located downstream of
the distal promoter rather than upstream, as reported for other systems
(18, 22). Therefore, its orientation to the distal promoter is also reversed. However, O6, which failed to block the formation of O1-protein complexes, also contains this entire sequence. This observation might be explained by a requirement of an additional two
nucleotides at the 5'-flanking region of SOX core-binding site for the
formation of the DNA-protein complex.
To obtain further insight into complex formation, we performed EMSAs
using unlabeled mutant oligonucleotides to compete with 32P-labeled O2 for the complex formation with NMB nuclear
extracts. We designed four mutated oligos; M7 and M8 have a mutation in each side of the Sox-binding consensus site, the 5' flanking region was
mutated in M6, and the 3'-flanking region was mutated in M9 (Fig.
4A). As shown in Fig.
4B, the probe O2 containing the complete Sox-binding site
and both 5'- and 3'-flanking regions has formed DNA-protein complex
bands in the presence of NMB nuclear extracts. The M6 competitor
effectively blocked the appearance of the major migrating bands, but M9
did not affect the bands. In general, both flanking regions of the core
consensus-binding site are required for protein binding in many
transcription factors (22, 23). Together these data indicate that the
specific sequence of four bases in the flanking region downstream of
the core is required for high affinity binding (lane 7 in
Fig. 4B). In addition, the presence of at least two bases
without sequence specificity at the upstream flanking region is
required for the DNA-protein complex formation (lanes 7 and
8 in Fig. 3C and lane 4 in Fig.
4B). As expected, the competitor M7 with the mutated
5'-core-binding site, could not abrogate the DNA-protein complex.
However, M8 with the mutated 3'-core-binding site could compete
partially but not as strongly as M6 and the self-competitor O2,
indicating that the 5'-core-binding site is more important than the
3'-core-binding site for high affinity binding. The DNA-complexes were
not inhibited by nonspecific competitors, Sp1 (24) and Ikaros (25)
consensus sequences, confirming the specificity of the DNA-protein
interaction (Fig. 4B). Taken together, these results suggest
that NMB nuclear extracts contain proteins that bind
sequence-specifically to the identified Sox binding motif of the
mor distal promoter.
DNA-Protein Interactions in the Positive Cis-acting Element of
the Mouse mor Gene Determined by DNase I Footprinting--
The
relationship between in vivo positive modulatory activity
and in vitro DNA-protein interaction(s) was initially
assessed by DNase I footprinting using a probe extending between
nucleotides Isolation of Transcription Factors That Interact with the Positive
Cis-acting Element Region in the mor Distal Promoter--
To identify
transcription factors that bind to the positive element of the distal
promoter, we utilized the yeast one-hybrid system to screen an adult
mouse brain MATCHMAKER cDNA library (Clontech). The O1 sequence was used as bait
because it contained more of the flanking regions potentially important
for the transcription factor binding affinity. A double-stranded
oligonucleotide containing four tandem repeats of the O1 sequence was
subcloned into pHISi. The resulting plasmid, pHISi-4x40 was integrated
into the yeast YM4271 genome. Using this yeast strain, 3 × 106 independent colonies from the library were screened. A
total of five independent, histidine-positive clones were selected and sequenced. The results revealed that all five clones belonged to the
SOX family of transcription factors. Two of the clones contained
full-length sequences (2.7 kilobase pairs) encoding polypeptides with
more than 80% sequence identity to human Sox21. These clones were
designated mSox21 (GenBankTM accession number AY142959).
The other two clones were identical to mSox18 and also contained
full-length cDNA sequences, whereas the fifth clone proved to be a
truncated form of mSox6 lacking the first 299 amino acids but encoding
a DNA binding domain.
To verify that the three cloned SOX proteins bind to the O1-containing
promoter in vivo, the three independent cDNA clones were
transformed into yeast strains containing the control plasmid pHISi.
All three SOX proteins could specifically activate the HIS3 gene
containing the four tandem repeats of the O1 element but not the HIS3
gene lacking these repeats. This suggests that the cloned SOX proteins
bind specifically to this O1 cis-acting element and not to
the sequence of the HIS minimal promoter. To exclude the possibility
that the addition of the O1 element may form some specific sequences
with the minimal HIS3 promoter that favor binding of the SOX protein,
we used another reporter yeast strain containing pLacZi-4x40. A control
yeast strain without repeats of the cis-acting elements but
with pLacZi integrated into its genome was also used. The cloned SOX
plasmids were transformed separately into these yeast strains. All
three SOX clones in yeast strains containing pLacZi-4x40 were found to
be true positives after the LacZ expression test. In control yeast
strains containing pLacZi transformed with SOX plasmids, there was no
activation, indicating that these three cloned SOX proteins indeed have
specific strong binding activity to the four tandem repeats of the O1
cis-acting elements in vivo.
Cloned SOX Proteins Bind Specifically to the Positive Cis-acting
Element of mor Distal Promoter in Vitro--
The ability of all three
Sox cDNAs (Sox6, Sox18, and Sox21) to encode proteins was verified
by in vitro translation, and the products were analyzed on
SDS-PAGE. All the in vitro translated SOX proteins were
electrophoresed and found to be the correct size, as expected from
their calculated molecular weights (data not shown). To confirm whether
the isolated SOX proteins can indeed bind to the positive
cis-acting element, EMSAs were carried out using in
vitro translated SOX protein products. The SOX proteins were able
to shift the target O2 oligonucleotide probe. The specificity of this
DNA-protein interaction was verified by complete inhibition in the
presence of cold wild-type O2 oligonucleotide in an EMSA using the
SOX18 protein (Fig. 6A).
Furthermore, competitor M7 or M8 containing a mutation in the Sox
binding motif (lane 4 and 5 in Fig.
6A) failed to efficiently block the complex formation, whereas competitor M6 containing a mutation in the upstream flanking region of the core motif competed with the SOX18-DNA complex
(lane 3). As we have observed similarly in the EMSA of Fig.
4B using nuclear extracts of NMB cells, competitor M9
containing a mutation in the downstream flanking region of the core
motif was not able to block the complex (lane 6 in Fig.
6A). Similar results were obtained when using in
vitro translated SOX21 (Fig. 6B). The competitive EMSA
using the SOX6 protein has not been performed because of unavailability
of the full-length Sox6 cDNA, although in vitro translated SOX6 protein using the partial cDNA was still able to
bind to the O2 oligonucleotide probe (data not shown). We conclude that
the in vitro translated SOX18 and SOX21 proteins have a
higher binding preference to the identified Sox binding motif than
other Sox-binding sites within probe O2. However, the identified Sox binding motif alone cannot account for 100% of the total Sox binding activity, because the competitor M9 containing a mutation of the downstream flanking region of the core motif could not bind to the SOX
proteins seen in lanes 6 of Fig. 6, A and
B.
SOX18 Binds Preferentially to the Sox-binding Site of mor Promoter
among the Known Sox Binding Consensus Sequences from Several Different
Promoters--
To test the preferential binding of SOX18 to the known
SOX family binding consensus sequences from several gene promoters, four different Sox-related binding sequences for T cell factor (TCF)-MW56 (T cell receptor) (26), SOX5-BS12 (26), TCF1 The Identified Sox Binding Motif Is Required for the Regulation of
mor Distal Promoter Activity--
To further confirm the importance of
the identified Sox binding motif in the regulation of the mouse
mor distal promoter, two mutated constructs, pL1.3K/721m2
and pL1.3K/721m123, were cloned. In construct pL1.3K/721m2, the
identified Sox binding motif of the distal promoter was mutated,
whereas in construct pL1.3K/721m123, both the Sox binding motif and its
flanking regions were mutated. As shown in Fig.
8, A and B, when
compared with the wild-type distal promoter (pL1.3K/721), mutation of
the Sox binding motif alone (pL1.3K/721m2) resulted in a two-thirds
decrease in the relative luciferase activity. Additional mutation of
the flanking region together with the Sox binding motif
(pL1.3K/721m123) caused a slight further decrease of the promoter
activity. The promoter construct pL1.3K/731 lacking the Sox-binding
site showed 60% less activity than the wild-type promoter as shown in
Fig. 8A and also in Fig. 1B, indicating
consistency of the experiments. These results suggest that the
identified Sox binding motif is important for up-regulating the mouse
mor distal promoter activity.
SOX Proteins Activate Transcription through the Sox Binding Motif
of mor Distal Promoter--
To determine how the cloned SOX proteins
regulate the transcription of the mouse mor gene, cDNAs
for full-length mSox18 or mSox21 were cloned into a mammalian
expression vector. These DNAs were co-transfected with the mouse distal
promoter linked to the luciferase reporter gene into NMB cells
endogenously expressing mor. Cells transfected with the
mutant reporter construct pL1.3K/721m123 served as controls. As shown
in Fig. 8C, mSox18 can specifically trans-activate the
distal promoter activity (pL1.3K/721) in a concentration-dependent manner that is consistent with the
previous reports that SOX18 is a trans-activator (29, 30). Mutations of
the Sox binding motif and its flanking region resulted in a significant
decrease of the trans-activation activity by mSox18. This decrease is
rather obvious when cells were transfected with low concentrations of
mSox18. On the other hand, mSox18 still has some trans-activation
activity when co-transfected with mutant reporter constructs of either
the identified Sox binding motif or its flanking region alone (data not
shown). This indicates that the identified Sox binding motif together
with its flanking region is important for the trans-activation by
co-expressed mSox18. In addition, other multiple Sox-binding sites
might be co-regulated by SOX18, although the physical interactions were
not shown with the SOX proteins. Similar results were also obtained
after co-transfection of cloned Sox18 and the distal promoter reporter
plasmid into Chinese hamster ovary cells, which do not express
mor endogenously (data not shown).
For the transcription analysis of mSox21, as in mSox18, co-transfection
with a fixed non-saturated amount of the reporter pL1.3K/721 and
increasing amounts of the expression plasmid pc1-SOX21 resulted in
increased luciferase activity in a concentration-dependent manner (Fig. 9A). Five
hundreds nanograms of mSox21, the maximum amount tested in this study,
showed the highest promoter activity on pL1.3K/721, with a gradual
increase in the activity, whereas 100 ng of mSOX18 showed 90% activity
compared with the maximal promoter activity, as seen with 500 ng of
mSOX18. This indicates that SOX18 at low expression levels is a more
potent activator of mor promoter than Sox21. When these two
proteins were co-expressed in NMB cells, Sox21 did not significantly
enhance Sox18-induced activation of the promoter in pL1.3K/721 (Fig.
9B) (at most a 15% increase), suggesting that the amount of
either DNA (500 ng) used alone might have been sufficient for maximum
promoter activity, and/or these two SOX proteins might act
independently in our system. Although the full-length cDNA of mSox6
was unavailable, the partial cDNA clone of mSox6 containing an
N-terminal and internal deletions but retaining HMG domain was inserted
into the mammalian expression vector. The resultant plasmid, pc1-Sox6,
did not show any significant effect when similarly co-transfected with
the distal promoter construct into NMB cells, indicating the
requirement of the full-length cDNA clone for the further
study.
In the present report, we identified two transcription factors,
SOX18 and SOX21, as potent transcriptional activators of the mouse µ opioid receptor (mor) gene. This regulatory activity is mediated by a Sox-binding site located in the downstream region of the
mor distal promoter. Deletion or mutation of this Sox
binding motif resulted in about a two-thirds decrease of the mouse
mor distal promoter activity in transient transfection
assays. Nuclear extracts from NMB cells endogenously expressing
mor could specifically bind to this Sox binding motif, and a
very high concentration of nuclear extract from mouse brain generated a
protected region in this area against DNase I. We were able to isolate
three SOX proteins (Sox6, Sox18, and Sox21) from mouse brain by the
yeast one-hybrid system using the cis-acting sequence.
Mutations of the Sox binding motif and its flanking regions blocked the
transactivation effects of mSox18. Thus, the identified Sox binding
motif plays an important role in the regulation of mor
distal promoter, and we have identified mSOX18 and mSOX21 as possible
candidates for this regulatory effect.
Cloning of these SOX proteins from the adult mouse brain cDNA
library indicates that the expression of these SOX genes is not only
restricted to early central nervous system development (31-34), but
these genes are also expressed in the adult mouse brain. Although none
of these proteins is strictly expressed in the central nervous system
(31-34), the co-existence of SOXes and MOR in the central nervous
system provides the possibility that these SOXes might function in
regulating mor gene expression.
Because of the unavailability of mouse cell lines that express
endogenous mor, we used nuclear extracts from the human
neuroblastoma NMB cell line as the source of transcription factors in
EMSAs. Although the SOX proteins of human and mouse are slightly
different, their amino acid sequences are highly conserved, especially
in the HMG DNA binding domain (35). In the case of Sox18, human Sox18
contains an HMG box with 98% amino acid identity to the HMG domain of
mouse Sox18 (34). Therefore, the DNA binding properties of these SOX
proteins in NMB nuclear extracts should be highly similar to the
corresponding transcription factors from mouse. We had tried to use
nuclear extracts from mouse brain in EMSAs. However, because of strong
background binding, we could not clearly distinguish any band
representing specific Sox binding. In DNase I footprinting analysis, we
could only detect the DNA-protein interactions on Sox-binding sites in
very high concentrations of nuclear extract from mouse brain. This may
reflect the relatively low amounts of mor expressed in the
brain as well as presumably, of the transcription factors regulating
them, which are not ubiquitous in their activity.
SOX proteins are found throughout the animal kingdom and have been
shown to be intricately involved in the regulation of the development
of germ layer formation, the central nervous system and other organs,
and cell-type specification (35). SOX factors are characterized by the
presence of a SRY box, a 79-amino acid protein motif (12, 35) that
encodes an HMG-type DNA binding domain. Thus, the SOX family falls into
a subclass of HMG box proteins, the members of which show highly
restricted tissue distribution and bind to specific sequences at high
affinities (12). The HMG domain has been known to interact with the
minor groove of the DNA helix and induce a dramatic bend in the DNA
molecules (35, 36), in contrast to the majority of types of DNA binding domains that have access to DNA through the major groove. These HMG-box
proteins are classified into two broad categories; those proteins that
carry multiple HMG domains (ribosomal transcription factor UBF
(upstream binding factor) and the abundant non-histone chromosomal
proteins HMG1 and HMG2) and show little DNA sequence preference and
those having a single HMG domain that bind DNA in a sequence-specific
manner including the TCF/lymphoid enhancer binding factor (LEF) family
and the SOX proteins (36). To date, more than 20 SOX proteins have been
identified in vertebrates, and they have been grouped into Groups A-F,
Group A being assigned to SRY (sex-determining region of Y chromosome),
which is the prototype of Sox genes, encoding the mammalian
testis-determining factor (37). SOX18 belongs to the F sub-group of SOX
proteins along with SOX7 and SOX17. SOX21 and SOX14 are classified into subgroup B2 (18). Within an individual group, the amino acid sequence
identity of the DNA binding (HMG-box) domain remains very high (over
90%), although it decreases to ~60% between distant groups. Then,
how do individual SOX proteins regulate the target gene temporally and
spatially? The present theory is that combinatorial protein
interactions of several SOX proteins and other transcription factors is
often necessary to promote target gene expression (13, 38-40). SOX2
and the POU domain transcription factor, OCT-3, bind adjacent sites and
participate together for the transactivation of the FGF4 gene through
protein-protein interactions in teratocarcinoma cells (38, 41), whereas
either factor alone is ineffective. Three different SOX proteins, a
long form of SOX5, SOX6, and SOX9, are co-expressed in chondrocytes and
cooperatively activate the chondrocyte-specific enhancer of the type II
collagen gene (39). The activation is facilitated by the dimerization
of the long form of SOX5 and SOX6. SOX6 contains a leucine zipper motif
that allows dimerization of the protein, and homodimers fail to bind DNA (40). These data suggest that, in testis, SOX6 may bind to another
protein as a heterodimer to show transactivation properties. Coincidentally, it was reported that a POU domain protein OCT-1 could
regulate the mouse mor distal promoter activity by binding to a functional OCT-1 binding site (a negative cis-acting
element), which is 94 bp upstream of the identified Sox binding motif
(42). It would be interesting to investigate whether these two sites could cooperate with each other to confer cell- or tissue-specific regulation of the mouse mor expression.
In our EMSAs, we observed the sequence-specific DNA-protein complexes
on the identified Sox binding motif with nuclear extract of NMB cells
(Fig. 2). Interestingly, the identified Sox binding motif and its
flanking regions together are responsible for the complex formation.
Because a mutation of 4 bp downstream of the core binding motif
abolished the formation of the major band in EMSAs (Figs. 4 and 6), it
is unlikely that either of them alone could account for the major band
formation. On the contrary, there would be some extent of cooperation
between the identified Sox-binding motif and its flanking regions. As
previously mentioned, there are theoretically three additional
Sox-binding sites adjacent to the 5' side of the identified Sox binding
motif. Because the O2 probe lacking the first SOX site still was able
to form the same major bands (Figs. 2C and 4B) as
O1 probe containing all four Sox-binding sites, the first Sox-binding
site is unlikely to be a major site for influencing the major band
formation. Based on the results from EMSAs using mutant or deleted
oligonucleotides (Fig. 3C, 4B, and 6), the
flanking sequences of the core-binding site also affect the formation
of SOX protein-DNA complexes to the Sox binding motif. In addition, the
requirement of specific flanking sequences for SOX protein binding was
confirmed by another EMSA using different Sox binding consensus
sequences of several SOX proteins (Fig. 7). The only competitor TCF1 It is very interesting to note that both mor and Sox genes
are associated with different effects depending on sex. Recent studies
indicate that the relative efficacy of µ opioids (e.g. morphine) as an antinociceptive agent is greater in male than in female
rodents and monkeys (45, 46). Perinatal exposure to opioid drugs
produced changes in binding and density of µ opioid receptor that
differed regionally and that were mostly different as a function of sex
(47). Sex-related differences in the experience of both clinically and
experimentally induced pain have been widely reported (48). Several
papers have reported a critical role for SOX proteins in sex
determination (49) as well as in the proper development of the central
and/or peripheral nervous system (12, 32, 50). In addition, Sox18 is
expressed in fetal brain (34) and also expressed weakly in adult brain
(33). The Sox21 gene is highly conserved and specifically expressed in
the brain (51, 52). Sox6 is also specifically expressed in the
developing nervous system (32). Collectively, it is feasible that SOX
proteins may mediate sex differential responses of the opioid system
through the mor distal promoter. Therefore, further studies
will help to elucidate the mechanism of association between the sex
differential response of the opioid system and opioid receptor gene
regulation by SOX proteins.
In conclusion, the foregoing observations indicate that an activating
cis-acting element can be used to regulate the target gene
transcription (mor) by transcriptional activators like SOX18 and SOX21. Here we contribute to the characterization of the µ opioid
receptor transcriptional machinery by identifying two members of the
SOX family as strong transcriptional activators of this gene. Future
experiments on the regulatory processes that control the activation
effect of the positive cis-acting element on the mor distal promoter by SOX proteins will help to understand
how the mor gene is expressed temporally, spatially, and
developmentally as well as gender-dependently in brain.
746 and
707 in
mor distal promoter was used as the bait because it acts as
a functional promoter element and binds several DNA-binding proteins.
From an adult mouse brain cDNA library, five cDNA clones
encoding three Sox gene family (Sry like high mobility
group (HMG) box gene) transcriptional factors, mSOX18,
mSOX21, and mSOX6, were isolated. Electrophoretic mobility shift assays
confirmed the presence of a binding site for SOX proteins in the
731/
725 region. Additionally, we have also established that the
flanking regions outside the core Sox-binding site play an essential
role in high affinity binding. DNase I footprint analysis indicates
that proteins from mouse brain interact with the Sox-binding site
within the mor distal promoter. Finally, we demonstrated
that overexpression of mSOX18 and/or mSOX21 was able to up-regulate
mouse mor distal promoter activity in
mor-expressing neuronal cells (NMB). These data
indicate that SOX proteins might contribute to the transcriptional
activity of the mor gene and suggest that µ opioid
receptor could mediate some of the developmental processes in which SOX
proteins are included.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and
. All three types of
receptors belong to the superfamily of G-protein-coupled receptors (2),
and the µ opioid receptor
(mor)1 is known to
play the essential role in morphine-induced analgesia, tolerance, and
dependence as indicated from pharmacological studies. This has also
been confirmed by additional in vivo pharmacological analyses of mice where mor was deleted by homologous
recombination (3).
721 and
687 possesses a strong
inhibitory effect against the distal promoter transcriptional function
(11). Because the exact role of the distal promoter in mor
expression is largely unknown, our objective in the present study was
to identify cis-acting DNA elements and their
trans-acting factors that are associated with transcription
from the mouse mor distal promoter. Secondly, we sought to
determine the possible role of the distal promoter in regulating
mor expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
745 to
706-bp sequence (the
positive regulatory element) of the mouse mor distal
promoter were ligated into the SacI-XbaI sites of
pHISi and EcoRI-SalI sites of pLacZi to generate
pHISi-4x40 and pLacZi-4x40, respectively (40 denotes the sequence
between
745 and
706). These two bait constructs were then
linearized with XhoI and NcoI, respectively, and
integrated into the genome of yeast strain YM4271. The resultant yeast
cells with the integrated pHISi-4x40 were tested for growth on minimal
medium lacking histidine (His
) in the presence of
increasing concentrations of 3-amino-1,2,4-triazole. Background growth
was inhibited in the presence of 30 mM
3-amino-1,2,4-triazole, and this concentration was then used when yeast
cells were transformed with a mouse brain cDNA library for
one-hybrid screening. Five positive transformants grown on
His
plates were selected. To exclude false positive
clones, plasmids recovered from these five clones were used to
transform yeast cells harboring the pLacZi-4x40 construct. Positive
transformants grown on His- and leucine-negative (Leu
)
medium containing 30 mM 3-amino-1,2,4-triazole were
streaked onto a nylon filter and incubated by placing the filter on the same medium at 30 °C for 2 days. The filter was then soaked in liquid nitrogen for 10 s and placed on a Whatman No. 3MM filter that had been presoaked in Z buffer (60 mM
Na2HPO4, 60 mM
NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM
-mercaptoethanol, pH 7.0) containing 0.01%
5-bromo-4-chloro-3-D-galactopyranoside (X-gal) and
incubated at 30 °C for 2 h. Plasmids from 10 positive blue
clones were sequenced, and their homology was analyzed using the BLAST algorithm.
1326 is underlined) and
all the corresponding reverse primers bearing essential Kozak sequences
(5'-CCATGGTG-3') and NcoI site (located at start codon of
luciferase). The resulting fragments were substituted into the
BfrI (
1105)-NcoI fragment of the pL1.3K/444
plasmid (11). The mutant reporter plasmids pL1.3K/721m2 and
pL1.3K/721m123 were constructed by PCR using pL1.3K/687 as the template
the same forward primer, mor5', as described above and reverse primer,
5'-GGGCCATGGCTGTTGTCGACAACAATTGTCTTTTTCTAA-3', for pL1.3K/721m2 and
5'-GGGCCATGGAGTACATCGATTCTGATTGTCTTTTTCTAAAAGAGAAAA-3' for pL1.3K/721m123 (the NcoI site is underlined, the
start codon is italicized, and mutated sites are in bold). The PCR
products were inserted into a TA cloning vector pCR2.1 (Invitrogen) and subsequently subcloned into the BfrI-NcoI sites
of pL1.3K/721. The mammalian expression plasmids, pc1-Sox18 or
pc1-Sox6, were constructed by inserting the
EcoRI-XhoI fragment of the original library
clones, pACT2-Sox18 or pACT2-Sox6, into the same sites of
pcDNA1.1/amp (Invitrogen), respectively. To construct pc1-Sox21, the BamHI-XhoI fragment of the original clone was
inserted into the same sites of pcDNA1.1/amp. The integrity of all
constructs was confirmed by restriction enzyme analysis and sequencing.
-galactosidase gene under the SV40 promoter was included in each
transfection for normalization. The luciferase and galactosidase
activities of each lysate were determined as described by the
manufacturers (Promega and Tropix, respectively). For co-transfection
assays, the procedures were the same as above, except the plasmids were a mixture of the given amount of pc1-Sox18 or pc1-Sox21 and 1 µg of a
corresponding reporter plasmid, pL1.3K/721 or pL1.3K/721m123.
-32P]ATP. The end-labeled DNA probes were incubated
with NMB nuclear extract or 8 µl of in vitro translated
products in a final volume of 20 µl of EMSA buffer (10 mM
Tris, pH 7.5, 5% glycerol, 1 mM EDTA, pH 7.1, 50 mM NaCl, 1 mM dithiothreitol, 1 mM
EDTA, and 0.1 mg/ml poly(dI-dC)) at room temperature for 30 min. For
oligonucleotide competition analysis, a 25-100-fold molar excess of
cold competitor oligonucleotide was added to the mixture before adding
the probe. The reaction mixtures were electrophoresed in 4%
polyacrylamide nondenaturing gel in 0.5 × TBE buffer (45 mM Tris borate and 1 mM EDTA) at 4 °C and
visualized by autoradiography.
812 to
575 bp was generated by PCR using a
32P-labeled primer for the coding strand and using
pL1.3K/508 (11) as template to create the 5'-labeled 236-bp probe. PCR
primers used to prepare the probe for footprinting were: sense,
5'-GAGAAATTGAAGGAGTGGGGGCACA-3' (bases
812 to
788) and antisense,
5'-ATACCTCAACACTCTTCCGACTCA-3' (bases
575 to
557). All the
oligonucleotides and probes were purified by PAGE. DNase I footprinting
was performed according to the recommended manual using the Core
Footprinting System (Promega). Nuclear extract from mouse brain was
prepared according to the method described by Sonnenberg et
al. (16). Binding reactions were carried out for 20 min on ice in
a final volume of 50 µl. The binding solutions contained 100 fmol of
labeled probe and the indicated amounts of the mouse brain nuclear
extract in a final buffer concentration of 10 mM Tris-HCl,
pH 7.5, 40 mM NaCl, 1 mM dithiothreitol, and 1 µg of poly(dI-dC). After incubation, MgCl2 and
CaCl2 were added to final concentrations of 5 and 2.5 mM, respectively, then 0.3 unit of DNase I (Promega) was
added. The incubation was continued for 4 min at room temperature. The digestion was stopped by using 90 µl of stop solution containing 20 mM EDTA, 1% SDS, 0.2 M NaCl, and 250 µg/ml
tRNA. DNA was extracted by using phenol-chloroform (1:1) and ethanol
precipitation before loading onto a 6% sequencing polyacrylamide gel
for electrophoresis.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
721
and
775 and a negative cis-acting element between the
nucleotides
687 and
721. Both of these regulatory elements are
present within the 5'-distal promoter regulatory sequences of
mor promoter. To define the exact position of the positive
cis-acting element, a further detailed 3'-deletional mutagenesis was performed in the region of
775 to
721 (Fig. 1). As shown in Fig. 1, a deletion of
just 4 bp from the 3' end (pL1.3K/725) exhibited a 50% decrease in
luciferase activity relative to the pL1.3K/721 construct. A further
deletion to
731 resulted in an additional 25% decrease in promoter
activity. These results indicate that the deleted segment of
730 to
721 is important for the positive cis-acting element.
Further deletions resulted in a slight increase in promoter activity,
indicating the complexity of regulation of distal promoter
activity.
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Fig. 1.
Identification of the positive cis-acting
element using 3' deletional analysis of distal promoter of the µ opioid receptor (mor) gene.
A, schematic representation of a series of 3' deletion
constructs of mouse mor distal promoter. Fragments of distal
promoter with varying lengths of the 3' regulatory regions were
inserted into the promoter-less luciferase vector, pGL3-Basic. The
numbers in the name of each construct refer to the number of the
nucleotide to the translation start site (designated +1) at the 5' end
and 3' ends of each inserted fragment, respectively. LUC
represents the luciferase gene. B, 3'-deletion analysis of
mouse distal promoter activity in transient transfection and luciferase
assays in NMB cells. The promoter activity of each construct was
expressed as relative luciferase activity, and transfection
efficiencies were normalized to -galactosidase activity by
co-transfection of the internal control plasmid pCH110. The activities
of the luciferase reporter were expressed as n-fold relative
to the activity of pGL3-Basic, which was assigned an activity value of
1.0. The data shown are the means of three independent experiments with
at least two different plasmid preparations. Error bars
indicate the range of standard errors.
730 to
721 could recruit transcription factors to regulate the promoter activity, we next performed EMSAs using nuclear extracts from
mor-expressing NMB cells. We selected the segments O1 (
746 to
707) and O2 (
741 to
718) (Fig.
2A) as probes, because they both covered the region of
730 to
721 but with different lengths of
their flanking sequences. As shown in Fig. 2, there are several protein-DNA complexes formed with the probe O1 and O2. The higher molecular weight complexes were efficiently inhibited by a 100-fold molar excess of cold self-competitors, indicating these complexes were
sequence-specific, whereas the fastest running band with the O2 probe
was not abrogated with self-competitor (Fig. 2C), indicating
the presence of nonspecific interaction. In summary, the O1 and O2
sequences, containing the positive cis-acting element, can
form sequence-specific DNA-protein complexes with NMB nuclear extracts,
suggesting the presence of endogenous transcription factors. Based on
the EMSA and luciferase reporter assay results, we hypothesize that the
positive cis-acting element plays a role in regulating
distal mor promoter activity.
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Fig. 2.
EMSA analysis of the cis-acting
elements of mor distal promoter. A,
schematic diagram representing the 5'-regulatory region of mouse
mor gene, from nucleotide 1326 to the translation start
site designated +1. Arrows indicate the distal and proximal
transcription initiation sites (TIS). The box
indicates the previously identified 34-base pair negative
cis-acting element from
721 to
687 (11). The solid
left end of the box indicates the critical sequence for
mediating the negative effects. The identified Sox binding motif is
shown as an open oval. The positions of the oligos O1 and O2
arbitrarily selected for EMSAs are as indicated. B, EMSA was
performed with nuclear extracts (NE) from NMB cells. The
double-stranded O1 was selected as probe and 32P-labeled.
Lane 1, probe alone; lanes 2 and 3,
probe plus 5 µg of NMB nuclear extracts; lane 3, 100 molar
ratio excess of unlabeled O1 as a self-competitor. The
sequence-specific DNA-protein complexes are indicated by the symbols
>, sequence nonspecific complexes are indicated by
asterisks. C, NMB nuclear extracts bind to O2
sequence. EMSA was performed similar to Fig. 2B, except that
a shorter oligonucleotide O2 was 32P-labeled and used as
the probe in EMSA. Lane 1, probe alone; lanes 2 and 3, probe plus 5 µg of NMB nuclear extracts; lane
3, 100 molar ratio excess of unlabeled O2 as a
self-competitor.
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Fig. 3.
The Sox binding property of the identified
cis-acting element. A, the O1 sequence of the
mor distal promoter has four putative Sox-binding sites
(underlined or lined above on the O1 sequence).
The identified Sox binding motif in this study is also
underlined and in bold. The modified
oligonucleotides, O2 to O7, are used as cold competitors in EMSA. The
numbers in parentheses at both ends of O1
sequence represent the location of the sequence in mor
promoter. B, EMSAs were performed as in Fig. 2. Lane
1, O1 probe alone; lanes 2-5, probe O1 and 5 µg of
NMB nuclear extracts (NE). A published consensus Sox
binding sequence, 5'-GATCCGCGCCTTTGTTCTCCCCA-3'
(SSC, Sox binding motif is underlined) (19)
was used as a competitor in 20 molar ratio excess (lane 3),
40 molar ratio excess (lane 4), and 100 molar excess
(lane 5). The sequence-specific DNA-protein complexes are
indicated by the symbol >. C, A putative Sox-binding motif
( 731 to
725) within the O1 and O2 element is important for NMB
nuclear extract binding. EMSA was performed with nuclear extracts from
NMB cells. The O1 fragment was used as the probe in the absence or
presence of 100-fold molar excess of unlabeled competitors. Lane
1, O1 probe alone; lanes 2-8, 5 µg of nuclear
extract; lane 2, absence of competitor; lane 3,
cold competitor O2; lane 4, cold competitor O3; lane
5, cold competitor O4; lane 6, cold competitor O5;
lane 7, cold competitor O6; lane 8, cold
competitor O7. The major bands representing the sequence-specific
DNA-protein complexes are indicated by the symbol <.
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Fig. 4.
EMSA analysis of a Sox-binding motif using O2
mutant competitors. A, the O2 sequence was used as wild-type
probe. The numbers in parentheses at both ends of
the O2 sequence represent the location of the sequence in the
mor promoter. The Sox binding motif is in bold.
For mutant sequences, M6-M9, mutated sites are italicized
and underlined. B, the probe O2 was
32P-radiolabeled, and EMSA was performed with NMB nuclear
extracts. Competition with unlabeled mutant sequences was carried out
at 100-fold molar excess. Lane 1, O2 probe alone; lane
2, the control binding reaction in the absence of unlabeled
competitor; lanes 3-7, different competitors as indicated
at the top of each lane. The nonspecific
competitors Sp1 or Ikaros were used in lane 8 or
9, respectively, indicating the sequence specific
interaction. The bands representing the sequence-specific DNA-protein
complex are indicated by the symbol <.
812 and
575. As shown in Fig.
5, at a very high concentration of
nuclear extract from mouse brain (80 µg), characteristic DNase I-hypersensitive sites below nucleotides
739 and above
725 and a
protected region (
739 to
725) against DNase I digestion were observed. In contrast, relatively lower concentrations of the nuclear
extracts (20 or 40 µg) hardly altered the DNase I digestion pattern
of the probe (Fig. 4, compare lanes 2 and 3 with
lane 4). This may be the reason why we did not observe the
DNA-complex formation from mouse brain in EMSA, suggesting very low
abundance of the endogenous transcription factor(s) that bind to this
area in adult mouse brain.
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Fig. 5.
DNase I footprinting. Mouse brain
proteins bind to the mor distal promoter at the Sox-binding
sites. DNase I footprint analysis of the promoter template from 776
to
711 bp was performed with mouse brain nuclear extracts. The
template is labeled at the coding strand. Lane M,
A + T dideoxy-termination sequencing reaction as indicated A + T
ladder; lane 1, no protein added; lanes 2-4,
increasing amount of mouse brain nuclear extracts, as indicated at the
top of each lane. The positions of DNase I protection site
and Sox-binding sites are indicated by a gray box and
bracket, respectively.
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Fig. 6.
In vitro translated SOX proteins
specifically bind to the identified Sox binding motif. A,
EMSAs were performed as indicated under "Experimental Procedures."
Briefly, double-stranded 32P-labeled O2
cis-acting element was incubated with 8 µl of SOX18
proteins obtained by in vitro transcription and translation,
in the presence or absence of various unlabeled double-stranded
oligonucleotide competitors. Lane 1 is the control reaction
in absence of competitor. Lane 2 is with O2 self-competitor.
Competitor oligonucleotides are indicated at the top of each
lane and were used in 25-fold molar ratio excess (lanes
3-6). B, in vitro translated SOX21 was used
instead of SOX18.
(T cell
receptor
chain enhancer) (27), and SRY (sex region of the Y
chromosome) (28) proteins were chosen for EMSA (Fig.
7A). Based on the sequence
similarities among the Sox-binding sites between O2 and the related
oligonucleotides (Fig. 7A), the competitor TCF1
containing the same core sequences as O2 showed the highest binding
inhibition, i.e. a 40% reduction in band intensity relative to the band seen with only the O2 probe (Fig. 7B). The
competitors, TCF-MW56 and SOX5-BS12, differing by 2 and 4 bases,
respectively, from the core sequence of O2, are less effective at
competing with the probe, showing 19 and 18% reductions in band
intensity, respectively. The lowest competition was observed with SRY
sequence, an 8% reduction in intensity, which is a similar competition
ratio to the nonspecific binding sequence Sp1 (3% reduction). These results indicate that SOX18 has a preferential interaction with the
cis-acting element of the mor promoter compared
with other Sox-related binding sites, and the flanking sequences of the
binding site may also be important for the DNA-protein interaction as described above (Figs. 3, 4, and 6).
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Fig. 7.
SOX18 binds preferentially to the Sox-binding
site of mor promoter compared with the known Sox
binding consensus sequences from several different promoters.
A, sequence alignment of Sox-related binding sites from four
different promoters with O2 sequence and their relative binding
affinities compared with O2. The core binding sequences are
underlined and in bold. The relative band
intensity for the nonspecific competitor Sp1 is 97%, indicating the
sequence-specificity of the interaction. The competitors were added to
the reaction at concentrations 25 times higher than the labeled probe
O2. The band intensity was measured by PhosphorImager Storm 840 (Molecular Dynamics), and the relative values were calculated compared
with the O2 band. B, EMSA with in vitro
translated Sox18. The O2 oligonucleotide was radiolabeled and used as a
probe. Lane 1, O2 probe alone; lane 2, the
control binding reaction without competitor; lane 3,
self-competitor O2; lane 4, TCF-MW56 (T cell receptor) (26,
53); lane 5, SOX5-BS12 (26); lane 6, TCF1 (T
cell receptor
chain enhancer) (27); lane 7, SRY (sex
region of the Y chromosome) (28). The nonspecific competitor Sp1 was
used in lane 8.
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Fig. 8.
The identified Sox binding motif is important
for activation of the mor distal promoter by cloned
mSOX18. A, wild-type and mutational reporter constructs used
in the transient transfection and luciferase assays. The reporter
constructs are: wild-type, pL1.3K/721; deletion mutant pL1.3K/731;
mutant pL1.3K/721m2, mutated in the Sox-binding site; mutant
pL1.3K/721m123, which mutated both the Sox-binding site and its
flanking regions. The × mark in the filled oval
indicates the mutated Sox-binding site or its flanking sequences.
LUC represents the luciferase reporter gene. B,
mutational analysis of mouse mor distal promoter activity by
transient transfection assays in NMB cells. Transfection efficiencies
were normalized as described in Fig. 1. The activities of the
luciferase reporter were expressed as n-fold relative to the
activity of pGL3-Basic, which was assigned an activity value of 1.0. The data shown are the means of three independent experiments with at
least two different plasmid preparations. Error bars
indicate the range of standard errors. C, NMB cells were
co-transfected with the indicated amounts of pc1-Sox18 and either
wild-type pL1.3K/721 or mutant pL1.3K/721m123 plasmid. The relative
luciferase activity was calculated as the activity of each reporter in
the absence or presence of the indicated amount of pc1-Sox18 and
normalized by -galactosidase activity as described above.
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Fig. 9.
Sox18-induced activation of the
mor distal promoter is enhanced by SOX21.
A, NMB cells were co-transfected with pL1.3K/721 and the
increasing amounts of pc1-Sox21. Luciferase activities, normalized to
-galactosidase activity from a co-transfected LacZ vector (pCH110),
are expressed as fold activation of the luciferase activity of the
pL1.3K/721 plasmid, which is arbitrarily defined as 1.0. Error
bars indicate the range of standard errors. B, NMB
cells were co-transfected with 0.5 µg of pc1-Sox18, 0.5 µg of
pc1-Sox21, or 0.5 µg of pc1-Sox18 plus 0.5 µg of pc1-Sox21 together
with 1 µg of pL1.3K/721. Luciferase activities are expressed as
described in A. Results are the means of three independent
experiments. Error bars indicate the range of standard
errors. The empty vector pcDNA1.1/amp (Invitrogen) was added to
make an equal amount of 2 µg of DNA for each transfection.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
containing the same core-binding site as O2 but having different
flanking sequences competed for about 40% with the probe O2. The
others showed minor interactions, although they have very similar core
binding sequences compared with O2 (Fig. 7A). It was
previously reported that SOX proteins achieve DNA sequence specificity
through subtle preferences for flanking nucleotides and that this is
likely to be dictated by signature amino acids in their HMG domains
(43, 44). For example, the optimal SOX9 binding sequence, AGAACAATGG,
contained a core DNA binding element AACAAT, flanked by 5'-AG and 3'-GG nucleotides. The 5'-AG- and 3'-GG-flanking nucleotides enhance binding
by SOX9 HMG domain but not by the HMG domain of another SOX factor,
SRY. For SRY, different 5'- and 3'-flanking nucleotides are preferred
(43). Therefore, in the Sox binding motif of mor distal
promoter, the presence of at least two bases at the 5'-flanking region
and a specific four bases at 3'-flanking region of the core-binding
site are required for SOX protein binding (e.g. Sox18 and Sox21).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Li-Na Wei, Dr. Jane L. Ko, Dr. Santosh Talreja, Joan M. Tetrault, and Dr. Ursula D'Souza for helpful suggestions and manuscript review.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Research Grants DA00546, DA01583, DA05695, and K05-DA70554 and by the A&F Stark Fund of the Minnesota Medical Foundation.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY142959.
To whom correspondence should be addressed: Dept. of Pharmacology,
University of Minnesota Medical School, 6-120 Jackson Hall, 321 Church
St. S. E., Minneapolis, MN 55455. Tel.: 612-626-6539; Fax:
612-625-8408; E-mail: hwang025@umn.edu.
Published, JBC Papers in Press, November 22, 2002, DOI 10.1074/jbc.M208780200
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ABBREVIATIONS |
---|
The abbreviations used are: mor, µ opioid receptor; EMSA, electrophoretic mobility shift assay; SRY, sex-determining region of Y chromosome; HMG, high mobility group; Sox, Sry like HMG box; TCF, T cell factor.
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