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.
Trang 1receptor 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: ktakeda@mail.tains.tohoku.ac.jp
(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 transactivatranscrip-tion
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.
Trang 2neural 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
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 EDNRBgene 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,
Trang 3we 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, EDNRBD2, 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 detecprotec-ted
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 EDNRBD1 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
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 indicated32P-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.
Trang 412-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
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 initiiniti-ation 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).
Trang 5assays (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 expresexpres-sion 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¢- or 3¢-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 activactiv-ity 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.
Trang 61.9- 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
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 (–).
Trang 7EDNRB 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
Trang 8C
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 transcriptposit-ional initiatposit-ion 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 indicatrepresent-ed 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.
Trang 9(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.
Trang 10cells, 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
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
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.