Báo cáo khoa học: SOX10, in combination with Sp1, regulates the endothelin receptor type B gene in human melanocyte lineage cells pptx

SOX10, in combination with Sp1, regulates the endothelin
receptor type B gene in human melanocyte lineage cells
Satoru Yokoyama, Kazuhisa Takeda and Shigeki Shibahara
Department of Molecular Biology and Applied Physiology, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, Miyagi,
Japan
Waardenburg syndrome (WS) is an auditory–pigmen-
tary disorder, which is characterized by varying combi-
nations of sensorineural hearing loss, heterochromia
iridis, and patchy abnormal pigmentation of the hair
and skin [1]. WS is associated with the deficiency of
neural crest-derived melanocytes, and is classified into
four types, depending on the presence or absence of
additional symptoms [2–9]. WS type1 (WS1) and type
2 (WS2) are distinguished by the presence or absence
of dystopia canthorum, respectively. The presence of
limb abnormalities distinguishes WS type 3 (WS3)
from WS2. WS type 4 (WS4), referred to as Hirsch-
sprung’s disease type 2 or Shaa–Waardenburg syn-
drome, is characterized by the presence of the
aganglionic megacolon. WS1 and WS3 are caused by
mutations in the PAX3 gene [2], and some cases of
WS2 are associated with mutations in the microphthal-
mia-associated transcription factor (MITF) gene [3] or
SLUG (SNAI2) gene [4]. WS4 is due to mutations in
the endothelin receptor type B (EDNRB) gene [5,6],
the endothelin 3 (EDN3) gene [7,8], or the Sry-box 10
(SOX10) gene [9].
Keywords
endothelin receptor type B; melanocytes;
SOX10; Sp1; Waardenburg syndrome
Correspondence

K. Takeda, Department of Molecular Biology
and Applied Physiology, Tohoku University
School of Medicine, 2–1 Seiryo-machi,
Aoba-ku, Sendai, Miyagi 980–8575, Japan
Fax: +81 22 7178118
Tel: +81 22 7178114
E-mail:
(Received 15 November 2005, revised 20
February 2006, accepted 27 February 2006)
doi:10.1111/j.1742-4658.2006.05200.x
Waardenburg syndrome (WS) is an auditory–pigmentary disorder that
exhibits varying combinations of sensorineural hearing loss and abnormal
pigmentation of the hair and skin. WS type 4 (WS4), a subtype of WS, is
characterized by the presence of the aganglionic megacolon and is associ-
ated with mutations in the gene encoding either endothelin 3, endothelin
receptor type B (EDNRB), or Sry-box 10 (SOX10). Here, we provide evi-
dence that SOX10 regulates the expression of EDNRB gene in human
melanocyte-lineage cells, as judged by RNA interference and chromatin im-
munoprecipitation analyses. Human melanocytes preferentially express the
EDNRB transcripts derived from the conventional EDNRB promoter.
SOX10 transactivates the EDNRB promoter through the cis-acting ele-
ments, the two CA-rich sequences and the GC box. Moreover, a transcrip-
tion factor Sp1 enhances the degree of the SOX10-mediated transactivation
of the EDNRB promoter through these cis-acting elements. Furthermore,
we have shown that the EDNRB promoter is heavily methylated in HeLa
human cervical cancer cells, lacking EDNRB expression, but not in mel-
anocytes and HMV-II melanoma cells. The expression of EDNRB became
detectable in HeLa cells after treatment with a demethylating reagent,
5¢-aza-2¢-deoxycytidine, which was further enhanced in the transformed
cells over-expressing SOX10. We therefore suggest that SOX10, alone or in

combination with Sp1, regulates transcription of the EDNRB gene, thereby
ensuring appropriate expression level of EDNRB in human melanocytes.
Abbreviations
ChIP, chromatin immunoprecipitaion; EDN, endothelin; EDNRA, endothelin receptor type A; EDNRB, endothelin receptor type B; EMSA,
electrophoretic mobility shift assay(s); ENS, enteric nervous system; HMG, high mobility group; MITF, microphthalmia-associated
transcription factor; PAX3, paired box gene 3; siRNA, small interfering RNA; SOX10, Sry-box 10; WS, Waardenburg syndrome; YDBS,
Yemenite deaf–blind hypopigmentation syndrome.
FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS 1805
Several lines of evidence have suggested the functional
relationships between these WS-related transcriptional
factors. Expression of Mitf, a basic helix-loop-helix-
leucine zipper protein, is critical for the development of
melanocytes [10] and is preceded by the expression of
Pax3 and Sox10 in melanoblasts located dorsal to the
neural tube and migrated along the dorsolateral path-
way [11,12]. PAX3 affects the development of melano-
cytes in culture by regulating MITF expression [13].
Likewise, SOX10 activates the MITF gene promoter
[14–17].
Recently, Sox10 has been shown to regulate Ednrb
expression in the precursors of the enteric nervous sys-
tem (ENS) through the ENS enhancers, which contain
the Sox10-binding sites, in the mouse Ednrb gene [18].
In fact, Sox10 mRNA and Ednrb mRNA exhibit over-
lapping expression patterns in neural crest derivatives
in wild-type mice [19], whereas the Ednrb expression
is reduced in the dominant megacolon (Dom) mouse,
which carries the truncated mutation of Sox10 gene
[19]. The Dom mouse represents a model for human
congenital megacolon [19,20].

The SOX genes encode transcription factors with a
high-mobility group box (HMG box) as a DNA-bind-
ing motif [21]. SOX10 is defective in some cases of
WS4 [9,22,23] and in patients with Yemenite deaf–
blind hypopigmentation syndrome (YDBS) [24]. YDBS
is a rare disorder characterized by severe early hearing
loss, microcornea and colobomata, and cutaneous pig-
mentation abnormalities. These two syndromes exhibit
a remarkable difference in the phenotype of the enteric
nervous system; namely, aganglionic megacolon is
associated with WS4, but not with YDBS.
The mutations in the EDNRB gene are associated
with WS4, which is inherited in a dominant [6] or
recessive [5] mode. EDNRB belongs to a superfamily
of G protein-coupled receptors [25]. Its ligand, endo-
thelin (EDN), is a highly potent vasoconstricting pep-
tide of 21 amino acid residues and consists of three
subtypes EDN1, EDN2, and EDN3 [26]. EDNRB has
a high affinity for all three EDNs, whereas endothelin
receptor type A (EDNRA), a subtype of EDNR, pos-
sesses a higher affinity for EDN1 and EDN2 than
EDN3 [25,27,28]. The Ednrb gene is expressed postnat-
aly in various tissues, including the myenteric plexus,
mucosal layer, ganglia, and blood vessels of the sub-
mucosa of the colon [29,30]. Importantly, the Edn3-
Ednrb signaling is required for the terminal migration
of melanoblasts and the precursors of the ENS [31,32].
However, little is known about the regulatory mechan-
ism of Ednrb expression in melanocytes.
We have investigated the hierarchy between SOX10

and EDNRB in melanocyte-lineage cells. EDNRB
mRNA consists of at least four transcripts in human
melanoma cells, which are derived from different pro-
moters [33], termed conventional EDNRB, EDNRB D1,
EDNRBD2, and EDNRBD3. Each of the conventional
EDNRB, EDNRBD1 and EDNRBD2 transcripts
encodes the same EDNRB protein of 442 amino acids,
while the EDNRBD3-encoded protein contains addi-
tional N-terminal amino acids (89 residues), followed
by the EDNRB protein [33]. Here, we show that
the conventional EDNRB mRNA is preferentially
expressed in melanocytes, and that SOX10 activates
the conventional EDNRB promoter, alone or in com-
bination with a transcription factor Sp1. Furthermore,
the EDNRB promoter is heavily methylated in HeLa
human cervical cancer cells, which do not express the
EDNRB gene, but not methylated in melanocytes.
Notably, enforced expression of SOX10 induces the
expression of EDNRB in HeLa cells only when HeLa
cells were treated with a demethylating reagent, 5¢-aza-
2¢-deoxycytidine. The present study suggests that
SOX10 is responsible for appropriate expression of the
EDNRB gene in human melanocyte-lineage cells.
Results and discussion
SOX10 is required for the expression
of EDNRB gene
To investigate the expression profiles of SOX10 and
EDNRB mRNAs in normal human epidermal melano-
cytes (NHEM) and human melanoma cells, we carried
out northern blot analysis (Fig. 1A). SOX10 and

EDNRB mRNAs are coexpressed in NHEM and four
human melanoma cell lines. EDNRB mRNAs were
detected as two major bands of about 4300 and 1800
nucleotides, which are generated by the use of alternat-
ive polyadenylation sites [34]. To explore the hierarchy
among SOX10, EDNRB, and MITF, we carried out
the RNA interference analysis against SOX10 in
HMV-II melanoma cells (Fig. 1B). We initially con-
firmed that the expression of SOX10 protein was
reduced by the small interfering RNA (siRNA) against
SOX10 (65% reduction), but not by the LacZ siRNA
(Fig. 1B). Because SOX10 has been known as a trans-
activator for MITF gene [14–17], MITF could be used
as a positive control for the SOX10 siRNA. The
expression of MITF protein was reduced by the
SOX10 siRNA (69% reduction), but not changed by
the LacZ siRNA. These results confirm the regulatory
role of SOX10 in the expression of MITF, thereby
indicating that the SOX10 siRNA worked properly in
HMV-II cells. Likewise, EDNRB protein was reduced
by the SOX10 siRNA (46% reduction). Subsequently,
Regulation of EDNRB by SOX10 S. Yokoyama et al.
1806 FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS
we examined the expression of EDNRB mRNA in
those HMV-II cells by northern blot analysis, showing
that the expression of EDNRB mRNA was reduced in
HMV-II cells when the SOX10 siRNA was transfected
(upper band 15% and lower band 35% reduction)
(Fig. 1C). We repeated a similar experiment with the
SOX10 siRNA and confirmed the reduced expression

of EDNRB mRNA (upper band 10%, lower band
24% reduction) (data not shown). These results sug-
gest that SOX10 is required for the expression of
EDNRB gene in human melanocyte-lineage cells.
Under the conditions used, there were no noticeable
changes in the viability of the cells transfected with the
SOX10 siRNA, despite that SOX10 is required for
melanocyte survival. It is conceivable that the reduced
level of SOX10 in the experiment does not affect cell
survival. Alternatively, certain genes may compensate
for the down-regulation of SOX10 expression.
Identification of a major species of EDNRB gene
transcripts
EDNRB mRNA consists of at least four transcripts
with different 5¢-ends (conventional EDNRB,
EDNRBD1, EDNRB D2, and EDNRBD3), which are
derived from three promoters of the human EDNRB
gene [33] (Fig. 2A). Thus, EDNRB mRNA may consist
of eight isoforms, because each EDNRB transcript
may have the two different 3¢-ends (see Fig. 1), gener-
ated by the use of alternative polyadenylation sites
[34]. However, because of small differences in the
size of each transcript, it is practically impossible to
identify the four transcripts by northern blot analysis.
We therefore performed S1 nuclease mapping analysis
to identify EDNRB transcripts expressed in NHEM
and HMV-II melanoma cells (Fig. 2B). Three protec-
ted fragments of 436, 407, and 228 bases were detected
in NHEM and HMV-II cells but not in HeLa cervical
cancer cells. The two fragments of 436 and 407 bases

are preferentially detected and consistent with the con-
ventional EDNRB mRNA, transcribed from the two
adjacent transcriptional initiation sites [35]. The faint
signal of 228 bases represents the expression of
EDNRBD2 mRNA or EDNRBD3 mRNA. In contrast,
the signal for the EDNRB D1 transcript of 997 bases
was undetectable. These results indicate that the con-
ventional EDNRB mRNA is abundantly expressed in
human melanocytes. Furthermore, the alternative pro-
moters have not been reported in the mouse Ednrb
gene. In the present study, we thus focused on the
conventional promoter of the EDNRB gene, which
is termed, the EDNRB promoter, unless otherwise
specified.
Functional analysis of the EDNRB gene promoter
in melanocyte-lineage cells
We first analyzed the promoter activity of the EDNRB
gene by transient transfection assays in HMV-II mel-
anoma cells and HeLa cervical cancer cells (Fig. 3A).
The deletion study showed that the promoter activities
of the EDNRB reporter constructs were higher in
HMV-II cells than in HeLa cells by about twofold,
except for a construct pGL3-E ()12), carrying the
ABC
Fig. 1. MITF and EDNRB genes are regulated by SOX10. (A) Northern blot analysis of SOX10 and EDNRB mRNA. Total RNA was prepared
from the indicated cell lines at the top of the panel. Autoradiograms of the RNA blots hybridized with the indicated
32
P-labeled cDNA probes
are shown. The bottom panel shows the expression of 28S ribosomal RNA visualized by ultraviolet transilluminator (internal control). EDNRB
mRNA is detected as two bands, about 4300 nucleotides and 1800 nucleotides as described previously [34]. (B) Effects of siRNA against

SOX10 on the expression of SOX10, MITF and EDNRB protein. Whole cell extracts were prepared from HMV-II human melanoma cells
transfected with each siRNA against SOX10 (siSOX10) or LacZ (siLacZ), or untransfected cells (–). The bottom panel shows a-tubulin as an
internal control. (C) The effect of siRNA against SOX10 on the expression of EDNRB mRNA. Total RNA was prepared from the indicated
cells same as (B). b-actin mRNA was used as an internal control.
S. Yokoyama et al. Regulation of EDNRB by SOX10
FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS 1807
12-base pairs promoter region. Likewise, the promoter
activity of the EDNRB promoter was higher in normal
human epidermal melanocytes and other human
melanoma cell lines, 624mel (Fig. 3B), G361 and
SK-MEL-28 (data not shown) than that detected in
HeLa cells (Fig. 3B). Thus, the 5¢-flanking region
between )105 and )12 is required for the basal promo-
ter activity of the EDNRB gene in melanocytes-lineage
cells and may confer the marginal cell specificity on
the ENDRB promoter. There was noticeable difference
in the promoter activities in melanoma cells between
pGL3-E ()3002) and pGL3-E ()105) (Fig. 3A), which
suggests the presence of the negative elements for the
promoter activity in the deleted region. Such a differ-
ence in the promoter activity was also detected in
HeLa cells. On the other hand, the EDNRB promoter
contains the three potential SOX10 sites (Fig. 3A),
which correspond to the enteric nervous system (ENS)
enhancer in the mouse Ednrb gene [18]. The expression
levels of pGL3-Em, containing the mutations at the
three potential SOX10 sites, were lower than those of
a wild-type construct, pGL3-E ()3022), but the expres-
sion level of pGL3-Em is significantly higher in
HMV-II cells than that in HeLa cells. This observation

was also seen in normal human epidermal melano-
cytes, and 624mel human melanoma cell lines exam-
ined (Fig. 3B), and other human melanoma cell lines,
G361 and SK-MEL-28 (data not shown). These results
suggest that the potential SOX10 sites are dispensable
for melanocytes lineage-specific expression of EDNRB,
which is consistent in part with the report in the trans-
genic mouse analysis of the Ednrb gene [18].
Transactivation of the EDNRB gene promoter
by SOX10 and Sp1
We then analyzed the effect of SOX10 on the promoter
function of the EDNRB gene by transient cotransfection
A
B
Fig. 2. Identification of the major transcripts of the EDNRB gene in human melanocytes. (A) Schematic representation of the promoters of
the EDNRB gene. The EDNRB gene is shown as a line and four arrows show the transcriptional initiation sites of each transcript. The open
boxes of each transcript show the 5¢-untranslated region. The shadow boxes and dotted boxes indicate the protein-coding region of each
transcript. The transcripts derived from the conventional EDNRB, EDNRBD1 and the EDNRBD2 promoters code for the same protein. The
protein encoded by EDNRBD3 mRNA has D3-specific N-terminal amino acids (dotted boxes), followed by the common coding region (shaded
boxes). The splicing sites are shown by a gap. The number shown on the transcripts represents the position from the transcriptional initi-
ation site (+1) of the conventional EDNRB mRNA. The conventional EDNRB mRNA has two transcriptional initiation sites (+1 and +30) [35].
The S1 probe, shown at the bottom, contains the EDNRB gene sequences (line) and the vector sequence (dotted line). The end-labeled site
of S1 probe is the XcmI site (position +436 in the antisense strand of EDNRB cDNA), and is indicated with an asterisk. The predicted frag-
ments are shown below the transcripts (boxes), together with each predicted size. (B) S1 nuclease mapping analysis of EDNRB transcripts.
An autoradiogram of the S1 nuclease mapping analysis is shown. Total RNA was prepared from the indicated cell lines at the top of panel.
The S1 probe and four predicted fragments are indicated as arrows. Size markers were end-labeled with 1-kb DNA Ladder and 100-bp DNA
Ladder (New England Biolabs, Beverly, MA, USA).
Regulation of EDNRB by SOX10 S. Yokoyama et al.
1808 FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS
assays (Fig. 4A). SOX10 significantly increased the

expression levels of reporter constructs, containing the
promoter region between )105 and )12. The localized
promoter region ()105 and ) 12), which is also respon-
sible for the marginal melanocyte-lineage specificity of
the EDNRB promoter, contains the GC box, a consen-
sus sequence of the binding site for Sp1 (Fig. 4B).
Moreover, Sp1 has been reported to interact with
SOX10 [36,37]. We also confirmed the interaction of
SOX10 and Sp1 by in vitro pull-down assay (data not
shown). We therefore analyzed whether Sp1 influences
the function of the EDNRB promoter. An Sp1 expres-
sion plasmid was coexpressed with SOX10 expression
plasmid in HeLa cells, which endogenously express Sp1
protein [38]. We thus confirmed the over-expression of
Sp1 protein in HeLa cells, when transfected with Sp1
expression plasmid (Fig. 4C). Sp1 or SOX10 alone
increased the promoter activity of pGL3-E ()3022)
A
B
Fig. 3. The EDNRB gene promoter shows the melanocyte lineage cell-specific activity. (A) Functional analysis of the EDNRB gene promoter
in melanoma cells. The left panel shows the reporter plasmids used. The number shown at the 5¢-or3¢-end of each construct represents the
position from the transcriptional initiation site (+1). The open circles on the EDNRB gene show the potential SOX10 sites, which correspond
to the enteric nervous system (ENS) enhancers in mice [18]. The internal deletion site is shown as a gap. X shows the mutation of each
potential SOX10 site. Relative luciferase activity in transient transfection assays is shown on the right. The cell lines used are HeLa cells
(open bars) and HMV-II cells (filled bars). Luciferase activity was normalized by each internal control activity (pRL-TK). Relative luciferase activ-
ity is shown as the ratio to the normalized luciferase activity obtained with pGL3-E ()3022) in HeLa cells. Data are mean ±
SD of at least
three independent experiments. The activity with * or ** is significantly higher in HMV-II cells than the value obtained with each reporter
plasmid in HeLa cells, P < 0.01 or P < 0.05. (B) Functional analysis of the EDNRB gene promoter in melanocytes-lineage cells. The left panel
shows the reporter plasmids used. Relative luciferase activity in transient transfection assays is shown on the right. The cell lines used are

HeLa cells, HMV-II and 624mel melanoma cells (dotted bars), and normal human epidermal melanocytes (NHEM) (shadow bars). Luciferase
activity was normalized by each internal control activity (pRL-TK). Relative luciferase activity is shown as the ratio to the normalized luciferase
activity obtained with pGL3-E ()3022) in HeLa cells. Data are mean ±
SD of at least three independent experiments. The activity with * is
significantly higher in melanoma cells and NHEM than the value obtained with each reporter plasmid in HeLa cells, P < 0.01 or P < 0.05.
S. Yokoyama et al. Regulation of EDNRB by SOX10
FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS 1809
1.9- or 3.9-fold, respectively (Fig. 3C). The combination
of SOX10 and Sp1 led to an 8.0-fold increase, suggest-
ing that SOX10 and Sp1 synergistically transactivate
the EDNRB promoter. Furthermore, SOX10, alone or
in combination with Sp1, significantly increased the
expression of pGL3-Em, containing the mutations at
the three potential SOX10 sites in the putative ENS
enhancer. It is therefore conceivable that these potential
SOX10 sites may be dispensable for the SOX10-medi-
ated transactivation. Taken together, the localized
promoter region ()105 and )12) is responsible not only
for the marginal melanocyte-lineage specificity of the
A
B
C
Fig. 4. SOX10 and Sp1 synergistically trans-
activate the EDNRB promoter. (A) Effects of
SOX10 on the EDNRB promoter. The right
panel shows the result of the transient
transfection assay in HeLa cells. The co-
transfection with empty vector (–) or SOX10
expression vector (SOX10) is shown as a
white bar or a shadow bar, respectively. Rel-

ative luciferase activity is shown as the ratio
to the normalized luciferase activity obtained
with cotransfection of pGL3-E ()3002) and
empty vector. Data are mean ±
SD of at
least three independent experiments. The
activity with * is significantly higher than the
value obtained with cotransfection of each
reporter plasmid and empty vector,
P < 0.01. (B) Effects of Sp1 on the SOX10-
mediated transactivation of the EDNRB pro-
moter. Shown is the nucleotide sequence of
the localized promoter region, in which a GC
box is underlined. HeLa cells were cotrans-
fected with each reporter shown to the left,
and an empty vector (open bars), Sp1
expression vector (dotted bars), SOX10
expression vector (shadow bars), or both of
Sp1 and SOX10 expression vectors (filled
bars). Relative luciferase activity is shown
as the ratio to the normalized luciferase
activity obtained with cotransfection of
pGL3-E ()3002) and the empty vector. Data
are mean ±
SD of at least three independent
experiments. The activity with * is signifi-
cantly higher than the value obtained with
each reporter plasmid and empty vector,
P < 0.01. (C) Over-expression of Sp1 and
SOX10 in HeLa cells. Enforced-expression

of Sp1 and ⁄ or SOX10 was assessed in
HeLa cells transfected with Sp1 and ⁄ or
SOX10 expression vector by western blot
analysis. Note that the amount of Sp1 pro-
tein was increased in the cells transfected
with Sp1 expression vector compared to
that in mock-transfected HeLa cells shown
as (–).
Regulation of EDNRB by SOX10 S. Yokoyama et al.
1810 FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS
EDNRB promoter but also for the SOX10-mediated
transactivation.
Identification of the cis-acting elements
responsible for the SOX10-mediated
transactivation of the EDNRB promoter
Within the region between )105 and )12 of the EDNRB
promoter, there is no consensus sequence, 5¢-(A ⁄ T)
(A ⁄ T)CAA(A ⁄ T)-3¢, for SOX10 binding. Instead, there
are the two CA-rich sequences (CA1 and CA2) and
the GC box (5¢-CCGCCC-3¢) (GC1). Notably, the
CA2 is overlapping with the GC box (Fig. 5A). It has
been reported that SOX10 binds the CA-rich sequence
in the neuronal nicotinic acetylcholine receptor b four
subunit gene promoter [39]. We therefore examined
whether CA1, GC1, or CA2 is involved in the basal
promoter activity and ⁄ or the SOX10-mediated transac-
tivation of the EDNRB promoter (Fig 5B,C). Each
base change at CA1, GC1 or CA2 resulted in the signi-
ficant decrease in the promoter activity in normal mel-
anocytes and HMV-II melanoma cells (Fig. 5B). The

GC box is especially important for the basal promoter
activity. Likewise, each base change abolished the
SOX10-mediated transactivation of the EDNRB pro-
moter (Fig. 5C), indicating that the three elements,
CA1, GC1 and CA2, are responsible for the transacti-
vation of the EDNRB promoter by SOX10.
SOX10 binds to the CA-rich sequences and the
GC box of the EDNRB promoter in vitro
We carried out electrophoretic mobility shift assay
(EMSA) using the labeled probes, which include CA1
(CA1 probe) or GC1 and CA2 (GC1 ⁄ CA2 probe)
(Fig. 5A,D). These oligonucleotide probes were incu-
bated with the lysates containing SOX10 protein syn-
thesized by in vitro transcription ⁄ translation system.
The CA1 probe was specifically bound by recombinant
SOX10 (Fig. 5D, left panel). The formation of the
SOX10–DNA complexes was inhibited by CA1 probe,
but the degree of inhibition with a competitor contain-
ing mutated CA1 (mCA1) was lower than that of CA1
probe. When we used the consensus SOX10-binding
site (cSOX10) as a competitor, which contains the
SOX10-binding site in human MITF gene promoter
[14–17], the formation of the SOX10-DNA complexes
was reduced. The SOX10–DNA complexes were not
detected with the lysates in the case of an empty vector
as a negative control. Thus, SOX10 binds to CA1 in
the EDNRB promoter. Unexpectedly, the degree of
competition with the mutated CA1 was similar to that
with cSox10 competitor, which may be due to addi-
tional SOX10 binding sites in the region of CA1

probe. Likewise, we showed that SOX10 specifically
bound to GC1 and CA2, as the SOX10–DNA com-
plexes were competed by the GC1 ⁄ CA2 probe or the
cSOX10 probe, but not by a mutated GC1 and ⁄ or
CA2 (mGC1 ⁄ CA2, GC1 ⁄ mCA2, or mGC1 ⁄ mCA2)
(Fig. 5D, right panel). Taken together, these results
suggest that SOX10 binds to the three cis-acting ele-
ments, CA1, GC1, and CA2, in the EDNRB promoter,
which is consistent with the results of the cotransfec-
tion assays.
Synergistic activation of the EDNRB promoter
by SOX10 and Sp1 through the two CA-rich
sequences and the GC box
We analyzed the functional significance of CA1, GC1,
and ⁄ or CA2 in the EDNRB promoter activity
(Fig. 6A). Each base change at CA1 and ⁄ or GC1 sig-
nificantly reduced the degree of activation caused by
SOX10 and Sp1, compared to the parent reporter plas-
mid, pGL3-E ()3022). The base change at CA2
showed 35% reduction compared to E ()3022) (P ¼
0.006). These results suggest that SOX10 and Sp1
synergistically transactivate the EDNRB promoter
through the two CA-rich sequences and the GC box.
To examine whether Sp1 binds to GC1, we performed
EMSA using the labeled GC1 ⁄ CA2 probe and recom-
binant human Sp1 protein (Fig. 6B). The Sp1-DNA
complexes were detected, and their formation was
completely competed by a wild-type GC1 ⁄ CA2 probe,
mutated CA2 probe (GC1 ⁄ mCA2), or a consensus
Sp1-binding sequence (cSp1). Interestingly the complex

formation was competed by mGC1 ⁄ CA2 probe, but
its competition ability was lower than that of the
GC1 ⁄ CA2 probe. Furthermore, mGC1⁄ mCA2 probe
did not inhibit the complex formation. These results
suggest that Sp1 recognizes the region containing GC1
and CA2. The binding of Sp1 to GC1 may be influ-
enced by the overlapping CA2.
We then examined the simultaneous binding of
SOX10 and Sp1 proteins to the GC1 ⁄ CA2, but were
unable to detect the complexes, containing both
SOX10 and Sp1 proteins (data not shown). It is con-
ceivable that the in vitro binding conditions are not
suitable for simultaneous binding of the two proteins
to the GC1 ⁄ CA2.
SOX10 and Sp1 bind to the EDNRB promoter
in vivo
To investigate whether SOX10 and ⁄ or Sp1 bind to the
EDNRB gene in vivo, chromatin immunoprecipitaion
S. Yokoyama et al. Regulation of EDNRB by SOX10
FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS 1811
A
B
C
D
Fig. 5. Identification of the cis-acting ele-
ments responsible for the SOX10-mediated
transactivation of the EDNRB promoter. (A)
Schematic representation of the EDNRB
promoter. The number represents the posit-
ion from the transcriptional initiation site

(+ 1) of the EDNRB gene. The two CA-rich
sequences and the GC box are marked. The
CA1 probe (line) and the GC1 ⁄ CA2 probe
(dotted line) are used in electrophoretic
mobility shift assay of Fig. 4(D). (B) Require-
ment of CA1, GC1, and CA2 for the melano-
cyte lineage cell-specific activity. Base
changes were introduced into CA1, GC1,
or CA2 of a parent plasmid, pGL3-E
()3022) reporter vector. X shows the muta-
tion introduced into CA1, GC1, or CA2.
HeLa cells (open bars), HMV-II cells (filled
bars), or normal human epidermal melano-
cytes (NHEM) (shaded bars) were transfected
with each reporter shown to the left. Other
conditions are represented as indicated in
Fig. 3(A,C). Effects of the base change at
CA1, GC1, or CA2. The transactivation of
the EDNRB promoter by SOX10 was
assessed in the case of each mutation
shown as X. Other conditions are represent-
ed as indicated in Fig. 3(B). (D) Electro-
phoretic mobility shift assays (EMSA) for
binding of SOX10 to the EDNRB promoter.
Shown are the autoradiographs of EMSA
with the CA1 probe (left) or the GC1 ⁄ CA2
probe (right). Lanes shown as (–) indicate no
protein or no competitor. The mock shows
the lysate containing a parent vector,
pIVEX3.2-MCS. SOX10 shows the lysate

containing SOX10 protein synthesized by
the in vitro transcription ⁄ translation
Escherichia coli lysate system. The compe-
titors used are wild-type probe or the
mutated probe (shown as Fig. 5A). The
consensus SOX10-binding site of human
MITF gene promoter is used as a compet-
itor (cSOX10). The arrows show the
SOX10–DNA complexes.
Regulation of EDNRB by SOX10 S. Yokoyama et al.
1812 FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS
(ChIP) assay was performed in HMV-II cells
(Fig. 7A). The PCR primer sets were designed to
amplify the DNA segments containing CA1, GC1, and
CA2 of the EDNRB promoter, which is responsible for
the SOX10-mediated transactivation. ChIP assay
revealed that the DNA segments of the EDNRB pro-
moter were amplified when precipitated with rabbit
anti-SOX10 IgG or goat anti-Sp1 IgG in HMV-II
cells, but not noticeably amplified when precipitated
with normal rabbit IgG (negative control) or normal
goat IgG (negative control) (Fig. 7B). These results
indicate that SOX10 and Sp1 bind to the region con-
taining CA1, GC1, and CA2 in the EDNRB promoter
in vivo. We could not detect the amplified fragments of
GAPDH gene as a negative control, when precipitated
with rabbit anti-SOX10 IgG or goat anti-Sp1 IgG.
SOX10 activates the EDNRB promoter in the
demethylation status
It has been reported that the EDNRB promoter is

located in the CpG islands, which are target sites of
DNA methylation [40–42]. In fact, the EDNRB pro-
moter is DNA-methylated in several types of tumor
A
B
Fig. 6. Sp1 is involved in the SOX10-medi-
ated transactivation of the EDNRB promot-
er. (A) Effects of Sp1 on the transactivation
of the EDNRB promoter. HeLa cells were
cotransfected with each reporter shown on
the left, and an empty vector (open bars),
Sp1 expression vector (dotted bars), SOX10
expression vector (shadow bars), or both of
Sp1 and SOX10 expression vectors (filled
bars). Relative luciferase activity is shown
as the ratio to the normalized luciferase
activity obtained with cotransfection of
pGL3-E ()3002) and the empty vector. Data
are mean ±
SD of at least three independent
experiments. The activity with # or ### is
significantly lower than the value obtained
with cotransfection of pGL3-E ()3002),
SOX10, and Sp1, #P < 0.01 or
###P < 0.001. (B) EMSA for the in vitro
binding of Sp1 to the EDNRB promoter.
Shown is the autoradiograph of the EMSA
by using the GC1 ⁄ CA2 probe. The recomb-
inant Sp1 was used. The competitors used
are wild-type probe or the mutated probe

(shown as Fig. 4A). The consensus Sp1
binding site is used as a competitor (cSp1)
[56]. The arrows show the Sp1–DNA com-
plexes.
S. Yokoyama et al. Regulation of EDNRB by SOX10
FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS 1813
cells, leading to gene silencing [40–43]. Moreover, the
GC1, identified as one of the key regulatory elements,
includes the CpG dideoxynucleotides, which are poten-
tial targets of DNA methylation (Fig. 8A). We there-
fore investigated the DNA methylation status of the
EDNRB promoter in normal human epidermal mel-
anocytes (NHEM), HMV-II melanoma cells, and
HeLa cells. The EDNRB promoter is heavily methyla-
ted in HeLa cells, but not in NHEM and HMV-II
cells (Fig. 8A). Subsequently, we examined whether
enforced expression of SOX10 induces the endogenous
EDNRB expression in HeLa cells. We established
FLAG-tagged SOX10 (F⁄ SOX10)-expressing stable
transformants from HeLa cells, and then chose the sta-
ble transformants #6 and #8, which appear to express
F ⁄ SOX10 more abundantly than other transformants
(Fig. 8B). Expression of EDNRB mRNA was unde-
tectable in cells transformed with the empty vector
(Mock) and the F ⁄ SOX10-expressing cells, as judged
by RT-PCR analysis (Fig. 8C). However, in the mock-
transformed cells, expression of EDNRB mRNA
became detectable after treatment with a demethylating
reagent, 5¢-aza-2¢-deoxycytidine (5¢-aza-dC) (Fig. 8C).
Importantly, the expression levels of EDNRB mRNA

were higher in F ⁄ SOX10-expressing cells after treat-
ment with 5¢-aza-dC (Fig. 8C). These results indicate
that SOX10 transactivates the endogenous EDNRB
promoter in the demethylation status. The induction of
the EDNRB expression by 5¢-aza-dC alone in mock-
transformed cells may be directed by Sp1, which is
expressed in HeLa cells [38].
Thus, the degree of the methylation in the EDNRB
promoter determines the transcription levels of the
EDNRB gene. However, we were unable to assess the
contribution of the methylation status to the promoter
activity by the transient expression assay, which could
account in part for a small difference in the melano-
cyte lineage-specific promoter activity. Alternatively,
the human EDNRB gene contains an additional mel-
anocyte-enhancer, which is however, not carried by the
reporter constructs used, containing the 3-kb length of
human EDNRB gene promoter, although the 1.2-kb
length of mouse Ednrb gene promoter is sufficient for
melanocyte-specific activity [18].
Implications
SOX10 interacts with Sp1 and activates the EDNRB
promoter. Sox10 also interacts with Pax3 and activates
the c-RET promoter [44,45]. The mutation in the
c-RET gene is responsible for the pathogenesis of
aganglionic megacolon [46]. Ubiquitously expressed
Sp1 may affect the pathogenesis of WS4 by cooper-
ating directly with SOX10 or by influencing the inter-
action of SOX10 with PAX3.
It should be noted that WS4 shows the phenotypic

variability [9,22]. In one family case of WS4, for exam-
ple, the proband and his sister are heterozygous for
the Q377X mutation of SOX10, but only the proband
has an aganglionic megacolon [22]. These observations
suggest the presence of modifier genes for the EDNRB
gene, and the expression of the modifier genes may be
influenced by the environmental factors, thereby lead-
ing to the phenotypic variability of WS4. One of such
modifiers might be Sp1, the function of which is
modulated growth factors [47] or metals [48].
In summary, we have provided evidence that
SOX10, alone or in combination with Sp1, may acti-
vate transcription of the human EDNRB gene, which
contributes to melanocyte lineage cell-specific expres-
sion, and that the regulation of EDNRB expression by
SOX10 requires the demethylation status of its promo-
ter. The regulatory network involving SOX10 and Sp1
may ensure the fine-tuning of EDNRB expression,
which contributes the homeostasis of human melano-
cytes. Future research on the genetic network of WS
genes will help clarify the pathogenesis of WS.
A
B
Fig. 7. Binding of SOX10 and Sp1 to the EDNRB promoter in vivo.
(A) Strategy for chromatin immunoprecipitation assay. Arrows indic-
ate the PCR primers used for the EDNRB gene promoter. (B)
In vivo binding of SOX10 and Sp1 to the EDNRB promoter. HMV-II
cells were fixed with formaldehyde, precipitated with anti-SOX10,
anti-Sp1, normal rabbit IgG, or normal goat IgG, and subjected to
PCR for the EDNRB promoter (top panel) or GAPDH gene (bottom

panel) as a negative control. The amplified DNA segments were
visualized with ultraviolet transilluminator.
Regulation of EDNRB by SOX10 S. Yokoyama et al.
1814 FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS
Experimental procedures
Cell culture
Human normal epidermal melanocytes (NHEM) were
obtained from Kurabo (Kurabo, Osaka, Japan) and cul-
tured in Medium 154S (Kurabo) containing human melano-
cyte growth supplement (Kurabo) at 37 °C under 5%
CO
2
⁄ 95% room air. HMV-II human melanoma cells were
obtained from RIKEN Cell Bank and cultured in nutrient
mixture Ham’s F12 medium containing 10% fetal bovine
serum at 37 °C under 5% CO
2
⁄ 95% room air. Human mel-
anoma cell lines, G361 and SK-MEL-28, were obtained
from Cell Resource Center for Biomedical Research (Insti-
tute of Development, Aging and Cancer, Tohoku Univer-
sity, Miyagi, Japan). 624mel human melanoma cells were
provided by Y. Kawakami (Division of Cellular Signaling,
Institute for Advanced Medical Research, School of Medi-
cine, Keio University, Tokyo, Japan). G361 and 624mel
cells were cultured in RPMI-1640 medium supplemented
with 10% fetal bovine serum. SK-MEL-28 cells were cul-
tured in minimum essential medium Eagle supplemented
with 10% fetal bovine serum. HeLa human uterine cervical
cancer cells and COS-7 monkey kidney cells were grown in

Dulbecco’s modified Eagle’s medium supplemented with
10% fetal bovine serum.
RNA interference
The small interfering RNA (siRNA) against SOX10 (927–
1753 numbered in accession number NM_006941) or LacZ
were generated by BLOCK-iT
TM
Complete Dicer RNAi
Kit protocol (Invitrogen, Carlsbad, CA, USA). HMV-II
melanoma cells were cultured for 24 h after plating in 6-cm
dishes and then transfected with each siRNA against
SOX10 or LacZ by Lipofectamine
TM
2000 protocol (Invitro-
gen). The cells were incubated for 3 days and then each si-
RNA was transfected again. After further 3 days, the cells
were harvested for northern blot analysis and western blot
analysis.
A
B C
Fig. 8. SOX10 activates transcription of the EDNRB gene in the demethylation status. (A) Methylation status of the EDNRB promoter. DNA
methylation status was determined in normal human epidermal melanocytes (NHEM), HMV-II melanoma cells, and HeLa cervical cancer
cells. Note that the EDNRB promoter is heavily methylated in HeLa cells, lacking EDNRB mRNA expression. (B) Isolation of stable transform-
ants, which express SOX10. HeLa cells were transfected with FLAG-tagged SOX10 (F ⁄ SOX10) or the empty vector (Mock), and four inde-
pendent clones were isolated, named #1–4 for Mock-transformed cell lines and #5–8 for F ⁄ SOX10. The western blot analysis with
anti-FLAG IgG (top panel) or anti-a-tubulin IgG (bottom panel) is shown. (C) Demethylation induces expression of the endogenous EDNRB
gene. Indicated stable transformants (#1, 2, 6, and 8) were untreated or treated with a demethylating reagent, 5¢-aza-dC for 7 days. The
stained gels of the RT-PCR products, obtained at logarithmic phase, are shown. The induction of EDNRB mRNA was enhanced in F ⁄ SOX10-
expressing cells.
S. Yokoyama et al. Regulation of EDNRB by SOX10

FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS 1815
Northern blot analysis
Total RNA was prepared by TRIzol reagent (Invitrogen)
and subjected to northern blot analysis as described previ-
ously [49]. The probes used were the human SOX10 cDNA
fragment (278–1753 numbered in accession number
NM_006941) and the human EDNRB cDNA fragment
(550–1268 numbered in accession number NM_003991) and
the human b-actin cDNA (124–1050 numbered in accession
number X00351). These DNA fragments were labeled with
[a-
32
P]dCTP using a BcaBEST labeling kit (Takara biomed-
ical, Shiga, Japan) and were used as hybridization probes.
Radioactive signals were detected by exposing the filters to
X-ray films (X-AR5; Kodak) or with a Bioimage Analyzer
(BAS1500; Fuji Film). The exposure time to X-ray films
varied depending on the experiments. The intensities of
hybridization signals were determined by photo-stimulated
luminescence with a Bioimage Analyzer.
Western blot analysis
Whole cell extracts were prepared from the cells transfected
with siRNA or the expression plasmid for SOX10 or Sp1
by the method of Schreiber et al. [50] and then subjected to
western blot analysis using anti-SOX10 IgG (Sigma-Aldrich
Japan, Tokyo, Japan), anti-MITF polyclonal IgG [51], anti-
Sp1 IgG (Santa Cruz Biotechnology, Santa Cruz, CA,
USA), anti-FLAG IgG (Invitrogen), or anti-a-tubulin IgG
(NeoMarkers, Fremont, CA, USA), as described previously
[51]. The relative amount of protein was determined by

NIH Image 1.63 f software. The amount of SOX10, MITF,
or EDNRB protein is normalized by that of a-tubulin
protein.
S1 nuclease mapping analysis
Total RNA was isolated from NHEM, HMV-II cells, and
HeLa cells with TRIzol reagent. An S1 probe used was the
fragment of the EDNRB gene (1–1438 numbered in acces-
sion number D13162), end-labeled with [c-
32
P] ATP at
XcmI site, located at the position 1438 in the antisense
strand of the EDNRB gene. The S1 probe contains the
pGEM-T Easy vector (Promega, Madison, WI, USA)
sequence, the 5¢-flanking region and exon 1 of the EDNRB
gene (Fig. 2A). S1 nuclease mapping analysis was per-
formed with 20 lg of total RNA from NHEM, HMV-II,
or HeLa cells, or yeast tRNA (negative control), as des-
cribed previously [52,53].
Plasmids construction
A human SOX10 expression vector, pcDNA3-SOX10, was
described previously [17]. A human Sp1 expression vector,
pCIneo-Sp1, was a gift from Dr Kojima (The Institute of
Physical and Chemical Research, Tsukuba Life Science Cen-
ter, Tsukuba, Ibaraki, Japan) [54]. To construct the FLAG-
tagged SOX10 expression vector, p3XFLAG-SOX10, the
HindIII ⁄ XbaI fragment of pcDNA3-SOX10 was inserted
into p3XFLAG-CMV7.1 vector (Sigma-Aldrich Japan). The
5¢-flanking region (position )3022 to +160 from the tran-
scription initiation site of the conventional EDNRB mRNA)
of the human EDNRB gene was generated by genomic

polymerase chain reaction (PCR) and was inserted into the
luciferase reporter plasmid pGL3-Basic (Promega), yielding
pGL3-E ()3022). Likewise, deletion constructs, pGL3-E
()1002), pGL3-E ()678), pGL3-E () 493), pGL3-E ()105),
and pGL3-E ()12) were generated. The internal deletion
plasmid, pGL3-ED, was constructed by deleting the
XmnI ⁄ MscI fragment from pGL3-E ()3022). Base changes
were introduced into pGL3-E ()3022) using QuickChange
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA,
USA) according to manufacturer’s instruction. The resulting
mutant constructs, pGL3-EmCA1, pGL3-EmGC1, pGL3-
EmCA2, and pGL3-Em3, carry a single or a combination of
the following base changes; CA1 (CACACCCC) was chan-
ged to mCA1 (CtCgagCC), GC1 (CCGCCC) to mGC1
(CCGaaC), and CA2 (CCACTGC) to mCA2 (CCcCgGg).
The mutant construct, pGL3-Em, carries the mutation of
three potential SOX10 sites; TTCAAT, ATTGAT, and
GTTGAA were changed into cTCgAg, ATTaAT, and
GTcGAc, respectively.
Transient transfection assays
The activities of the human EDNRB gene promoters were
assessed by transient expression of firefly luciferase genes
in HeLa cervical cancer cells, or human melanoma cells
HMV-II, G361, SK-MEL-28 or 624mel, and normal human
epidermal melanocytes as described previously [53]. Cells
used were cultured for 24 h after plating in 12-well dishes
and then transfected with each reporter plasmid (120 ng),
pRL-TK (20 ng), and SOX10 cDNA (50 ng), Sp1 cDNA
(50 ng), or an empty expression vector by FuGENE 6
(Roche Diagnostics, Mannheim, Germany). Total amount

of plasmids transfected was 500 ng per well. pRL-TK con-
tains the herpes simplex virus thymidine kinase promoter
region upstream of Renilla luciferase (Promega). The cells
were harvested 24 or 48 h after transfection, and then lucif-
erase activity was measured by Dual-Luciferase Reporter
Assay System (Promega). Firefly luciferase activity was nor-
malized with each Renilla luciferase activity.
EMSA
SOX10 cDNA was subcloned in the pIVEX2.3-MCS
(Roche Diagnostics), which was used for the production of
recombinant SOX10 protein by the RTS100 (Roche Diag-
nostics) in vitro transcription ⁄ translation Escherichia coli
Regulation of EDNRB by SOX10 S. Yokoyama et al.
1816 FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS
lysate system according to manufacturer’s instruction
(Roche Diagnostics). A parent vector pIVEX2.3-MCS was
also used as a negative control. The recombinant human
Sp1 protein was purchased from Promega. EMSA was per-
formed as previously described [55]. In brief, double-stran-
ded oligonucleotides, CA1 probe (5¢-CATTCCCTCCCTG
GCACACCCCTTCCAGAACGCCCC-3¢) and GC1 ⁄ CA2
probe (5¢-GATCCCCTTCCAGAACGCCCCGCCCCACT
GCATATTATTTACCCCTCCA-3¢) were end-labeled with
[c-
32
P] ATP and used as probes. The SOX10-containing
lysates were preincubated for 30 min on ice with or without
a competitor in the absence of a probe and then incubated
with the probe for 30 min on ice in 20 lL solution contain-
ing 1 mgÆmL

)1
bovine serum albumin (BSA), 0.1 lg poly
(dI-dC), 10 mm 4-(2-hydroxyethyl) piperazine-1-ethanesulf-
onic acid (Hepes) (pH 7.5), 50 mm NaCl, 5 mm MgCl
2,
0.5 mm ethylenediamine-N,N,N¢,N¢-tetraacetic acid (EDTA),
0.1 mm dithiothreitol (DTT), and 5% glycerol. The reac-
tion mixture was loaded on 5% polyacrylamide gels
(PAGE) and run in 0.5 · TBE buffer (50 mm Tris, 1 mm
EDTA, 48.5 mm boric acid). The gels were dried and auto-
radiographed to detect mobility shift. Competitors used
were mCA1 (5¢-CATTCCCTCCCTGGCtCgagCCTTCCA
GAACGCCCC-3¢), mGC1 (5¢-CCCCTTCCAGAACGCCC
CGaaCCACTGCATATTATTTAC-3¢), mCA2 (5¢-GAACG
CCCCGCCCCcCgGgATATTATTTACCCC-3¢), mGC1 ⁄
mCA2 (5¢-CCCCTTCCAGAACGCCCCGaaCCcCgGgAT
ATTATTTACCCC-3¢), a consensus Sp1 binding site
(cSp1) (5¢)ATTCGATCGGGGCGGGGCGAGC-3 ¢) [56],
and a consensus SOX10 binding site (cSOX10) (5¢-AGAG
AAATACCATTGTCTATTAATACT-3¢).
Chromatin immunoprecipitation (ChIP) assay
ChIP was performed according to the methods, reported
previously [51,57]. Briefly, formaldehyde was added to the
culture medium of HMV-II cells at the final concentration
of 1%; after 10 min the cells were washed with phosphate-
buffered saline, collected, and sedimented at 5000 g for
5 min. The pellet was resuspended in 5 mm of 1,4-piperaz-
inediethanesulfonic acid (pH 8.0), 85 mm KCl, 0.5% Noni-
det P-40, and protease inhibitor cocktail (Sigma-Aldrich
Japan) for 5 min. Nuclei were collected by centrifugation at

5000 g for 5 min, then resuspended in 50 mm Tris ⁄ HCl
(pH 8.1), 10 mm EDTA, and 1% sodium dodecyl sulfate
(SDS), and incubated on ice for 10 min. The chromatin
was fragmented by sonication to 1000–600 basepairs. For
immunoprecipitation, the solution was diluted with immu-
noprecipitation dilution buffer (0.01% SDS, 1.1% Triton
X-100, 1.2 mm EDTA, 16.7 mm Tris ⁄ HCl, pH 8.1, and
167 mm NaCl) and incubated with anti-SOX10 IgG (Sigma-
Aldrich Japan), anti-Sp1 IgG (Santa Cruz Biotechnology),
normal rabbit IgG (Santa Cruz Biotechnology), or normal
goat IgG (Santa Cruz Biotechnology) at 4 °C overnight.
Protein G MicroBeads (Miltenyi Biotec, Gladbach,
Germany) were then added to the mixture and rotation was
continued for 2 h. After immune complexes were washed,
cross-link reversal step was performed by addition of
300 mm NaCl and heating at 65 °C overnight. DNA was
ethanol precipitated, treated with proteinase K, extracted
with phenol ⁄ chloroform, and finally ethanol precipitated,
which was subjected to PCR for the amplification of the
EDNRB gene promoter region. In the case of the EDNRB
promoter region, the primers used were 5¢-CATTCCCTCC
CTGGCACACCCCTTCCAGAACGCCCC-3¢ as a for-
ward primer and 5¢-CGAGCCAAGTCGCTGCAAACGCT
AATACCG-3¢ as a reverse primer. GAPDH gene was ampli-
fied as a negative control. The primers used were 5¢-TGG
TATGACAACGAATTTG-3¢ as a forward primer and 5¢-
TCTACATGGCAACTGTGAGG-3¢ as a reverse primer.
The PCR condition was 42 cycles of 98 °C for 20 s, 55 °C for
30 s, and 72 °C for 1 min. The identity of the amplified seg-
ment was confirmed by sequencing.

Methylation analysis
In order to analyze the methylation status in 5¢-flanking
region of the EDNRB gene, we prepared genomic DNA
from NHEM, HMV-II, and HeLa cells. Methylation status
was determined by the method of bisulfite-modified DNA
[41]. This technique depends on chemical modification of
the genomic DNA with sodium bisulfite, where unmethylat-
ed cytosine residues are converted to uracil residues, but
methylated cytosines remain unchanged. The PCR primers
used for the EDNRB gene promoter were 5¢-GGAGTT
TTGTTTGGGATTTTTATT-3¢ (sense) and 5¢-AAACTC
CTTCCTAATACCCT-3¢ (antisense). The amplified seg-
ments were subcloned into the pGEM-T Easy vector
(Promega), and the nucleotide sequence of the insert frag-
ment was determined with ABI3100 analyzer (Applied Bio-
systems, Foster, CA, USA.).
5¢-Aza-2¢-deoxycytidine treatment
For construction of another FLAG-tagged SOX10 expres-
sion vector, pIRES-F ⁄ SOX10, the SacI ⁄ SacI and SacI ⁄
XbaI fragments of p3XFLAG-SOX10 were inserted into
pIRESneo vector (Clontech, Mountain View, CA, USA).
The stable-transformed HeLa cells with pIRESneo (Mock)
or pIRES-F ⁄ SOX10 (F ⁄ SOX10) were selected with G418
(350 lgÆmL
)1
) (Clontech).
HeLa transformants (Mock or F ⁄ SOX10) were seeded in
a 60-mm dish 24 h prior to treatment with 5¢-aza-2¢-de-
oxycytidine (5¢-aza-dC) (1 lm) (Sigma-Aldrich Japan). Cul-
ture medium and drug were replaced every 24 h. After

7 days, cells were harvested for RNA extraction. The total
RNA was used for RT-PCR. The primers used were as fol-
lows:
EDNRB: 5¢-TTGTGTCCTGCCTTGTGTTC-3¢ (sense)
and 5¢-CAGAATCCTGCTGAGGTGAA-3¢ (antisense);
S. Yokoyama et al. Regulation of EDNRB by SOX10
FEBS Journal 273 (2006) 1805–1820 ª 2006 The Authors Journal compilation ª 2006 FEBS 1817
SOX10: 5¢-CCCAAGCTTATGGCGGAGGAGCAGGAT
CTATCGGAGGTG-3¢ (sense) and 5¢-GCTCTAGATGA
GGTGGGCAAGGAACAGGGCACACAGGCT-3¢ (anti-
sense); and GAPDH: 5¢-ACCACAGTCCATGCCATCAC
3¢ (sense) and 5¢-TCCACCACCCTGTTGCTGTA-3¢ (anti-
sense). The PCR condition was 42 cycles (SOX10), 45
cycles (EDNRB) or 30 cycles (GAPDH) of 94 °C for 30 s,
55 °C for 30 s, and 72 °C for 1 min. The identity of the
amplified segment was confirmed by sequencing.
Statistical analysis
All data are mean ± sd of at least three independent
experiments. Two-tailed Student’s t-test was used for com-
parison between the two groups. Differences between mean
values were considered significant when P < 0.05.
Acknowledgements
This work was supported in part by Grants-in-Aid for
Scientific Research (B) (to S.S.), for Scientific Research
(C) (to K.T.), for Scientific Research on Priority Area
(to S.S.), and for Exploratory Research (to S.S.) from
the Ministry of Education, Science, Sports, and Cul-
ture of Japan, and by the 21st Century COE Program
Special Research Grant ‘the Center for Innovative
Therapeutic Development for Common Diseases’ from

the Ministry of Education, Science, Sports and Culture
of Japan.
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