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6(,.& "%$'% .&2*&&+I

J�ai d�abord étudié l�interaction entre MITF et ;VCS !(" .66+&*!"#2.!.'('.*& %' .66+&*-,*'I

J�ai pu confirmer la coK!"#2.!.'('.*& 0(&$ ,( ="(2'.*& ($$*2.#% i ,( 2B"*6('.&% %&'"% :>QS %'

,%$ $*+$K+&.'#$ A<QS7 4:5C95O %' 4:5C95P 0% ;VCSI <*+" #8(,+%" ,% "a,% 0% ;VCS

0(&$ ,% 6#,(&*6%7 F% 6% $+.$ 2*&2%&'"#% $+" ,% "a,% 0% A<QS7 ,( !,+$ /"(&0% $*+$K+&.'# 0%

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dans d�autres complexes de remodelage. J�ai infecté plusieurs lignées cellulaires d%

!(

6#,(&*6% (8%2 0%$ 8%2'%+"$ ,%&'.8."(+_ %_!".6(&' 0%$ $BC;5 0."./#$ 2*&'"% A<QSI E%

silencing de BPTF conduit à l�arrêt de la prolifération, à des modifications de la morphologie,

i +&% 6.'*$% 0#=%2'+%+$%7 (8%2 (!!(".'.*& 0% 2%,,+,%$ 6+,'.K&+2,##$ %' i ,( $#&%$2%&2%7

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A<QS G&*2GK0*H& !"#$%&'%&' 0% &*6-"%+$%$ 2("(2'#".$'.@+%$ *-$%"8#%$ 0(&$ ,%$ 2%,,+,%$

.&=%2'#%$ %& !("(,,h,% (8%2 0%$ 8%2'%+"$ %_!".6(&' 0%$ $BC;5 2*&'"% :>QSI <("

immunoblot, j�ai montré qu�une perte de BPTF n�affecte pas l�expression de MITF de façon

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5=.& 0% '%$'%" 2%''% .0#%7 Fk(. %==%2'+# 0%$ %_!#".%&2%$ 0k5C;K4%@ $+" ,%$ 2%,,+,%$ $BA<QS %'

$B:>QS i ,k(.0% 0% 0%+_ $BC;5$ 0.$'.&2'$ !*+" 2B(@+% =(2'%+" (=.& 0k%_2,+"% ,%$ %==%'$ B*"$ K

2.-,%I E% $#@+%&Y(/% ( 6*&'"# +& 2B%8(+2B%6%&' $./&.=.2('.= %&'"% ,%$ /h&%$ "#/+,#$ !("

MITF et BPTF. 42 % des gènes dont l�expression est augmentée et 30 % des gènes dont

l�expression est diminuée suite a+ $BA<QS $*&' "#/+,#$ 0% ,( 6n6% =(Y*& $+.'% (+ $B:>QSI

J�ai analysé de manière plus approfondie les gènes régulés en commun et j�ai pu démontrer

@+% ZXo 0%$ /h&%$ (+/6%&'#$ %' `\o 0%$ /h&%$ 0.6.&+#$ $*&' ($$*2.#$ i 0%$ $.'%$ 0%

,.(.$*& 0% :>QS7 $+//#"(&' @+% ,%+" "#/+,('.*& !(" :>QS %$' 0."%2'%I fk(. #/(,%6%&' "%6("@+#

@+% :>QS %' A<QS 2*K"#/+,%&' 0% &*6-"%+_ /h&%$ .6!,.@+#$ 0(&$ ,( 6./"('.*& %' 0%

l�invasion. ZEB1 est l'un des facteurs de transcription connus pour réguler la transition

#!.'B#,.*K 6#$%&2BD6('%+$% MQL:N7 %& (/.$$(&' 2*66% +& "#!"%$$%+" 0% ,( LK2(0B#".&%I

L�TEM est l'un des événements majeurs dans le développement de tumeurs métastatiques,

par lequel les cellules cancéreuses acquièrent des propriétés invasives. Or, l�expression de

cLAO %$' =*rtement augmentée lors de la perte d�expression de MITF out de BPTF. Ces

0%+_ =(2'%+"$ 2**!h"%&' 0*&2 !*+" "#!".6%" $*& %_!"%$$.*&I

<*+" 0#'%"6.&%" $. A<QS ( +& "a,% .&0#!%&0(&' 0% :>QS 0(&$ ,% 6#,(&*6%7 Fk(. "#(,.$# ,%

$.,%&2.&/ 0% A<QS 0(&$ ,%$ 2%,,+,%$ 1205Lu, une lignée invasive n�exprimant pas MITF. La

suppression de BPTF conduit à l�arrêt de leur prolifération et au changement de

morphologie, tandis que l�élimination de MITF n'a eu aucun effet, comme prévu .En

"%8(&2B%7 ,% $.,%&2.&/ 0% A<QS 0(&$ !,+$.%+"$ ,./&#%$ &*&Kmélanome n�affecte pas leur

prolifération de façon significative. L�ensemble de ces résultats montrent que BPTF/;VCS

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essentiel dans d�autres type$ 2%,,+,(."%$I

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include Merkel and Meissner�s corpuscles (for touch), Pacinian corpuscles (for pressure),

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#(

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#&

?Y)'N5)N=2>)W8992<950)

I. Hair follicle structure

J(." =*,,.2,% 6(.& $'"+2'+"%$ ("% 'B% !(!.,,(7 6('"._7 B(." $B(='7 "**' $B%('B7 B(." =.-%"7

(&0 'B% -+,/%I QB% B(." =*,,.2,% .$ 2*&'(.&$ ( !%"6(&%&' !*"'.*&7 $+-0.8.0%0 .&'* +!!%" MV<<N

(&0 ,*H%" !%"6(&%&' !*"'.*& ME<<N7 (&0 ( '"(&$.%&' !*"'.*& MS./+"% ZNI QB% '"(&$.%&' !*"'.*&

*= 'B% B(." =*,,.2,% 6(G%$ +! '* 'H* 'B."0$ *= 'B% 6('+"% B(." =*,,.2,% .$ 2*&$'"+2'%0 (&0

0.$.&'%/"('%0 .& %8%"D B(." 2D2,%7 HB.,% 'B% !%"6(&%&' !*"'.*& "%6(.&$ 2*&$'(&' M4'%&& (&0

<(+$ XWWONI )

) The papilla .$ (& *8(, $'"+2'+"% 2*&&%2'%0 '* 'B% 2*&&%2'.8% '.$$+% $%"8.&/ ($ (

2(!.,,("D (22%$$ !*.&'I )

) The hair matrix, %&8%,*!.&/ 'B% !(!.,,(7 .$ 2*&$.$'%0 *= %!.'B%,.(, 2%,,$ (&0

6%,(&*2D'%$I 9%,, 0.8.$.*& .& 'B% 6('"._ %&(-,%$ 'B% !"*0+2'.*& 2%,,$ .&2,+0%0 .& 'B% =*"6('.*&

*= 'B% (,, 'B% 6(.& $'"+2'+"%$ *= 'B% B(." =.-%" (&0 'B% .&&%" "**' $B%('BI) )

The root sheath .$ 6(0% *= (& %_'%"&(, (&0 .&'%"&(, "**' $B%('BI QB% %_'%"&(, $B%('B

.$ 2*&$.$'%0 *= %6!'D "%2'(&/+,(" 2%,,$7 *"./.&('.&/ ="*6 G%"('.&*2D'%$ 'B(' '%"6.&(,,D

0.==%"%&'.('%7 %_'"+0% 'B%." *"/(&%,,%$ (&0 -%2*6% './B',D !(2G%0 H.'B -+&0,%$ *= OWK&6

=.,(6%&'$ ($$%6-,%0 ="*6 2D$'%.&%K".2B B(." G%"('.&$7 HB.2B -%2*6% !BD$.2(,,D 2"*$$K,.&G%0

'* /.8% 'B% B(." $B(=' B./B '%&$.,% $'"%&/'B (&0 =,%_.-.,.'DI )

) The hair bulge .$ ,*2('%0 .& 'B% ,*H%" !%"6(&%&' !*"'.*& *= 'B% B(." =*,,.2,%I >' .$

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Human conditions related to the genes involved in NC-melanocyte regulatory

network

!" Waardenburg syndrome (WS). #$%& &'()*+,- %& . $-*-)%/.*' )%&+*)-* /$./ 0.1&-&

$'2+2%3,-(/./%+( +4 /$- $.%* .() -'-&5 2./0$' )-2%3,-(/./%+( +4 /$- &6%( .() $-.*%(3

%,2.%*,-(/" 7.&-) +( /$- .))%/%+(.8 &',2/+,&5 9: %& 08.&&%4%-) %(/+ 9:;5 9:<5 9:= .()

9:>" 9:; .() = %& 0.1&-) ?' @.A= ,1/./%+(&5 .() .))%/%+(.8 &',2/+,& %(081)- +018.*

)-4+*,./%+( 4+* 9:;5 .() &-B-*- )-4-0/& %( /$- 8%,? ,1&08-& 4+* 9:=" 9:< )%&28.'& /$-

/'2%0.8 9: &',2/+,& 8%&/-) .?+B-" !/ %& 0.1&-) ?' ,1/./%+(& %( MITF5 SOX10, EDN3/EDNRB

.() B-*' *.*-8' SNAIL2. #$- SOX10 ,1/./%+( .8&+ 0.1&-& . &'()*+,- 0.88-) C-,-(%/-

)-.4D?8%() $'2+2%3,-(/./%+( &'()*+,- ECF7:G5 0+(&%)-*-) /+ ?- .& . 9:<" 9:>5 .8&+

6(+H( .& 9..*)-(?1*3�:$.$ +* I%*&0$&2*1(3 9..*)-(?1*3 &'()*+,-&5 )%&28.'& . *.(3-

+4 2$-(+/'2-& *-,%(%&0-(/ +4 ?+/$ 9: E2%3,-(/ )-4%0%-(0%-& .() )-.4(-&&G .()

I%*&0$&2*1(3 )%&-.&- EI:JKG E.3.(38%+(%0 ,-3.0+8+(G5 .() %/ %& 0.1&-) ?' SOX10 .()

EDN3/EDNRB ,1/./%+(& E@%(3.18/ -/ .8" <L;LG"

!!" Teitz syndrome. !/ %& . &'()*+,- 08+&-8' *-8./-) /+ /$- 9:<5 .() %/ %& 0.1&-) ?'

MITF ,1/./%+(&" I+H-B-*5 %/ %& 0$.*.0/-*%M-) ?' . 1(%4+*, )%81/%+( +4 2%3,-(/./%+( .() (+/

/$- 2./0$' )-2%3,-(/./%+( &--( %( 9: E@%(3.18/ -/ .8" <L;LG"

!!!" Piebaldism" !/ %& . )-2%3,-(/./%+( )%&+*)-*5 0$.*.0/-*%M-) ?' 2./0$-& +4 H$%/- &6%("

#$- $-.*%(3 .() -'- 2%3,-(/./%+( .*- (+*,.8" !/ %& 0.1&-) ?' KIT .() SNAIL2 ,1/./%+(&

E:.(0$-MDN.*/%( -/ .8" <LL=G"

!"

"

7" :%3(.8%(3 2./$H.'& %(B+8B-) %( ,-8.(+0'/- )-B-8+2,-(/

I. The Wnt signaling pathway in melanocyte development

#$- 9(/ &%3(.8%(3 2./$H.' $.& ?--( &$+H( /+ $.B- . 0*10%.8 *+8- %( ,-8.(+?8.&/ .()

,-8.(+0'/- 0-88 8%(-.3-" 9O# .*- &-0*-/-) 38'0+2*+/-%(& /$./ $.B- ,18/%28- *+8-& %( (-1*.8

0*-&/ %()10/%+(5 &2-0%4%0./%+( .() ,-8.(+0'/- )%44-*-(/%./%+(" 9(/ &%3(.8%(3 0+,2*%&-& &-B-*.8

,+)-& +4 .0/%B./%+(5 %(081)%(3 0.(+nical Wnt/ D0./-(%( 2./$H.'5 .() (+(D0.(+(%0.8 9(/PJ.<Q

.() 28.(.* 0-88 2+8.*%/' E@J@G 2./$H.'&"

In the best understood canonical WNT/ D0./-(%( &%3(.8%(3 2./$H.'5 9O# 8%3.()&

?%() /+ /$- *-0-2/+* R*%MM8-) ERMG5 .() .0/%B./- /$- &%3(.8%(3 2./$H.' *-&18/%ng in D0./-(%(

accumulation and translocation into the nucleus. D0./-(%( %& . &/*10/1*.8 2*+/-%( .&&+0%./-)

to adherens junctions. In the absence of the Wnt signal, cytoplasmic D0./-(%( %& )-3*.)-)

by a D0./-(%( )-&/*10/%+( 0+,28-A5 0+,2+&-) +4 SA%(5 .)-(+,./+&%& 2+8'2+&%& 0+8% ES@JG5

protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1!

(CK1!) Phosphorylation of Dcatenin within this complex by CK1! and GSK3 targets it for

1?%T1%/%(./%+( .() &1?&-T1-(/ 2*+/-+8'/%0 )-&/*10/%+( ?' /$- 2*+/-+&+,.8 ,.0$%(-*'" 7%()%(3

+4 9(/ /+ %/& *-0-2/+* 0+,28-A 0+,2+&-) +4 /$- RM .() /$- 8+HD)-(&%/'D8%2+2*+/-%(D*-8./-)

2*+/-%(UPV EWK@UPVG /*%33-*& . &-*%-& +4 -B-(/& /$./ )%&*12/& /$- S@JPSA%(PX:Y= 0+,28-A

/$./ %& *-T1%*-) 4+* /$- /.*3-/-) destruction of D0./-(%(" #$- ?%()%(3 +4 9(/ /+ /$- RMPWK@UPV

0+,28-A %()10-& . 0+(4+*,./%+(.8 0$.(3- +4 /$- *-0-2/+* -(.?8-& /$- %(/-*.0/%+( +4 /$- RM

H%/$ /$- F%&$-B-88-) EF&$G 2*+/-%( /$./ 4.0%8%/./-& /$- ?%()%(3 +4 SA%( /+ . 0+(&-*B-)

&-T1-(0- %( /$- 0'/+28.&,%0 /.%8 +4 WK@UPV5 /$1& )%&*12/%(3 /$- )-&/*10/%+( 0+,28-A EJ8-B-*&

.() O1&&- <L;<G. As a result, D0./-(%( .001,18./-& %( /$- 0'/+28.&, .() /*.(&8+0./-& %(/+

/$- (108-1&5 H$-*- %/ %(/-*.0/& H%/$ ,-,?-*& +4 /$- 8',2$+%) -($.(0-* ?%()%(3 4.0/+* ;P#D0-88

&2-0%4%0 4.0/+* EW-4;P#04G 4.,%8' +4 /*.(&0*%2/%+( 4.0/+*& EO-8&+( .() O1&&- <LL>G. Wnt/ D

0./-(%( .() WZR; %(/-*.0/ 41(0/%+(.88' %( /$- (108-1&5 *-&18/%(3 %( D0./-(%( ,-)%./-)

/*.(&0*%2/%+(.8 *-318./%+( E7-$*-(& -/ .8" ;[[VG"

!( B%/*+ &/1)%-& +4 018/1*-) ?+/$ ,+1&- .() .B%.( (-1*.8 0*-&/ 0-88 018/1*-& .0/%B./%+(

of the Wnt signaling, either by treatment with Wnt or activation of D0./-(%( 2*+,+/-)

2%3,-(/ 0-88 )-B-8+2,-(/ EF1(( -/ .8" <LLL5 \%( -/ .8" <LL;G" !( B%B+5 %( M-?*.4%&$ .

constitutive activation of D0./-(%( %( /$- OJ 2*+,+/-& /$- 4+*,./%+( +4 ,-8.(+0'/-& H$%8-

/$- (-1*.8 0-88 8%(-.3-& .*- &122*-&&-)" ]( /$- 0+(/*.*'5 12+( /$- 9(/ &%3(.8%(3 %($%?%/%+(5

/$- OJJ& .&&1,- /$- (-1*.8 *./$-* /$.( ,-8.(+0'/- 4./- EF+*&6'5 N++( .() K.%?8- ;[[^G"

#H+ 9(/ 4.0/+*&5 9(/; .() =.5 .*- (-0-&&.*' 4+* ,-8.(+?8.&/ )-B-8+2,-(/ %( /$- -,?*'+

EF+*&6' -/ .8" ;[[^5 !6-'. -/ .8" ;[[_G. In the mouse, the D0./-(%( -A2*-&&%+( %( /$-

#"

"

migratory NCC population is regulating cell specification. Ectopic expression of D0./-(%( %(

/$- 2*-,%3*./+*' OJ *-&18/& %( )-B-8+2,-(/ +4 /$- (-1*.8 8%(-.3-& .() .?+8%&$-& ,-8.(+0'/-

)-B-8+2,-(/" EW-- -/ .8" <LL>G. On the other hand, constitutive D0./-(%( %( /$- -.*8'

,%3*./+*' OJJ 2+218./%+( *-&18/& %( &/*+(3 *-)10/%+( +4 /$- (-1*.8 8%(-.3-&5 ?1/ 8-.)& /+ /$-

-0/+2%0 ,-8.(+?8.&/ 4+*,./%+( EI.*% -/ .8" <L;<G. The ectopic expression of D0./-(%( %( /$-

8./-* )-B-8+2,-(/.8 &/.3-&5 1&%(3 /$- #'*DJ*- ,+)-85 -(.?8%(3 /$- 0+(&/%/1/%B- -A2*-&&%+( +4

D0./-(%( %( /$- .8*-.)' -&/.?8%&$-) ,-8.(+?8.&/& )%) (+/ .8/-* /$-%* 2*+8%4-*./%+( *./- EF-8,.&

-/ .8" <LL_5 I.*% -/ .8" <L;<G"

N%/4 %& . /.*3-/ 9(/ 2./$H.' %( B.*%+1& &2-0%-& E9%)81() -/ .8" <LL<G" !( M-?*.4%&$5 /$-

*-)10-) #JR &%3(.8%(3 +* ,1/./%+(& %( /$- 2*+,+/-* ?%()%(3 &%/- *-&18/-) %( 8+&& +4 N%/4

-xpression, indicating Dcatenin is required for Mitf transcription activation Wnt/ D0./-(%(

.0/%B./-& N%/4 ?' ?%()%(3 /+ /$- #JRPWZR; ?%()%(3 &%/- %( /$- Mitf 2*+,+/-* EF+*&6'5 K.%?8-

.() N++( <LLLG" !( 018/1*-) ,-8.(+0'/-&5 9(/D=. %()10-& /$- -A2*-&&%+( +4 -()+3-(+1&

Mitf ?' /*.(&.0/%B./%(3 /$- N!#R 2*+,+/-* B%. /$- WZRD;D?%()%(3 &%/-" E#.6-). -/ .8" <LLL?G"

R1*/$-*,+*-5 N!#R 2$'&%0.88' %(/-*.0/& H%/$ WZRD;5 .0/%B./%(3 +4 /$- Dct 2*+,+/-*" #$- ?IWID

W` *-3%+( +4 N!#RDN %& *-&2+(&%?8- 4+* /$- 1(%T1- %(/-*.0/%+( H%/$ WZRD;5 .& %/ %& (+/

%(/-*.0/%(3 H%/$ #JR; EC.&1,+/+ -/ .8" <LL<G" !( .))%/%+(5 N!#R %& 0++2-*./%(3 H%/$ WZR; /+

regulate its own expression. The two factors act synergistically and the interaction with the D

0./-(%( %& %,2+*/.(/ 4+* /$-%* 0++2-*./%+(" N+*-+B-*5 N!#R *-0*1%/-) +( /$- N 2*+,+/-*

41(0/%+(& .& . (+(DFOSD?%()%(3 0+4.0/+* 4+* WZRD; E:.%/+ -/ .8" <LL<G" !/ $.& .8&+ ?--(

&$+H( N%/4 %(/-*.0/& directly with D0./-(%(5 .() B%. /$%& %(/-*.0/%+(5 N%/4 .0/& /+ &-T1-&/-* D

catenin form the Wnt/ D0./-(%( &%3(.8%(3 /.*3-/D3-(-& %( +*)-* /+ .0/%B./- /*.(&0*%2/%+( +4 /$-

N%/4D&2-0%4%0 /.*3-/ 2*+,+/-*& E:0$-2&6' -/ .8" <LLVG"

#$- (+(D0.(+(%0.8 9(/ 28.(.* 0-88 2+8.*%/' E@J@G 2./$H.' %& .0/%B./%(3 /$- &,.88 X#@

$')*+8.&-& ?-8+(3%(3 /+ /$- K$+ &12-*4.,%8' E,+&/ &/1)%-) .*- K.0; .() K$+SG5 B%. 31.(%(-

-A0$.(3- 4.0/+* EXZRG" ](- +4 /$- 41(0/%+(& +4 /$- K$+ X#@.&-& %& .0/%B./%(3 K$+D

.&&+0%./-) 6%(.&- EK]JYG5 +(- +4 /$- ,.a+* 0'/+&6-8-/+( *-318./+*& EX+*)+( .() O1&&-

<LLV5 Y+,%'. .() I.?.& <LL^G" F-8-/%+( +4 K.0; %( -,?*'+(%0 ,+1&- ,-8.(+?8.&/& *-&18/&

%( .?-**.(/ ,%3*./%+(5 .() .8&+ )-4-0/& %( 0-88 0'08- 2*+3*-&&%+( .() 0'/+6%(-&%& EW% -/ .8"

<L;;.G" R1*/$-*,+*-5 K.0; $.& .( %,2+*/.(/ *+8- )+H(&/*-., +4 ,1/.(/ OK.&bV;Y %( /$-

&1*B%B.8 +4 /$- ,-8.(+0'/-& EW% -/ .8" <L;<.G" S(+/$-* 4.0/+* %( /$%& 2./$H.'5 @DK-A;5 . K.0D

&2-0%4%0 K$+ X#@.&- 31.(%(- (108-+/%)- -A0$.(3- 4.0/+* EXZRG5 %& 0*10%.8 4+* -,?*'+(%0

,-8.(+?8.&/ ,%3*./%+(5 &%(0- %/& )-8-/%+( *-&18/& %( . H$%/- ?-88' 2$-(+/'2- EW%()&.' -/ .8"

<L;;G"

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II. The KIT (c-Kit)/KITL (SCF) signaling pathway

Kit E.8&+ 6(+H( .& c-KitG %& . *-0-2/+* /'*+&%(- 6%(.&- /$./ $.& ?--( &$+H( /+ ?-

-(0+)-) ?' /$- 8+01& W (Chabot et al. 1988)" #$- 8%3.() .0/%B./%(3 /$- 6%(.&-5 Y!#W EY%/

8%3.()5 .8&+ 6(+H( .& &/-, 0-88 4.0/+* E:JRG5 :/--8 4.0/+* +* ,.&/ 0-88 3*+H/$ 4.0/+*DNXRG %&

-(0+)-) ?' Sl(Steel) 8+01& EJ+2-8.() -/ .8" ;[[LG" S0/%B./%+( +4 Y!# *-0-2/+* ?' Y!#W 8%3.()

*-&18/& %( *-0-2/+* )%,-*%M./%+( .() %/& &1?&-T1-(/ .1/+2$+&2$+*'8./%+(" Y!# &%3(.8%(3 %&

*-T1%*-) 4+* /$- ,-8.(+?8.&/ &1*B%B.8 .() ,%3*./%+( EJ.?8-5 \.06&+( .() :/--8 ;[[U5

N.06-(M%- -/ .8" ;[[_5 9-$*8-DI.88-* .() 9-&/+( ;[[UG" S ,+1&- 8%(- H%/$ 2+%(/ ,1/./%+(&

%( 0DY%/ )%&28.'& ,18/%28- )-4-0/& *-8./-) /+ /$- (-1*.8 0*-&/D)-*%B-) 2%3,-(/ 0-88&5

$-,./+2+%-/%0 0-88&5 .() 2*%,+*)%.8 3-*, 0-88&" !()--)5 $+,+M'3+/- ,%0- .*- ?8.06D-'-)5

H$%/-5 )-.45 &/-*%8- .() .(-,%0 EK1.(5 `$.(3 .() X.+ <LLUG"

Kitl 2*+)10-& /H+ Y!#W 2*+/-%(&5 . /*.(&,-,?*.(- .() . &+81?8- 4+*," #$-

/*.(&,-,?*.(- 4+*, %& *-T1%*-) 4+* ,-8.(+0'/- 2*-01*&+* &1*B%B.8 %( /$- )-*,%&5 H$%8- /$-

&+81?8- 4+*, %& (--)-) )1*%(3 -.*8' ,-8.(+?8.&/ )%&2-*&.8 +( /$- 8./-*.8 ,%3*./%+( 2./$H.'

4+*, /$- (-1*.8 0*-&/5 .() .8&+ 4+* /$-%* %(%/%.8 &1*B%B.8 H%/$%( /$- (-1*.8 0*-&/ 2*-,%3*./%+(

.*-. E9-$*8-DI.88-* .() 9-&/+( ;[[UG" ZA2-*%,-(/& 1&%(3 . ,+(+08+(.8 .(/%DY!# .(/%?+)'

ESJY<G .8&+ *-B-.8-) .( %,2+*/.(/ *+8- +4 :JRPY!# &%3(.8%(3 %( /$- 2+&/(./.8 ,-8.(+0'/-&

EO%&$%6.H. -/ .8" ;[[;5 ]61*. -/ .8" ;[[UG" N-8.(+0'/- &/-, 0-88& EN:JG &1*B%B-

%()-2-()-(/8' +4 0D6%/ &%3(.8%(3 %( . /*.(&3-(%0 ,+1&- ,+)-8" I+H-B-*5 N:J& ,%3*./%+( +1/

+4 /$- $.%* 4+88%08-& %& Y!#D&%3(.8%(3 )-2-()-(/ EY1(%&.). -/ .8" ;[[^G" 7+/0$6.*-B. -/ .8"

)-,+(&/*./-) /$- .(/%D0DY!# .(/%?+)' 0+,28-/-8' .?+8%&$-) /$- /*.(&%-(/ ,-8.(+0'/-

2+218./%+(5 -B%)-(0-) ?' /$- H$%/- 0+./ +4 /$- .(%,.8&5 ?1/ /$- ,-8.(+0'/- &/-, 0-88& H-*-

%(/.0/5 &$+H%(3 0DY!# %& (+/ *-T1%*-) 4+* /$-%* 41(0/%+( E7+/0$6.*-B. -/ .8" <LL;G" K-0-(/8'5 .

&/1)' 2-*4+*,-) %( M-?*.4%&$ .%,-) /+ )-0%2$-* H$./ 2.*/ +4 /$- ,-8.(+0'/- *-3-(-*./%+( %&

0DY%/ *-318./%(35 -&/.?8%&$,-(/ +4 /$- N:J (%0$-5 2*+)10/%+( +4 ).13$/-* 0-88& +* 2*+8%4-*./%+(

.() )%44-*-(/%./%+( +4 /$- ).13$/-* 0-88&" #$- Y%/ ,1/.(/& H-*- (+/ .?8- /+ *-3-(-*./- /$-

N:J&5 .() /$- 1()-8.'%(3 0.1&- +4 /$%& 2$-(+/'2- H.& &$+H( /+ ?- /$- .?-**.(/

-&/.?8%&$,-(/ +4 /$- N:J& E]cK-%88'D@+8 .() \+$(&+( <L;=G"

III. Endothelin 3 (Edn3) / Endothelin receptor B (EdnrB) signaling

#$- -()+/$-8%( &%3(.8%(3 &'&/-, 0+(&%&/& +4 /$- X 2*+/-%( 0+128-)D*-0-2/+*&5

-()+/$-8%( *-0-2/+*& S EZ()*SG .() 7 EZ()*7G5 .() /$*-- 8%3.()& ?%()%(3 /$- *-0-2/+*&5

-()+/$-8%(D ;5 < .() =" Z)(*? ?%()& .88 /$*-- %&+2-2/%)-& H%/$ -T1.8 .44%(%/' E:.8).(.D

J.?+B-*)- .() Y+& <L;LG" Z)(; +* Z)(= ?%()%(3 /+ Z)(*? *-&18/& %( /$- .0/%B./%+( +4 ,18/%28-

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&%3(.8%(3 2./$H.'&5 &10$ .& 2*+/-%( 6%(.&- J E@YJG5 NS@Y .() JKZ7PSDJKZ7 E7+$, -/ .8"

;[[U5 :./+D\%( -/ .8" <LL^G"

#$- %(/-*.0/%+( +4 ZFO= H%/$ ZFOK7 %& -&&-(/%.8 4+* )-B-8+2,-(/ +4 (-1*.8 0*-&/D

)-*%B-) 0-88 8%(-.3-&5 %(081)%(3 ,-8.(+0'/-& .() -(/-*%0 (-1*+(&5 -B%)-(0-) ?' &/1)%-& +(

,%0- 0.**'%(3 Ednrb ,1/./%+(& E7.'(.&$ -/ .8" ;[[>5 I+&+). -/ .8" ;[[>5 W--5 W-B+*&- .()

:$%( <LL=5 K-%) -/ .8" ;[[VG" Z)(*? %& *-T1%*-) 4+* /$- ,-8.(+?8.&/ 2*+8%4-*./%+(5 ,%3*./%+(

.() &1*B%B.8 Z;L .() Z;<"U5 H$%0$ 0+**-&2+()& /+ /$- 2-*%+) /$- OJJ& .*- .?+1/ /+ 8-.B-

/$- 2*-,%3*./%+( .*-." :%(0- /$- Z)(*? ,1/.(/ 2$-(+/'2- 0+18) ?- *-&01-) ?' .0/%B./%(3 /$-

Z)(*? 3-(- .& 8./- .& Z;L5 %/ .22-.*& /$./ Z)(*? %& (+/ *-T1%*-) 2*%+* /+ /$- ,-8.(+?8.&/

,%3*./%+(" R1*/$-*,+*-5 /$- *-&01- +4 /$- 2$-(+/'2- H.& (+/ -44%0%-(/ .4/-* Z;<"U5 &133-&/%(3

Z()=PZ)(*7 %& (+/ *-T1%*-) .4/-* ,-8.(+?8.&/& 8-.B- /$- OJ E:$%( -/ .8" ;[[[G" S(/+/$-*

&/1)' .8&+ &$+H-) . &-B-*- 2$-(+/'2- ./ Z;<"U 1&%(3 0+,?%(-) /*.(&3-(%0 ,+)-8 $.*?+*%(3

,1/./%+( %( Ednrb .() Z)(*?DW.0` *-2+*/-* 8%(- EW-- -/ .8" <LL=G"

Z()=PZ)(*7 &%3(.8%(3 $.& ?--( &$+H( /+ 0++2-*./- H%/$ :+A;L )1*%(3 -(/-*%0

(-*B+1& &'&/-, .() ,-8.(+0'/- )-B-8+2,-(/" S(%,.8& /$./ H-*- $-/-*+M'3+1& 4+* ?+/$

,1/./%+(& -A$%?%/-) .( %(0*-.&- %( H$%/- &2+//%(3 .() 8+&& +4 ,-8.(+0'/-& %( /$- %((-* -.*

E:/.(0$%(. -/ .8" <LLVG"

IV. TGF signaling pathway

The transforming growth factor beta (TGF ) pathway is involved in multiple acpects of

0-88 ?%+8+3'5 %(081)%(3 2*+8%4-*./%+(5 )%44-*-(/%./%+( .() .2+2/+&%&5 .() %& %,2+*/.(/ ?+/$ %( /$-

-,?*'+(%0 )-B-8+2,-(/ .() %( /$- .)18/"

The TGF sig(.8%(3 2./$H.' %(B+8B-) /H+ 6%()& +4 *-0-2/+*&5 /'2- ! .() /'2- !!"

W%3.() ?%()%(3 %()10-& $-/-*+)%,-*%M./%+( %(B+8B%(3 /H+ /'2- !! .() /H+ /'2- ! *-0-2/+*&5

*-&18/%(3 %( 2$+&2$+*'8./%+( +4 . 38'0%(-D&-*%(- EX:GD*%0$ *-3%+( %( /$- /'2- ! *-0-2/+* ?' /$-

/'2- !! *-0-2/+*" #$- )+H(&/*-., /.*3-/ +4 /$%& &%3(.8%(3 %& /$- :,.) 2./$H.'5 4+* /$-

2$+&2$+DX: *-3%+( &-*B-& .& . ?%()%(3 &%/- 4+* /$- :,.) 2*+/-%(& EKD:,.)&G" KD:,.)

2*+/-%(& .*- )%B%)-) %( /H+ )%&/%(0/ ?*.(0$-&d /$- #XR?D:,.) 2./$H.' EKD:,.)<P=G +* /$-

7N@D:,.) 2./$H.' EK:,.);P UP^G" #XR? &%3(.8%(3D,-)%./-) :,.) 2./$H.' %& 41*/$-*

.0/%B./-) ?' :,.) .(0$+* 4+* *-0-2/+* .0/%B./%+( E:SKSG" 7%()%(3 +4 KD:,.)&

2$+&2$+*'8./-& /H+ &-*%(-& %( /$- JD/-*,%(1& +4 /$- 2*+/-%( ?' /$- /'2- !5 *-&18/%(3 %( KD:,.)

)%&&+0%./%+( 4*+, /$- *-0-2/+* 0+,28-A .() ?%()%(3 /+ /$- 0+,,+( :,.) E0+:,.)G5 :,.)>"

#$- KD:NSFP0+:NSF 0+,28-A-& /*.(&8+0./- %( /$- (108-1&5 H$-*- /$-' /.6- 2.*/ *-318./%+(

/$-%* /.*3-/ 3-(-& E9*.(. <L;=G"

In melanocytes, TGF 1 cooperates with the KIT/KITL signaling to promote

&"

"

,-8.(+?8.&/ 2*+8%4-*./%+( .() )%44-*-(/%./%+(" #$- .(/%DTGF 1 antibody inhibits the cell growth,

.() *-&18/& %( *-2*-&&%+( +4 /$- -A2*-&&%+( +4 /$- Y!# 2*+/-%( .() ,KOS EY.H.6.,% -/ .8"

<LL<G. TGF has also been shown to have a crucial role in melanocyte differentiation and

&1( /.((%(3 *-&2+(&-5 &%(0- %( /$- .?&-(0- +4 ef *.)%./%+(5 keratinocytes secrete TGF

H$%0$ ?8+06& ,-8.(+0'/- )%44-*-(/%./%+( B%. @Sg= %($%?%/%+( EC.(3 -/ .8" <LL^G" !( /$-

melanocyte stem cells, TGF signaling is activ./-) ./ /$- 2+%(/ H$-( &/-, 0-88& *-D-(/-* /$-

T1%-&0-(/ &/./- EO%&$%,1*. -/ .8" <L;LG5 )%&01&&-) 41*/$-* %( /$- &-0/%+( +( ,-8.(+0'/- &/-,

0-88& ?-8+H"

J" #*.(&0*%2/%+( 4.0/+*& *-318./%(3 /$- ,-8.(+0'/- 8%(-.3-

N-8.(+0'/- 0-88 8%(-.3- %& *-318./-) ?' . H%)- 0+88-0/%+( +4 /*.(&0*%2/%+( 4.0/+*&

%(B+8B-) %( (-1*.8 0*-&/ )-B-8+2,-(/5 .() .*- ,-)%./+*& +4 /$- 0+((-0/%+( ?-/H--( ,18/%28-

&%3(.8%(3 2./$H.'& 0+(/*+88%(3 ,-8.(+0'/- )-B-8+2,-(/5 )%44-*-(/%./%+(5 &1*B%B.8 .()

,-8.(+0'/- &/-, 0-88 ,.%(/-(.(0-"

I. SOX transcription factors

#$- :*'D*-8./-) INX ?+A E:+AG 4.,%8' 0+(&%&/& +4 /*.(&0*%2/%+( 4.0/+*& /$./ 0+(/.%( .

0+(&-*B-) INX ?+A FOSD?%()%(3 )+,.%(" :+A 3-(-& $.B- ,18/%28- *+8-& %( 0-88 &2-0%4%0./%+(

.() )%44-*-(/%./%+(5 ,.(' +4 /$-, 41(0/%+(%(3 /+3-/$-* H%/$ 0-88 /'2-D&2-0%4%0 /*.(&0*%2/%+(

4.0/+*& E7+H8-&5 :0$-2-*& .() Y++2,.( <LLLG"

SOX10

:+A;L %& -A2*-&&-) -.*8' )1*%(3 (-1*.8 0*-&/ )-B-8+2,-(/h %/ &/.*/& /+ ?- -A2*-&&-) %(

/$- OJJ .& /$-' &/.*/ ,%3*./%(3 4*+, /$- 2*-,%3*./%+( .*-. %( /$- OJ EI.*% -/ .8" <L;<G"

:]g;L ,1/./%+(& $.B- ?--( .&&+0%./-) /+ ,18/%28- $1,.( 0+()%/%+(&5 %(081)%(3

9:>5 I:JK .() CF7:" e&%(3 /$- ,1/.(/ :+A;L ,+1&- ,+)-8 &-B-*.8 &/1)%-& $.B- &$+H(

0*10%.8 *+8-& +4 :+A;L %( 2-*%2$-*.8 (-*B+1& &'&/-, .() ,-8.(+0'/- )-B-8+2,-(/" E7*%/&0$ -/

.8" <LL;G" N+*-+B-*5 /$- ,1/.(/ -,?*'+& 0+,28-/-8' 8.06 ,-8.(+?8.&/ 2+218./%+( .() /$-

OJJ 2*%,.*' 018/1*-& 3-(-*./-) 4*+, /$-&- -,?*'+& .*- 1(.?8- /+ 2*+)10- ,-8.(+0'/-&

E@+//-*4 -/ .8" <LL;G"

:+A;L 0++2-*./-& H%/$ +/$-* 0*10%.8 /*.(&0*%2/%+( 4.0/+*& -A2*-&&-) )1*%(3

,-8.(+0'/-5 &10$ .& N%/4 .() @.A=" N%/4 2*+,+/-* 0+(/.%(& &-B-*.8 :]g ?%()%(3 &%/-&5

48.(6%(3 . @Sg= ?%()%(3 &%/-" 7+/$ :+A;L .() @.A= .0/%B./- Mitf 2*+,+/-*5 H$-*-.& :+A;L

.0/%B./%+( E;LLAG %& (+/.?8' $%3$-* /$.( /$- +(- H%/$ @.A= EUAG" I+H-B-*5 /$-&- /H+ 4.0/+*&

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"

.0/%B./- /$- 2*+,+/-* &'(-*3%&/%0.88' E<LLAG H$-( 0+D/*.(&4-0/-)" #$-&- *-&18/& &133-&/ /$./

N!#R %& )+H(&/*-., +4 :]g;L .() @Sg=5 .() /$./ /$- $'2+2%3,-(/./%+( .() )-.4(-&&

+?&-*B-) %( 9: %()%B%)1.8& H%/$ ,1/./%+(& %( /$-&- /$*-- 3-(-& .*- .88 18/%,./-8' 0.1&-) ?' .

)%&*12/%+( %( N!#R 41(0/%+( E@+//-*4 -/ .8" <LLLG"

:+A;L .() @.A= .8&+ ?%() ,-8.(+0'/-D&2-0%4%0 Mitf -($.(0-*5 *-&18/%(3 %( &'(-*3%&/%0

.0/%B./%+( +4 Mitf" F1- /+ . &2-0%4%0 -($.(0-* +*3.(%M./%+(5 @.A= .() :+A;L (--) /+ ?%()

%()%B%)1.88' %( +*)-* /+ .0$%-B- &'(-*3%&," #$- ,1/./%+( %( /$- /*.(&.0/%B./%+( )+,.%( +4

:+A;L .?*+3./-& @.A=D:+A;L %(/-*.0/%+(5 .() 8-.)& /+ 8+&& +4 N%/4 -A2*-&&%+(" EW.(3 .()

Z2&/-%( <LL=5 @+//-*4 -/ .8" <LLLG"

S2.*/ 4*+, /$- *+8- +4 :]g;L %( ,-8.(+0'/- )%44-*-(/%./%+(5 %/ &-*B-& .& . 0+D.0/%B./+*

+4 . &-/ +4 N%/4 /.*3-/ 3-(-& 0*%/%0.8 4+* 2%3,-(/./%+(5 %(081)%(3 F0/5 #'*5 .() #*2;" :+A;L .()

N%/4 )%*-0/8' .0/%B./- Dct /*.(&0*%2/%+( +( /$-%* +H(5 %)-(/%4'%(3 Dct .& /$- )%*-0/ :+A;L /.*3-/

%( ,-8.(+0'/-&" N+*-+B-*5 :+A;LD)-2-()-(/ .0/%B./%+( +4 Dct %& &'(-*3%&/%0.88' -($.(0-) ?'

N%/4 EW1)H%35 K-$?-*3 .() 9-3(-* <LL>G"

:+A;L .() N%/4 .*- 0+D-A2*-&&-) %( ,%3*./%(3 ,-8.(+?8.&/& %( /$- ,+1&- -,?*'+& ?1/ /$-%*

-A2*-&&%+( )%44-*& %( 0-88& +4 /$- (-H?+*( %((-* -.* E9./.(.?- -/ .8" <LL<G" N+*-+B-*5 :+A;L

%& (+/ -A2*-&&-) %( /$- $.%* 4+88%08- N:J&5 ?1/ %/ %& -A2*-&&-) %( /$- )%44-*-(/%./-) 0-88& %( /$-

$.%* ?18? E]&.H. -/ .8" <LLUG" K-0-(/8'5 :]g;L $.& .8&+ ?--( &$+H( /+ ?- %,2+*/.(/ %( /$-

N:J 2++8 *-(-H.8" S ,+1&- ,+)-8 0+(&/%/1/%B-8' -A2*-&&%(3 :+A;L H.& 1&-) /+ .&&-&& /$-

*+8- +4 -0/+2%0 -A2*-&&%+( +4 :+A;L +( /$- ,-8.(+0'/- 8%(-.3-" :1&/.%(-) :+A;L -A2*-&&%+(

0.1&-& 2*-,./1*- )%44-*-(/%./%+( +4 /$- N:J&" #$%& 8-.)& /+ -A$.1&/%+( +4 /$- N:J 2++8 .()

*-&18/& %( . 2*-,./1*- $.%* 3*.'%(35 0+(4%*,%(3 /$- %,2+*/.(0- +4 2*-0%&- *-318./%+( +4 :+A;L

-A2*-&&%+( %( .88 .&2-0/& +4 ,-8.(+0'/- 0-88 8%(-.3- ?%+8+3' EI.**%& -/ .8" <L;=G"

SOX2"

ZA2*-&&%+( +4 :+A< %& .&&+0%./-) /+ )-B-8+2,-(/ +4 /$- ,-8.(+0'/-& .*%&%(3 4*+,

:0$H.(( &/-, 0-88 2*-01*&+*&" ZA2*-&&%+( +4 :+A< %& 3*.)1.88' )%,%(%&$-) )1*%(3

)-B-8+2,-(/ +4 )-B-8+2,-(/ +4 ?+/$ (-1*.8 0*-&/D .() :J@&" #$- -A2*-&&%+( +4 :+A< $.&

.8&+ ?--( &$+H( /+ ?- %(B-*&-8' 0+**-8./-) H%/$ N%/4 -A2*-&&%+(5 .() %/ $.& ?--( &133-&/-)

:+A< .() N%/4 0+D*-2*-&& +(- .(+/$-*" :J@D)-*%B-) ,-8.(+0'/-& .*%&- 4*+, . &,.88

2+218./%+( +4 (-*B- .&&+0%./-) ,-8.(+?8.&/& .() /$-%* ,%3*./%+( %& *-318./-) ?' *-0-2/+* 7

EZ)(*?G .() 9(/U. &%3(.8%(3" ES).,-'6+ -/ .8" <L;<G"

K-0-(/8'5 . 2+&&%?8- .8/-*(./%B- +*%3%( .() ,%3*./+*' 2./$H.' +4 %( /$- /*1(6 2*+2+&-&

,-8.(+0'/-& )%44-*-(/%./- 4*+, :0$H.(( 0-88 2*-01*&+*& E:J@&G &%/1./-) %( (-*B-&"

ES).,-'6+ -/ .8" <LL[5 71)%5 @.//-*&+( .() @.*%0$' <L;;G" #$- 0*.(%.8 ,-8.(+0'/-& .*-

)-*%B-) ?+/$ 4*+, (-1*.8 0*-&/D .() :J@D)-*%B-) 2*+3-(%/+*& *-318./-) ?' %(/-*.0/%+(& +4

!("

"

:]g< .() N!#R" O-*B-D.*%&%(3 ,-8.(+0'/-& .*- 41*/$-* *-318./-) ?' -()+/$-8%( *-0-2/+* 7

EZ)(*?G .() 9(/U. &%3(.8%(3 ES).,-'6+ -/ .8" <L;<G"

II. PAX transcription factors

f-*/-?*./- @.A 3-(-& .*- *-8./-) /+ /$- Drosophila 2.%*-)D*18- 3-(-5 paired5 H$%0$ -(0+)-&

. 2*+/-%( H%/$ /H+ FOS ?%()%(3 )+,.%(&5 . 2.%*-) )+,.%( E@FG .() . 2.%*-)D8%6-

$+,-+)+,.%( EIFG"

@Sg=

@.A= %& -A2*-&&-) B-*' -.*8' %( /$- )-B-8+2,-(/ +4 /$- (-1*.8 0*-&/ .() %/ $.& . *+8-

%( (-1*.8 0*-&/ &2-0%4%0./%+( EN-18-,.(& .() 7*+((-*DR*.&-* <LL>G" OJJ& ,%3*./%(3 %( /$-

)+*&+8./-*.8 2./$H.' )%44-*-(/%./- %(/+ /$- ,-8.(+?8.&/ 8%(-.3-5 .() ,.%(/.%( @.A=

-A2*-&&%+(" @.A= %& +(- +4 /$- 4.0/+*& %( . /*.(&0*%2/%+(.8 (-/H+*6 E.8+(3 H%/$ :+A;L .()

N%/4G 0*10%.8 4+* ,-8.(+0'/- 8%(-.3- &1*B%B.8 .() )%44-*-(/%./%+(5 N:J& .() ,-8.(%( &'(/$-&%&

E7+%&&' .() O+*)81() ;[[_5 :+,,-* <LLUG" #$- *+8- +4 @.A= %( ,-8.(+0'/-& %( -A-*/-)

/$*+13$ %/& )%*-0/ ?%()%(3 /+ /$- 2*+,+/-*& +4 ,18/%28- ,-8.(+0'/- &2-0%4%0 3-(-&5 &10$ .& /$-

Mitf E9./.(.?- -/ .8" ;[[^G .() Trp1 EX.8%?-*/ -/ .8" ;[[[G .() Dct EW.(3 -/ .8" <LLUG"

@.A= 0.( .0/ ?+/$ .& .( .0/%B./+* +* . *-2*-&&+*5 .() :+A;L $.& ?--( &$+H( /+

-($.(0- @.A= .0/%B%/'" @.A= .() :+A;L ?%() c-RET .() Mitf -($.(0-*& .() &'(-*3%&/%0.88'

.0/%B./- /$- -A2*-&&%+( +4 /$-&- /.*3-/ 3-(-&5 ?1/ /$- ,-0$.(%&,& /+ ,+)18./- /$-%*

-A2*-&&%+( )%44-*" !( /$- 0.&- +4 c-RET -($.(0-*5 +(8' @.A= FOSD?%()%(3 %& &144%0%-(/ 4+*

&'(-*3%&/%0 .0/%+( +4 :+A;L .() @.A=5 H$%8- +( /$- Mitf -($.(0-*5 ?%()%(3 +4 ?+/$ 4.0/+*& %&

*-T1%*-) 4+* &'(-*3%&/%0 .0/%B./%+( EW.(3 .() Z2&/-%( <LL=G" R1*/$-*,+*-5 :+A;L .() @.A=

&'(-*3%&/%0.88' .0/%B./- Mitf H$-( ?+1() /+ /$- 0+(&-(&1& &%/-& H%/$%( %/& 2*+,+/-* E@+//-*4 -/

.8" <LLLG" @.A= ?%()%(3 /+ . 0%& *-318./+*' -($.(0-* 8+0./-) 12&/*-., +4 Mitf %(B+8B-& %/&

2.%*-) .() $+,-+)+,.%( EJ+**' .() e()-*$%88 <LLU5 9./.(.?- -/ .8" ;[[^G"

@.A= .8&+ ?--( &$+H( /+ $.B- . 0*10%.8 *+8- %( N:J ,.%(/-(.(0-" @.A= 0++2-*./-&

H%/$ :+A;L /+ .0/%B./- N%/4" S/ /$- &.,- /%,- %/ .0/& .& . *-2*-&&+* +4 /$- F0/ -($.(0-*5

0+,2-/%(3 H%/$ N%/4 4+* /$- ?%()%(3" @.A= %& 41*/$-* -A-*/%(3 *-2*-&&%+( +4 /$- F0/ -($.(0-*

?' *-0*1%/%(3 *-2*-&&+* X*3>" #$- *-2*-&&%+( ,-)%./-) ?' @.A= $.& ?--( &$+H( /+ ?-

*-8%-B-) /$*+13$ .0/%B./%+( +4 /$- 9(/ &%3(.8%(3 %( /$- -,?*'+(%0 ,-8.(+?8.&/& EW.(3 -/ .8"

<LLUG" !( 0+(/*.&/5 @.A= $.& ?--( &$+H( /+ ?- -A2*-&&-) %( /$- N:J& /+3-/$-* H%/$ F0/"

7-&%)-& F0/5 @.A= %& /$- +(8' +/$-* 2*+/-%( &$+H( /+ ?- -A2*-&&-) %( /$- N:J %( /$- W@@

E]&.H. -/ .8" <LLUG"

K-0-(/8'5 . (+B-8 &/*./-3' +4 2%3,-(/./%+( %()10/%+( $.& ?--( *-2+*/-)" RXR<P:#S#=

2./$H.' $.& ?--( &$+H( /+ ,+)18./- @.A= -A2*-&&%+( %( *-&2+(&- /+ ef7 *.)%./%+(" R34<

$.& ?--( &$+H( /+ ?- &-0*-/-) ?' /$- 6-*./%(+0'/-& %( *-&2+(&- /+ ef75 /$- &%3(.8 H$%0$

&/%,18./-) :#S#= 2$+&2$+*%8./%+( E@D:#S#=G" R1*/$-*,+*-5 @D:#S#= )%*-0/8' /.*3-/& @.A=

!)"

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.() .0/%B./-& %/& -A2*-&&%+(" @.A=dd#'*DJ*- ,+1&- ,+)-8 &$+H-) %(0*-.&-) 2%3,-(/./%+(

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2*+)10/%+( EF+(3 -/ .8" <L;<G

@SgV

@.AV $.& ?--( &$+H( /+ ?- %,2+*/.(/ 4+* )-B-8+2,-(/ +4 /$- *-/%(. EN.*T1.*)/ -/ .8"

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F" N-8.(+0'/- 0+8+(%M./%+( +4 /$- -2%)-*,%& .() /$- $.%* 4+88%08-&

!( /$- ,+1&-5 %( .88 $.%* 0+B-*-) *-3%+(& /$- ,-8.(+0'/-& .*- 0+(4%(-) /+ /$- $.%*

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/$- $.%* 4+88%08-& ./ B-*' -.*8' &/.3- +4 /$-%* ,+*2$+3-(-&%&" ](0- /$-' 0+8+(%M- /$-

-2%)-*,%&5 ,-8.(+?8.&/& 2*+8%4-*./- -A/-(&%B-8' %( /$- 4%*&/ <> $+1*&" :%(0- 3%B-( .*-. +4 &6%(

0.( &122+*/ 8%,%/-) (1,?-* +4 ,-8.(+0'/-&5 /$- (1,?-*& +4 ,-8.(+0'/-& *-,.%( 0+(&/.(/

H$%8- /$-' *-)%&/*%?1/- ?-/H--( -2%)-*,%& .() /$- )-*,%&" #$-' 0+8+(%M- /$- $.%* 4+88%08-& %(

*-318.* &2.0%(35 *-&18/%(3 %( %(0*-.&-) (1,?-* +4 ,-8.(+?8.&/& *-&%)%(3 %( /$- $.%* 4+88%08-&"

S/ Z;[ %(/-*4+88%018.* ,-8.(+?8.&/& 1()-*3+ . &-0+() *+1() +4 2*+8%4-*./%+(" 7' @>5 +B-* [Li

+4 /$- ,-8.(+0'/-& .*- 0+(4%(-) /+ /$- $.%* 4+88%08-&" W+0.8%M./%+( +4 ,-8.(+?8.&/& /+ $.%*

4+88%08-& %& .( .0/%B- 2*+0-&& .() %/ *-T1%*-& Y!#PY!# 8%3.() &%3(.8%(3 /+ -($.(0- ,%3*./%+( +4

/$- ,-8.(+?8.&/& %(/+ /$- $.%* 4+88%08-& E\+*).( .() \.06&+( <LLLG"

!*"

"

R%31*- _ d F-B-8+2,-(/5 ,%3*./%+( .() $+,%(3 +4 /$- ,-8.(+0'/- 8%(-.3-

#$- ,-8.(+0'/- 8%(-.3- %& )-8.,%(./-) 4*+, /$- (-1*.8 0*-&/ .() &2-0%4%-) 1()-* 0+(/*+8 +4 9(/

&%3(.8%(3 .() :+A;L -A2*-&&-) %( /$- (-1*.8 0*-&/ .() %,,-)%./-8' .4/-* /$- OJJ& 8-.B- /$- OJ"

R1*/$-* ,%3*./%+( .() &1*B%B.8 +4 /$- ,-8.(+?8.&/& %& *-318./-) ?' -()+/$-8%( = &%3(.8%(3" R%(.8 $+,%(3

&/-2 %& *-318./-) ?' Y!#PY!#W &%3(.8%(3 ES).2/-) 4*+, EW.*1-5 Y1,.&.6. .() X+)%(3 <LL=GG"

! "

"

Z" N-8.(+0'/- &/-, 0-88&

X-(-*.88'5 0-88& (--) /+ 4184%88 /H+ 0+()%/%+(& /+ ?- 0+(&%)-*-) &/-, 0-88&" R%*&/ %& /$-

&/-, 0-88 &-84D*-(-H.8h .& /$-' )%B%)-5 /$-' ,.%(/.%( /$- &/-, 0-88 2+218./%+(" :-0+()5 /$-'

2*+B%)- /$- &2-0%.8%M-) .() )%44-*-(/%./-) ).13$/-* 0-88& +4 /$-%* &2-0%4%0 /%&&1- /'2-"

N-8.(+0'/- &/-, 0-88& EN:JG .*- . &+1*0- +4 /*.(&%-(/ .,28%4'%(3 0-88& .()

)%44-*-(/%./-) ,-8.(+0'/-&5 .() $.B- ?--( &/1)%-) ,+&/ -A/-(&%B-8' %( /$- ,+1&-" S(

.)B.(/.3- /+ &/1)'%(3 /$- ,1*%(- ,-8.(+0'/- %& /$./ 3*+H/$ .() .2+2/+&%& .*- 2.%*-) /+ /$-

3*+H/$ 0'08- +4 /$- $.%* 4+88%08-" #$- &/-, 0-88& H-*- 4%*&/ %)-(/%4%-) 1&%(3 /$- 7*)e

%(0+*2+*./%+( &/.%(%(35 &%(0- /$-&- 0-88& .*- &8+H 0'08%(3 .() H-*- )-&0*%?-) .& 8.?-8D

*-/.%(%(3 0-88& EJ+/&.*-8%&5 :1( .() W.B6-* ;[[LG" #$- F0/DW.0` /*.(&3-(%0 ,+1&- ,+)-8

$.& ?--( 1&-) /+ &/1)' N:J& %( B%B+5 /.6%(3 .)B.(/.3- +4 /$- 4.0/ F0/ %& +(- +4 /$- *.*-

N:J ,.*6-*& %)-(/%4%-) /+ )./- EN.06-(M%- -/ .8" ;[[_G"

I. Melanocyte stem cell discovery and establishment of the niche

S *-,.*6.?8- &/1)' ?' O%&$%,1*. -/ .8" $.& ?--( /$- 4%*&/ /+ )-,+(&/*./- /$- 0-88& %(

/$- W@@ .& /$- N:J& /+3-/$-* H%/$ /$- *+8- /$- (%0$- %( /$- &/-, 0-88 )-/-*,%(./%+(" 7' 1&%(3

/$- .(/%DY%/ .(/%?+)'5 /$- .1/$+*& $.B- )-28-/-) /$- )%B%)%(3 .() 2%3,-(/D2*+)10%(3 0-88&5

H%/$+1/ .44-0/%(3 T1%-&0-(/ 1(2%3,-(/-) N:J&" S4/-* /$- 4%*&/ $.%* 0'08-5 /$-*- H.& . 2.*/%.8

*-0+B-*' +4 2%3,-(/./%+(5 4*+, /$- $.%* 4+88%08-& /$./ *-/.%(-) F0/DW.0` -A2*-&&%+( %( /$-

?183-5 (.,-8' %( /$- )+*&.8 +B-* $.%*& .() %( B-(/*.8 &-(&+*' $.%*&5 H$%0$ )-B-8+2 -.*8%-*

/$.( +/$-* $.%*&" R1*/$-*,+*-5 /$- W.0`Q 0-88& 0+8+(%M-) /$- ?183- .*-. .& -.*8' .& @L"U .()

&1*B%B-) %( Y%/D%()-2-()-(/ ,.((-*5 H%/$ 8+H +* 1()-/-0/.?8- Y%/ -A2*-&&%+(" #$- 7*)eD8.?-8

*-/.%(%(3 0.2.0%/' +4 /$- F0/DW.0`Q 0-88& &%/1./-) %( /$- W@@ H.& .8&+ &$+H(5 0+(4%*,%(3

/$-&- 0-88& .& /$- T1%-&0-(/5 &8+H 0'08%(3 0-88&"

!( +*)-* /+ 0+(4%*, /$- F0/DW.0`Q 0-88& %( /$- W@@ .*- %()--) &/-, 0-88&5 /$- .1/$+*&

%,28.(/-) )%44-*-(/ 2+*/%+(& +4 /$- $.%* Ee@@5 W@@ .() ?18?G %(/+ .8?%(+ (-+(./.8 ,%0-5 /+

*-0+(&/%/1/- /$- $.%* 4+88%08-&" ](8' /$- W@@ %,28.(/& 2*+)10-) 2%3,-(/-) $.%*5 )-,+(&/*./%(3

/$- W@@ *-3%+( /+ ?- /$- &/-, 0-88 (%0$-5 .() /$- F0/DW.0`Q 0-88& /+ ?- N:J& EO%&$%,1*. -/

.8" <LL<G"

II. Proliferation and differentiation of the MSCs

S2.*/ 4*+, /$- N:J5 /$- $.%* 4+88%08- %& .8&+ . (%0$- 4+* -2%/$-8%.8 &/-, 0-88& EZ:JG"

#$- Z:J& .*- ,18/%2+/-(/ .() 0.( 3%B- *%&- /+ ,18/%28- 0-88 /'2-&5 %(081)%(3 6-*./%(+0'/-& .()

!!"

"

4+88%018.* 0-88&" 7-0.1&- /$-&- /H+ &/-, 0-88 2+218./%+(& *-&%)- ./ /$- &.,- 8+0./%+(5 %/ %&

2+&&%?8- /$-*- H+18) ?- %(/-*.0/%+(& ?-/H--( /$-," #$- $.%* 4+88%08- #$- IR 1()-*3+-&

0'8%0-& )*%B-( ?' /$- 2*+8%4-*./%+( .() )%44-*-(/%./%+( +4 -2%/$-8%.8 &/-, 0-88& EZ2:J&G *-&%)%(3

%( /$- ?183- .*-. .& H-88 .& /$- &-0+().*' $.%* 3-*, E&IXG E!/+ -/ .8" <LL>G"

!( . &/1)' 0+()10/-) ?' K.??.(% -/ .85 /$- .1/$+*& )-,+(&/*./- %(/-*.0/%B./%+(

?-/H--( /$- /H+ &/-, 0-88 2+218./%+(& H%/$%( /$- (%0$-" R1*/$-*,+*- /$- &/1)' )-,+(&/*./-&

/$- *+8- +4 /$- 9(/ &%3(.8%(3 2./$H.' %( /$- ?%+8+3' +4 /$- ?+/$ +4 /$- &/-, 0-88 /'2-&"

During telogen, D0./-(%( 0+18) ?- )-/-0/-) +(8' %( /$- 0'/+28.&, +4 #*2<Q N:J& %(

/$- ?183-D&IX .*-." S/ /$- .(.3-( +(&-/5 ?+/$ Z:J& .() N:J& /$./ 0+D8+0.8%M- %( /$- &IX

display nuclear D0./-(%(5 %()%0./%(3 .0/%B- 0.(+(%0.8 9(/ &%3(.8%(3" #$- .1/$+*& 1/%8%M-) /H+

)%44-*-(/ &/*./-3%-& /+ %(B-&/%3./- /$e role of Wnt/ D0./-(%( &%3(.8%(3" R%*&/ H.& /$-

constitutive activation of D0./-(%( 1&%(3 #'*DJ*-ZK#<:: D0./-(%(48E-A=GPQ ,+1&- 8%(-" #$-

mutant MSCs displayed ectopic accumulation of D0./-(%( %( /$- (108-1&5 H$%0$ *-&18/-) %(

/$- 2*-,./1*- )%44-*-(/%./%+( +4 /$- N:J&5 .() -0/+2%0 2%3,-(/./%+( %( /$- ?183- .*-." S4/-*

&-B-*.8 $.%* 0'08-&5 /$- ,1/.(/ ,%0- &/.*/-) /+ )%&28.' 2*-,./1*- $.%* 3*.'%(35 &133-&/%(3

/$./ 0+(&/%/1/%B- 9(/ &%3(.8%(3 )*%B-& N:J 2*-,./1*- )%44-*-(/%./%+(5 /$-%* 8+&& +4 &-84D*-(-H.8

0.2.0%/'5 .() 18/%,./-8' 8-.)& /+ -A$.1&/%+( +4 /$- N:J 2++8"

D0./-(%( )-28-/%+( %( /$- N:J& %( /$- /*.(&3-(%0 ,+1&- ,+)-8 .8&+ *-&18/-) %(

2*-,./1*- $.%* 3*.'%(3" #$%& *-&18/-) %( .?-**.(/ )%44-*-(/%./%+( +4 /$- ?18? ,-8.(+0'/-&"

I+H-B-*5 /$- ?183- N:J& H-*- &/%88 2*-&-(/ .() 1(2%3,-(/-)" R1*/$-*,+*-5 /$- 9(/ 8%3.()&

expressed by ESCs activate Wnt signaling the MSCs during early anagen. D0./-(%(

&/.?%8%M./%+( %( Z:J& 2*+,+/-& ,-8.(+0'/- 2*+8%4-*./%+(5 H$%0$ %& ,-)%./-) ?' Z)(=PZ)(*7

&%3(.8%(3 EK.??.(% -/ .8" <L;;G"

III. MSC quiescence, maintenance in undifferentiated state and self-renewal

9$%8- 9(/ &%3(.8%(3 2./$H.' %& %,2+*/.(/ %( N:J 2*+8%4-*./%+( .() )%44-*-(/%./%+(5

&-B-*.8 +/$-* &%3(.8%(3 2*+/-%(& .() 2./$H.'& $.B- . *+8- %( N:J T1%-&0-(0-5 ,.%(/-(.(0-

%( 1()%44-*-(/%./-) &/./- .() /$-%* &-84D*-(-H.8 &10$ .& #XF signaling pathway, Bcl2, and

/$- O+/0$ 2./$H.'"

TGF signaling proteins are expressed during catagen and promote apoptosis that

*-&18/& %( 0+,28-/- *-3*-&&%+( +4 /$- $.%* &$.4/" !( /$- 0-88& +4 /$- ?183-5 -A2*-&&%+( +4 /$-

(108-.* 2$+&2$+D:,.)< 0.( be detected, indicating activated TGF signaling. The exposure

to TGF signaling pathway promotes cellDcycle arrest, both in vitro and in vivo. In vitro, TGF

%()10-& 0-88 0'08- .**-&/5 .() %/ .8&+ .?*+3./-& 2%3,-(/ 2*+)10/%+( ?' )+H(D*-318./%(3 N!#R

.() %ts downstream target genes required for melanogenesis. In the mouse, TGF signaling

%& *-T1%*-) /+ ,.%(/.%( /$- N:J 2++8 ?' *-318./%(3 /$- *-D-(/*' +4 /$- 0-88& %( /$- T1%-&0-(/

!#"

"

state. Conditional ablation of TGF signaling by one of the TGF signaling, TGRD? /'2- !!

*-0-2/+* E#XR?K!!G %( /$- ,-8.(+0'/- 8%(-.3- %( /$- ,+1&- 0.1&-& %(0+,28-/- ,.%(/-(.(0-

+4 N:J& .() *-&18/& %( ,%8) $.%* 3*.'%(3 EO%&$%,1*. -/ .8" <L;LG" #$- 2*+0-&& +4

-&/.?8%&$,-(/ +4 /$- T1%-&0-(/ &/./- %& )-2-()-(/ +4 708<" #$- ,1/.(/ ,+1&- $.*?+*%(3

,-8.(+0'/-D&2-0%4%0 -A2*-&&%+( +4 ,1/.(/ 708< .8&+ *-&18/& %( 2*+3*-&&%B- $.%* 3*.'%(3 .()

0+,28-/- 8+&& +4 /$- N:J& %( /$- ?183- *-3%+( )1- /+ .2+2/+&%& .& /$-' *-D-(/-* /$-

T1%-&0-(/ &/./-" I+H-B-*5 /$- 708< .?8./%+( $.& ,+*- 2*+(+1(0-) 2$-(+/'2-5 &%(0- /$- $.%*

3*.'%(3 %& 4.&/-* .() ,+*- 0+,28-/- EO%&$%,1*.5 X*.(/-* .() R%&$-* <LLUG"

#$- O+/0$ &%3(.8%(3 2./$H.' 0+,2+(-(/& 0+,2*%&- /$- &1*4.0- *-0-2/+*& O+/0$;D>5

.() 8%3.()& \.33-)D;5 < .() F-8/.D8%6- EF88G;D=" W%3.() ?%()%(3 /+ /$- *-0-2/+* &/%,18./-&

2*+/-+8'/%0 08-.B.3-5 *-&18/%(3 %( /$- *-8-.&- +4 /$- %(/*.0-8818.* )+,.%( +4 O+/0$ EO!JG5 H$%0$

%& /*.(&8+0./%(3 /+ /$- (108-1& .() ?%()%(3 /+ J:W /*.(&0*%2/%+( 4.0/+*&5 %( ,+1&- 6(+H( .&

*-0+,?%(./%+( &%3(.8 ?%()%(3 2*+/-%(D\ EK7@D\G5 .0/%B./%(3 B.*%+1& /.*3-/ 3-(-&5 %(081)%(3

/*.(&0*%2/%+( +4 *-2*-&&+*& ?-8+(3%(3 /+ /$- IZ: EI.%*'PZ($.(0-* +4 :28%/G 4.,%8' EK.)/6-5

:0$H-%&31/$ .() @-.* <LLUG"

!( ,-8.(+0'/-&5 /$- 0+()%/%+(.8 6(+06+1/ +4 /$- O+/0$; .() O+/0$< *-0-2/+*& 1&%(3

/$- #'*DJ*- ,+1&- 8%(- )+-& (+/ &$+H .(' )-4-0/& %( ,-8.(+?8.&/ 2*+8%4-*./%+( .()P+*

,%3*./%+( )1*%(3 /$- -,?*'+(%0 )-B-8+2,-(/ E:0$+1H-' -/ .8" <LL_G"

#$- *-&18/ +4 0+()%/%+(.8 .?8./%+( +4 K7@D\ H.& . &/*%6%(3 8+&& +4 0+./ 0+8+* 4*+, ?%*/$5

.() /$- 2$-(+/'2- H.& 41*/$-* .00-8-*./-) ?' )-2%8./%+(" I%&/+8+3%0.8 .(.8'&%& +4 /$- ,1/.(/

.(%,.8& &$+H-) . )*.,./%0 *-)10/%+( +4 /$- ,-8.(+0'/-& %( /$- $.%* 4+88%08-& ./ @> .()

B%*/1.88' 0+,28-/- .?&-(0- %4 /$- ,-8.(+0'/-& ./ @=>" !( .))%/%+(5 O+/0$ &%3(.8%(3 $.& . B%/.8

*+8- %( /$- ,.%(/-(.(0- +4 /$- N:J& %( /$- ,1/.(/ .(%,.8&5 -B%)-(0-) ?' 0+,28-/- .?&-(0-

+4 F0/D-A2*-&&%(3 0-88& %( /$- ?183-" Z0/+2%0 -A2*-&&%+( +4 I-&; 1()-* /$- 0+(/*+8 +4 F0/

2*+,+/-* H.& .?8- /+ *-&01- /$- ,1/.(/ 2$-(+/'2-5 %()%0./%(3 I-&; %& /$- 2*-)+,%(.(/ I-&

/*.(&0*%2/%+( 4.0/+* 3+B-*(%(3 /$- O+/0$ &%3(.8%(3D.0/%B./-) /*.(&0*%2/%+( *-318./%+( %(

,-8.(+0'/-&" EN+*%'.,. -/ .8" <LLVG"

S))%/%+(.8 &/1)%-& 1&%(3 K7@D\65 O+/0$; .() O+/0$< 0+()%/%+(.8 .?8./%+( 1&%(3 /$-

#'*DJ*- ,%0- $.B- &$+H( ?+/$ +4 /$- *-0-2/+*& .*- -&&-(/%.8 4+* *-3-(-*./%+( +4 /$- 2%3,-(/

0-88 2+218./%+( )1*%(3 /$- $.%* 0'08-" 7+/$ $+,+M'3+1& O+/0$; .() O+/0$< ,1/.(/& .*- B-*'

&%,%8.* /+ /$- K7@D\ ,1/.(/5 H$%8- /$- )+1?8- 6(+06+1/ +4 /$- *-0-2/+*& -A$%?%/& -B-( ,+*-

.00-8-*./-) 2*+0-&& +4 0+./ 0+8+* 8+&& E:0$+1H-' -/ .8" <LL_G" R1*/$-* 0$.*.0/-*%M./%+( +4 /$-

K7@D\ ,1/.(/ %( 0+,?%(./%+( H%/$ F0/D8.0` -A2*-&&%+( &$+H-) &-B-*.8 .))%/%+(.8 *+8-& +4 /$-

O+/0$ &%3(.8%(3 %( ,-8.(+0'/-&" #$- .?&-(0- +4 /$- O+/0$ &%3(.8 0.1&-& N:J 2*-,./1*-

)%44-*-(/%./%+( %( /$- ?183-" R1*/$-*,+*-5 O+/0$D)-4%0%-(/ ,./*%A ,-8.(+0'/-& 0.((+/ 2*+)10-

2%3,-(/" R%(.88'5 N:J& .() ,-8.(+0'/-& -A$%?%/-) -0/+2%0 8+0.8%M./%+( %( /$- ,1/.(/ .(%,.8&5

!$"

"

&10$ .& /$- .*-. H%/$%( /$- )-*,.8 2.2%88.-5 +* /$- )-*,%& %( B%0%(%/' +4 /$- W@@ ES1?%(D

I+1M-8&/-%( -/ .8" <LL^G"

!

"

R%31*- ^ d N-8.(+0'/- &/-, 0-88 -&/.?8%&$,-(/ .() ,.%(/-(.(0-

!# #$- *-318./+*' (-/H+*6 0+(/*+88%(3 /$- ,-8.(+0'/- 8%(-.3- %(081)-& -A2*-&&%+( +4 F0/ .() @.A= 4*+, B-*' -.*8' &/.3-& +4 ,-8.(+0'/- )-B-8+2,-(/ .() %& ,.%(/.%(-) %( /$- ,-8.(+0'/- &/-, 0-88& E.).2/-) 4*+, E]&.H. <LL^GG" "# F%44-*-(/ ,-0$.(%&, *-318./%(3 ,-8.(+0'/- &/-, 0-88 ,.%(/-(.(0- .() &-84D*-(-H.8" !($%?%/%+( +4 /$- Y!# &%3(.8%(3 .() 8+&& +4 0.(+(%0.8 9(/ &%3(.8%(3 *-&18/& %& 8+&& +4 ,18/%2+/-(0' +4 /$- N:J&" #$- MSCs remain in the bulge, but are unable to generate progeny. TGF and Notch signaling loss, +B-*.0/%B- 9(/ &%3(.8%(3 .() 3-(+/+H%0 &/*-&& *-&18/& %( 2*-,./1*- )%44-*-(/%./%+( +4 /$- .() -A$.1&/%+( +4 /$- N:J 2++8 E.).2/-) 4*+, EW.(35 N.&0.*-($.& .() :$-. <L;=GG"

!%"

"

R" #$- *+8- +4 ,-8.(+0'/-& %( 2%3,-(/./%+(

@%3,-(/./%+( +4 /$- &6%( %& . ,18/%D&/-2 2*+0-&&5 +001**%(3 ?+/$ %( /$- -2%)-*,%& .()

/$- $.%* 4+88%08-&" !/ &/.*/& H%/$ /$- &'(/$-&%& +4 ,-8.(%( %( ,-8.(+&+,-&5 /$- &2-0%.8%M-)

,-8.(+0'/-&5 4+88+H-) ?' /$-%* /*.(&4-* /+ 6-*./%(+0'/-& ?' /$- 1&- +4 ,-8.(+0'/- )-()*%/-&"

I. The melanin unit � epidermal and follicular melanocyte interactions

#$- 41(0/%+(.8 +*3.(%M./%+( +4 /$- 6-*./%(+0'/-& .() /$- ,-8.(+0'/-& %( /$- -2%)-*,%&

%( /-*,-d �the epidermal melanin unit� EI..&& -/ .8" <LLUG" Y-*./%(+0'/-& &/%,18./-

,-8.(+0'/- 3*+H/$ )-()*%0%/' .() 2%3,-(/ 2*+)10/%+( ?' &-0*-/%(3 ,18/%28- 4.0/+*& &10$ .&

growth factors (FGF, SCF, HGF), hormones like !DMSH, and cytokines such as TGF

ER%31*- [G"

!( /$- $.%* 4+88%08-&5 /$- ,-8.(+0'/-& /+ 6-*./%(+0'/-& *./%+ %& ;dU E:8+,%(&6% -/ .8"

<LLUG" @%3,-(/./%+( +4 /$- $.%* 4+88%08- %& ,+*- 0+,28-A .() %/ %(B+8B-) 0++*)%(./-) .0/%+( +4

/$- 4+88%018.* ,-8.(+0'/-&5 6-*./%(+0'/-& %( /$- ,./*%A .() 4%?*+?8.&/& *-&%)%(3 %( /$- )-*,.8

papilla. The pigmentation system in the follicle is called �hair melanin unit� or �follicular

melanin unit�. Hair melanocytes are larger and have more dendritic proj-0/%+(& /$.( /$-%*

-2%)-*,.8 0+1(/-*2.*/& E:8+,%(&6% -/ .8" <LLU5 #+?%( <L;;G" Z2%)-*,.8 ,-8.(+0'/-& .*- 8+(3D

8%B%(3 0-88&5 H$%8- $.%* 4+88%08- ,-8.(+0'/-& )%- /$*+13$ .2+2/+&%& ./ /$- -() +4 -B-*' /$- $.%*

0'08-" #$- 2%3,-(/ 2*+)10/%+( %& %(%/%./-) .() +(3+%(3 +(8' )1*%(3 /$- .(.3-( 2$.&-h %/ %&

&%3(%4%0.(/8' *-)10-) %( /$- 0./.3-(5 .() 0+,28-/-8' .?&-(/ )1*%(3 /-8+3-( E#+?%( <L;;5

O%&$%,1*. <L;;G"

II. Molecular basis of the suntan response

ZA/-*(.8 &%3(.8& +4 ,-8.(+3-(-&%& %( /$- -2%)-*,%&5 .8&+ 6(+H( .& &1(/.( *-&2+(&-5

2*%,.*%8' %(081)- ef *.)%./%+(D,-)%./-) 2*+)10/%+( +4 !D,-8.(+0'/-Dstimulating hormone (!D

MSH). The gene encoding !DN:I %& 2*+D+2%+,-8.(+0+*/%( E@]NJG5 . ,18/%0+,2+(-(/

precursor for ! DN:I E,-8.(+/*+2%0G5 SJ#I E.)*-(+0+*/%0+/*+2%0G5 .() /$- +2%+%) 2-2/%)- D

-()+*2$%( EY%22-(?-*3-* -/ .8" ;[[VG" !DN:I %& 2*+)10-) .() &-0*-/-) 4+88+H%(3 ef ?' ?+/$

6-*./%(+0'/-& .() ,-8.(+0'/-& %( /$- &6%( E:0$.1-* -/ .8" ;[[>G. The levels of POMC/ !D

N:I .*- 3*-./8' -8-B./-) .& . *-&2+(&- /+ /$- ef *.)%./%+(5 .() /$- ,.%( &-(&+* .()

-44-0/+* +4 /$%& *-&2+(&- %& 2U= EJ1% -/ .8" <LL_G" N-8.(+0+*/%(D;D*-0-2/+* ENJ;KG %&

-A2*-&&-) in melanocytes and is activated upon stimulation with the !DN:I" NJ;K $.&

?--( &$+H( /+ ?- %,2+*/.(/ %( /.((%(3 .() 2%3,-(/./%+( %( $1,.(& Ef.8B-*)- -/ .8" ;[[UG"

TGF signaling has been shown to be crucial for regulation of the tanning response" !( /$-

!&"

"

absence of the UV radiation, TGF is secreted by the keratinocytes and is repressing PAX3,

*-&18/%(3 %( 8+H -A2*-&&%+( +4 N!#R .() 2%3,-(/./%+( *-8./-) 3-(-&" e2+( ef *.)%./%+(5

TGF repression of PAX3 is released through p53 activation of APD;5 .0/%(3 .& . *-2*-&&+*

of TGF EC.(3 -/ .8" <LL^G. Additionally, the signaling cascade !MSH/MC1R is also

*-318./-) ?' N!#R5 &%(0- NJ;K %& . )%*-0/ N!#R /.*3-/ ES+6% .() N+*+ <LL<G"

The !DN:IPNJ;K &%3(.8%(35 /$*+13$ XD2*+/-%( 0+128-) *-0-2/+*5 .0/%B./-& /$-

.)-('8./- 0'08.&- ESJG .() *-&18/& %( 2*+)10/%+( +4 0SN@" :%3(.8%(3 B%. 0SN@ $.& ,18/%28-

)+H(&/*-., /.*3-/&" #$-&- %(081)- 2*+/-%( 6%(.&- S E@YSG5 NS@YPZKY E,%/+3-( .0/%B./-)

2*+/-%( 6%(.&-P-A/*.0-8818.* &%3(.8D*-318./-) 6%(.&-G &%3(.8%(3 2./$H.'" @$+&2$./%)'8 !(+&%/+8D

=D6%.(&- E@!=YG 2./$H.' %& %( /1*( %($%?%/-) ?' /$- 0SN@ &%3(.8%(3" #$- .0/%B./-) @YS 41*/$-*

2$+&2$+*'8./-) /$- 0SN@ *-&2+(&- -8-,-(/D?%()%(3 2*+/-%( EJKZ7G" JKZ7 %& . /*.(&0*%2/%+(

factor that is principally responsive to the !DN:IPSN@0 /$./ %& 3+%(3 /$*+13$ /$- &1*4.0-

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/$- N!#R 2*+,+/-* *-B-.8-) . 0'08%0 SN@ E0SN@G *-&2+(&%B- -8-,-(/ EJKZG ./ 2+&%/%+( D;>L

/+ D;>_ /+ /$- /*.(&0*%2/%+( &/.*/ &%/- E7-*/+8+//+ -/ .8" ;[[^.5 R1&- -/ .8" ;[[[G" Z8-B./-)

0SN@ 8-B-8& .*- %()10%(3 /$- JKZ7 ?%()%(3 /+ /$- 2*+,+/-*& +4 N!#R5 H$%0$ %& .0/%B./%(3 /$-

-(M',-& %(B+8B-) %( 2%3,-(/ 2*+)10/%+(5 /'*+&%(.&-5 #*2; .() F0/5 /$*+13$ $%3$8' 0+(&-*B-)

ND?+A %( /$-%* 2*+,+/-*& E7-*/+8+//+ -/ .8" ;[[^.5 7-*/+8+//+ -/ .8" ;[[V5 7-*/+8+//+ -/ .8"

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#$- NS@YPZKY &%3(.8%(3 2./$H.' %& .8&+ %()10-) ?' 0SN@ &%3(.8%(3 .() %& 41*/$-*

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%/& )-3*.)./%+( ?' /$- 2*+/-+&+,.8 ,.0$%(-*'5 .() /$1& %($%?%/%(3 ,-8.(+3-(-&%&" #$%&

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E71&0. .() 7.88+//% <LLLG"

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<LLLG"

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!'"

"

/*.(&0*%2/%+( 4.0/+*" S0/%B./-) e:R; ?%()& /'*+&%(.&- 2*+,+/-* .() 2*+,+/-& %/& -A2*-&&%+(

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*.)%./%+(D,-)%./-) /.((%(3 *-&2+(&-" O.,-8'5 /$- .0/%B./%+( +4 @]NJ5 NJ;K .() /$-

,-8.(+3-(-&%& -(M',-& %& .?+8%&$-) %( /$- .?&-(0- +4 e:R; EJ+**- -/ .8" <LL>G"

III. Melanin synthesis and melanosome production

#H+ ,.%( /'2-& +4 ,-8.(%( .*- &'(/$-&%M-) H%/$%( ,-8.(+&+,-&5 2$-+,-8.(%( .()

-1,-8.(%(" N-8.(%( 2*+)10/%+( $.& ,18/%28- ?-(-4%0%.8 /*.%/& %(081)%(3 .?&+*2/%+( +4 ef

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J+&/%( .() I-.*%(3 <LL_5 K%8-' ;[[_G"

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*-&18/%(3 %( 2*+)10/%+( +4 WD=5>D)%$')*+ A'2$-('8.8.(%(- EF]@SG" F]@S %& /$-( +A%)%M-)5

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)%$')*+A'%()+8- EFI!G5 H$%0$ 1()-*3+-& +A%)./%+( .() 2+8',-*%M./%+( 3%B%(3 *%&- /+ /$-

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#("

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R%31*- [ d #.((%(3 *-&2+(&- � 6-*./%(+0'/-D,-8.(+0'/- %(/-*.0/%+( 8-.)%(3 /+ 2%3,-(/ 2*+)10/%+(

Y-*./%(+0'/-& .() ,-8.(+0'/-& %(/-*.0/ %( /$- -2%)-*,%&5 H$-*- 12+( ef *.)%./%+( 6-*./%(+0'/-&

secrete multiple signaling molecules, including KITL, !MSH, TGF and others resulting %( +(&-/ +4

2%3,-(/ 2*+)10/%+(" #$- )-/.%8& +4 /$- 2%3,-(/ 2*+)10/%+( *-.0/%+(& .*- )-&0*%?-) %( /$- /-A/" E.).2/-)

4*+, E]&.H. <LL^GG"

+,"

-./0123.456"7043"85./47039:451"

$%&'()*)+%

#)"

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IV. Role of melanocytes in diversity of human pigmentation

I1,.( &6%( $.*?+*& .88 /'2-& +4 ,-8.(%(5 .() /$- *./%+ ?-/H--( /$-, )-/-*,%(-&

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E,%0*+2$/$.8,%0G .() $.) (.,-) /$- 8+01& mi" #$- 3-(- 0+**-&2+()%(3 /+ /$%& 8+01&5 Mitf,

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2*+,+/-*& 0+)- 4+* (%(- )%44-*-(/ %&+4+*,& /$./ $.B- ?--( %)-(/%4%-) /+ )./- -A2*-&&-) 4*+,

/$- mi 8+01&d N!#R � S5 75 J5 F5 Z5 I, J, M and Mc. The first 5� exon differs for all the

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# "

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[" #$- 0+8+*-) ?+A-& *-2*-&-(/ -A+(& ?-8+(3%(3 /+ /$-%* *-&2-0/%B- %&+4+*,&" N-8.(+0'/- &2-0%4%0

%&+4+*, N %& )-2%0/-) %( /$- ?+//+, +4 /$- 2.(-8 E,.3-(/. ;N -A+(G" #$- ?IWI )+,.%( %& )-2%0/-) ?'

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II. The MITF protein structure

#$- N%0*+2$/$.8,%.D.&&+0%./-) /*.(&0*%2/%+( 4.0/+* EN!#RG %& . ,-,?-* +4 /$-

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1&1.88' *-,.%(& +( /$- &1*4.0- 4+* . 8+(3 /%,- ?-4+*- 2-(-/*./%(3 %(/+ /$- )--2-* 8.'-*& +4

/$- &6%(" #$%& /'2- %& *-2+*/-) ,+*- +4/-( %( /$- -8)-*8' 2-+28-5 .22-.*%(3 +( *-3%+(& +4 /$-

?+)' /$./ .*- 0$*+(%0.88' -A2+&-) /+ /$- &1(5 &10$ .& /$- 4.0-5 -.*&5 .*,& .() 122-* /*1(6"

Acral lentiginous melanoma (ALM) %& .8&+ &2*-.)%(3 +( /$- &1*4.0- +4 /$- &6%( %( /$-

?-3%((%(3 +4 %/& )-B-8+2,-(/" #$- 0$.*.0/-*%&/%0 4-./1*- +4 /$%& /'2- %& %/ .22-.*& 1()-* /$-

(.%8& +* +( /$- &+8-& +4 /$- 4--/ +* 2.8,&5 .& ?8.06 +* ?*+H( *-3%+(&" #$%& /'2- +4 ,-8.(+,.

%& /$- ,+&/ 0+,,+( %( S4*%0.(DS,-*%0.(& .() S&%.(&5 .() /$- 8-.&/ 0+,,+( .,+(3

J.10.&%.(&" !/ 0.( .8&+ +4/-( 2*+3*-&& ,+*- T1%068' /$.( ::N .() WN"

Nodular melanoma (NM) %& +4/-( %(B.&%B- ./ /$- /%,- +4 /$- )%.3(+&%&" !/ %& ,+&/ 0+,,+( %(

/$- -8)-*8'" #$- /'2%0.8 8+0./%+(& %(081)- /$- /*1(65 .*,&5 8-3&5 .() /$- &0.82 *-3%+( %( ,-("

#$%& /'2- %& /$- ,+&/ .33*-&&%B- +4 .88 /$- /'2-& +4 ,-8.(+,.&5 .() %& *-2+*/-) %( ;Li /+

;Ui +4 0.&-&"

$%&'()+' 15.%6!7108'10(9

+:1'10)(

";!< =;!2 >?@

2:.%/A070'& *./%'B0(9 +%&'()+' VLi <Li j;i

C%(109) +'&09(' ;Li ;Ui <^i

!7/'& &%(1090():* +%&'()+' <Li ;Li =Vi

=)B:&'/ +%&'()+' _Li ;<i j<i

#.?8- < d #$- 2-*0-(/.3- +4 .001*.(0- +4 )%44-*-(/ .0/%B./%(3 ,1/./%+(& E7KSR5OKS:5 Y!#G %( /$- ,.%(

,-8.(+,. /'2-&

S).2/-) 4*+, EY.??.*.$ .() J$%( <LLUG

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F" S ,+8-018.* ,+)-8 +4 ,-8.(+,. 2*+3*-&&%+(

#$- J8.*6 ,+)-85 . )-/.%8-) &/1)' +4 2*-01*&+* 8-&%+(& 8-.)%(3 /+ /$-

)-B-8+2,-(/ .() /$- 2*+3*-&&%+( +4 ,-/.&/./%0 ,-8.(+,. -,2$.&%M-& /$- &/-2H%&-

/*.(&4+*,./%+( +4 ,-8.(+0'/-& /+ ,-8.(+,.h %/ )-&0*%?-& .88 /$- $%&/+8+3%0 0$.(3-&

.00+,2.('%(3 )%44-*-(/ 2$.&-& +4 2*+3*-&&%+( EJ8.*6 -/ .8" ;[^>G ER%31*- ;=G"

#$- ,+)-8 *-0.2%/18./-& &%A )%44-*-(/ &/-2& +4 )%&-.&- 2*+3*-&&%+(d

Benign nevus5 0$.*.0/-*%M-) ?' ,-8.(+0'/- 2*+8%4-*./%+( .() ?-(%3( 8-&%+(&" #$-&- (-B%

0+(/.%( ,-8.(+0'/-& /$./ H%88 %( ,+&/ 0.&-& 1()-*3+ /-*,%(.8 )%44-*-(/%./%+("

Hyperplastic nevus, 0$.*.0/-*%M-) .& ,-8.(+0'/%0 $'2-*28.&%.5 $.& .& %/& ,.%( 4-./1*- /$-

.?-**.(/ )%44-*-(/%./%+( +4 /$- ,-8.(+0'/-&" #$-&- (-B% 0+(/.%( %(0*-.&-) (1,?-* +4

,-8.(+0'/-&5 .*- 8+0./-) .8+(3 /$- ?.&.8 8.'-*5 .() /$-%* 3*+H/$ %& 8%,%/-)"

Dysplastic nevus5 0+(&%&/-) +4 )'&28.&/%0 0-88& /$./ $.B- .0T1%*-) /$- 2*+2-*/%-& +4 .?-**.(/

3*+H/$" :10$ &/*10/1*-& .*- %)-(/%4%-) .& .&',,-/*%05 H%/$ %**-318.* ?+*)-*&5 %(0*-.&%(3 %(

)%.,-/-* .() $.B- ,18/%28- 0+8+*& H%/$%( /$- (-B1&"

Radial-growth phase %& 0$.*.0/-*%M-) ?' .( %(/*.D-2%)-*,.8 0-88 3*+H/$" #$-&- 8-&%+(& &$+H

0'/+,+*2$+8+3%0.8 4-./1*-& +4 0.(0-*" #$-' &/.*/ /+ 2-(-/*./- /$- 2.2%88.*' )-*,%&5 ?1/ 4.%8 /+

4+*, 0+8+(%-& %( &+4/ .3.*"

Vertical-growth phase, 0+(&%)-*-) /+ ?- /$- /%22%(3 2+%(/ +4 ,-/.&/./%0 ,-8.(+,.

)-B-8+2,-(/5 %& 0$.*.0/-*%M-) ?' )-*,.8 %(B.&%+(" #$-&- 8-&%+(& 0+(/.%( /$- 0-88& /$./ $.B-

.0T1%*-) /$- .?%8%/' /+ %(B.)- /$- )-*,%&5 -A2.() /$*+13$ /$- 2.2%88.*' .() *-/%018.* )-*,%&

.() 4./" #$-' .*- 0.2.?8- +4 3*+H%(3 %( &+4/ .3.* .() 4+*, /1,+* ,+)18-& H$-( %(a-0/-) %(/+

(1)- ,%0-"

Metastasis, /$- 4%(.8 &/-2 %( /$- ,+)-85 -(0.2&18./-& /$- &100-&&418 &2*-.) +4 0-88& %( +/$-*

.*-.& +4 /$- &6%(5 /$-%* %(/*.B.&./%+( %(/+ ?8++) .() 8',2$./%0 B-&&-8& .() .?%8%/' /+ 4+*,

,-/.&/./%0 4+0% %( +/$-* +*3.(&"

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R%31*- ;= d N+)-8 +4 ,-8.(+,. 2*+3*-&&%+(

!( /$- ,+)-8 +4 ,-8.(+,. 2*+3*-&&%+(5 ,-8.(+0'/-& .*- *-2*-&-(/-) %( ?81-5 .() 2.&&%(3 /$*+13$

)%44-*-(/ 2$.&-& +4 ,-8.(+,. )-B-8+2,-(/" !( /$- 8+H-* 2.(-8 .*- .88 /$- ,1/./%+(& *-0+*)-) /+

0+(/*%?1/- /+ ,-8.(+,. 2*+3*-&&%+(" I+H-B-*5 (+/ -B-*' 0.&- +4 ,-8.(+,. 2*+3*-&&%+( 3+-& /$*+13$

.88 /$- )-2%0/-) &/-2& EN%88-* .() N%$, <LLVG"

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I. Benign nevus formation � BRAF and NRAS as driver mutations

!( /$- J8.*6 ,+)-85 /$- %(%/%.8 &/.3- +4 ,-8.(+0'/- /*.(&4+*,./%+( %& . ?-(%3( (-B1&

ER%31*- ;=G" #$- ,.%( 0.1&- +4 /$- (-B1& 4+*,./%+( %& .( .?(+*,.8 .0/%B./%+( +4 /$-

NS@YPZKY &%3(.8%(3 2./$H.'" #$- ,+&/ 0+,,+( ,-0$.(%&, +4 /$%& 2./$H.' %& . &+,./%0

3.%(D+4D41(0/%+( ,1/./%+(& +4 K.& 2*+/-%(& E,+&/ 0+,,+( OKSRG5 +* K.4 2*+/-%(& E,+&/

0+,,+( 7KSRG5 H$%0$ .*- 2*-&-(/ %( [Li +4 ,-8.(+,. 0.&-& E`$1.(3 -/ .8" <LLUG"

!(%/%.8 4%()%(3& &133-&/-) /$./ ZKY $'2-*.0/%B./%+( %& . *-&18/ +4 OKS: ,1/./%+(5 &%(0-

%/ $.& ?--( *-2+*/-) %( ?-/H--( ;UD=Li +4 ,-8.(+,.& EB.( Z8&.& -/ .8" ;[[UG" OKS: %& .

2.*/ +4 KS: 4.,%8' +4 &,.88 X#@.&-& E.8+(3 H%/$ IKS: .() YKS:G" #$- ,+&/ 0+,,+(

,1/./%+( +4 OKS: %& . &1?&/%/1/%+( +4 381/.,%(- /+ 8'&%(- +* .*3%(%(- ./ 2+&%/%+( V; EbV;Y5

bV;KG EF.B%-& -/ .8" <LL<5 Z88-*$+*&/ -/ .8" <L;;G"

!/ H.& 8./-* )%&0+B-*-) /$./ ,+&/ 0+,,+(8' ,1/./-) 0+,2+(-(/ +4 /$%& 2./$H.' %&

7KSR5 +(- +4 /$- /$*-- K.4 2*+/-%(& E.8+(3 H%/$ SKSR .() JKSRG" !/ %& ,1/./-) %( ULD_Li +4

/$- 0.&-& H%/$ /$- 2$+&2$+D,%,%0 &1?&/%/1/%+( +4 B.8%(-D/+D381/.,%0 .0%) ./ /$- 2+&%/%+( VLL

EfVLLZG *-2*-&-(/%(3 /$- B.&/ ,.a+*%/' +4 /$- .0/%B./%(3 ,1/./%+(& EF.B%-& -/ .8" <LL<G" !(

,-8.(+,.&5 OKS: .() 7KSR ,1/./%+(& .*- %( 3-(-*.8 ,1/1.88' -A081&%B-5 %()%0./%(3 -%/$-*

+4 /$- /H+ ,1/./%+(& %& &144%0%-(/ /+ 0+(&/%/1/%B-8' .0/%B./- /$- NS@Y &%3(.8%(3 2./$H.' EX+-8

-/ .8" <LLVG"

ZB-( /$+13$ 7KSR .0/%B./%+( %& /$- ,+&/ 2*-B.8-(/ 2*%,.*' ,1/./%+( )*%B%(3

,-8.(+,. )-B-8+2,-(/5 7KSR ,1/./%+(& +001* ./ . &%,%8.* 4*-T1-(0' %( ?-(%3( (-B% .() %(

2*%,.*' .() ,-/.&/./%0 ,-8.(+,.& E@+88+06 -/ .8" <LL=G" #$- -A28.(./%+( 4+* /$%& 0.,- 4*+,

. &/1)' )-,+(&/*./%(3 /$./ +(0+3-(%0 7KSR /*%33-*& . 2*+3*., +4 +(0+3-(-D%()10-)

&-(-&0-(0-5 0.1&%(3 0-88& /+ -(/-* %**-B-*&%?8- 3*+H/$ .**-&/5 .() *-,.%( &/./%0 EF$+,-( -/

.8" <LL[5 N%0$.8+38+1 -/ .8" <LLUG" #$%& &133-&/& /$./ (-B% .0T1%*- .))%/%+(.8 ,1/./%+(& %(

+*)-* /+ ?'2.&& /$- &-(-&0-(0- ?8+06 .() )-B-8+2 . ,.8%3(.(/ 8-&%+("

II. Dysplastic nevus formation � CDKN2A and PTEN inactivation

N.%( 0$.*.0/-*%&/%0 +4 )'&28.&/%0 (-B% .*- ,1/./%+(& %( /$- CDKN2A .() PTEN,

0.1&%(3 /$- %(.0/%B./%+( +4 /$- 2*+/-%( /$-&- 8+0% -(0+)-" S 3-*,8%(- ,1/./%+( )-.0/%B./-&

JFYO<S %( <UD>L i +4 4.,%8%.8 ,-8.(+,. E#$+,2&+(5 :0+8'-* .() Y-44+*) <LLUG5 H$%8-

@#ZO %& ,1/./-) %( <UDULi +4 (+(D4.,%8%.8 ,-8.(+,. E915 X+-8 .() I.81&6. <LL=G"

CDKN2A %& . /1,+* &122*-&&+* 3-(- &%/1./-) ./ /$- [2<; 8+01&" !/ %& 0+,28-A .() %/

-(0+)-& /H+ )%&/%(0/ 2*+/-%(&5 !OY>S .() SKR /$*+13$ .8/-*(./%B- 2*+,+/-*& .() 4%*&/ -A+(&

-(0+)-& /H+ /1,+* &122*-&&+* 2*+/-%(& EJ$%(5 @+,-*.(/M .() F-@%($+ ;[[^G ER%31*- ;>G"

$$"

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#$- &-0+() -A+( /$./ %& &$.*-) %& /*.(&8./-) %( )%44-*-(/ *-.)%(3 4*.,- 4+* /$- /H+ 2*+/-%(&5

*-()-*%(3 /H+ 2*+/-%(& H%/$ (+ .,%(+ .0%) $+,+8+3'5 ?1/ (-B-*/$-8-&& $.B%(3 . 0*10%.8 *+8- %(

.(/%/1,+*.8 *-&2+(&-&"

!OY>S E2;V!OY>SG5 /$- 4+1()%(3 ,-,?-* +4 !OY> E%($%?%/+* +4 0'08%(D)-2-()-(/ 6%(.&-

>G 4.,%8' +4 2*+/-%(&5 %($%?%/& /$- 0-88 0'08- %( /$- X; 2$.&- ?' %($%?%/%(3 0'08%(D)-2-()-(/

6%(.&-& EJFY&G >PV" #$-&- JFY& 2$+&2$+*'8./- .() %(.0/%B./- *-/%(+?8.&/+,. EK?G 2*+/-%(5

/$-*-?' .88+H%(3 /$- : 2$.&- -(/*' E:-**.(+5 I.((+( .() 7-.0$ ;[[=G" W+&& +4 !OY>S

2*+,+/-& K? %(.0/%B./%+( /$*+13$ %/& $'2-*2$+&2$+*'8./%+( .() .0/%B./%+( +4 /*.(&0*%2/%+(

4.0/+* Z<R;5 *-&18/%(3 %( .( 1(*-&/*.%(-) 0-88 0'08- 2*+3*-&&%+(" !OY>S $.& .( %,2+*/.(/ *+8-

%( 0+(/*+8 +4 0-8818.* &-(-&0-(0- &%(0- /$*+13$ !OY>SDK7 2./$H.' E:B%)-*&6.'. -/ .8" <LL=G"

SKR E.8/-*(./%B- *-.)%(3 4*.,-G 2*+/-%( E2;>SKR %( $1,.(& .() 2;[SKR %( ,%0-G $.& .

*+8- %( /$- %($%?%/%+( +4 NFN<D,-)%./-) 1?%T1%/%(./%+( .() 2*+/-.&+,- )-3*.)./%+( +4 2U="

!(.0/%B./%+( +4 SKR5 /$*+13$ %(.0/%B./%+( +4 2U=5 .44-0/& 0-88 0'08- 0+(/*+85 FOS ).,.3-

*-&2+(&- .() /$- *-318./%+( +4 0-88 )-./$ EJ$%(5 @+,-*.(/M .() F-@%($+ ;[[^5 Y.,%a+ -/ .8"

;[[^5 `$.(35 g%+(3 .() C.*?*+13$ ;[[^G" !( 0+(/-A/ +4 ,-8.(+,.5 SKR %(.0/%B./%+(

.?+8%&$-& +(0+3-(- %()10-) &-(-&0-(0- .() *-()-*& /$- ,-8.(+,. 0-88& 0.2.?8- +4

,.8%3(.(/ /*.(&4+*,./%+(" S8/$+13$ 2U= $.& . 8+H 4*-T1-(0' +4 ,1/./%+(&5 %/ %& )+H(&/*-.,

+4 /$- SKR5 H$%0$ 41(0/%+(.88' *-28.0-& 2U= 8+&& E:$.*28-&& .() J$%( <LL=G"

@$+&2$./.&- .() /-(&%( $+,+8+31- E@#ZOG %& . 8%2%) 2*+/-%( 2$+&2$./.&- /$./ %&

&%/1./-) +( 0$*+,+&+,- ;LT5 . *-3%+( 6(+H( /+ ?- &1?a-0/-) /+ 8+&& +4 $-/-*+M'3+&%/'

EW]IG %( ,.(' $1,.( 0.(0-*& %(081)%(3 ,-8.(+,. E7.&/%.( <LL=5 91 -/ .8" <LL=G" !/

*-318./-& -A/*.0-8818.* 3*+H/$ &%3(.8& ?' (-3./%B- *-318./%+( +4 2$+&2$+%(+&%/+8 =D6%(.&-

E@!=YGDSY# 2./$H.'" @!=Y 0./.8'M-& 2$+&2$./%)'8%(+&%/+8 /*%2$+&2$./- E@!@=G 2*+)10/%+(5

H$%0$ &-*B-& .& .( %(/*.0-8818.* &-0+() ,-&&-(3-*5 8-.)%(3 /+ 2$+&2$+*'8./%+( +4 SY#5

H$%0$ %& 6(+H( /+ 2*+,+/- 0-88 0'08- 2*+3*-&&%+( .() %($%?%/ .2+2/+&%&" SY# *-318./-&

(1,-*+1& 4.0/+*&5 &10$ .& 7SF E708< S(/.3+(%&/ +4 )-./$G5 JJOF; E0'08%( F;G5 R]g] .()

ORD#B EF%88+( .() N188-* <L;L5 :+-(3.& .() W+H- <LL=G"

#$- @!= 6%(.&-�SY# 2./$H.' %& +4/-( $'2-*.0/%B- %( ,-8.(+,." SY# %& 2*-&-(/ %( /$-

0-88& %( /$*-- %&+4+*,&5 SY#;5 < .() =" !( ,-8.(+,.5 SY#= %& . 2*-)+,%(.(/ %&+4+*,5 .() %&

2*-&-(/ %( >LDVLi +4 /$- 0.&-&" SY#= %($%?%/%+( %( ,-8.(+,. 0+(/-A/ %& *-&18/%(3 %(

%(0*-.&-) .2+2/+&%& .() %($%?%/%+( +4 /1,+* )-B-8+2,-(/" !()--)5 SY#= &%8-(0%(3 ?' &%KOS

8-.)& /+ )-0*-.&-) &1*B%B.8 +4 ,-8.(+,. 0-88&" E:/.$8 -/ .8" <LL>G" Z8-B./-) 2$+&2$+DSY#

8-B-8& %(B-*&-8' 0+**-8./- H%/$ 2./%-(/ &1*B%B.8 EF.%5 N.*/%(6. .() W% <LLUG5 H$%8- .88-8%0 8+&& +*

.8/-*-) -A2*-&&%+( +4 @#ZO 0+,2*%&-& <Li D >Li +4 ,-8.(+,. /1,+*&5 *-&2-0/%B-8' EX+-8

-/ .8" <LLV5 @+88+06 -/ .8" <LL<G"

N18/%28- &/1)%-& &$+H /$./ 7KSRfVLLZ ,1/./%+( .() 8+&& +4 @#ZO5 +* OKS:bV;Y

$%"

"

,1/./%+( .() 8+&& +4 !OY>S 0++2-*./- /+ 2*+,+/- /$- )-B-8+2,-(/ +4 ,+*- ,.8%3(.(/ &/.3-&

+4 ,-8.(+,. EX+-8 -/ .8" <LLV5 #&.+ -/ .8" <LL>?5 C+1 -/ .8" <LL<G" #$%& %& 41*/$-* &122+*/-)

?' ,1*%(- ,-8.(+,. ,+)-8&5 H$-*- OKS:bV;Y %( 0+,?%(./%+( H%/$ !OY>. 8+&& ES06-*,.((

-/ .8" <LLUG5 +* 7KSRfVLLZ %( 0+,?%(./%+( H%/$ @#ZO 8+&& EF.(6+*/ -/ .8" <LL[G *-&18/& %(

)-B-8+2,-(/ +4 ,.8%3(.(/ ,-8.(+,."

III. Radial growth phase (RGF) � expansion within the epidermis

#$- 0$.*.0/-*%&/%0 4-./1*- +4 /$- *.)%.8 3*+H/$ 2$.&- %& /$- ?'2.&& +4 &-(-&0-(0-5

%,,+*/.8%M./%+( .() 2*+8%4-*./%+( +4 /$- 0-88& .8+(3 /$- 8%(- +4 /$- ?.&-,-(/ ,-,?*.(-5

$+*%M+(/.88' %( /$- -2%)-*,%&" 7-&%)-& /$- NS@Y .0/%B./%+( ,-(/%+(-) .?+B- .& /$- 2*%,.*'

2./$H.' /+ &/%,18./- 0-88 2*+8%4-*./%+(5 0-88& %( /$- *.)%.8 3*+H/$ 2$.&- 2*+8%4-*./- -%/$-* )1-

/+ JJOF; E0'08%( F;G +* JFY> .,28%4%0./%+(" F1*%(3 /$- *.)%.8 3*+H/$ 2$.&- 0-88& 3*+H

2*-)+,%(.(/8' 08+&- /+ /$- ?.&.8 ,-,?*.(-5 &%(0- /$-%* 3*+H/$ %& &/%88 )-2-()-(/ +( /$-

-A+0*%(- 4.0/+*& 2*+)10-) ?' /$- 6-*./%(+0'/-&"

CCND1, 0+)%(3 4+* . 0'08%( F;5 %& . 2*+/+D+(0+3-(- H%/$ .( %,2+*/.(/ *+8- %(

(1,-*+1& 0.(0-*&5 %(081)%(3 ,-8.(+,." S,28%4%0./%+( +4 /$- CCND1 8+01& .& H-88 .&

JJOF; 2*+/-%( +B-*-A2*-&&%+( %& )-/-0/-) %( <Li +4 ,-8.(+,. 0.&-&5 .() %/ %& ,+&/

0+,,+( %( .0*.8 ,-8.(+,.5 H%/$ E>>iG" R1*/$-*,+*-5 .?8./%+( +4 JJOF; %( ,-8.(+,. 0-88&

?' &%KOS %()10-& .2+2/+&%& .() *-)10-& /1,+* 3*+H/$ %( ,+1&- A-*+3*.2$&" E:.1/-* -/ .8"

<LL<G"

JFY> ,1/./%+(& .*- *.*-5 ?1/ JFY>K<>J ,1/./%+( *-()-*& /$- 2*+/-%( %(&-(&%/%B- /+

!OY>S *-318./%+(5 H$%8- 2*-&-*B%(3 /$- %(/-*.0/%+( H%/$ 0'08%( F;5 0.1&%(3 . 0+(&/%/1/%B-

.0/%B./%+( +4 /$- 6%(.&-" S8&+5 . &,.88 2-*0-(/.3- +4 4.,%8%.8 ,-8.(+,. 0+(/.%(& 3-*,8%(-

,1/./%+(& +4 JFY> EJ-$. -/ .8" ;[[^5 `1+ -/ .8" ;[[VG"

K.&PK.4PNZYPZKY 2./$H.' .8&+ *-318./-& /$- 0-88 &%3(.8& )+H(&/*-., 4*+,

*-0-2/+*& .0/%B./-) ?' :JR5 RXR +* IXR 4.0/+*&5 +* ?' Y!#" !()%B%)1.88'5 /$-&- 4.0/+*& -B+6-

. H-.6 +* /*.(&%-(/ ZKY .0/%B./%+( E7+$, -/ .8" ;[[U5 9-88?*+06 -/ .8" <LL<G" #$- 0+,?%(-)

.0/%B%/' +4 /$-&- 4.0/+*& +* /$- ,1/./%+(& +4 /$- *-0-2/+*& ,%3$/ *-&18/ %( ,-8.(+,.

)-B-8+2,-(/5 ?1/ /$%& ,-0$.(%&, +4 .0/%B./%+( %& *.*- %( ,-8.(+,.& E9%88,+*-D@.'(- -/ .8"

<LLUG" Y!# ,1/./%+(& .*- 2*-&-(/ %( +(8' <i +4 ,-8.(+,.&5 .8&+ 3-(-/%0 .,28%4%0./%+( +4 Y!#

$.& ?--( &$+H( %( /$- 0.&- +4 .0*.85 ,10+&.8 .() ,-8.(+,. +( /$- 4.0-" S &1?3*+12 +4

,-8.(+,.& +B-*-A2*-&&%(3 Y!# .() JFY> +44-* .() -A28.(./%+( +4 . ,-0$.(%&, +4

,.8%3(.(/ /*.(&4+*,./%+( %( ,-8.(+mas that don�t carry BRAF or NRAS mutations E:,.88-'

-/ .8" <LL^G

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R%31*- ;> d N.%( &%3(.8%(3 2./$H.'& .() 2*+/-%(& ,1/./-) %( ,-8.(+,.

#$- ,.%( 2./$H.'& )-*-318./-) %( ,-8.(+,.5 NS@Y .() @!=Y E*-2*-&&-) ?' @#ZOG" #$- 3-(-

*-318./%+( (-/H+*6 %( /$- (108-1& %(B+8B-& Z<R5 JFY>PV .() JFY< 2*+,+/%(3 /$- 0-88 0'08- ?'

%(.0/%B./%(3 K7" JFY>PV 3-/ *-2*-&&-) ?' !OY>S" SKR %& *-2*-&&%(3 NFN<5 H$%0$ %& (-3./%B-8'

*-318./%(3 2U= &/.?%8%/'" #$- JFYO<S 8+01&5 0+)%(3 ?+/$ !OY>S .() SKR %& )-2%0/-) H%/$ /$-

.8/-*(./%B-8' &28%0-) -A+(& E.).2/-) 4*+, EN%88-* .() N%$, <LLV5 O-8&+( .() #&.+ <LL[GG"

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IV. Vertical growth phase (VGF)

f-*/%0.8 3*+H/$ 2$.&- %& 0+(&%)-*-) /+ ?- . 0*10%.8 /%22%(3 2+%(/ %( ,.8%3(.(/

,-8.(+,. )-B-8+2,-(/" S/ /$%& &/.3- ,-8.(+,.& .0T1%*- %(B.&%B- 2+/-(/%.8" e&%(3

%(a-0/%+(& %(/+ (1)- ,%0-5 I-*8'( -/ .8 $.& ?--( .?8- /+ &$+H /$- fXR %(a-0/-) ,-8.(+,.&

4+*, /1,+*&5 H$%8- KXR %(a-0/-) ,-8.(+,.& )+ (+/ EI-*8'( -/ .8" ;[^UG"

9$%8- ,-8.(+,. 0-88& .*- %( /$- -.*8%-* 2$.&-& +4 )-B-8+2,-(/5 /$-' .*- &/%88

)-2-()-(/ +( 6-*./%(+0'/-& 4+* 3*+H( 4.0/+*& E7+3-(*%-)-* .() I-*8'( <LL<G" #+ ?'2.&& /$%&

*-T1%*-,-(/5 ,+*- .)B.(0-) ,-8.(+,.& )-B-8+2 .1/+0*%(- .0/%B./%+( ?' &-0*-/%(3 B.*%+1&

3*+H/$ 4.0/+*&" #$-&- %(081)- @FXR E2-88-/D)-*%B-) 3*+H( 4.0/+*G5 ?RXR5 !XRD; E%(&18%(

grown factor 1), TGF (transforming growth factor ), chemokines, VEGF, HGF, EGF EW% -/

.8" <LL=G"

!( +*)-* /+ ?- .?8- /+ %(B.)- /$- &1**+1()%(3 /%&&1-&5 /$- 0-88& 1()-*3+ /$- 2*+0-&& +4

-2%/$-8%.8D/+D,-&-(0$',.8 /*.(&%/%+( EZN#G" ZN#D8%6- E2&-1)+DZN#G 2*+2-*/%-& %( /$- 0+(/-A/

+4 ,-8.(+,. *-4-*& /+ 8+&& +4 0+(/.0/ H%/$ 6-*./%(+0'/-& .() )-B-8+2,-(/ +4 ,%3*./%+( .()

%(B.&%B-(-&& E7-((-// <LL^G" :-B-*.8 2./$H.'& $.B- ?--( &$+H( /+ %()10- ZN#5 .88 +4

H$%0$ 41(0/%+( /$*+13$ /$*-- /*.(&0*%2/%+( 4.0/+* 4.,%8%-& %(081)%(3 /$- :(.%8 E:OS!W5 :WeXG5

`-? E`Z7;5 `Z7<G .() ?.&%0 $-8%AD8++2D$-8%A EZ>_5 #9!:#;G" #$- .8/-*-) 3-(- -A2*-&&%+(

(-/H+*6 %( /$- 0-88& 1()-*3+%(3 ZN# %(0*-.&-& -A2*-&&%+( +4 /$-&- /*.(&0*%2/%+( 4.0/+*&5 .()

/$%& %( /1*( )+H( *-318./%(3 -A2*-&&%+( +4 ZD0.)$-*%( E@-%(.)+5 ]8,-). .() J.(+ <LL_5

]8,-). -/ .8" <LL_G" F+H(D*-318./%+( +4 ZD0.)$-*%( .() -A2*-&&%+( +4 OD0.)$-*%( %& +(- +4

$.88,.*6& +4 ZN# EI&1 -/ .8" ;[[V5 #$%-*' -/ .8" <LL[G"

K-0-(/8'5 2%B+/.8 %,2+*/.(0- +4 /$- `Z7; .() `Z7< /*.(&0*%2/%+( 4.0/+*& %(

,-8.(+0'/- 8%(-.3- .() ,-8.(+,. $.& ?--( &$+H(" `-?< /*.(&3-(%0 6(+06+1/ ,+1&- 8%(-

H.& 1&-) /+ .?8./- `-?< &2-0%4%0.88' %( ,-8.(+0'/-&" `-?< 8+&& .44-0/-) ,-8.(+?8.&/

,%3*./%+( .() ,-8.(+0'/- )%44-*-(/%./%+(5 *-&18/%(3 %( .( .?8./%+( +4 /$- ,-8.(+0'/- 0-88

8%(-.3- .() . 8+&& +4 0+./ 0+8+*" W+&& +4 `-?< %( /$- ,-8.(+0'/- 8%(-.3- *-&18/& %( .

)+H(*-318./%+( +4 N%/4 .() ,-8.(+0'/- )%44-*-(/%./%+( ,.*6-*&5 H$%0$ -A28.%(-) /$- 8+&& +4

0+./ 0+8+* 2$-(+/'2-" S))%/%+(.88'5 /$- 8+&& +4 N%/4 8-) /+ &%3(%4%0.(/ 12*-318./%+( +4 `-?;

E+B-* ;LAG %( ,-8.(+,. 0-88 8%(-&" R1*/$-*,+*-5 &-B-*.8 N%/4 ?%()%(3 &%/-& 0+18) ?- )-/-0/-)

%( /$- B%0%(%/' +4 /$- `-?; 8+01&5 %()%0./%(3 `-?; %& . )%*-0/ N%/4 /.*3-/" N+*-+B-*5 /$- 8+&& +4

`-?< -A2*-&&%+( %& %,28%0./-) $1,.( ,-8.(+,. )-B-8+2,-(/5 -B%)-(0-) ?' /$- 2+&%/%B-

0+**-8./%+( ?-/H--( 8+&& +4 `-?< -A2*-&&%+( .() 2./%-(/ &1*B%B.8 EF-(-06-* -/ .8" <L;>.G"

S(+/$-* &/1)' .8&+ *-2+*/-) .))%/%+(.8 4.0/+*& %(B+8B-) %( ZN# /+ ?- %,2+*/.(/ 4+* ,-8.(+,.

)-B-8+2,-(/" :OS!W< .() `Z7< $.B- ?--( &$+H( /+ ?- -A2*-&&-) %( ,-8.(+0'/-& .() /+

$.B- .(/%D/1,+*.8 2*+2-*/%-&5 &%(0- /$-&- /H+ 2*+/-%(& .0/%B./- N!#R .() .& . *-&18/

%("

"

,-8.(+0'/-& 1()-*3+ /-*,%(.8 )%44-*-(/%./%+(" !( *-&2+(&- /+ OKS:P7KSR .0/%B./%+(5 ZN#

/*.(&0*%2/%+( 4.0/+* (-/H+*6 *-+*3.(%M-& .() .& . *-&18/5 #9!:#; .() `Z7; .*- 12*-318./-)"

#$%& &H%/0$ 0++2-*./-) H%/$ 7KSR .() *-&18/& %( ZD0.)$-*%( 8+&&5 -($.(0-) %(B.&%+( .()

2*+3*-&&%+( /+ . ,+*- ,.8%3(.(/ 2$-(+/'2- EJ.*.,-8 -/ .8" <L;=G"

7-&%)-& /$- +(&-/ +4 ZN#5 /$- .?%8%/' /+ &122*-&& .2+2/+&%& %& +(- +4 /$- $.88,.*6& +4

/$- 0-88& %( /$- fXR5 &%(0- /$-' .*- (+ 8+(3-* )-2-()-(/ +( &1*B%B.8 &%3(.8& 4+*, /$-

&1**+1()%(3 0-88&" #$- 2*%,.*' 0$.(3-& /$./ &122*-&& .2+2/+&%& .*- $%3$8' &-8-0/-) %(

advanced melanoma. Apoptosis can occur via mitochondria dependent intrinsic, and �death

receptor� dependent extrinsic pathways. In the case of melanoma, defective regulation of the

%(/*%(&%0 2./$H.' %& /$- 0.1&- +4 ,-8.(+,. *-&%&/.(0-"

#$- 2./$H.'& /$- 8-.) /+ *-)10-) .2+2/+&%& +(0- )-*-318./-) %(081)- /$- %(/*%(&%0

.2+2/+/%0 2./$H.' B%. 8+&& +4 S2.4; .() *-)10-) .0/%B%/' +4 2U= E:+-(3.& .() W+H- <LL=G"

S))%/%+(.88'5 &1*B%B%(3 &%3(.8%(3 2./$H.'& /$./ .*- +4/-( ,1/./-) %( ,-8.(+,. %(081)- 8+&& +4

@#ZO5 *-&18/%(3 %( .0/%B./%+( +4 @!=YPSY# &%3(.8%(35 .() .8&+ ORD#B activation that can

2+/-(/%./- &1*B%B.8 .() +(0+3-(-&%&" OKS: &%3(.8%(3 +B-*-A2*-&&%+( 0.( .8&+ .44-0/ 0-88

&1*B%B.8 &%(0- %/ $.& ?--( &$+H( %/ 0.( .0/ /$*+13$ @!=Y E7-((-// <LL^5 :+-(3.& .() W+H-

<LL=G" S))%/%+(.88'5 R.0/+*& #7g< .() #7g= *-2*-&& 2<;J!@; .() SKR5 %,2%(3%(3 +4 /$-%*

*+8-& .& /1,+* &122*-&&+*&" #7g= .8&+ $.& . *+8- %( ZD0.)$-*%( *-2*-&&%+( .() ,-8.(+,.

%(B.&%B-(-&& EK+)*%31-M -/ .8" <LL^G" :-*%(- #$*-+(%(- 6%(.&- ;; E:#Y;;G %& %(B+8B-) %( 2<;

.0/%B./%+( /$*+13$ 2U= %( ,18/%28- 0-88 /'2-&5 %& 4+1() ,1/./-) %( ;Li +4 ,-8.(+,. 0.&-&

E7-((-// <LL^G"

Wnt/ D0./-(%( 2./$H.' $.& .8&+ ?--( &$+H( /+ ?- %,2+*/.(/ %( /$- )-B-8+2,-(/ +4

more aggressive melanoma phenotype. Constitutive activation of D0./-(%( %& 4*-T1-(/8'

4+1() %( ,+*- /$.( =L i +4 ,.8%3(.(/ ,-8.(+,.5 .() /$- .0/%B./%+( %& 0.1&-) ?' )-8-/%+( +4

D0./-(%( -A+( = EK%,, -/ .8" ;[[[G"

To better understand how D0./-(%( .0/& %( ,-8.(+,.5 F-8,.& -/ .8" 0*-./-)

/*.(&3-(%0 ,%0- -A2*-&&%(3 . &/.?%8%M-) mutant D0./-(%( E?0./kG5 8.06%(3 /$- -A+(=5 /$./5 .&

,-(/%+(-) ?-4+*-5 *-()-*& %/ *-&%&/.(/ /+ X:Y= 2$+&2$+*'8./%+( .() &1?&-T1-(/ )-3*.)./%+("

By using this model, the authors have shown D0./-(%( 0++2-*./-& H%/$ OKS: /+ 2*+,+/-

melanoma. Also, D0./-(%( )+-& (+/ .44-0/ ,-8.(+0'/- 2*+8%4-*./%+( %( 018/1*-5 ?1/ ?'2.&&-&

/$- *-T1%*-,-(/ 4+* JFYO<S ,1/./%+(& ?' *-2*-&&%(3 /$- JFYO<S 2*+,+/-* EF-8,.& -/ .8"

<LL_G"

Wnt/ D0./-(%( &%3(.8%(3 $.& .8&+ ?--( &$+H( /+ *-318./- -A2*-&&%+( +4 @]e )+,.%(

/*.(&0*%2/%+( 4.0/+* 7KO< %( ,-8.(+,." 7KO< %& $%3$8' 12*-318./-) %( ,-8.(+,. .() %&

involved in proliferation and survival. Regulation BRN2 expression by D0./-(%( $.& ?--(

&$+H( %( ,-8.(+,. 0-88 8%(-& .() %( /*.(&3-(%0 ,%0-" N+*-+B-*5 &%8-(0%(3 +4 7*(< %(

%)"

"

melanoma cell lines overexpressing D0./-(%( &%3(%4%0.(/8' *-)10-& /$- 2*+8%4-*./%+( *./- +4

/$-&- 0-88& EX++).88 -/ .8" <LL>G"

Paradoxically, although D0./-(%( -A2*-&&%+( %& &--( %( /$- 0+(/-A/ +4 0.(0-*& &10$

.& 0+8+( +* 8%B-* 0.(0-* /+ $.B- . 2++* 2*+3(+&%& E7+'- -/ .8" <L;L5 W%1 -/ .8" <L;LG5 %(

,-8.(+,. $%3$ (108ear D0./-(%( %& .&&+0%./-) H%/$ 3++) 2*+3(+&%& EJ$%-( -/ .8" <LL[G"

Increased expression of D0./-(%( &122*-&&-& ,-8.(+,. 0-88 %(B.&%+( ES*+M.*-(. -/ .8"

<L;;G5 . 2*+0-&& /$./ ,.' ?- -A28.%(-) ?' /$- 12D*-318./%+( +4 N!#R 4.B+*%(3 . 2*+8%4-*./%B-

phenotype. Nevertheless, it has also been reported that D0./-(%( 0.( 4.B+* 81(3 ,-/.&/.&%&

%( /$- OKS:D?0./k ,+1&- ,+)-85 H$%8- *-2*-&&%(3 ,%3*./%+( EX.88.3$-* -/ .8" <L;<G. Thus, D

0./-(%( &%3(.8%(3 .44-0/& ,.(' .&2-0/& +4 ,-8.(+,. 0-88 2$'&%+8+3' .() in vitro 0-88 ?.&-)

,+)-8& )+ (+/ .8H.'& *-0.2%/18./- /$- ,+*- 0+,28-A -44-0/& &--( in vivo.

V. Melanoma metastasis

N-/.&/./%0 ,-8.(+,.& .*- 4188' /*.(&4+*,-)5 %,,+*/.85 $.B- 3.%(-) /$- .?%8%/' /+

&122*-&& .2+2/+&%& .() $.B- %(0*-.&-) ,%3*./+*' .() %(B.&%B- 2*+2-*/%-&" !( +*)-* /+

&100-&&4188' )%&&-,%(./- /$*+13$+1/ /$- ?+)'5 /$-&- 0-88& (--) /+ -(/-* ?8++) .() 8',2$./%0

B-&&-8& /$*+13$ . 2*+0-&& +4 %(/*.B.&./%+(5 /*.B-8 /+ )%&/.(/ +*3.(& H%/$%( /$- ?8++) 48+H5

.//.0$ /+ /$- B-&&-8 H.88 .() -A%/ /$*+13$ /$- 2*+0-&& +4 -A/*.B.&./%+( .() -&/.?8%&$ .

&-0+().*' /1,+*"

R.0/+*& %(B+8B-) %( ZN# %( /$- fXR +4 ,-8.(+,. )-B-8+2,-(/ 0+(/%(1- /+ $.B-

0*10%.8 *+8-& %( /$- -&/.?8%&$,-(/ +4 ,-8.(+,. ,-/.&/.&%&" X-(-& &10$ .& :OS!W .()

:WeX5 *-318./-) ?' /$- @!=YPSY# 2./$H.' .*- %(B+8B-) %( %(/*.B.&./%+( .() -A/*.B.&./%+(

2*+0-&&" :OS!W .() :WeX )+H(*-318./- -2%/$-8%.8 ,.*6-* ZD0.)$-*%(5 .() 12*-318./-

,-&-(0$',.8 ,.*6-*& &10$ .& f%,-(/%(5 %,28%0./-) %( ,%3*./%+( .() %(B.&%+( EX12/. -/ .8"

<LLU5 @+&-* -/ .8" <LL;G" R1*/$-*,+*-5 .0/%B./-) 4+*, +4 SY# 2*+,+/-& +B-*-A2*-&&%+( +4

fZXR5 . 6-' *-318./+* +4 .(3%+3-(-&%&5 *-&18/%(3 %( 2*+8%4-*./%+( .() *-+*3.(%M./%+( +4 ?8++)

B-&&-8 -2%/$-8%1, EN1*16-&$5 F%B- .() \.'&+( <L;LG"

](- +4 /$- @!=Y /.*3-/&5 2$+&2$+%(+&%/%)-D)-2-()-(/ 6%(.&-D; E@FY;G $.& *-0-(/8'

?--( *-2+*/-) /+ 2*+,+/- ,-8.(+,. )-B-8+2,-(/ .() /+ ?- 0*10%.8 4+* ,-/.&/.&%&

)-B-8+2,-(/" @FY; %& . &-*%(-P/$*-+(%(- 2*+/-%( 6%(.&- /$./ 2$+&2$+*'8./-& 2*+/-%( 6%(.&-&

S E@YSG5 7 E@Y75 %(081)%(3 @Y7PSY#G .() J E@YJG 4.,%8%-&" @FY; %(.0/%B./%+( 1&%(3 .

,+1&- ,+)-85 +* 2$.*,.0+8+3%0.8 %($%?%/%+( )-8.'& ,-8.(+,. )-B-8+2,-(/ .() %($%?%/&

,-/.&/.&%& H$-( 0*+&&-) /+ 7*.4fVLLZdd@/-(DPD ,-8.(+,. ,+)-8" !()--)5 @FY; ,1/.(/ ,%0-

)-B-8+2 &%3(%4%0.(/8' 8-&& ,-/.&/.&%& %( 8',2$ (+)-5 81(3& .() &28--( E:0+*/-3.3(. -/ .8"

<L;>G"

%*"

"

S ,+)- +4 9(/ &%3(.8%(3 &$+H( /+ ?- %(B+8B-) %( )-B-8+2,-(/ +4 ,-8.(+,.

,-/.&/.&%& %& 9(/ 28.(.* 0-88 2+8.*%/' E@J@G 2./$H.'" #$%& ,+)- +4 9(/ &%3(.8%(3 %(B+8B-&

K$+ 4.,%8' +4 X#@ $')*+8.&-& /$./ 28.' ,.(' ,.a+* *+8-& %( 0'/+&6-8-/+( *-318./%+(" #$- /H+

,-,?-*& +4 /$- 4.,%8' /$./ $.B- ?--( /$- ,+&/ -A/-(&%B-8' &/1)%-) .*- KSJ; .() KI]S

E71**%)3- .() 9-((-*?-*3 <LL>G"

#$- *+8- +4 KSJ; %( ,.8%3(.(/ ,-8.(+,. $.& *-0-(/8' ?--( *-2+*/-)" S &/1)' 1&%(3

-A+,- &-T1-(0%(3 %)-(/%4%-) *-01**-(/ &+,./%0 ,1/./%+(& %( KSJ; %( ,.8%3(.(/ ,-8.(+,.

EY*.1/$.,,-* -/ .8" <L;<G" S))%/%+(.88'5 . ,1/./%+( +4 KSJ;5 &1?&/%/1/%+( +4 2*+8%(- <[ /+

&-*%(- EKSJ;@<[:G %()10-& &2+(/.(-+1& .0/%B./%+( +4 /$- X#@D.&-5 8-.)%(3 /+ %(0*-.&-)

?%()%(3 +4 %/& )+H(&/*-., -44-0/+*&" J+(&-T1-(/8'5 ,-8.(+0'/- 2*+8%4-*./%+( .() ,%3*./%+(

%(0*-.&-& .() .00+1(/& 4+* KSJ; 0.(0-*D%()10%(3 2*+2-*/%-& EF.B%& -/ .8" <L;=G"

e2&/*-., *-318./+* +4 /$- X#@.&-&5 XZR @KZg; $.& %,2+*/.(/ *+8- %( ,-8.(+?8.&/

,%3*./%+(" !( /$- ,+1&- ,-8.(+,. ,+)-85 @KZg; .?8./%+( $.& ?--( &$+H( /+ $.B-

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0*+&&-) /+ /$- ,+1&- ,-8.(+,. ,+)-8 )%&28.'-) (+ 81(3 ,-/.&/.&%&5 -B-( /$+13$ 2*%,.*'

/1,+*& )-B-8+2-) ./ /$- &.,- *./- .& %( /$- 0+(/*+8 EW%()&.' -/ .8" <L;;G" S))%/%+(.88'5

.(+/$-* XZR5 @KZg< $.& ?--( &$+H( /+ ?- 4*-T1-(/8' ,1/./-) %( ,-8.(+,. E7-*3-* -/ .8"

<L;<G"

#$- *+8- +4 (+(D0.(+(%0.8 9(/PJ.<Q &%3(.8%(3 /$+13$ 9O#US $.& .8&+ ?--( &$+H( /+

$.B- . *+8- %( ,-/.&/./%0 ,-8.(+,." 9#OUS H.& &$+H( /+ )%*-0/8' .44-0/ ,-/.&/./%0

,-8.(+,. &%(0- %/& +B-*-A2*-&&%+( -($.(0-& /$-%* ,+/%8%/' .() %(B.&%B- 2*+2-*/%-&

E9--*.*./(. -/ .8" <LL<G" 9O#USP@YJ &%3(.8%(3 ,-)%./-& ,-8.(+,. ,+/%8%/' .() ,-/.&/.&%&

?' %($%?%/%(3 ,-/.&/.&%& *-2*-&&+*& .() 2*+,+/%(3 ZN# EF%&&.(.'.6- -/ .8" <LL_G"

#XRD and Sonic hedgehog pathways regulate melanoma tumorigenesis and

,-/.&/.&%&" @*+8%4-*./%B- ,-8.(+,. 0-88& .*- &-(&%/%B- /+ #XRD induced inhibition of

2*+8%4-*./%+( .& .0/%B./%+( +4 /$%& 2./$H.' *-2*-&&-& N!#R -A2*-&&%+( EI+-6 -/ .8" <LLVG" !(

,-8.(+,. 0-88&5 #XRD regulates expression of GLI2, a transcription factor downstream of

/$- &+(%0 $-)3-$+3 2./$H.' EF-((8-* -/ .8" <LL[G" !(0*-.&-) XW!< -A2*-&&%+( %( ,-8.(+,.

/1,+*& %& .&&+0%./-) H%/$ 8+&& +4 ZD0.)$-*%( -A2*-&&%+(5 .( %(0*-.&-) 0.2.0%/' in vitro .()

4+*,./%+( +4 ?+(- ,-/.&/.&-& in vivo ES8-A.6% -/ .8" <L;LG" R1*/$-*,+*-5 XW!< .8&+

0++2-*./-& H%/$ `Z7; %( *-2*-&&%+( +4 JFI; %( ,-8.(+,. 0-88&5 8-.)%(3 /+ ,+*- .33*-&&%B-

/1,+* 2$-(+/'2- E@-**+/ -/ .8" <L;=G" XW!< .() N!#R &$+H *-0%2*+0.8 -A2*-&&%+( 2.//-*(& %(

. H%)- *.(3- +4 ,-8.(+,. 0-88& in vitro E\.B-8.1) -/ .8" <L;;G" XW!< ?%()& /+ .() *-2*-&&-&

/$- N!#R 2*+,+/-*" !( .))%/%+( $+H-B-*5 .0/%B./%+( +4 /$- #XRD pathway also downD

*-318./-& 0SN@P@YS .0/%B%/' 8-.)%(3 /+ . *-)10/%+( %( JKZ7 /*.(&0*%2/%+(.8 .0/%B%/' .()

$-(0- )+H(D*-318./%+( +4 N!#R -A2*-&&%+( E@%-**./ -/ .8" <L;<G"

% "

"

Z" f-*&./%8- *+8-& +4 N!#R %( ,-8.(+,.

I. Phenotype switching

#+ 3.%( .( +B-*B%-H +4 /$- 38+?.8 3-(- -A2*-&&%+( 2*+4%8- %( ,-8.(+,.&5 I+-6 -/ .8"

.(.8'M-) . &1?&/.(/%.8 (1,?-* +4 ,-8.(+,. 0-88 8%(-& .() &$+*/D/-*, $1,.( /1,+* 018/1*-&"

#$%& .(.8'&%& 3.B- *%&- /+ /$- 2$-(+/'2-D&H%/0$%(3 $'2+/$-&%&5 &%(0- /$- .1/$+*& *-B-.8-)

/$- -A%&/-(0- +4 /H+ )%&/%(0/ 0-88 2+218./%+(& H%/$%( ,-8.(+,.&" #$- 2$-(+/'2%0 )%44-*-(0-&

+4 /$-&- /H+ 2+218./%+(&5 +(- 2*+8%4-*./%B- .() /$- +/$-* %(B.&%B-5 H-*- 0+**-8./-) /+ 38+?.8

0$.(3-& %( 3-(- -A2*-&&%+( 2.//-*(& %)-(/%4'%(3 3-(- -A2*-&&%+( &%3(./1*-& 4+* /$- )%44-*-(/

&/./-& EI+-6 -/ .8" <LL^G" K-0-(/8'5 .(.8'&%& +4 .?+1/ ULL ,-8.(+,. &$+*/D/-*, /1,+*

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DF.I!0%!&$2!%#>&:!:+>9+$&07!2*'/!#2!&2!#'/&+;+:!+T>+%&1+7$#99B!&7!2&@L]!+T>+%&1+7$2-!9+#:!$0!

+7$%B!&7$0!2+7+2'+7'+,g-!eS

!

a+! /#;+! >%+;&0*29B! %+>0%$+:! #! E+701+! A&:+! #7#9B2&2! 0H! DF.I! $#%E+$! E+7+2! &7! eS,D+9!

1+9#701#! '+992eS

! </%01#$&7! &11*70>%+'&>&$#$&07! _</F"`! '0*>9+:! $0! :++>! 2+J*+7'&7E!

&:+7$&H&+:!c,5SSS!E+701&'!DF.I!4&7:&7E!2&$+2!#770$#$+:!$0!*>A#%:2!0H!eSSS!70;+9!>0$+7$&#9!

$#%E+$! E+7+2 ! F7$+E%#$&07!0H!</F"^2+J!:#$#!A&$/! @L]^2+J! H0990A&7E! 2&@L]!1+:&#$+:!DF.I!

(70'(:0A7! 2/0A+:! $/#$! DF.I! :&%+'$9B! #7:! >02&$&;+9B! %+E*9#$+2! E+7+2! &7;09;+:! &7! 6L]!

%+>9&'#$&07! #7:! %+>#&%! #7:! 1&$02&2 ! F7! '07$%#2$-! DF.I! %+>%+22+2! E+7+2! &7;09;+:! &7! &7;#2&07!

2*'/!#2!D<]DR5

!#7:!3W<R!_#920!(70A7!#2!@#)"\,P

`!#7:!]DU.)5 !./*2!$/%0*E/!&$2!:*#9!

H*7'$&072!#2!#7!#'$&;#$0%!#7:!%+>%+220%-!DF.I!>%010$+2!>%09&H+%#$&07!4*$!%+>%+22+2!&7;#2&07 !

.0!4+$$+%!*7:+%2$#7:!$/+!109+'*9#%!H*7'$&07!0H!DF.I-!A+!*2+:!$#7:+1!#HH&7&$B!>*%&H&'#$&07!$0!

&209#$+!DF.I!H%01!eS,D+9!1+9#701#!'+992!#7:!1#22^2>+'$%01+$%B!$0!&:+7$&HB!&$2!&7$+%#'$&7E!

>#%$7+%2 !a+!%+>0%$!/+%+!$/+!H&%2$!'01>%+/+72&;+!'/#%#'$+%&2#$&07!0H!$/+!DF.I!&7$+%#'$01+!#7:!

A+! &7;+2$&E#$+! H*7'$&07#9! &7$+%#'$&072! 0H! DF.I! A&$/! #! 70;+9! H0%1! 0H! $/+! K@?,! '/%01#$&7^

%+10:+99&7E! '01>9+T! $/#$! #%+! %+J*&%+:! H0%! >%09&H+%#$&07! 0H! 1+9#701#! '+992! &7! ;&$%0! #7:!

1+9#70'B$+2!&7!;&;0!&7!1&'+ !

:-,?)',/

DF.I!#220'&#$+2!A&$/!1*9$&>9+!'01>9+T+2!&7;09;+:!&7!$%#72'%&>$&07!#7:!6L]!%+>9&'#$&07!#7:!

%+>#&%!

,5,!

!

a+!+2$#49&2/+:!eS,D+9!'+99!9&7+2!2$#49B!+T>%+22&7E!#7!L^$+%1&7#9!I)]?^W]!+>&$0>+!$#EE+:!

DF.I!_I^W^DF.I-!I&E !,]`!#7:!>+%H0%1+:!$#7:+1!#HH&7&$B!>*%&H&'#$&07!0H!209*49+!7*'9+#%!#7:!

'/%01#$&7!#220'&#$+:!H%#'$&072!H%01!9&7+!_)`h!A/+%+!I^W^DF.I!+T>%+22&07! &2!'01>#%#49+! $0!

$/#$!0H!+7:0E+70*2!DF.I,C-!5C

!L*1+%0*2!>%0$+&72!A+%+!042+%;+:!2+9+'$&;+9B!#220'&#$+:!A&$/!

I^W^DF.I! &7! &11*70>%+'&>&$#$&072! H%01! +#'/! H%#'$&07! A/&9+! #9102$! 70! >%0$+&72! A+%+!

:+$+'$+:!&7!$/+!&11*70>%+'&>&$#$&072!H%01!$/+!*7$#EE+:!eS,D+9!'+992!_I&E !,K` !

F11*70>%+'&>&$#$&072! H%01! #99! H%#'$&072! A+%+! #7#9B2+:! 4B! 1#22! 2>+'$%01+$%B! %+;+#9&7E!

>+>$&:+2! H0%! #! 9#%E+! 7*14+%! 0H! >%0$+&72! &7! H%#'$&072! H%01! I^W^DF.I-! A/+%+#2! 1#7B! H+A+%!

>+>$&:+2!A+%+!>%+2+7$! &7! $/+!*7$#EE+:!'07$%09! _3*>>9+1+7$#%B!.#49+!3,` !.A0! &7:+>+7:+7$!

>*%&H&'#$&072! #7:! 1#22^2>+'$%01+$%B! #7#9B2+2! A+%+! >+%H0%1+:! #7:! 079B! >%0$+&72! &:+7$&H&+:!

2>+'&H&'#99B! &7! $/+!I^W^DF.I!>%+'&>&$#$&072!A&$/!70!>+>$&:+2! &7! $/+!'07$%09!+T>+%&1+7$2!#%+!

:&2'*22+: ! F7! #::&$&07-! 1#7B! %&40^7*'9+0>%0$+&7! >#%$&'9+-! 2>9&'+0201+! '01>07+7$2! #7:!

'/#>+%07+!>%0$+&72!A+%+!H0*7:!2>+'&H&'#99B!&7!$/+!DF.I^'07$#&7&7E!H%#'$&072-!4*$!#2!$/+2+!#%+!

'01107!'07$#1&7#7$2-!$/+B!/#;+!70$!4++7!'072&:+%+:!&7!$/+!2*42+J*+7$!#7#9B2&2 !

"%0$+&72!>%+;&0*29B!2*EE+2$+:!$0!&7$+%#'$!A&$/!DF.I!A+%+!&:+7$&H&+:!4B!0*%!>%0'+:*%+ !.INK-!

.INC!#7:!.IN<!A+%+!&:+7$&H&+:!&7!40$/!$/+!209*49+!#7:!'/%01#$&7!#220'&#$+:!H%#'$&072!_.#49+!

,!#7:!3*>>9+1+7$#%B!.#49+!3,` !./+2+!>%0$+&72!4+907E!$0!$/+!2#1+!4W)W!D&.!2*4H#1&9B!#7:!

#%+!(70A7!DF.I!/+$+%0:&1+%&2#$&07!>#%$7+%2Rg

. Similarly, we detected the MITF cofactor ^

'#$+7&7! _<.LLK,`! &7! 40$/! H%#'$&072RP

! U*%! >%0'+:*%+! $/+%+H0%+! &:+7$&H&+:! >%+;&0*29B!

:+2'%&4+:!DF.I!>#%$7+%2 ./+!'/%01#$&7^%+10:+99&7E!H#'$0%!K@?,!&2!%+>0%$+:!$0!&7$+%#'$!A&$/!

DF.Id-! C5

-! /0A+;+%! $/+! 7#$*%+! 0H! $/+! K@?,^'07$#&7&7E! '01>9+T! /#2! 70$! 4++7! :+2'%&4+: !

K@?,! _3D]@<]R`! #2! A+99! #2! $/+! "K@D,! _K]I,gS`-! 3D]@<<5! _K]I,QS`-! 3D]@<65!

_K]IPSK`-!#7:!]<.)P]!_K]IeC]`!2*4*7&$2!0H!$/+!K@?,!'/%01#$&7^%+10:+99&7E!'01>9+T!

A+%+!:+$+'$+:! &7!40$/! H%#'$&072! _.#49+!,! #7:!3*>>9+1+7$#%B!:#$#!2+$!3,`!#907E!A&$/!<W6Q!

%+>0%$+:! $0! #220'&#$+! A&$/! K@?,! &7! /*1#7! 7+*%#9! '%+2$! '+992C ! ./&2! 2*EE+2$2! $/#$! DF.I!

&7$+%#'$2!A&$/!#!K@?,^<W6Q^'07$#&7&7E!'01>9+T!2>+'&H&'! $0!7+*%#9!'%+2$!:+%&;+:!'+992! _2++!

4+90A` !

a+! #920! &:+7$&H&+:! 70;+9! >0$+7$&#9! DF.I! >#%$7+%2 ! ./+! K".I-! 3D]@<],! _3LI5)`-!

3D]@<]e!_3LI5W`!#7:!@KK"R!'01>07+7$2!0H!$/+!LO@I!'/%01#$&7^%+10:+99&7E!'01>9+T!

#220'&#$+:! A&$/! DF.I! 2>+'&H&'#99B! &7! $/+! '/%01#$&7! #220'&#$+:! H%#'$&07 ! <01>07+7$2! 0H! $/+!

6L]! :#1#E+! %+2>072+! 1#'/&7+%B! #220'&#$+:! A&$/! DF.I! &7'9*:&7E! X@<<e! #7:! X@<<P!

_8*gS!#7:!8*QS`-!6L]^:+>+7:+7$!>%0$+&7!(&7#2+!_"@86<`-!K@<]5!#2!A+99!#2!D3W5!#7:!

D3WP ! a+! &:+7$&H&+:! $/+! WN<.! :01#&7^'07$#&7&7E! NC^9&E#2+! WN@<5-! A/&'/! /#2! 4++7!

,55!

!

&1>9&'#$+:! &7! 6L]! %+>#&%R-! #2! A+99! #2! OK@e! #! 2+'07:! WN<.! :01#&7^'07$#&7&7E! NC^9&E#2+!

A&$/! H*7'$&072! &7! 40$/! 6L]! %+>#&%! #7:! $%#72'%&>$&07Q-! 5S

! LNO@)R! #! (70A7! WN@<5!

&7$+%#'$&7E! >%0$+&7! 5! A#2! #920! H0*7:! #907E! A&$/! $/+! :+^*4&J*&$&7#2+! +7bB1+2! O3"Q! #7:!

O3",,! $/#$! A+%+! >%+H+%+7$&#99B! %+>%+2+7$+:! &7! $/+! 3LN ! F7! '07$%#2$-! O3",C! $/#$! /#2! 4++7!

2/0A7! $0! %+E*9#$+! DF.I! 2$#4&9&$BP,

! A#2! 70$! :+$+'$+:! &7! 0*%! +T>+%&1+7$2 ! D#7B! 0H! $/+2+!

&7$+%#'$&072! A+%+! ;+%&H&+:! 4B! &11*70490$! #2! WN@<5-! K@?,-! O3"Q-! O3",,-! X@<<e! #7:!

X@<<P! A+%+! :+$+'$+:! &7! $/+! I^W^DF.I! &11*70>%+'&>&$#$&072! H%01! $/+! 209*49+! 7*'9+#%!

H%#'$&07-!4*$!70$!&7!$/+!*7$#EE+:!'07$%092!_I&E !,<`-!A&$/!+7%&'/1+7$!0H!O3"Q!#7:!O3",,!&7!

$/+! 3LN! '01>#%+:! $0! $/+! <]N! #7:! $/+! 2+9+'$&;+! >%+2+7'+! 0H! WN@<5! &7! $/+! 3LN !

]9$+%7#$&;+9B-! A+! $%#72H+'$+:! eS,D+9! '+992! A&$/! #! ;+'$0%! +T>%+22&7E! I^DF.I! 0%! #! '07$%09!

+1>$B!;+'$0%!#7:!>+%H0%1+:!#7$&^!I9#E! &11*70>%+'&>&$#$&07 ! F7! $/+2+! +T>+%&1+7$2-!I^DF.I!

2>+'&H&'#99B!'0^>%+'&>&$#$+2!A&$/!+7:0E+70*2!.@@]"!#7:!LNO@)R!_3*>>9+1+7$#%B!I&E !3,` !

a+! &:+7$&H&+:! 2+;+%#9! 0$/+%! '01>9+T+2! &7$+%#'$&7E! A&$/! DF.I ! I0*%! 2*4*7&$2! 0H! .IFFF<-! #!

@L]! >09B1+%#2+! FFF! '0H#'$0%! A+%+! :+$+'$+:! 2*EE+2$&7E! $/#$! DF.I! 1#B! %+E*9#$+! "09! FFF!

$%#72'%&>$&07 ! ]9$+%7#$&;+9B-! #! H%#'$&07! 0H! .IFFF<! &2! 90'#$+:! #$! E+701&'! 90'&! '#99+:! NT$%#!

.IFFF<!2&$+2!_N.<2`!&7!#42+7'+!0H!$/+!"09!FFF!1#'/&7+%B!A/+%+!&$!1#B!0%E#7&b+!/&E/+%!0%:+%!

'/%01#$&7!2$%*'$*%+!&7!#220'&#$&07!A&$/!2*4*7&$2!0H!$/+!'0/+2&7!'01>9+TP-!CC-!RS

!F7!#''0%:#7'+!

A&$/! $/&2! &:+#-! $/+! 3D<],-! 3D<C-! 3.]?5! #7:! "63e! '0/+2&7! 2*4*7&$2! A+%+! #920! H0*7: !

./*2-!DF.I!1#B!#220'&#$+!A&$/!.IFFF<!#7:M0%!'0/+2&7!#$!#!2*42+$!0H!E+701&'!2&$+2!$0!%+E*9#$+!

/&E/+%!0%:+%!'/%01#$&7!2$%*'$*%+ !

We identified the TRIM28 (TIF1 , KAP1) co^%+>%+220%!>%0$+&7!$/#$!4+907E2!$0!#!2*4^H#1&9B!

of TRIM proteins comprising TRIM24 (TIF1!) and TRIM33 (TIF1") 22. These proteins form

1*9$&^>%0$+&7!'01>9+T+2!#907E!A&$/!W6]<,!#7:!W6]<5!#7:!$/+!W",!>%0$+&725C

-!#99!0H!A/&'/!

A+%+! #920! &:+7$&H&+: ! .@@]"! &2! #! >%+;&0*29B! '/#%#'$+%&b+:! '0H#'$0%! H0%! 'DV<Cg-! RC

! a+!

&:+7$&H&+:! .@@]"-! @OfK),-! @OfK)5-! #7:! K]IeC]! &7! $/+! DF.I! &7$+%#'$01+ ! ./+2+!

>%0$+&72! H0%1!#!'01>9+T!A&$/!.@@]"!$/#$!#'$2!#2!#!DV<!'0H#'$0%RR

-!4*$!A+!:&:!70$!:+$+'$!

?<Le!0%!0$/+%!(70A7!2*4*7&$2!0H!3]?]!#220'&#$+:!A&$/!DF.I !./+!>%+'&2+!'01>02&$&07!0H!

$/&2!DF.I^.@@]"!'01>9+T! $/+%+H0%+! %+1#&72! $0!4+!:+$+%1&7+: ! F7$+%+2$&7E9B-!1*$#$&072! &7!

.@@]"! /#;+! %+'+7$9B! 4++7! #220'&#$+:! A&$/! /*1#7! 1+9#701#eP

! ./&2! 042+%;#$&07! #7:! &$2!

&7$+%#'$&07!/+%+!A&$/!DF.I!2*EE+2$!#!'%&$&'#9!%09+!&7!1+9#701#E+7+2&2 !

F7!#::&$&07!$0!$%#72'%&>$&07!'01>9+T+2-!$/+!@I<,-!@I<5-!@I<R!#7:!@I<e!2*4*7&$2!0H!6L]!

%+>9&'#$&07!H#'$0%!<!#220'&#$+!A&$/!DF.I!#907E!A&$/!$/+!D<DC-!D<De!#7:!D<DQ!2*4*7&$2!

0H!$/+!D<D!'01>9+T!$/#$!H0%12!#$!6L]^%+>9&'#$&07!0%&E&72 !]8]"g!#7:!]8]"g)!A+%+!#920!

,5C!

!

&:+7$&H&+:!#7:!]8]"g!/#2!4++7!2/0A7!$0!&7$+%#'$!A&$/!$/+!D<D!'01>9+T!,R

-!#9$/0*E/!$/+2+!

>%0$+&7! (&7#2+! ]! #7'/0%&7E! >%0$+&72! /#;+! #::&$&07#9! H*7'$&072!g ! ./+! (&7#2+")8,-! $/#$! /#2!

4++7!2/0A7!$0!4+!+22+7$&#9!H0%!$/+!1&$02&2!0H!1+9#701#!'+992!RQ

-!A#2!H0*7:!&7!$/+!'/%01#$&7^

#220'&#$+:! H%#'$&07! 2*EE+2$&7E! $/#$! &$! 1#B! >/02>/0%B9#$+! DF.I! 07! '/%01#$&7 ! I&7#99B-! A+!

:+$+'$+:! 1*9$&>9+! 2*4*7&$2! 0H! $/+! 7*'9+#%! >0%+! '01>9+T! 2*EE+2$&7E! $/#$! DF.I! 2*4^'+99*9#%!

90'#9&b#$&07!1#B!4+!%+E*9#$+:!4B!7*'9+#%^'B$0>9#21&'!2/*$$9&7E !.0E+$/+%-!$/+2+!:#$#!:+2'%&4+!

#!'01>%+/+72&;+!2+$!0H!DF.I!&7$+%#'$&7E!>%0$+&72 !

a+! #920! 70$+:! $/#$! $/+! E+7+2! '0:&7E! 1#7B! 0H! $/+! DF.I! &7$+%#'$&07! >#%$7+%2! #%+! #220'&#$+:!

A&$/!DF.I^0''*>&+:!2&$+2!2*EE+2$&7E!$/#$!$/+&%!+T>%+22&07!1#B!4+!%+E*9#$+:!2>+'&H&'#99B!&7!$/+!

1+9#70'B$+!9&7+#E+!4B!DF.I !F7!$/+!'#2+!0H!$/+!K@?,!'01>9+T-!$/+!E+7+2!+7'0:&7E!K@?,-!

K]I,gS-!K]IPSK!#7:!<W6Q!A+%+!#99! #220'&#$+:!A&$/!DF.I!4&7:&7E!2&$+2! _3*>>9+1+7$#%B!

I&E !35` !./+! X@<<e-!X@<<P-!"@86<-!D3W5-!D3WP-!O3"Q-!O3",,-!OK@e-!LNO@)R-!

")8,-! .INK-! #7:! .IN<! E+7+2! #920! #%+! #220'&#$+:! A&$/! DF.I! 4&7:&7E! 2&$+2! i_,g-! eS

! #7:!

*7>*49&2/+:!042+%;#$&072j !

DF.I!&7$+%#'$2!A&$/!#!K@?,^<W6Q!'07$#&7&7E!'01>9+T !

F$!/#2!>%+;&0*29B!4++7!%+>0%$+:!$/#$!DF.I!&7$+%#'$2!A&$/!K@?,-!4*$!$/+!7#$*%+!0H!$/+!K@?,!

'01>9+T! &7$+%#'$&7E! A&$/! DF.I! /#2! 70$! 4++7! :+2'%&4+: ! 3+;+%#9! %+9#$+:! K@?,^'07$#&7&7E!

'01>9+T+2!/#;+!4++7!:+2'%&4+:Re-!eC

!K#2+:!07!$/+!1#22!2>+'$%01+$%B!%+2*9$2-!$/+!'01>9+T!$/#$!

&7$+%#'$2!A&$/!DF.I!102$!'902+9B!%+2+149+2!$/+!"K]I!;#%&#7$!A&$/!$/+!>%+2+7'+!0H!"K@D, !

.0!&7;+2$&E#$+!$/+!7#$*%+!0H!$/+!K@?,!'01>9+T+2!&7!eS,D+9!'+992-!+T$%#'$2!A+%+!>%+'&>&$#$+:!

A&$/! #7! #7$&^K@?,! #7$&40:B! 2/0A&7E! '0^>%+'&>&$#$&07! 0H! K@?,! A&$/! ]@F65-! 3D]@<<,!

_K]I,ee`-! ]<.)P]-! "K@D,-! #7:! 3D]@<<5! _K]I,QS`! A/+%+#2! 3D]@<65! _K]IPSK`!

#7:! ]@F6,K! A+%+! 70$! '0^>%+'&>&$#$+:! _3*>>9+1+7$#%B! I&E ! C]` ! K@?,! $/+%+H0%+! &7$+%#'$2!

A&$/! $/+2+! 2*4*7&$2! $0! H0%1! #! "K]I^$B>+! '01>9+T! &7! eS,D+9! '+992 ! L+;+%$/+9+22-! 70$#49B!

#42+7$! H%01! $/+! $#EE+:^DF.I! 1#22^2>+'$%01+$%B! #7#9B2&2! #%+! $/+! ]@F6,]MK! #7:! ]@F65!

2*4*7&$2 ! ]2! $/+2+! #%+! >%0$+&72! 0H! 9#%E+! 109+'*9#%! 1#22-! &$! &2! *79&(+9B! $/#$! $/+B! A+%+!

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0H!.33!A&$/!$/+!"09!FF!>#*2+!2&$e located immediately 5� of the first downstream nucleosome.

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(Beverly, MA, USA) was used according to the manufacturer�s instructions to

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%+1#&7&7E!>+99+$!A#2!:&E+2$+:!A&$/!1&'%0'0''#9!7*'9+#2+!#7:!207&'#$+: !.#EE+:!>%0$+&72!A+%+!

&11*70>%+'&>&$#$+:! A&$/! #7$&^I9#E! D5^#E#%02+! _3&E1#`-! +9*$+:! A&$/! I9#E! >+>$&:+! _S e!

1EM1)`-! H*%$/+%! #HH&7&$B! >*%&H&+:! A&$/! #7$&^W]! #7$&40:B^'07k*E#$+:! #E#%02+! _3&E1#`-! #7:!

+9*$+:!A&$/!W]!>+>$&:+!_,!1EM1)` !./+!W]!#7:!I9#E!>+>$&:+2!A+%+!H&%2$!4*HH+%+:!A&$/!eS1D!

.%&2^W<9!_>W!g e`-!$/+7!:&9*$+:!$0!R!1EM1)!&7!.?NL!,eS!4*HH+%!_5S!1D!.%&2!#$!>W!Q Pe-!,eS!

1D!L#<9-!C!1D!DE<95-!S ,!1D!N6.]-!,Sl!E9B'+%09-!S S,l!L"RS`-!#7:!2$0%+:!#$! ^5Sv<!

*7$&9! *2+ ! K+$A++7! +#'/! 2$+>-! 4+#:2! A+%+! A#2/+:! &7! .?NL! ,eS! 4*HH+% ! <01>9+T+2! A+%+!

%+209;+:! 4B! 363^"]?N! #7:! 2$#&7+:! *2&7E! $/+! 3&9;+%! Y*+2$! (&$! _F7;&$%0E+7` ! D#22^

2>+'$%01+$%B! A#2! >+%H0%1+:! #$! $/+! .#>9&7! K&090E&'#9! D#22! 3>+'$%01+$%B! I#'&9&$B! _W#%;#%:!

D+:&'#9!3'/009-!K02$07-!D]` !

<+99!'*9$*%+!#7:!9+7$&;&%#9!&7H+'$&072 !

D+9#701#!'+99!9&7+2!38^D+9^5g!#7:!eS,D+9!A+%+!E%0A7!&7!@"DF!,PRS!1+:&*1!_3&E1#-!3$!

)0*&2-!DU-!O3]`!2*>>9+1+7$+:!A&$/!,Sl!H0+$#9!'#9H!2+%*1!_I<3` !5dC.!'+992!A+%+!E%0A7!&7!

Dulbecco�s modified Eagle�s medium supplemented with 10% FCS and

>+7&'&99&7M2$%+>$01B'&7! _Q e! pEM19` ! W+%1+2^C]! '+992! A+%+! E%0A7! &7! @"DF! ,PRS! 1+:&*1!

_3&E1#`! 2*>>9+1+7$+:! A&$/! ,Sl! I<3-! 5SS7D!."]-! 5SS>D! '/09+%#! $0T&7-! ,S7EM19! /*1#7!

2$+1!'+99! H#'$0%! _F7;&$%0E+7`!,S!7D!+7:0$/+9&7^,! _K#'/+1`!#7:!>+7&'&99&7M2$%+>$01B'&7! _Q e!

pEM1)` !]99!9+7$&;&%#9!2/@L]!;+'$0%2!A+%+!04$#&7+:!H%01!3&E1#!_D&22&07!2/^@L]!2+%&+2`!&7!

$/+! ")8S! ;+'$0% ! ./+! H0990A&7E! '072$%*'$2! A+%+! *2+: ! 2/K@?,! _.@<LSSSSS,eeRd`! #7:!

2/DF.I! _.@<LSSSSS,d,,d`\! I0%! 9+7$&;&%*2! &7H+'$&07-! ;&%*2! A#2! >%0:*'+:! &7! 5dC.! '+992!

#''0%:&7E!$0!$/+!1#7*H#'$*%+%2!>%0$0'09!:+2'%&4+: !

.%#72H+'$&072-!+T$%#'$!>%+>#%#$&07!#7:!#7$&40:&+2 !

Transient and stable transfections were performed with 5 #g of expression vectors and using

I*?NLN!P!%+#E+7$!_@0'/+`!H0990A&7E!$/+!1#7*H#'$*%+%2!&72$%*'$&072 !D+:&*1!A#2!%+>9#'+:!

5R!/0*%2!#7:!'+99!A+%+!'099+'$+:!Rg!/0*%2!#H$+%!$%#72H+'$&07 !<+992!9B2&2!A#2!>+%H0%1+:!*2&7E!

)36K!eSS!4*HH+%!_eSS1D!8<9-!5e1D!.%&2!#$!>W!Q d-!,Sl!E9B'+%09-!S\Se!l!L"^RS-!,1D!

6..!#7:!>%0$+#2+!&7/&4&$0%!'0'($#&9` !O>!$0!C!1E!0H!A/09+!'+99!+T$%#'$2!A+%+!:&9*$+:!&7!)63K!

A&$/0*$!8<9!$0!04$#&7!#!H&7#9!'07'+7$%#$&07!0H!,SS1D!8<9!#7:!&7'*bated for 12h with 5 #g of

specific antibody and 50#l Slurry of protein^?! 2+>/#%02+! _?N! W+#9$/'#%+` ! K+#:2! A+%+!

A#2/+:!$/%++! $&1+2! &7!)36K!CSS-! $A&'+! &7!)36K!,eS!#7:!40&9+:! &7!)#+119&!4*HH+%!4+H0%+!

>%0$+&7! 2+>#%#$&07! 4B! 363�"]?N ! I0%! H9#E! &11*70>%+'&>&$#$&072-! +T$%#'$2! A+%+! &7'*4#$+:!

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with 50#l Slurry of Anti^I9#E!D5^#E#%02+!#HH&7&$B!E+9!_3&E1#`!#7:!A#2/+:!2&1&9#%9B!>%&0%!$0!

+9*$&07! A&$/! I9#E! >+>$&:+! _S-e! 1EM1)` ! F11*70490$2! A+%+! >+%H0%1+:! A&$/! $/+! H0990A&7E!

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0H! #! n^2'0%+! '01>*$+:! #2! Z! bw_T^#)/$, where x is the observed value (number of 10$&H!

occurrence), # is the mean of the number of occurrences of motifs (computed over controls)

and $ is the standard deviation of the number of occurrences or co^0''*%%+7'+2! 0H! 10$&H2!

_'01>*$+:!0;+%!'07$%092` !n^2'0%+2!A+%+!2*42+J*+7$9B!$*%7+:!&7$0!>^;#9*+2!#7:!#!1*9$&^$+2$&7E!

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>+99+$!4B!'+7$%&H*E#$&07!#$!Rv< !./+!7*'9+#%!209*49+!H%#'$&07!A#2!04$#&7+:!4B!&7'*4#$&07!0H!$/+!

>+99+$! &7! /&E/! 2#9$! 4*HH+%! _H&7#9! L#<9! '07'+7$%#$&07! 0H! CSS1D`! #7:! $/+7! 2+>#%#$+:! 4B!

'+7$%&H*E#$&07! #$! Rv< ! .0! 04$#&7! $/+! 7*'9+#%! &7209*49+! H%#'$&07! _'/%01#$&7! H%#'$&07`-! $/+!

%+1#&7&7E!>+99+$!A#2!:&E+2$+:!A&$/!1&'%0'0''#9!7*'9+#2+!#7:!207&'#$+: !.#EE+:!>%0$+&72!A+%+!

&11*70>%+'&>&$#$+:! A&$/! #7$&^I9#E! D5^#E#%02+! _3&E1#`-! +9*$+:! A&$/! I9#E! >+>$&:+! _S e!

,gC!

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1EM1)`-! H*%$/+%! #HH&7&$B! >*%&H&+:! A&$/! #7$&^W]! #7$&40:B^'07k*E#$+:! #E#%02+! _3&E1#`-! #7:!

+9*$+:!A&$/!W]!>+>$&:+!_,!1EM1)` !./+!W]!#7:!I9#E!>+>$&:+2!A+%+!H&%2$!4*HH+%+:!A&$/!eS1D!

.%&2^W<9!_>W!g e`-!$/+7!:&9*$+:!$0!R!1EM1)!&7!.?NL!,eS!4*HH+%!_5S!1D!.%&2!#$!>W!Q Pe-!,eS!

1D!L#<9-!C!1D!DE<95-!S ,!1D!N6.]-!,Sl!E9B'+%09-!S S,l!L"RS`-!#7:!2$0%+:!#$!^5Sv<!

*7$&9! *2+ ! K+$A++7! +#'/! 2$+>-! 4+#:2! A+%+! A#2/+:! &7! .?NL! ,eS! 4*HH+% ! <01>9+T+2! A+%+!

%+209;+:! 4B! 363^"]?N! #7:! 2$#&7+:! *2&7E! $/+! 3&9;+%! Y*+2$! (&$! _F7;&$%0E+7` ! D#22^

2>+'$%01+$%B! A#2! >+%H0%1+:! #$! $/+! .#>9&7! K&090E&'#9! D#22! 3>+'$%01+$%B! I#'&9&$B! _W#%;#%:!

D+:&'#9!3'/009-!K02$07-!D]` !

<+99!'*9$*%+!#7:!9+7$&;&%#9!&7H+'$&072 !

D+9#701#!'+99!9&7+2!38^D+9^5g!#7:!eS,D+9!A+%+!E%0A7!&7!@"DF!,PRS!1+:&*1!_3&E1#-!3$!

)0*&2-!DU-!O3]`!2*>>9+1+7$+:!A&$/!,Sl!H0+$#9!'#9H!2+%*1!_I<3` !gggD+9!#7:!DL.!A+%+!

E%0A7! @"DF! ,PRS! 1+:&*1-! 2*>>9+1+7$+:! A&$/! ,Sl! /+#$^&7#'$&;#$+:! H0+$#9! '#9H! 2+%*1!

_I<3&` !,5Se)O!A+%+!E%0A7!&7!1+:&*1!aRgd!_D<6K!,eC!M!)NFKUfF.n!)^,e!_CZ,`-!3&E1#-!

3$!)0*&2-!DU-!O3]`-!2*>>9+1+7$+:!A&$/!Rl!/+#$^&7#'$&;#$+:!H0+$#9!'#9H!2+%*1!_I<3&` !5dC.!

'+99s were grown in Dulbecco�s modified Eagle�s medium supplemented with 10% FCS and

^C]! '+992! A+%+! E%0A7! &7! @"DF! ,PRS! 1+:&*1!

_3&E1#`! 2*>>9+1+7$+:! A&$/! ,Sl! I<3-! 5SS7D!."]-! 5SS>D! '/09+%#! $0T&7-! ,S7EM19! /*1#7!

2$+1!'+99! H#'$0%! _F7;&$%0E+7`!,S!7D!+7:0$/+9&7^,! _K#'/+1`!#7:!>+7&'&99&7M2$%+>$01B'&7! _Q e!

^@L]!2+%&+2`!&7!

$/+! ")8S! ;+'$0% ! ./+! H0990A&7E! '072$%*'$2! A+%+! *2+: ! 2/K".I! _.@<LSSSSC,d,e5-!

.@<LSSSSC,d,eC`! -! 2/DF.I! _.@<LSSSSS,d,,d`-! 2/3D]@<],! _.@<LSSSSCSCPRC-!

.@<LSSSSCSCPRR`-! 2/3D]@<]e! _.@<LSSSSCegRQR-! .@<LSSSSCegRdQ` ! I0%! 9+7$&;&%*2!

&7H+'$&07-! ;&%*2! A#2! >%0:*'+:! &7! 5dC.! '+992! #''0%:&7E! $0! $/+! 1#7*H#'$*%+%2! >%0$0'09!

:+2'%&4+: !!

.%#72H+'$&072-!+T$%#'$!>%+>#%#$&07!#7:!#7$&40:&+2 !!

Transient and stable transfections were performed with 5 #g of expression vectors and using

I*?NLN!P!%+#E+7$!_@0'/+`!H0990A&7E!$/+!1#7*H#'$*%+%2!&72$%*'$&072 !D+:&*1!A#2!%+>9#'+:!

5R!/0*%2!#7:!'+992!A+%+!'099+'$+:!Rg!/0*%2!#H$+%!$%#72H+'$&07 !<+992!9B2&2!A#2!>+%H0%1+:!*2&7E!

)36K!eSS!4*HH+%!_eSS1D!8<9-!5e1D!.%&2!#$!>W!Q d-!,Sl!E9B'+%09-!S\Se!l!L"^RS-!,1D!

6..!#7:!>%0$+#2+!&7/&4&$0%!'0'($#&9` !O>!$0!C!1E!0H!A/09+!'+99!+T$%#'$2!A+%+!:&9*$+:!&7!)63K!

A&$/0*$!8<9!$0!04$#&7!#!H&7#9!'07'+7$%#$&07!0H!,SS1D!8<9!#7:!&7'ubated for 12h with 5 #g of

specific antibody and 50#l Slurry of protein^?! 2+>/#%02+! _?N! W+#9$/'#%+` ! K+#:2! A+%+!

A#2/+:!$/%++! $&1+2! &7!)36K!CSS-! $A&'+! &7!)36K!,eS!#7:!40&9+:! &7!)#+119&!4*HH+%!4+H0%+!

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>%0$+&7! 2+>#%#$&07! 4B! 363�"]?N ! I0%! H9#E! &11*70>%+'&>&$#$&072-! +T$%#'$2! A+%+! &7'*4#$+:!

with 50#l Slurry of Anti^I9#E!D5^#E#%02+!#HH&7&$B!E+9!_3&E1#`!#7:!A#2/+:!2&1&9#%9B!>%&0%!$0!

+9*$&07! A&$/! I9#E! >+>$&:+! _S-e! 1EM1)` ! F11*70490$2! A+%+! >+%H0%1+:! A&$/! $/+! H0990A&7E!

#7$&40:&+2Z! DF.I! _F7$+%'/&1-! D3^QQ,^"`-! #'$&7! _F?KD<-! 56Q`-! K".I! #7$&40:B! A#2! #! E&H$!

H%01!<#%9!a*!#7:!h0+!)#7:%B-!3D]@<],!#7:!3D]@<]e!#7$&40:&+2!A+%+!#!E&H$!H%01!"+$+%!

K+'(+% !

</%01#$&7^&11*70>%+'&>&$#$&07!

K@?,! </F"! +T>+%&1+7$2! A+%+! >+%H0%1+:! 07! 7#$&;+! D7#2+^:&E+2$+:! '/%01#$&7 ! eT,SQ! $0!

eT,Sg! H%+2/9B! /#%;+2$+:! eS,D+9! '+992! A+%+! %+2*2>+7:+:! &7! 51)! &'+^'09:! /B>0$&7&'! 4*HH+%!

_S CD!3*'%02+-!PS1D!8<9-!,e1D!L#<9-!e1D!DE<95-!S ,1D!N6.]-!,e1D!.%&2^W<9!_>W!

Q e`-!S e1D!6..-!S ,1D!"D3I-!>%0$+#2+! &7/&4&$0%!'0'($#&9`!#7:!'B$0>9#21&'! H%#'$&07!A#2!

%+9+#2+:!4B! &7'*4#$&07! A&$/!51)!0H! 9B2&2^4*HH+%! _S CD!2*'%02+-!PS!1D!8<9-!,e1D!L#<9-!

e1D!DE<95-!S ,1D!N6.]-!,e1D!.%&2^W<9! _>W!Q e`-!S e1D!6..-!S ,1D!"D3I-!"F<-!

S el! _;M;`! F?N"])! <]^PCS`! H0%! ,S! 1&7*$+2! 07! &'+ ! ./+! 2*2>+72&07! A#2! 9#B+%+:! 07$0! #!

2*'%02+!'*2/&07!_, 5D!2*'%02+-!PS!1D!8<9-!,e1D!L#<9-!e1D!DE<95-!S ,1D!N6.]-!,e!

1D!.%&2^W<9!_>W!Q e`-!S e1D!6..-!S ,1D!"D3I-!"F<`!#7:!'+7$%&H*E+:!H0%!5e!1&7*$+2!#$!

RQSS! %>1! &7! #! 2A&7E! %0$0% ! ./+! 7*'9+#%! >+99+$! A#2! %+2*2>+7:+:! &7! :&E+2$&07! 4*HH+%! _S C5D!

2*'%02+-eS1D!.%&2^W<9!_>W!Q e`-!R1D!DE<95-,1D!<#<95-!S ,1D!"D3I`!#7:!2*4k+'$+:!$0!

D&'%0'0''#9!L*'9+#2+!:&E+2$&07!H0%!e!1&7*$+2!#$!CQv< !./+!%+#'$&07!A#2!2$0>>+:!4B!#::&$&07!

0H! N6.]! #7:! 2*2>+72&07! '/&99+:! 07! &'+! H0%! ,S! 1&7*$+2 ! ./+! 2*2>+72&07! A#2! '9+#%+:! 4B!

'+7$%&H*E#$&07!#$!,S-SSS!%>1!_Rv<`!H0%!,S!1&7*$+2!#7:!2*>+%7#$#7$!_'/%01#$&7`!A#2!*2+:!H0%!

H*%$/+%! >*%>02+2 ! </F"! A#2! >+%H0%1+:! 07! *>! $0! PS! pE! 0H! 7#$&;+! '/%01#$&7! +22+7$&#99B! #2!

>%+;&0*29B!:+2'%&4+: !

)+E+7:2!$0!3*>>9+1+7$#9!I&E*%+2 !!

3*>>9+1+7$#9!I&E*%+!, !?+7+2!+7'0:&7E!2*4*7&$!0H!LO@I!#%+!#220'&#$+:!A&$/!DF.I^0''*>&+:!

2&$+2 ! O<3<! 2'%++72/0$! 0H! $/+! &7:&'#$+:! 90'&! 2/0A&7E! DF.I! 0''*>&+:! 2&$+2! &7:&'#$+:! 4B!

#%%0A2 !!

3*>>9+1+7$#9!I&E*%+!5 !3+9+'$&;+!%+J*&%+1+7$!H0%!K".I!H0%!$/+!>%09&H+%#$&07!#7:!2*%;&;#9!0H!

1+9#701#! '+992 ! ] ! a+2$+%7! 490$! 2/0A&7E! (70'(:0A7! 0H! K".I! #7:! DF.I! &7! 38^D+9^5g!

'+992 !K !?%0A$/!'*%;+2!0H!38^D+9^5g!#7:!D7$!'+992!H0990A&7E!K".I!(70'(:0A7 !< !"/#2+!

'07$%#2$!1&'%02'0>B!0H!38^D+9^5g!#7:!D7$!'+992!H0990A&7E!K".I!(70'(:0A7 !D#E7&H&'#$&07!

X5S !6 !"/#2+!'07$%#2$!1&'%02'0>B!0H!gggD+9!'+992!H0990A&7E!K".I!(70'(:0A7 !N !?%0A$/!

,ge!

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707^1+9#701#! '+992! H0990A&7E! K".I! (70'(:0A7 ! I ! "/#2+! '07$%#2$! 1&'%02'0>B! 0H! $/+!

&7:&'#$+:!'+99!9&7+2!H0990A&7E!K".I!(70'(:0A7 !

3*>>9+1+7$#9! I&E*%+! C ! ]7! DF.I^&7:+>+7:+7$! %09+! H0%! K".I! &7! ,5Se)*! '+992 ! ] ! a+2$+%7!

490$!2/0A&7E!(70'(:0A7!0H!K".I!#7:!#42+7'+!0H!DF.I!&7!,5Se)*!'+992 !K !?%0A$/!'*%;+2!

0H!,5Se)*!'+992!H0990A&7E!K".I!(70'(:0A7 !< !"/#2+!'07$%#2$!1&'%02'0>B!0H!,5Se)*!'+992!

H0990A&7E!K".I!(70'(:0A7 !D#E7&H&'#$&07!X5S !!

3*>>9+1+7$#9! I&E*%+! R ! N22+7$&#9! %09+! 0H! $/+! LO@I! '#$#9B$&'! 2*4*7&$2! 3D]@<],! #7:!

3D]@<]e! &7! eS,1+9! '+992 ! ] ! a+2$+%7! 490$! 2/0A&7E! (70'(:0A7! 0H! 3D]@<],! #7:!

3D]@<]e! &7! eS,D+9! '+992 ! K ! "/#2+! '07$%#2$! 1&'%02'0>B! 0H! eS,D+9! '+992! H0990A&7E!

3D]@<],!#7:!3D]@<]e!(70'(:0A7 !D#E7&H&'#$&07!X5S !!

3*>>9+1+7$#9!I&E*%+!e ! DF.I!#7:!K".I! %+E*9#$+!:&2$&7'$-! 4*$! 0;+%9#>>&7E! E+7+!+T>%+22&07!

>%0E%#11+2! &7!eS,D+9!#7:!W+%1+2^C]!'+992 !f+77!:&#E%#12! &99*2$%#$+! $/+!0;+%9#>!4+$A++7!

*>!#7:!:0A7^%+E*9#$+:!E+7+2!&7!$/+!$A0!'+99!$B>+2 !3+;+%#9!+T#1>9+2!0H!'011079B!%+E*9#$+:!

*>!#7:!:0A7^%+E*9#$+:!E+7+2!#%+!&7:&'#$+: !!

3*>>9+1+7$#9!I&E*%+!P !"%+1#$*%+!E%+B&7E!0H!1&'+!9#'(&7E!K>$H! &7!$/+!1+9#70'B$+2!9&7+#E+ !

]^K !"/0$0E%#>/2!0H!1&'+!0H!$/+!&7:&'#$+:!E+70$B>+2!#7:!>02$^7#$#9!:#B2!4+H0%+!072+$!0H!/#&%!

E%0A$/ !<^6 !"/0$0E%#>/2!0H!,R!#7:!5,!:#B^09:!1&'+!0H! $/+!&7:&'#$+:!E+70$B>+2!&99*2$%#$&7E!

$/+!'/#%#'$+%&2$&'2!0H!$/+!H&%2$!'0#$ !

3*>>9+1+7$#9! I&E*%+! Q ! 6&1&7&2/+:! 1+9#7049#2$! >%09&H+%#$&07! &7! K>$H^1*$#7$! 1&'+ ! ]^K !

"/0$0E%#>/2! 0H! %+>%+2+7$#$&;+! K>$H90TMm! #7:! K>$H90TM90T! N,e e! H0+$*2+2! &7! $/+! 6'$^)#'n!

4#'(E%0*7:! H0990A&7E! )#'n! 2$#&7&7E! $0! ;&2*#9&b+! 1+9#7049#2$2 ! "#7+9! ]! 2/0A2! $/+! ;+7$%#9!

>0%$&07! #7:!>#7+9! K! $/+! $%*7(!#7:!#! b001!0H! %+>%+2+7$#$&;+! '+992! H%01! $/+! $%*7(! %+E&07 !< !

Y*#7$&H&'#$&07!0H!)#'n^9#4+99+:!1+9#7049#2$2!&7!$/+!&7:&'#$+:!%+E&072 !]7!+T#1>9+!0H!$/+!E%&:!

*2+:!&2!2/0A7!0;+%!$/+!9&14!#7:!>#A !!!!

3*>>9+1+7$#%B!6#$#2+$!, !NT'+9!2>%+#:2/++$!0H!E+7+2!2>+'&H&'#99B!#7:!'011079B!%+E*9#$+:!4B!

K".I! #7:! DF.I! (70'(:0A7! &7! eS,D+9! '+992! #907E! A&$/! $/+! #>>%0>%&#$+! E+7+! 07$090EB! #2!

:+2'%&4+:!&7!I&E*%+!C]-!K!#7:!< !!

3*>>9+1+7$#%B!6#$#2+$! 5 !NT'+9! 2>%+#:2/++$!0H!E+7+2!A&$/!#220'&#$+:!DF.I!0''*>#7'B!#7:!

%+E*9#$+:!&7!2/K".I!#907E!A&$/!$/+!#>>%0>%&#$+!E+7+!07$090EB!#2!:+2'%&4+:!&7!I&E*%+!C6 !

3*>>9+1+7$#%B! 6#$#2+$! C !NT'+9! 2>%+#:2/++$!0H! E+7+2! %+E*9#$+:!4B! 2/K".I!#7:! 2/DF.I! &7!

W+%1+2!C]!'+992!#907E!A&$/!'01107!#7:!2>+'&H&'!E+7+2!#7:! $/+&%!07$090EB!#2!:+2'%&4+:! &7!

I&E!R6 !

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3*>>9+1+7$#%B! 6#$#2+$! R ! NT'+9! 2>%+#:2/++$! 0H! E+7+2! %+E*9#$+:! 4B! 2/K".I! #7:! 2/DF.I!

'01107! &7!eS,D+9! #7:!W+%1+2!C]!'+992! #907E!A&$/! '01107!#7:! 2>+'&H&'!E+7+2! #7:! $/+&%!

07$090EB!#2!:+2'%&4+:!&7!3*>>9+1+7$#9!I&E*%+!e !!

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Identification of a ZEB2-MITF-ZEB1 transcriptionalnetwork that controls melanogenesis and melanomaprogression

G Denecker1,2, N Vandamme1,2, O Akay1,2, D Koludrovic3, J Taminau1,2, K Lemeire2, A Gheldof1,2, B De Craene1,2, M Van Gele4,

L Brochez4, GM Udupi5,6, M Rafferty6, B Balint6, WM Gallagher5,6, G Ghanem7, D Huylebroeck8,9, J Haigh2,10, J van den Oord11,

L Larue12, I Davidson3, J-C Marine13,14 and G Berx*,1,2

Deregulation of signaling pathways that control differentiation, expansion and migration of neural crest-derived melanoblasts

during normal development contributes also to melanoma progression and metastasis. Although several epithelial-to-

mesenchymal (EMT) transcription factors, such as zinc finger E-box binding protein 1 (ZEB1) and ZEB2, have been implicated in

neural crest cell biology, little is known about their role in melanocyte homeostasis and melanoma. Here we show that mice

lacking Zeb2 in the melanocyte lineage exhibit a melanoblast migration defect and, unexpectedly, a severe melanocyte

differentiation defect. Loss of Zeb2 in the melanocyte lineage results in a downregulation of the Microphthalmia-associated

transcription factor (Mitf) and melanocyte differentiation markers concomitant with an upregulation of Zeb1. We identify

a transcriptional signaling network in which the EMT transcription factor ZEB2 regulates MITF levels to control melanocyte

differentiation. Moreover, our data are also relevant for human melanomagenesis as loss of ZEB2 expression is associated with

reduced patient survival.

Cell Death and Differentiation advance online publication, 25 April 2014; doi:10.1038/cdd.2014.44

Melanocytes are specialized cells in the skin that produce

melanin, a pigment that is responsible for skin and hair color

and that provides protection against ultraviolet (UV) radiation.

During mouse embryogenesis, melanoblasts originate from

the neural crest and migrate along a dorsolateral pathway

from the neural tube to the developing dermis.1 Around

embryonic day (E) E11 they move into the epidermis and

eventually populate the developing hair follicle.2 Here they

separate into two distinct populations: the differentiated

pigmented melanocytes, which reside in the hair matrix, and

the non-pigmented melanocyte stem cells (MSC) in the bulge.

The latter cells are responsible for replenishing the hair follicle

with new melanocytes during each hair cycle. Genetic studies

in mice demonstrated the importance of several key players

(such as sex-determining region Y (SRY)-box 10 (Sox10),

paired-box 3 (Pax3), microphthalmia-associated transcription

factor (Mitf), endothelin 3/endothelin receptor B (Edn3/

Ednrb), Kitl/Kit, Slug, cellular myelocytomatosis oncogene

cellular homolog (cMyc) and b-Catenin (b-Cat)) for melano-

blast cell fate specification, proliferation, migration and

survival.2–4 The master regulator of the melanocyte develop-

ment is MITF, which is spatio-temporally controlled by several

key transcription factors such as SOX10, PAX3 and

b-catenin.5–7 Fundamentally, MITF induces gene expression

patterns that prompt a melanocyte to differentiate and initiate

pigment production by activating genes important for melanin

biosynthesis (such as Tyrosinase (Tyr), Dopachrome

tautomerase (Dct), Tyrosinase-related protein 1 (Tyrp1) and

1Unit of Molecular and Cellular Oncology, Inflammation Research Center, VIB, 9052 Ghent, Belgium; 2Department of Biomedical Molecular Biology, Ghent University,9052 Ghent, Belgium; 3Institut de Genetique et de Biologie Moleculaire et Cellulaire, CNRS, INSERM, Universite de Strasbourg, Illkirch, France; 4Department ofDermatology, Ghent University Hospital, 9000 Ghent, Belgium; 5UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College,Dublin 4, Ireland; 6OncoMark Limited, Nova UCD, Belfield Innovation Park, University College Dublin, Belfield, Dublin 4, Ireland; 7Institute Jules Bordet, Brussels,Belgium; 8Laboratory of Molecular Biology (Celgen), Department of Development and Regeneration, KU Leuven, 3000 Leuven, Belgium; 9Department of Cell Biology,Erasmus MC, 3015 GE Rotterdam, The Netherlands; 10Vascular Cell Biology Unit, Department for Molecular Biomedical Research, VIB, Ghent, Belgium; 11Departmentof Pathology, University Hospital Leuven, KU Leuven, Leuven, Belgium; 12Curie Institute, Developmental Genetics of Melanocytes, Centre National de la RechercheScientifique (CNRS) UMR3347, Institut National de la Sante et de la Recherche Medicale (INSERM) U1021, Orsay, France; 13Center for the Biology of Disease,Laboratory for Molecular Cancer Biology, VIB, Leuven, Belgium and 14Center for Human Genetics, KU Leuven, Leuven, Belgium*Corresponding author: G Berx, Unit of Molecular and Cellular Oncology, Inflammation Research Center, VIB, Technologiepark 927, B-9052 Ghent (Zwijnaarde),Belgium. Tel: +32 9 331 36 60; Fax: +32 9 331 36 09; E-mail: [email protected]

Received 18.9.13; revised 17.2.14; accepted 10.3.14; Edited by G Melino

Abbreviations: b-Cat, b-Catenin; BCL-2, B-Cell CLL/Lymphoma 2; bHLH, basic Helix Loop Helix; BRAF, viral RAF murine sarcoma viral oncogene homolog B1;Cdkn2a, cyclin-dependent kinase inhibitor 2A; ChIP, chromatin immunoprecipitation; c-MYC, cellular myelocytomatosis oncogene cellular homolog; CRE, cyclisationrecombination; CTRL, control; DCT, dopachrome tautomerase; E, embryonic day; EDN3/EDNRB, endothelin 3/endothelin receptor B; ERK, extracellular signal-regulated kinase; EMT, epithelial-to-mesenchymal transition; FL, floxed; HF, hair follicle; KD, knockdown; LacZ, b-D-galactosidase; MC1R, melanocortin 1 receptor;MCKO, melanocyte-specific knockout; MCWT, melanocyte-specific wild-type; MEK, MAPK/ERK kinase; MITF, microphthalmia-associated transcription factor;P, postnatal day; PAX3, paired-box 3; PGP, P-glycoprotein; PMEL, premelanosome protein; NHM, normal human primary melanocytes; NRAS, neuroblastoma RASviral oncogene homolog; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; S100b, S100 calcium-binding protein B; shRNA, short hairpin RNA;siRNA, short interfering RNA; SOX10, sex-determining region Y (SRY)-box 10; TG, transgenic; TGF-b, transforming growth factor b; TYRP1, tyrosinase-related protein1; TYR, tyrosinase; UV, ultraviolet; X-gal, 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside; ZEB, zinc finger E-box binding protein

Cell Death and Differentiation (2014), 1–12

& 2014 Macmillan Publishers Limited All rights reserved 1350-9047/14

www.nature.com/cdd

melanocortin 1 receptor (Mc1r)) and melanosome formation

(such as the premelanosome protein (Pmel)).8,9 Maintenance

of the melanocyte stem cell compartment is mainly determined

by transforming growth factor b (TGF-b), NOTCH signaling and

MITF-dependent BCL-2 activity.10,11 Deficiency in melanoblast

development and migration generally result in white spotting

phenotypes, whereas defects in melanocyte stem cell

maintenance result in progressive hair graying phenomena.3

Melanoma is amalignant tumor arising from themelanocyte

lineage and is the most therapy-resistant and deadly form of

skin cancer. Several pathways have been identified to have a

major role in the development of malignant melanoma, among

ZEB2 regulates melanocyte differentiation

G Denecker et al

2

Cell Death and Differentiation

which the RAS/RAF/MEK/ERK pathway is probably the most

studied and has recently become the target for anti-melanoma

therapy.12 Activating mutations in RAS (Q61K), with NRAS

being the most frequent, occur in 15% of cases of human

melanoma.12 Melanocyte-specific expression of NRASQ61K

together with cyclin-dependent kinase inhibitor 2A (Cdkn2a)

or p53 deficiency leads to accelerated melanoma formation in

mice.13,14 BRAF activating mutations (V600E) occur at

high frequencies (50–60%) in human melanoma. Specific

inhibitors of BRAFV600E are being successfully used in

melanoma therapy.15 However, patients become rapidly

resistant to this therapy, and combination therapies with

MEK inhibitors are being investigated.16,17 Drug resistance of

malignant melanoma cells has been associated with the

expression of the drug transporter P-glycoprotein (PGP) and

increasedmigratory and invasive behavior.18 EMT (Epithelial-

to-mesenchymal transition) has a key role in the collective

movement of normal cells during different stages of embryo-

nic development, whereas in adults it has been associated

with several pathologies, including fibrosis and cancer

progression.19,20 EMT is a process in which epithelial cells

lose their epithelial characteristics, gain mesenchymal

features and becomemigratory. Several pathways have been

shown to induce EMT, all of which function through the

induction of three families of transcription factors, the Snail

(SNAIL, SLUG), Zeb (ZEB1, ZEB2) and basic Helix Loop

Helix (bHLH) (E47, TWIST and others) families.21 Melano-

blasts undergo a migration process during embryogenesis

andmaturemelanocytes are periodically replenished from the

stem cells and undergo a constant cycling and migration

process. TheEMT transcription factor SLUG, has been shown

to have an important role in the melanocyte lineage, as

Slug-deficient mice have pigmentation abnormalities due to

melanoblast migration defects andSLUGmutations have also

been found in some cases of human piebaldism.22–24

Furthermore, homozygous deletions of the SLUG gene were

found in patients with type 2D of Waardenburg syndrome, an

auditory-pigmentary syndrome caused by a migration defi-

ciency of melanocytes and other neural crest-derived

cells.25,26 However, although it has been shown that siRNA-

mediated inhibition of Slug leads to the suppression of

metastasis in an orthotopic mouse model of melanoma,

SLUG expression has not been correlated with melanoma

metastasis.27 Recently, it became clear that in vitro activation

of the MEK-ERK signaling drives the reversion of the EMT

transcription factor expression pattern inmelanocytes with the

downregulation of SLUG and ZEB2 and the induction of

TWIST1 and ZEB1.28

ZEB2 is a transcription factor required for neural crest cell

development. It belongs to the Zeb family of zinc-finger-

homeodomain transcription factors and binds the E-box

sequences CACCT(G).29,30 We previously showed that condi-

tional Zeb2 expression in epithelial cells results in specific

downregulation of E-cadherin and gain of malignant features

characterized by amesenchymal gene expression profile, such

as N-cadherin and Vimentin.29,31 Furthermore, it has recently

become clear that during EMT not only epithelial characteristics

are abolished, but that cells are reprogrammed to different

extents.32 Mouse embryos deficient in Zeb2 exhibit defects

from E8.5 onwards, with early arrest of cranial neural crest cell

migration and absence of neural crest cells at the postotic vagal

level.33 Tissue specific disruption of Zeb2 very early in neural

crest cell development using a Wnt1-Cre-based approach,

results in abnormalities in craniofacial and heart development,

as well as defects in the peripheral nervous system.34

On the basis of these observations we hypothesized that

ZEB2 may have a key role in the process of melanogenesis.

To test this possibility we used a melanocyte-specific loss of

function approach in mice. Unexpectedly we find that in

addition to a role in proper melanoblast development, ZEB2 is

required for the correct differentiation of melanocytes through

its ability to act as an upstream regulator of Mitf. Our results

propose a model whereby in melanocytes ZEB2 is necessary

for Mitf expression and activity which in its turn is responsible

for Zeb1 repression. Measurement of nuclear ZEB2 levels

shows a significantly shorter melanoma-specific survival in

those patients with low ZEB2 expression.

Results

Melanocyte-specific ZEB2 deficiency causes congenital

loss of hair pigmentation. We show for the first time that

ZEB2 is expressed in the melanocytes of human skin

epidermis, as well as in the differentiated melanocytes of

mouse hair follicles (Figure 1a). ZEB2 is also expressed in

migrating melanoblasts of the mouse embryo and their

precursors, the neural crest cells.33,35 As both complete

knockout and Wnt1-Cre-mediated neural-crest-specific dele-

tion of Zeb2 are embryonic lethal (E9.5 and E12.5-postnatal

(P) 0, respectively),33,34 we conditionally deleted Zeb2 in the

melanocyte lineage by using the tyrosinase(Tyr)::Cre mouse

line, which allows CRE-mediated Zeb2 deletion under the

control of the Tyrosinase promoter starting at E10.5

(Supplementary Figure S1a).34,36 Homozygous melanocyte-

specific deletion of Zeb2 (ZEB2MCKO) caused a severe loss

of hair pigmentation compared with wild-type littermates

Figure 1 Zeb2 loss in the melanocyte lineage causes congenital loss of pigmentation and impairs melanoblast development. (a) ZEB2 is expressed in differentiatedmelanocytes in the human epidermis, and in the hair follicles of mouse skin. (b) Melanocyte-specific deletion of Zeb2 (ZEB2MCKO) causes congenital loss of hair pigmentation inhomozygous knockout mice (2 months old). This loss of pigmentation wasmore pronounced on the belly than on the back. (c) Determination of melanin pigment (mg/ml) in the dorsaland ventral hair of ZEB2MCWT and ZEB2MCKO mice (n¼ 7–8 for each group) at the age of 4–6 months. (d) ZEB2 is required for proper melanoblast development in homozygousembryos at E15.5. Whole-mount LacZ staining of E15.5 ZEB2MCWT;Dct-LacZ and ZEB2MCKO;Dct-LacZ embryos. (e) Comparison of melanoblast migration in the epidermis anddermis of dorsal and ventral areas of ZEB2MCWT;Dct-LacZ and ZEB2MCKO;Dct-LacZ embryos (n¼ 4 and 5, respectively). Results represent the number of melanoblasts per areaanalyzed. (f) Nuclear fast-red staining of LacZ-stained skin sections of ZEB2MCWT;Dct-LacZ and ZEB2MCKO;Dct-LacZ mice to visualize hair follicles (HF) (arrow: pigmented HF,arrowhead: LacZ-positive HF, *: LacZ-negative HF). (g) Quantitative analysis of the pigmented, LacZ-positive, non-pigmented LacZ-negative HF and LacZ-positive stem cells in thebulge area of ZEB2MCWT (n¼ 7) and ZEB2MCKO (n¼ 8) mice per 0.5mm analyzed. Data of ZEB2MCWT and ZEB2MCKOmice were compared by using unpaired Student’s t-test andare presented as means±95% CI. P-values are indicated with (Po0.001). Micrograph images were taken with a � 60/0.8 objective (a) or a � 10/0.25 objective (e and f)


G Denecker et al

3


(ZEB2MCWT). This hair pigmentation loss was present from

the first hair cycle and continued through adulthood

(Supplementary Figure S1b and Figure 1b). The melanin

content of the dorsal and ventral hair were reduced by 93 and

91%, respectively (Figure 1c). Light microscopic analysis

confirmed the disappearance of melanin pigment from the

hair shafts (Supplementary Figure S1c), but hair with mixed

pigmentation were also observed (data not shown).

The absence of ZEB2 impairs melanoblast development.

Congenital loss of hair pigmentation indicates a disturbance

of melanoblast migration or proliferation due to the absence

of ZEB2. To further analyze this phenotype in-depth, we

crossed conditional melanocyte-specific Zeb2 knockout mice

with Dct::LacZ melanoblast reporter mice37 and analyzed

the LacZ-positive melanoblast population in ZEB2MCWT and

ZEB2MCKO embryos at E15.5. Absence of Zeb2 severely

impaired melanoblast development (Figure 1d) and quanti-

fication of the LacZ-positive melanoblasts on the back and

belly of embryos further confirmed a significant reduction of

melanoblast numbers in both areas (Supplementary Figure

S1d). In agreement with an expected defect in migration,

melanoblast numbers decreased more on the belly (95%)

than on the back (70%) of the ZEB2-deficient embryos.

To visualize the distribution of melanoblasts in the dermis

and their subsequent migration to the epidermis,

we made transversal sections of E15.5 LacZ embryos

and quantified the melanoblasts in both areas. There was

no difference in the number of melanoblasts present in the

dermis between ZEB2MCKO compared with control embryos

(Figure 1e). However, in the epidermis of both the belly and

back there was a significant reduction of melanoblasts in the

ZEB2MCKO embryos, suggesting an important role for ZEB2

in this process. In addition, melanoblast of ZEB2MCKO

embryos had fewer cell protrusions, compatible with their

altered characteristics (Supplementary Figure S1e).

ZEB2 is necessary for proper melanocyte differentiation.

Although melanoblast development was severely affected,

30% of them reached the dorsal area at E15.5. This raised

the question whether the remaining melanoblasts could still

populate the hair follicles of ZEB2MCKO mice. Histological

analysis of ZEB2MCWT;Dct-LacZ and ZEB2MCKO;Dct-LacZ

skin from 5.5-day-old (P5) mice showed that some hair

follicles in ZEB2MCKO mice are still LacZ positive and/or

(hypo-)pigmented, indicating that ZEB2-deficient melano-

blasts can migrate and populate the bulb area of the hair

follicles (Figure 1f). However, quantitative analysis of the hair

follicles showed that there were significantly fewer pigmented

or LacZ-positive hair follicles present in ZEB2MCKO mice

(Figure 1g). Furthermore, ZEB2MCKO skin contained some

completely undifferentiated hair follicles that were neither

pigmented nor LacZ positive, which was not the case for

ZEB2MCWT skin (Figures 1f and g). Immunohistochemical

analysis of depigmented ZEB2-stained sections confirmed

nuclear ZEB2 presence in the differentiated melanocytes of

the bulb area, the migrating melanocytes in the epidermis

and importantly also in the melanocyte stem cells

of ZEB2MCWT mice, whereas ZEB2 was absent in all cells

of the melanocyte lineage of ZEB2MCKO skin (Supplementary

Figure S2a). Quantitative analysis of the melanocyte stem

cells revealed a reduction in LacZ-positive stem cells in

ZEB2MCKO mice compared with ZEB2MCWT mice (Figure 1g),

which agrees with the presence of fewer melanoblasts in the

epidermis in ZEB2MCKO embryos. To visualize melanocytes

independently of Dct promoter reporter activity, we analyzed

the expression of the general melanocyte marker S100

calcium-binding protein B (S100b), which does not depend

on the differentiation status of the melanocytes. S100b

staining showed that melanocytes were present in most of

ZEB2MCKO hair follicles (Figure 2a). Moreover, quantifying

S100-positive melanocytes per hair follicle showed that

the number of S100-positive ZEB2MCKO melanocytes was

reduced by 40% per hair follicle, which further points to a role

of ZEB2 in migration and/or survival of melanocytes

(Supplementary Figures S2b and c). Further immunohisto-

chemical analysis and quantification of positive cells of the

melanocyte-specific transcription factor MITF and one of its

regulators (i.e., PAX3) showed that in the absence of ZEB2

the amount of MITF-positive melanocytes was strongly

reduced, when compared with the total amount of melano-

cytes per hair follicle (S100-positive melanocytes), whereas

the amount of PAX3-positive melanocytes was not affected

(Figure 2a, Supplementary Figures S2b and c). The intensity

of MITF staining was also lower, indicating a decrease in

both the number of MITF-positive cells and in MITF protein

levels in still MITF-positive cells. These observations indicate

that ZEB2 is an important regulator of MITF-dependent

melanocyte differentiation. We analyzed the presence of

other terminal differentiation markers, such as TYRP1 and

tyrosinase enzymatic activity. Most ZEB2MCKO hair follicles

were indeed negative for these markers (Figure 2a and data

not shown). mRNA expression analysis of several melano-

cyte differentiation markers (Tyrp1, Tyr, Dct, Pmel, Mc1R

and Mitf) on whole skin mRNA of ZEB2MCKO and ZEB2MCWT

mice further showed that the differentiation status of

ZEB2MCKO melanocytes was affected, as all of these

markers are significantly downregulated in ZEB2MCKO skin

(Supplementary Figure S2d). Close examination of

the melanosomes in ZEB2-stained sections of ZEB2MCWT

Figure 2 Melanocyte-specific Zeb2 deficiency causes the formation of undifferentiated melanocytes in the bulge area of hair follicles. (a) Immunohistochemical staining ofsections of ZEB2MCWT and ZEB2MCKO;Dct-LacZ-positive skin sections with S100b, PAX3, MITF and TYRP1 antibodies. (b) Detailed microscopic analysis of melanosomes inthe hair shafts and the bulb area of the hair follicles, combined with immunohistochemical staining of ZEB2 on LacZ-positive skin sections of ZEB2MCWT and ZEB2MCKO;Dct-LacZ-positive mice. Insets show the altered morphology of melanosomes in the ZEB2MCKO sections compared with ZEB2MCWT. (c) Electron microscopic analysis ofmelanosomes in the bulb area of ZEB2MCWT and ZEB2MCKO mice demonstrates the absence or irregular morphology of melanosomes in the ZEB2MCKO hair follicles.(d) Genetic compensation of the loss of Zeb2 in the ZEB2MCKO;Dct-LacZ mice with melanocyte-specific overexpression of ZEB2 (ZEB2MCTG). Left panel: completecompensation of pigmentation in ZEB2MCKO ZEB2MCTG;Dct-LacZ mice compared with ZEB2MCKO;Dct-LacZ mice. Right panels: reappearance of pigmented melanosomesand ZEB2 expression in the ZEB2MCKO ZEB2MCTG;Dct-LacZ hair follicles. All microscopic analyses were done on skin sections of 5.5-day-old (a–c) or 13.5-day-old mice(d) and Immunohistochemical micrograph images were taken with a � 60/0.8 objective (a and d) or a � 100/1.25 objective (b)


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and ZEB2MCKO skin confirmed the absence of melanin in

most melanocytes of the bulb area and in hair shafts of

ZEB2MCKO skin, although in some hair follicles a few

remaining melanosomes were visible (Figure 2b). Electron

microscopy analysis showed that these melanosomes were

spherical with irregular borders, in contrast to the rod-shaped

melanosomes of ZEB2MCWT hair follicles (Figure 2c). These

results collectively show that melanocytes form but do not

differentiate properly, pointing to a crucial role for ZEB2

in melanocyte differentiation. These effects were strictly

ZEB2-dependent, as in a complementation experiment in

which we crossed the ZEB2MCKO mice with a conditional

Zeb2 transgenic strain (Rosa26-ZEB2TG/TG-IRES-GFP)

activated by the same Tyr-Cre as for the deletion of Zeb2,

melanogenesis was completely rescued (Supplementary

Figure S3a and Figure 2d). Such Zeb2 complemented

mice (ZEB2MCKO; ZEB2MCTG) regained pigmentation, and

histological sections demonstrated that ZEB2-positive

melanocytes are pigmented (Figure 2d), confirming the cell

autonomous action of ZEB2. Furthermore, in vivo mRNA

expression analysis of the melanocyte differentiation

markers (Tyrp1, Tyr, Dct, PmeL, Mc1R and Mitf) confirmed

the full functional complementation of Zeb2 deficiency with

transgenic Zeb2 (data not shown).

ZEB2 controls an MITF-ZEB1-dependent transcriptional

network essential for melanocyte differentiation. The

melanocyte differentiation block observed upon Zeb2 abla-

tion could also be due to a change in the functionality of the

melanoblasts or melanocyte stem cells. We therefore

investigated whether ZEB2 is required for the maintenance

of the differentiation program in melanocytes. To this end,

we transfected primary mouse melanocytes with an siRNA

pool targeting Zeb2 (siZEB2) or with a control siRNA pool

(siCTRL). Significant knockdown of Zeb2 caused nearly

complete loss of differentiation of the melanocytes

(Figure 3a) and a concomitant decrease in the steady-state

mRNA levels of Mitf and several genes of the melanin

synthesis pathway (Figure 3b). To examine whether EMT

was also affected, we assessed the expression levels of two

representative EMT target genes, E-cadherin (Cdh1; epithe-

lial marker) and Vimentin (Vim; mesenchymal marker).

Unexpectedly, Cdh1 was downregulated, whereas Vim

was upregulated (Figure 3b). Interestingly we found that

Zeb2 knockdown caused transcriptional upregulation of Zeb1

(Figure 3b). The latter results were confirmed at the protein

level in the immortalized melanocyte cell line Melan-a in

which Zeb2 knockdown caused a strong downregulation of

MITF and concomitant upregulation of ZEB1, and the

mesenchymal markers Vimentin (VIM) and Fibronectin

(FN1) (Figure 3c). These observations support the hypothe-

tical model of ZEB2 dictating MITF expression and activity.

In the absence of Zeb2, MITF expression is lost, coinciding

with ZEB1 upregulation (Figure 3d). This model is confirmed

in vivo as ZEB1 staining intensity was clearly increased in a

fraction of the undifferentiated ZEB2-deficient melanocytes

(Figure 3e), whereas ZEB1 could not be detected in

differentiated melanocytes of ZEB2MCWT mice. ZEB1 protein

was also undetectable in primary melanocytes of human

origin (Supplementary Figure S3b and see below). On the

other hand, ZEB1 was readily observed in the stem cells of

both ZEB2MCWT and ZEB2MCKO mice (Figure 3e). Thus, an

intricate balance between these two ZEB-family members

appears to be important in the transcriptional regulation

of melanocyte differentiation.

Strong nuclear ZEB2 expression in primary melanomas

is associated with better survival. A role for ZEB2 in the

regulation of MITF levels and/or activity, melanoblast

development and melanocyte differentiation raises

the possibility that deregulation of ZEB2 expression may

contribute to melanoma progression/metastasis. Consis-

tently, immunohistochemical analyses of ZEB2 in a cohort

of human benign naevi, primary and metastatic melanoma

samples revealed a very heterogeneous pattern

of expression particularly in primary melanoma, with areas

of high expression next to strong local downregulation

(Supplementary Figure S3c and Figure 4a). This hetero-

geneous downregulation of ZEB2 expression is reminiscent

to the profile of MITF expression in human melanoma.38

Interestingly we found that human melanoma metastases

express high nuclear ZEB2 levels suggesting reversible

loss of expression during melanoma dissemination

(Supplementary Figure S3c). We extended this study by

analyzing 178 primary melanoma samples on a tissue array.

This study showed that ZEB2 has significant prognostic

relevance in melanoma, as strong nuclear ZEB2 expression

is beneficial for the patients in terms of melanoma-specific

survival and recurrence-free survival (Figure 4b). In contrast,

weak nuclear ZEB2 expression was associated with a worse

patient outcome (Figure 4b). Therefore, ZEB2 might serve

as a gatekeeper controlling human melanoma progression

and provide a novel prognostic marker for melanoma.

ZEB2 loss results in reduced MITF expression, and is

associated with melanoma progression. We have

provided genetic evidence that ZEB2 regulates Mitf expre-

ssion levels in primary melanocytes in vivo (Figure 2a and

Supplementary Figure S2d). This was confirmed in mela-

noma cells, as Zeb2 knockdown in the B16 mouse

melanoma cell line caused a transcriptional decrease in

expression of Mitf and its well-established target genes

and a concomitant increase in ZEB1 expression (Figure 5a).

Figure 3 ZEB2 is necessary for proper differentiation of primary melanocytes. (a and b) Morphology of primary melanocytes after knockdown of Zeb2 by siRNAtransfection and relative mRNA expression of Zeb2, melanocytes-specific differentiation genes Tyrp1, Tyr, Dct, PmeL and Mc1R, the epithelial/mesenchymal markersE-cadherin/Vimentin and Zeb1. Scrambled siRNA was used as a control (siCTRL). (c) Western blot analysis after siRNA knockdown of Zeb2 in the primary Melan a cell line.(d) Hypothetical model of the ZEB2-MITF-ZEB1 balance in melanocytes (e) ZEB1 expression in dedifferentiated LacZ-positive melanocytes of the hair follicles of ZEB2MCKO

mice and in the melanocyte stem cells of both ZEB2MCWT and ZEB2MCKO mice. QPCR data were compared by using unpaired Student’s t-test (n¼ 3) and are presented asaverages±S.D. P-values are indicated with (**Po0.005 and *Po0.05). Micrograph images were taken with a � 20/0.2 objective (a) or a � 60/0.8 objective (e)


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Importantly, transfection of the Zeb2 KD cells with an Mitf

expression vector rescued the differentiation defect indicat-

ing that this phenotype is MITF-dependent (Figure 5b). This

expression switching of ZEBs was confirmed in short-term

culture human melanoma cells, in which we observed this

particular ZEB heterogeneity in NRAS- or BRAF-mutated

samples: 67% (8 out of 12) express both ZEB1 and ZEB2

whereas the remaining express either ZEB1 (2 out of 12)

Figure 4 ZEB2 expression in melanoma. (a) Heterogeneous ZEB2 expression in primary human melanoma with vertical growth phase. (b) Kaplan–Meier curves formelanoma recurrence-free survival, comparing human melanoma samples with strong nuclear ZEB2 expression (n¼ 83) and weak nuclear ZEB2 expression (n¼ 82) from amelanoma tissue array. Data were compared using the log-rank test and **P¼ 0.0065. A representative ZEB2 staining of both groups is shown in the right panel (*** very highnuclear ZEB2, ** high nuclear ZEB2, * low nuclear ZEB2). ZEB2 protein expression values were determined using an automated image analysis approach(IHC-MARK, Oncomark, Dublin, Ireland) designed to quantify immunohistochemically stained slides. Micrograph images were taken with a � 4/0.1 and � 20/0.4 objective(a) or a � 10/0.25 objective (b)

Figure 5 The ZEB2-MITF-ZEB1 transcriptional network. (a) Knockdown of Zeb2 by siRNA transfection of the B16 melanoma cell line. Scrambled siRNA was used as acontrol (siCTRL). Relative mRNA expression of endogenous Zeb2, the melanocyte-specific differentiation genes Mitf, Tyrp1, Tyr, Dct, Mc1R and Zeb1 after Zeb2 knockdownare shown. (b) Relative mRNA expression of subset of MITF target genes after siRNA knockdown of Zeb2 in the B16 melanoma cell line after compensation with Mitf.(c) Western blot analysis of ZEB2 and ZEB1 expression in short-term culture human melanoma cell lines with different NRAS (blue circles) or BRAF (red circles) mutated orWT (green circles) status. Primary human melanocytes were used as a reference. (-) specific ZEB1 band, (*) aspecific band. (d) Western blot analysis of ZEB2, MITF andZEB1 in a panel of human melanoma cell lines. Primary human melanocytes were used as a reference. (e) 501Mel cells were infected with lentiviral vectors expressing controlshRNA (shCTRL) or shRNA directed against MITF (shMITF). Left panel: western blot analysis shows strongly downregulated MITF expression after shMITF infection. Rightpanel: shMITF knockdown leads to increased ZEB1 mRNA expression. Two independent primer pairs located in exons 1–2 and 4–5 for the ZEB1 gene were used. (f) UCSCscreenshot of a wig file of an HA-ChIP-seq from 501Mel cells expressing a 3HA-Control vector or 3HA-tagged MITF at the ZEB1 locus. Several MITF binding sites areobserved (shown by arrows) both upstream and downstream of the ZEB1 gene. (g) Anti-HA-ChIP-qPCR from native 501Mel cells (CTRL) or 501MEL cells stablyexpressing 3HA-tagged MITF (HA-MITF). qPCR was performed with primers that amplify a region 1 kb upstream of the TYR transcription start site (TSS) as negative control,the TYR TSS as positive control and two sites from the ZEB1 locus 50 and 30 of the gene as indicated in f. QPCR data were compared by using unpaired Student’s t-test(n¼ 3, for a, e and g) or paired Student’s t-test (n¼ 5, for b) and are presented as averages±S.D. or are presented as log-transformed relative expression values with 95% CI.P-values are indicated with ***Po0.0005, **Po0.005 and *Po0.05


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or ZEB2 (2 out of 12) (Figure 5c and Supplementary

Figure S3d). Melanoma cells that are wild type for NRAS or

BRAF mainly express ZEB2 only (67%, 6 out of 9),

resembling the expression pattern of normal melanocytes

which also only express ZEB2. In addition, the analysis of a

cohort of human melanoma cell lines supported the tendency

of an inverse correlation between ZEB2 and MITF versus

ZEB1 expression (Figure 5d). These results were further


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confirmed on the human SKMel28 melanoma cell line in

which we performed siRNA-mediated KD of ZEB2, which

caused a strong downregulation of MITF and concomitant

upregulation of ZEB1 (Supplementary Figure S3e). Further-

more, knockdown of MITF in human melanoma 501Mel cells

results in upregulation of ZEB1 expression suggesting that

MITF contributes to the direct repression of ZEB1

(Figure 5e). Interestingly, MITF-ChIP-seq analysis recently

identified the ZEB1 locus as a potential MITF target in human

melanoma 501Mel cells (Figure 5f).39 The specificity of this

binding to the ZEB1 locus was confirmed by anti-HA-ChIP-

qRT-PCR for both the 50- and 30-end of the gene (Figure 5g).

Discussion

In this work we have identified an important novel function for

ZEB2 in melanocytes: ZEB2 regulates the expression of MITF

and thereby coordinates the development and differentiation

of themelanocyte lineage.Moreover we provide evidence that

ZEB2 is expressed in a heterogeneous manner in human

melanoma. These observations have important implications

for our understanding of melanoma biology as well as for

anti-melanoma therapy.

Emerging evidence indicates that the transcriptional

pathways critical for melanogenesis, like those controlled by

MITF, could be disturbed in pigmentation-associated

diseases and melanoma progression and metastasis.40 MITF

is considered to be themaster regulator of melanocytes as it is

essential for melanoblast survival and melanocyte-specific

lineage differentiation. On the basis of the proven role of Zeb2

in neural crest cell delamination, we expected a major defect

in melanoblast development.34 However, although mice

deficient for Zeb2 in the melanocyte lineage have an impaired

melanoblast development; a significant fraction of ZEB2-

deficient melanoblasts was still able to migrate to the

epidermis where they populate the hair follicles. Unexpect-

edly, the ZEB2-deficient melanocytes in the hair follicle bulb

area remain undifferentiated, causing a congenital loss of

pigmentation. These melanocytes have a very low MITF

expression, supporting the role of ZEB2 as a driver

for melanocyte differentiation. In vitro data further confirm

the crucial role of ZEB2 as a regulator of MITF and

melanocyte differentiation coinciding with an upregulation of

ZEB1. In this context, it is noteworthy that the loss of Zeb2 in

non-transformed melanocytes in vivo can result in ZEB1

misexpression. Furthermore the strongly reduced expression

of the melanocortin receptor upon Zeb2 loss potentially

contributes to the maintenance of the observed low Mitf

transcriptional levels.

EMT has a key role during different stages of embryonic

development including the formation and migration of the

neural crest cells. Neural crest cells are a multipotent,

migratory, transient cell population that migrate through the

vertebrate embryo to infiltrate different organs and differenti-

ate in various cell lineages including melanocytes.41 During

the progression to melanoma, melanocytes may acquire

multiple traits of high grademalignancy in context of additional

oncogenic changes by reactivating this silenced embryonic

program.25 In contrast to epithelial cells where EMT-inducing

transcription factors favor dedifferentiation and dissemination,

we shedded light on a more complex interaction between

ZEB2 and MITF in the melanocyte lineage. As ZEB2

expression favors MITF expression and thus differentiation

in melanocytes, the loss of Zeb2 in the melanocyte lineage

results in the formation of undifferentiated melanocytes with

loss ofMitf and upregulation of Zeb1. Together with our finding

that ZEB1 is expressed in melanocyte stem cells, it seems

that ZEB switching is a naturally occurring transcription factor

reprogramming that also can be facilitated by activated

oncogenic RAS/RAF signaling.28

Examination of human melanoma samples and cell lines

assessed the relevance of the identified effects upon in vivo

loss of ZEB2 in a clinical context. It seems that ZEB2,

particularly in the highly invasive vertical growth phase of

human melanoma, gets downregulated in a very heteroge-

neous manner. Importantly, nuclear ZEB2 expression in

melanomas is linked with good prognostic markers whereas

loss of ZEB2 is associated with poor melanoma-specific

survival. The partial or total clearance of ZEB2 in human

melanoma could be caused by the well-known negative

control of ZEB2 expression by the miR200 family. However

this reasoning is not supported by the observation that

expression of the miR200 family is lost during melanoma

progression ruling out their potential dominant action of ZEB2

repression, at least during melanoma progression.42,43

As MITF depletion forces melanocytes to senescence,39

ZEB1 may be a failsafe program for a melanoma cell to

overcome the senescent state, exemplifying why ZEB1

expression in human melanoma is inversely correlated with

MITF status. In this context, it is noteworthy that loss of ZEB2

in non-transformed melanocytes in vivo can result in ZEB1

misexpression, which potentially takes over in an alternative

way the function of ZEB2 on the expense of differentiation.

Our novel finding demonstrates that the switch from ZEB2 to

ZEB1 is associated with a reduction of MITF, the master

controller of differentiation, growth and migration in melano-

cytes. In melanoma, MITF has been described as a lineage

addiction oncogene that is controlling melanoma progression

following a rheostat model, in which high MITF expression is

required for proliferation whereas its reduced level results in

invasive behavior. We postulate that low MITF expression

favors melanoma invasion and metastasis, which corre-

sponds with the poor patient prognosis predicted by the loss

of ZEB2, as shown in our study.

Materials and MethodsMice. Mice were kept in accordance with the institutional guidelines regardingthe care and use of laboratory animals and all procedures were approved by theinstitutional ethics committee. Mice with the following genotypes have beendescribed elsewhere: conditional Zeb2fl/fl,44 Tyr::Cre36 and Dct::LacZ.37

Conditionally, Rosa-Zeb2TG/TG-IRES-GFP overexpressing mice were generatedusing G4 hybrid ES cells and the Gateway-compatible Rosa26 locus targetingvector, as previously described.45,46

Melanin content determination. Melanin was extracted with an alkalinesolution as previously described.47

Whole-mount LacZ staining of embryos and skin. To obtain E15.5embryos, timed matings were set up, and the day on which a vaginal plug wasdetected was set as E0.5. For skin samples, back skin of 5.5-day-old mice wasisolated. Embryos and back skin were washed in PBS and fixed in 4%paraformaldehyde for 3 h at room temperature (RT), after which they were washed


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with permeabilization solution (0.1 M phosphate buffer pH 7.4, 2 mM MgCl2,2.5mM EGTA, 0.01% sodium deoxycholate, 0.02% NP40, 0.005% BSA) andstained overnight at RT with permeabilization buffer containing 5mM potassiumferricyanide, 5 mM potassium ferrocyanide and 0.1% LacZ solution. The sampleswere washed in PBS and postfixed for 2 h in 4% paraformaldehyde at RT. Tocount embryonic melanoblasts externally, photos were taken with a Nikon AZ100Multizoom microscope (Nikon, Tokyo, Japan) and defined regions were analyzedfor LacZ-positive melanoblasts with Volocity software. For further immunohisto-chemical analysis of the back skin, samples were dehydrated, embedded inparaffin and sectioned at 6 mm.

Human tissues. Tissue samples were obtained from the Department ofDermatology, Universitair Ziekenhuis Gent, Ghent, Belgium, from theDepartment of Pathology, University Hospital Leuven, KU Leuven, Leuven,Belgium and from archival paraffin-embedded patient samples fromSt. Vincent’s University Hospital, Dublin, Ireland. All patient specimens wereused in accordance with institutional and national policies at the respectivelocations, with appropriate approval provided by the relevant Ethicscommittees at the respective institutions. All patient-related information wasanonymized.

Immunohistochemistry. Tumors, organs and skin were isolated and fixedovernight in 4% paraformaldehyde solution, dehydrated, embedded in paraffin and cutinto 5-mm sections. For histology, samples were stained with hematoxylin and eosin,or with nuclear fast red. For immunohistochemical staining, antigen retrieval was donein citrate buffer and endogenous peroxidases were blocked with 3% H2O2 in methanol.The sections were incubated with primary antibodies and stained with biotin-conjugated secondary antibodies followed by Streptavidin-HRP based development(substrate development with DAB or AEC). When necessary, the signal was amplifiedusing the Tyramide Signal Amplification (TSA) kit (Perkin Elmer Applied Biosystems,Zaventem, Belgium), indicated with asterisk. The following antibodies were used:mouse anti-ZEB2 (1/300*) (in-house monoclonal antibody, clone 7F7) for normalmouse sections, rabbit anti-ZEB2 (1/500*) (HPA003456, Sigma, Diegem, Belgium) forhuman sections, goat anti-ZEB1 (1/50) (sc-10572, Santa Cruz, Heidelberg, Germany)for mouse sections, rabbit anti-ZEB1 (1/10 000*) (gift from Professor D Darling) forhuman sections; rabbit anti-S100b (1/1000*) (ZO311, Dakopatts, Leuven, Belgium),rabbit TYRP1 (1/1000*) (gift received kindly from Professor V Hearing); mouse anti-MITF (1/300) (Ab 12039, Abcam, Cambridge, UK), mouse anti-PAX3 (1/200) (DSHB,Iowa city, IA, USA).

Tyrosinase assay. The Tyrosinase assay was performed as describedpreviously.47

Transmission electron microscopy. Back skin from 5.5-day-oldZEB2MCWT and ZEB2MCKO mice was immersed in a fixative solution of 2.5%glutaraldehyde, 3% formaldehyde and 0.02% CaCl2 in 0.1 M Na-cacodylate buffer,and processed as previously described.48 Ultrathin sections of a gold interferencecolor were cut using an ultra microtome (Leica EM UC6, Diegem, Belgium),post-stained with uranyl acetate and lead citrate in a Leica EM AC20, andcollected on Formvar-coated copper slot grids. They were viewed with a JEOL1010 transmission electron microscope (� 10 000 images) (JEOL, Tokyo, Japan).

Primary melanocyte cultures. Primary melanocytes were isolated from5.5-day-old pups as previously described.47

siRNA and plasmid transfections. To knock down Zeb2 in primaryNRAS transformed melanocytes, primary Melan a mouse melanocytes, B16-BL6mouse melanoma and SKMel28 human melanoma, we used siRNA pools formouse Zeb2 (Dharmacon, St. Leon Rot, Germany) and a scrambled siRNA pool(Dharmacon) as a control. Transfections were performed with HiPerfect (Qiagen,Antwerpen, Belgium) according to the manufacturer’s instructions. Five days aftersiRNA transfection, RNA and protein lysates were prepared. To overexpressmouse Mitf we used a mouse Mitf expression vector and an empty vector as acontrol. Transfections were performed with GenJet (Stratagene, Amsterdam,The Netherlands) according to the manufacturer’s instructions. Mitf transfectionswere done 1 day after siRNA knockdown of Zeb2.

Lentiviral transfections. To knock down MITF in 501Mel cells we used anshRNA for human MITF and a scrambled shRNA (shCTRL) as a control. The

shRNA vectors are pLKO based from Sigma, the Mission shRNA series. 501Melcells were infected with shRNA directed against MITF or the Mission shRNAcontrol sequence and selected for 4 days with 3.0mg/ml of puromycin.

ChIP analysis. ChIP and ChIP-Seq experiments were performed onchromatin from native 501Mel and on 501Mel cells stably expressing 3HA-taggedMITF to standard protocols as previously described.39

Real-time qPCR analysis. RNA was extracted from cell cultures or ears of15.5-day-old ZEB2MCWT and ZEB2MCKO mice by using RNeasy extraction columns(Qiagen). RNA was treated with 1 U of RNAse-free DNase RQ1 (Promega,Leiden, The Netherlands) per mg RNA for 30min at 37 1C in appropriate buffer.DNAse was inactivated by incubation in Promega stop solution for 10min at 65 1C.Bulk Mg2þ was removed by using Amicon ultra 0.5-ml centrifugal filters (Millipore,Brussels, Belgium) in two consecutive diluting washes. cDNA synthesis wasperformed with iScript (Bio-Rad, Hercules, CA, USA) following the manufacturer’sinstructions. Quantitative PCR was done using the Fast SYBR master mix kit(Applied Biosystems, Gent, Belgium) or SensiFastTM SYBR No-Rox kit (Bioline,Alpen aan de Rijn, The Netherlands) for the genes of interest and referencegenes. Primers were designed using Primer Express 1.0 Software (Perkin ElmerApplied Biosystems) or obtained from the literature.47,49 Plates were run on theLightCycler 480 (Roche, Vilvoorde, Belgium). The average threshold cycle oftriplicate reactions was used for all subsequent calculations using the deltaCTmethod. Graphs represent the average normalized relative expression values ofZEB2MCWT and ZEB2MCKO mice, with the WT average normalized relative to theexpression value at 4 days set to 1.

Western blot analysis. Cells were lysed in Laemli-lysis buffer (50mMTris-HCl pH 6.8, 10% glycerol, 2% SDS). After sonicating and centrifuging thesamples, 20mg of protein was separated on gel and transferred to a PVDFmembrane. Membranes were incubated with primary antibodies and appropriateHRP-labeled secondary antibodies (GE Healthcare, Diegem, Belgium). Detectionwas performed with the Western LightningT chemiluminescence reagentplus kit (Perkin Elmer, Waltham, MA, USA) or the Immobilion western HRPsubstrate (Millipore). The following antibodies were used: rabbit anti-ZEB2(1/1000) (HPA003456, Sigma); rabbit anti-ZEB1 (1/1000) (Gift from ProfessorDarling); mouse anti-b-Tubulin (1/1000) (T4026, Sigma); mouse anti-MITF(Ab 12039 Abcam); mouse anti-Vinculin (1/3000) (V9131, Sigma); mouseanti-Fibronectin (1/1000) (Abcam, ab23750).

Statistical analysis. Data were analyzed with GraphPad Prism 5 (GraphPadSoftware Inc., La Jolla, CA, USA) and R (The Comprehensive R Archive Network,http://www.cran.r-project.org).

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements. This research was funded by grants from the FWO,the Geconcerteerde Onderzoeksacties of Ghent University, the Stichtingtegen Kanker, the Association for International Cancer Research (Scotland),the EU-FP6 framework program BRECOSM LSHC-CT-2004-503224, theEU-FP7 framework programs TuMIC 2008-201662 and Target-Melanoma(www.targetmelanoma.com). Work in the lab of ID was supported by grantsfrom the Ligue National Contre le Cancer, the INCa, the Universite deStrasbourg and the ANR. The IGBMC high throughput sequencing facilityis a member of the ‘France Genomique’ consortium (ANR10-INBS-09-08).We acknowledge Riet DeRycke for expert electron microscopic analysis,Dr. Amin Bredan for critical reading of the manuscript and the membersof our research group for valuable discussions. We thank Professor D Darling forproviding the ZEB1 antibody, Professor K Hearing for providing the TYRP1antibody.

Author contributionsGD, NV, OA, DK, JT, KL, AG, BDC, BB, ID, JCM and GB performed theexperiments and analyzed the interpreted data. MVG, LB, GMU, MR, WMG, GG,DH, JvdO, LL and JH provided valuable reagents/material. GD and GB conceived-designed the project, analyzed the interpreted data and wrote the paper with inputsparticularly from NV, DH, LL, JH, JCM and all other authors.


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G Denecker et al

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