P oc. Na l. Acad. Sci. USA
Vol. 94, pp. 1177–1182, Feb ua y 1997
Biochemis y
A single zinc inge mo i in he silencing ac o REST ep esses
he neu al-speci ic ype II sodium channel p omo e
(ne ous sys em
y
ansc ip ion
y
ep esso s)
JOSE
´TAPIA-RAMI
´REZ*, BART J. L. EGGEN,MARIA J. PERAL-RUBIO,JUAN J. TOLEDO-ARAL†,AND GAIL MANDEL‡
Depa men o Neu obiology and Beha io and Ins i u e o Cell and De elopmen al Biology, S a e Uni e si y o New Yo k a S ony B ook, S ony B ook, NY
11794-5230
Communica ed by William Lenna z, S a e Uni e si y o New Yo k, S ony B ook, NY, Decembe 16, 1996 ( ecei ed o e iew No embe 18, 1996)
ABSTRACT The ype II ol age-dependen sodium chan-
nel is p esen in neu onal cells, whe e i media es he p op-
aga ion o ne e impulses. Res ic ed exp ession o he ype II
sodium channel gene o neu ons is due, a leas in pa , o
binding o he ep esso p o ein REST (also e med NRSF o
XBR) o he RE1 (also called NRSE) sequence in he ype II
sodium channel gene. P e ious s udies ha e shown ha a
domain in REST con aining eigh GL1-K u¨ppel zinc inge
mo i s media es DNA binding. Dele ional and GAL4- usion
gene analyses now e eal ep esso domains ha lie ou side o
he DNA-binding domain in bo h he amino and ca boxyl
e mini o REST. Mu a ional analysis u he iden i ies a
single zinc inge mo i in he ca boxyl- e minal domain as
being essen ial o ep essing ype II sodium channel epo e
genes. These s udies e eal wo domains in REST ha may
media e in e ac ions wi h o he p o eins in ol ed in es ic -
ing exp ession o a la ge se o genes o he e eb a e ne ous
sys em.
The abili y o gene a e ac ion po en ials is o en due o he
p esence o ol age-dependen sodium channels in he plasma
memb anes o he exci able cells. Sodium channels a e en-
coded by a la ge mul igene amily, and membe s o his amily
a e s uc u ally dis inc and exp essed in a issue-speci ic
manne ( e iewed in e . 1). The ype II sodium channel gene
(2, 3) is exp essed o high le els exclusi ely in neu ons in he
cen al ne ous sys em (4, 5). Because o his selec i e exp es-
sion pa e n, he ype II sodium channel has p o ided an
excellen model o s udies o he mechanisms egula ing
neu al-speci ic gene exp ession.
Type II sodium channel epo e genes con aining 1050 bp
o 59 lanking sequence a e exp essed in neu onal cell lines bu
no in nonneu onal cells, consis en wi h exp ession o he
endogenous gene. Dele ional analysis has iden i ied a 23-bp
elemen in he ype II sodium channel egula o y egion,
e med ep esso elemen 1 (RE1), ha p e en s exp ession o
ype II epo e genes in nonneu onal cell ypes (6, 7).
Remo al o he RE1 esul s in app oxima ely 80- old de e-
p ession o he ype II sodium channel epo e gene speci i-
cally in nonneu onal cell ypes. Rep esso elemen s wi h
sequences and unc ional p ope ies simila o hose o he ype
II sodium channel RE1 a e p esen in he egula o y egion o
se e al o he genes exp essed exclusi ely in he ne ous sys em
( e iewed in e . 8), including SCG10 (9, 10), synapsin (11), he
b
1 subuni o he nico inic ace ylcholine ecep o (12), he
musca inc M4 ecep o (13, 14), and neu al–glial cell adhesion
molecule (15). The widesp ead occu ence o RE1-like se-
quences in di e en genes sugges s a mo e global ole o
ep ession in es ic ing gene exp ession o he ne ous sys em.
Recombinan RE1-silencing ansc ip ion ac o (REST) is
su icien o ep ess epo e genes con aining RE1-like a ge
sequences in co ans ec ion analyses o neu onal cells (8, 16,
17). The deduced p ima y s uc u e o REST does no e eal
any amino acid homologies which would poin o ep esso
domains. Howe e , p e ious s udies sugges ed ha he DNA-
binding and ep esso domains o REST we e physically
dis inc . The REST DNA-binding domain was iden i ied o ig-
inally, in a gene ic sc een in yeas , as a clus e o eigh
GL1-K u¨ppel class C
2
H
2
(Cys
2
His
2
) zinc inge s (16). Exp es-
sion in skele al muscle cells o a po ion o REST con aining
hese zinc inge s esul ed in de ep ession o co ans ec ed
ype II epo e genes (16). This esul sugges ed ha he
amino- e minal zinc inge s we e ac ing as a dominan nega i e
mu an by in e e ing wi h binding o he endogenous REST
p o ein o he RE1 a ge si e. The esul u he sugges ed ha
he domains equi ed o ep ession mus be loca ed elsewhe e
in he molecule.
I is clea , om s udies o bo h p oka yo es and euka yo es,
ha se e al di e en mechanisms ha e e ol ed o ep essing
gene exp ession. Despi e he impo ance o nega i e gene
egula ion, he molecula componen s equi ed o his mech-
anism a e only beginning o eme ge. REST-media ed ep es-
sion is cell- ype speci ic, and REST ep esses minimal p o-
mo e s ha do no equi e ac i a o s o ansc ip ion (6, 9).
Thus, REST p o ides an excellen model o s udying he
molecula basis o his class o ep esso s. In his s udy we
sough o pe o m a s uc u e– unc ion analysis o REST o
he pu pose o elucida ing po en ially impo an domains by
which REST migh in e ac wi h he ansc ip ional machin-
e y.
We ha e iden i ied, using a dele ional and GAL4- usion
gene app oach, wo dis inc ep esso domains in REST. As
p edic ed by he ea lie s udies, exp ession o he DNA-
binding domain alone in neu onal PC12 cells did no esul in
ep ession o ype II epo e genes. Ra he , dis inc domains
in he amino and ca boxyl e mini we e iden i ied ha we e
each pa ially equi ed and su icien o ep ess he ype II
p omo e . The ca boxyl- e minal domain con ains a p edic ed
zinc inge mo i , and mu a ions ha des oy he inge s uc-
u e abolish ep ession. I is likely ha he wo REST domains
The publica ion cos s o his a icle we e de ayed in pa by page cha ge
paymen . This a icle mus he e o e be he eby ma ked ‘‘ad e isemen ’’ in
acco dance wi h 18 U.S.C. §1734 solely o indica e his ac .
Copy igh q1997 by THE NATIONAL ACADEMY OF SCIENCES OF THE USA
0027-8424y97y941177-6$2.00y0
PNAS is a ailable online a h p:yywww.pnas.o g.
Abb e ia ions: RE1, ep esso elemen 1; REST, RE1-silencing an-
sc ip ion ac o ; NRSF, neu al- es ic i e silencing ac o ; UAS, up-
s eam ac i a ing sequence; CMV, cy omegalo i us; CAT, chlo am-
phenicol ace yl ans e ase.
*P esen add ess: Depa amen o de Gene ica y Biologia Molecula ,
Cen o de In es igacion y de Es udios A anzados del Ins i u o
Poli ecnico Nacional, A . Ins i u o Poli ecnico Nacional No. 2508,
Col. San Ped o Zaca enco, Mexico, D.F. cp 07300.
†P esen add ess: Depa men o Physiology and Biophysics, School o
Medicine, Uni e si y o Se ille, 41009 Se ille, Spain.
‡To whom ep in eques s should be add essed.
1177
a e si es o in e ac ions wi h o he ac o s ha a e equi ed o
ep ession o he se o genes con aining RE1 sequences.
METHODS
Plasmid Cons uc ions. The plasmid REEX1 (amino acids
1–1097) is a de i a i e o he plasmid REST-EXPRESS (16)
gene a ed by subcloning a 4.03-kb EcoRI agmen con aining
he en i e REST coding sequence in o he EcoRI si e o
pcDNAI-Amp (In i ogen). Cons uc s encoding pa ial seg-
men s o he REST p o ein we e gene a ed as ollows: A
pa ial cDNA clone e med p73 (amino acids 73–545), con-
aining he deduced eigh zinc inge s o he DNA-binding
domain, has been desc ibed (16). REEX21 (amino acids
73–636) was c ea ed by a h ee-way liga ion o a HindIIIy
HincII agmen o p73 wi h a HincIIySphI agmen o
REEX1 be ween he HindIII and SphI si es o pcDNAI-Amp.
REEX21 ex ends p73 by 91 amino acids. REEX7, con aining
an in e nal dele ion be ween amino acids 636 and 786, was
cons uc ed by a h ee-way liga ion o an EcoRIyblun ed SphI
agmen o REEX1 wi h a blun ed Bs XIyXbaI agmen o
REEX1 inse ed be ween he EcoRI and XbaI si es o pc
DNAI-Amp. REEX9 (amino acids 1–1036) was gene a ed by
liga ion o an EcoRIySphI agmen o REEX1 and a SphIy
EaeI agmen o REEX1 be ween he EcoRI and No I si es
o pcDNAI-Amp. REEX8 (amino acids 73–1097) was c ea ed
by subcloning a P uIIyXbaI agmen o REEX1 be ween he
EcoRV and XbaI si es o pcDNAI-Amp. The o ien a ion o all
ecombinan REST cons uc s was con i med by es ic ion
analysis, and REST dele ional mu an s we e also checked by
sequence analysis o ensu e ha ameshi s had no occu ed.
The plasmid pSG424 con aining he GAL4 DNA-binding
domain (amino acids 1–147) was ob ained om S an Fields
(Uni e si y o Washing on, Sea le). To cons uc GAL4-N1,
he amino- e minal sequences o REST (amino acids 1–83)
we e ob ained by he polyme ase chain eac ion (PCR) using
REEX1 as he empla e. The PCR p oduc s we e diges ed wi h
BamHI and KpnI es ic ion enzymes and subcloned in o he
pSG424 ec o . GAL4-REEX1 was gene a ed by h ee-way
liga ion o a BamHIyP uII agmen o GAL4-N1 wi h a
P uIIyXbaI agmen o REEX1 be ween he BamHI and
XbaI si es o pSG424. GAL4-p73 (amino acids 62–545 o
REST) was gene a ed by i s liga ing a P uIIyXbaI agmen
o GAL4-REEX1 be ween he SmaI and XbaI si es o pSG424,
gene a ing GAL4-REEX8. Second, a ClaIyXbaI agmen o
p73 was liga ed be ween he ClaIyXbaI si es o GAL4-
REEX8, esul ing in GAL4-p73. To cons uc GAL4-C3,
REST amino acids om 1008 o 1097, con aining he single
zinc inge , we e ampli ied by he PCR and he agmen was
subcloned in o he pSG424 backbone a he BamHI si e. All o
he GAL4-REST usions we e sequenced ac oss he GAL4-
REST junc ion o ensu e ha he inse s we e in ame wi h
GAL4. All cons uc s gene a ed by he PCR we e ully se-
quenced o ensu e ha mu a ionsydele ions had occu ed
du ing he ampli ica ion eac ion.
The RE1-con aining ype II sodium channel-chlo amphen-
icol ace yl ans e ase (CAT) epo e gene, pSDK7, has been
desc ibed p e iously (7). The ups eam ac i a ing sequence
(UAS) ype II CAT epo e gene was gene a ed by subs i u -
ing he 23-nucleo ide ype II RE1 sequence in he CAT
epo e plasmid o i e copies o he UAS om he plasmid
pGAL4-TKCAT p o ided by Thomas Shenk, P ince on Uni-
e si y (18).
Si e-Di ec ed Mu agenesis. To gene a e a mu an REST
molecule wi h an amino acid change in he p edic ed ca boxyl-
e minal zinc inge mo i , a comme cial DNA mu agenesis ki
was used (Mo ph, 5 P ime 33 P ime). An oligonucleo ide
encoding a mu a ion in a cys eine esidue o he zinc inge was
syn hesized. This oligonucleo ide, 59-GCGGCTAAGG-
GAGATTTTGTTCGTATCTTCTGTGATCG-39, was used
o cons uc he mu an GAL4-C3M1. The agmen was
checked by sequence analysis. (The bold ace le e ep esen s
he change om wild- ype sequence.) The mu a ion esul s in
he subs i u ion o an a ginine esidue o a cys eine. To
gene a e an in ac REST molecule con aining he mu a ion in
he ca boxyl- e minal zinc inge , an EcoRIyEagI agmen o
REEX1 and an EaeIyXbaI agmen o GAL4-C3M1 we e
liga ed in o he pcDNA1-Amp ec o a he EcoRI and XbaI
si es.
T ansien T ans ec ions and CAT Assays. PC12 cells we e
g own as desc ibed p e iously (19). COS-1 cells we e g own in
Dulbecco’s modi ied Eagle’s Medium (DMEM) supplemen ed
wi h 10% bo ine cal se um (HyClone). COS-1 cells we e
ans ec ed wi h 8
m
g o plasmid DNA by ea men wi h
calcium phospha e (20). Fo he ans ec ions shown in Fig. 1,
PC12 cells we e elec opo a ed wi h mix u es o 10
m
go
epo e plasmid con aining ei he 5
m
g o emp y pcDNAI-
Amp ec o o 10
m
g o pcDNAI-Amp plasmids con aining
REST cDNA inse s. Fo he ans ec ions shown in Fig. 2,
PC12 cells we e elec opo a ed wi h mix u es o 10
m
go
epo e plasmid and 5
m
g o pSG424 o equimola ( o 5
m
go
pSG424) amoun s o GAL4-REST usion cDNA cons uc s.
To all mix u es, pBluesc ip II SK (S a agene) was added o
b ing he inal amoun o DNA o 20
m
g. Fo he ans ec ions
shown in Fig. 3, 10
m
g o epo e plasmid was mixed wi h
ei he 1
m
g o pcDNAI-Amp o an equimola amoun o
pcDNAI-Amp con aining REST inse s. The mix u e o cells
and DNA was chilled on ice o 5–10 min be o e elec opo-
a ion (250 mV, 960 mF). Following elec opo a ion he cells
we e chilled on ice o a u he 10 min be o e pla ing in
100-mm dishes. Medium was changed 24 h a e ans ec ion
and cells we e ha es ed a e 48 h . The p o ein concen a ion
o he cell lysa es was de e mined wi h he mic o BCA p o ein
assay eagen (Pie ce) in mic o i e pla es. Assays o CAT
ac i i y in lysa es o he ha es ed cells we e pe o med as
desc ibed p e iously (7). Rela i e ac i i y o he ex ac s was
calcula ed by de e mining he pe cen age o ace yla ed chlo -
amphenicol, using a Molecula Dynamics Phospho Image .
Fo each cons uc a leas wo di e en p epa a ions o
plasmid DNA we e used (Qiagen om Qiagen o Je S a om
Genomed). In each expe imen , cons uc s we e ans ec ed in
duplica e and di e en expe imen s we e pe o med se e al
imes as no ed in Resul s.
Wes e n Blo Analyses. Nuclea ex ac p epa a ion and
Wes e n blo ing o COS-1 cells ans ec ed wi h he app o-
p ia e cDNA cons uc s we e pe o med as desc ibed p e i-
ously (16), and he samples we e solubilized in Laemmli
sample bu e . A e sepa a ion on educing SDSypolyac yl-
amide gels, ei he 7% o 12% polyac ylamide, p o eins we e
ans e ed on o ni ocellulose memb anes. Blo s we e incu-
ba ed ei he wi h a polyclonal an i-GAL4 an ibody (Ups a e
Bio echnology) o an a ini y-pu i ied polyclonal an i-REST
an ibody. The an ibodies we e isualized by using he ECL
de ec ion me hod (NEN).
RESULTS
Two Dis inc Domains in REST A e In ol ed in Rep ession
o Type II Sodium Channel Repo e Genes in PC12 Cells.
Al hough no known ep esso consensus sequences we e ap-
pa en in he deduced p ima y sequence o REST, h ee
dis inc domains, depic ed in Fig. 1, we e iden i ied. These
domains, om amino e minus o ca boxyl e minus, consis ed
o he ollowing: (i) a clus e o eigh GLI-K u¨ppel ype zinc
inge s cons i u ing he DNA-binding domain (16, 17), (ii)a
no el ei e a ed p oline- ich mo i , and (iii) a single C
2
H
2
zinc
inge mo i in he ca boxyl e minus. He e, we ha e gene a ed
a amily o dele ion molecules o examine he po en ial in-
ol emen o hese mo i s in ansc ip ional ep ession by
REST. The REST cDNAs we e placed unde con ol o he
1178 Biochemis y: Tapia-Ramı´ ez e al. P oc. Na l. Acad. Sci. USA 94 (1997)
CMV 1E p omo e in he mammalian exp ession ec o
pcDNA1 (Fig. 1). The cons uc s we e co ans ec ed in o PC12
cells, which do no exp ess signi ican le els o he endogenous
REST gene, along wi h CAT epo e genes con aining he
ype II sodium channel RE1 sequence ups eam o he ype II
p omo e . CAT ac i i y esul ing om co ans ec ion o he
ype II epo e gene and he emp y ec o was se o 100%.
Co ans ec ion o PC12 cells wi h he ype II sodium channel
epo e gene and he ull-leng h REST cDNA (REEX1)
caused a g ea e han 7- old ep ession o epo e gene
exp ession (13.3% CAT ac i i y; Fig. 1). Because he domain
con aining he clus e o eigh zinc inge s is equi ed o DNA
binding o he RE1 sequence (16) his domain mus be
included in all o he REST dele ional mu an s. As expec ed,
exp ession o he REST DNA-binding domain alone (p73) did
no esul in ep ession o he ype II epo e gene. In ac ,
CAT ac i i y o his mu an was sligh ly g ea e han ha o he
con ol alue, pe haps ep esen ing a sligh dominan in e -
e ing e ec om compe i ion o RE1 binding by he low
le els o REST p o ein ha a e p esen in PC12 cells ( e . 16;
see also esul s wi h GAL4-p73 in Fig. 2B). Inclusion o a
egion adjacen o he DNA-binding domain caused only a
modes inc ease in ep esso ac i i y (75% CAT ac i i y; Fig.
1). The domain con aining he six ei e a ed p oline- ich
mo i s was also dele ed om he in ac REST molecule
(REEX7). The emo al o his domain did no in e e e wi h
ep esso ac i i y (Fig. 1), indica ing ha hese mo i s a e no
equi ed o he ep esso mechanism.
Exp ession o a unca ed REST molecule lacking he 60
e minal amino acids o REST ha includes he lone zinc
inge mo i (REEX9) esul ed in a pa ial de ep ession o he
ype II p omo e , causing an app oxima ely 3- old inc ease in
CAT ac i i y compa ed wi h ha media ed by he wild- ype
REEX1 molecule. Thus, o all o he ob ious domains e ealed
by elucida ion o he REST p ima y sequence, only he
ca boxyl- e minal zinc inge domain exhibi ed signi ican
ep esso ac i i y. Howe e , he equi emen o his domain
was only pa ial, sugges ing ha o he domains in REST we e
also equi ed o media e ep ession. In suppo o his in e -
p e a ion, a pa ial cDNA ex ending om he p edic ed ini-
ia o me hionine in NRSF o amino acid 585 also exhibi ed
ep esso ac i i y in ansien ans ec ion analyses (17). By
p ocess o elimina ion in compa ing he p edic ed s uc u es o
he pa ial NRSF p o ein and ull-leng h RESTyNRSF, an-
o he candida e o a ep esso domain was he amino e mi-
nus o REST ha was missing in he DNA-binding domain
cons uc , p73. Resul s o ans ec ions wi h a REST mu an
ha lacks hese amino acids (REEX8) showed ha he amino-
e minal domain was pa ially equi ed o ep ession o ype
II sodium channel epo e gene exp ession (37.3% CAT
ac i i y; Fig. 1). The amoun o ep ession exhibi ed by he
REST mu an s dele ed in he amino- and ca boxyl- e minal
domains we e oughly equi alen (app oxima ely 3- old e-
p ession o epo e gene ac i i y o each cons uc ).
To de e mine whe he he amino- and ca boxyl- e minal
domains we e su icien o media e ep ession, ep esso ac-
i i y o he indi idual domains was es ed by using hem
in- ame o cDNA coding o he DNA-binding domain o he
yeas ac i a o p o ein GAL4. Co espondingly, in he ype II
sodium channel epo e gene, he ype II RE1 was eplaced
wi h i e copies o he yeas UAS, he a ge si e o binding
by he GAL4 p o ein. The chime ic cDNAs and UAS epo e
genes we e hen co ans ec ed in o PC12 cells. The ac i i y o
he UAS ype II epo e gene coexp essed wi h he GAL4
DNA-binding domain alone was se a 100%.
To alida e his sys em o analyzing REST ep esso ac i -
i y, a chime ic p o ein con aining he ull-leng h REST p o ein
(GAL4-REEX1; Fig. 2A) was i s in oduced in o PC12 cells
along wi h he UAS epo e gene. As expec ed, CAT ac i i y
o GAL4-REEX1 was ep essed app oxima ely 4- old com-
pa ed wi h con ol CAT ac i i y (Fig. 2B). To de e mine
whe he he 83 amino- e minal (GAL4N1; Fig. 2A) o 88
ca boxyl- e minal (GAL4C3; Fig. 2A) amino acids we e su -
icien o media e ep ession in his sys em, hese domains we e
also used in- ame wi h he GAL4 DNA-binding domain.
Exp ession o hese REST domains esul ed in d ama ic
ep esso ac i i y, 9.5- old and 11- old, espec i ely (Fig. 2B),
indica ing ha hey we e indeed su icien o media e ep es-
sion o UAS epo e gene exp ession. In con as o hese
esul s, ans ec ions wi h he GAL4-p73 usion gene (Fig.
2A), encoding he eigh K u¨ppel ype zinc inge s in he
DNA-binding domain, did no esul in ep ession o epo e
gene ac i i y (Fig. 2B).
Mu a ion o he Single Zinc Finge in he Ca boxyl-
Te minal Domain Ab oga es Rep ession. Unlike he amino-
e minal domain, he ca boxyl- e minal domain ha was su -
icien o ep ess he ype II sodium channel p omo e con-
ained a ecognizable mo i , a single C
2
H
2
zinc inge . To es
FIG. 1. Domains in he amino and ca boxyl e mini o REST a e equi ed, in pa , o media e ep ession o ype II sodium channel epo e
genes. The amily o REST molecules exp essed unde con ol o he cy omegalo i us (CMV) p omo e , in PC12 cells, is shown wi h espec o
he p esence o known mo i s in he deduced p ima y s uc u e o REST ( op line). The do ed lines indica e he egions in REST ha we e dele ed.
A ca oon o he co ans ec ed epo e gene shows he posi ions o he ype II ep esso elemen 1 (RE1), ype II sodium channel p omo e ( ype
II) and CAT gene (bo om line). A ows indica e he s a si es o ansc ip ion in he exp ession plasmids. On he igh , he pe cen ac i i y o
CAT esul ing om ans ec ion o he di e en cons uc s is no malized o ha om he ec o alone, which is se o 100%. S anda d e o s o
he mean and (in pa en heses) he numbe s o expe imen s a e indica ed.
Biochemis y: Tapia-Ramı´ ez e al. P oc. Na l. Acad. Sci. USA 94 (1997) 1179
whe he his mo i was equi ed o ep ession by REST, one
o he cys eine esidues c i ical o he zinc inge s uc u e was
changed o an a ginine, and he mu a ed domain was used
in- ame wi h he GAL4-DNA-binding domain. The mu a ed
cons uc (GAL4C3M1) was co ans ec ed in o PC12 cells
along wi h he UAS ype II epo e gene (Fig. 2B). The zinc
inge mu a ion abolished he ep esso ac i i y no mally
obse ed wi h he co esponding wild- ype REST domain ( he
CAT ac i i y was equi alen o ha seen by exp ession o he
con ol GAL4 and GAL4-p73 cons uc s). Wes e n blo anal-
ysis o COS-1 cells ans ec ed wi h he GAL4 usion genes
indica ed ha he lack o ep ession by he mu a ed ca boxyl-
e minal zinc inge domain (GAL4-C3M1) and by he REST
p73 domain was no due simply o di e ences in he le els o
exp essed chime ic p o eins (Fig. 2C). Rep ession o epo e
gene ac i i y media ed by he GAL4-REST usion p o eins
equi ed he REST domains o be e he ed o he DNA
h ough he GAL4 DNA-binding domain because ans ec-
ions o he GAL4-REST cons uc s wi h a ype II epo e
gene lacking an UAS did no esul in ep ession o CAT
ac i i y (da a no shown).
The abo e s udies indica ed ha he single zinc inge mo i
was equi ed o ep ession media ed by he ca boxyl- e minal
REST domain. To de e mine whe he he zinc inge mo i was
also equi ed o ep ession wi hin he con ex o he in ac
REST molecule, he cys eine- o-a ginine poin mu a ion was
in oduced in o he ull-leng h REST molecule REEX1 and
co ans ec ed wi h he RE1- ype II sodium channel epo e
gene in o PC12 cells (Fig. 3). The poin mu a ion in REEX1
(REEX1M1) esul ed in a pa ial de ep ession o he ype II
p omo e (4.5- old inc ease in CAT ac i i y compa ed wi h
wild- ype REEX1; Fig. 3B). The esidual ep esso ac i i y is
likely due o he p esence o he amino- e minal agmen
shown abo e o be su icien o pa ially media e ep ession.
T ans ec ion o he wild- ype and mu an cDNAs in o COS-1
cells esul ed in he exp ession o a 200-kDa p o ein ha was
de ec ed by an an i-REST an ibody (Fig. 3C). The educed
ep esso ac i i y o mu a ed REST was no due o educed
accumula ion o he p o ein, because Wes e n blo analysis
indica ed simila le els o exp ession o he wild- ype and
mu an p o eins (Fig. 3C).
DISCUSSION
The molecula mechanisms esponsible o egula ing exp es-
sion o genes in he ne ous sys em a e unde s ood poo ly.
FIG. 2. Domains in he amino and ca boxyl e mini o REST a e su icien o media e ep ession, and a poin mu a ion in he zinc inge mo i
ab oga es ep esso ac i i y. (A) Schema ic ep esen a ion o he amily o GAL4-REST chime ical cDNAs exp essed wi h a UAS ype II-CAT
epo e gene in ansien ans ec ions o PC12 cells. The epo e gene con ains i e copies o he UAS. The do ed lines indica e egions in REST
ha we e dele ed. A ows indica e s a si es o ansc ip ion. The ca boxyl- e minal (C3), amino- e minal (N1), and mu a ed ca boxyl- e minal
(C3M1) agmen s o REST a e in- ame wi h he DNA-binding domain o he GAL4 p o ein. (B)(Le ) Rep esen a i e au o adiog am showing
hin-laye ch oma og aphy (TLC) ac iona ion o ace yla ed o ms o chlo amphenicol. Each sample is om a dish o cells ans ec ed ansien ly
wi h he GAL4-REST chime ical cDNA and he UAS ype II epo e gene. No e ha he CAT assay wi h he GAL4-p73 cons uc was om a
di e en TLC pla e. (Righ ) His og am showing compiled da a om independen expe imen s. S anda d e o s o he mean and he numbe s o
expe imen s a e indica ed. (C) Wes e n blo analysis o COS-1 cells ans ec ed wi h he indica ed GAL4-REST ca boxyl- e minal (C3 and C3M1)
and GAL4-p73 cons uc s. The amoun s o he exp essed p o eins (a owheads) show ha he inabili y o he mu a ed ca boxyl- e minal agmen
C3M1 and p73 o ep ess is no due o ins abili y o he exp essed p o ein.
1180 Biochemis y: Tapia-Ramı´ ez e al. P oc. Na l. Acad. Sci. USA 94 (1997)
Howe e , ecen s udies ha e shown ha a leas one o hese
mechanisms in ol es ansc ip ional ep ession media ed by
he DNA-binding p o ein REST. The disco e y ha REST is
also in ol ed in egula ing he exp ession o many o he genes
exp essed in he ne ous sys em, h ough a common RE1-like
gene ic elemen , unde sco es he impo ance o elucida ing
he molecula mechanism by which REST ep ession is me-
dia ed.
Euka yo ic ansc ip ion can be p e en ed by mechanisms
ha a e dependen upon (silencing) o independen o ( e-
p ession) ch oma in s uc u e ( o e iew see e . 21). The
obse a ion ha REST can inhibi epo e gene exp ession in
ansien ans ec ion analyses wi h plasmid DNA sugges s ha
ch oma in emodeling is no equi ed o i s abili y o block
ansc ip ion, and classi ies REST as a ep esso . Two well
cha ac e ized domains ha e been shown o media e he ac i -
i ies o o he ep esso p o eins, a domain ha is ich in alanine
esidues (22–24), and a K u¨ppel-associa ed box A (KRAB-A)
domain, ich in cha ged amino acids, ha is p esen in a la ge
numbe o zinc inge p o eins (25, 26). The deduced p ima y
s uc u e o ull-leng h REST ( e s. 16 and 27, and D. Ande -
son, pe sonal communica ion) does no con ain ei he o hese
mo i s.
The clus e o eigh GL1- K u¨ppel ype zinc inge s in REST
binds o he ype II RE1 sequence in i o and in i o (16, 17).
Despi e he abili y o bind DNA, hey a e no su icien o
media e ep ession. An addi ional C
2
H
2
zinc inge mo i
esides in he deduced ca boxyl e minus o REST. The
dele ional and GAL4 usion gene analyses pe o med in his
s udy indica ed ha , unlike he DNA-binding domain, his
domain was pa ially equi ed and su icien o media e e-
p ession o ype II sodium channel epo e genes. In ac , he
amoun o ep ession media ed by he GAL4-ca boxyl-
e minal usion p o ein was e en g ea e han ha media ed by
ep ession o he GAL4-REST chime a con aining he en i e
REST sequence. I is possible ha he zinc inge mo i in he
isola ed ca boxyl- e minal domain is mo e accessible o o he
componen s in ol ed in he ep ession mechanism han when
embedded in he in ac REST molecule. To exclude he
possibili y ha all small agmen s used o GAL4 will media e
ep ession in his sys em, we examined wo addi ional REST
agmen s o a size simila o ha o he ca boxyl- e minal
domain. The small GAL4-REST usion p o eins do no exhibi
ep esso ac i i y (da a no shown). Fu he mo e, he ca box-
yl- e minal domain con aining a single poin mu a ion in he
zinc inge mo i also does no exhibi ep esso ac i i y.
Zinc inge mo i s in p o eins a e usually associa ed wi h
DNA binding ( o e iew see e . 28). The zinc inge s can also
media e p o ein–p o ein in e ac ions (29–34), and some o he
p o eins ha con ain zinc inge mo i s a e ansc ip ional
ep esso s. Howe e , o ou knowledge, REST is he i s
example whe eby a zinc inge s uc u e is equi ed o media e
ep ession. Fo example, in he ansc ip ion ac o YY1,
al hough an iden i ied ep esso domain con ained wo zinc
inge s, mu a ional analysis indica ed ha he s uc u es o
hese zinc inge s we e no equi ed o ep ession (35). I may
be ha he zinc inge mo i in REST is su icien o media e
ep ession, al hough lanking amino acid sequences may also
con ibu e o he abili y o he minimal 23 amino acid mo i o
ep ess. In e es ingly, in he ansc ip ion ac o TFIIIA, he
linke sequence cha ac e is ic o K u¨ppel ype zinc inge s has
been shown o con e high-a ini y DNA binding on he zinc
inge domain (36). This linke sequence is p esen in he
K u¨ppel ype zinc inge s ha cons i u e he DNA-binding
domain in REST. The zinc inge mo i in he ca boxyl-
e minal domain o REST ha media es ep ession, as a single
s uc u e, does no ha e his linke sequence and does no
appea o bind DNA. Fo example, in ansien ans ec ion
analysis, chime ic GAL4 p o eins con aining he ca boxyl-
e minal domain o REST, and hus he zinc inge , do no
ep ess ype II sodium channel epo e genes con aining he
RE1 sequence in place o he UAS (J.T.-R. and G.M.,
unpublished esul s). Al hough we canno o mally exclude he
possibili y ha his zinc inge mo i binds o DNA (bu see e .
27), i does no appea o bind o he ype II RE1 sequence.
The dele ional and usion gene s udies he ein ha e e ealed
he p esence o wo dis inc ep esso domains loca ed a
opposi e ends o he REST molecule. Indeed, he REST
amino- e minal domain used o GAL4 is as e ec i e in
media ing ep ession o he ype II p omo e as is he ca boxyl-
e minal domain. Fu he , like he zinc inge domain, he
amino- e minal domain is also pa ially equi ed o ep es-
FIG. 3. The single C
2
H
2
zinc inge in he ca boxyl e minus o REST is su icien o media e ep ession o ype II sodium channel epo e
genes. (A) Schema ic ep esen a ion o he REST exp ession ec o and ype II-CAT epo e genes. Rela i e loca ions o dis inc domains in REST
a e indica ed. (B) Rep esen a i e au o adiog am (Le ) shows TLC ac iona ion o ace yla ed o ms o chlo amphenicol om PC12 cells
co ans ec ed wi h he di e en REST cons uc s shown and he RE1- ype II-CAT epo e gene. O he symbols a e he same as in Fig. 1. A
his og am (Righ ) shows a compila ion o CAT ac i i y om ou independen expe imen s using he REST cons uc s indica ed o he
au o adiog am. S anda d e o s o he mean a e shown. (C) Wes e n blo analysis o COS cells ans ec ed wi h he wild- ype (REEX1) and mu a ed
(REEX1M1) cDNAs. Uppe a owhead deno es o e exp essed REST p o ein (200 kDa) and lowe a owhead deno es c oss- eac ing endogenous
p o ein mig a ing a 116 kDa (16).
Biochemis y: Tapia-Ramı´ ez e al. P oc. Na l. Acad. Sci. USA 94 (1997) 1181

sion. No ob ious mo i s a e p esen wi hin he amino- e minal
sequences. Howe e , u u e s udies, such as compa isons wi h
REST homologues in o he species, may help cla i y he
unc ional mo i s.
Many ep esso complexes consis o a DNA-binding p o ein
in e ac ing wi h co ep esso s. Examples o such complexes a e
hy oid ho mone and e inoic acid ecep o s and he TRACs
(37, 38), MadyMaxysin3 (39), yeas TUP1ySSN6 and se e al
di e en DNA-binding p o eins (e.g., see e . 40), he imme-
dia e ea ly p o eins NAB1yNGF1AyKROX20 (41), and D o-
sophila Hai y- ela ed p o eins and G oucho (42, 43). I is likely
ha he amino- e minal and zinc inge domains now iden i-
ied in REST in e ac wi h co ep esso p o eins o wi h
p o eins ha a e pa o he ini ia ion complex.
We acknowledge g a e ully he con ibu ions o Julia G imes in all
aspec s o p epa a ion o he manusc ip and Simon Halegoua and
Paul B ehm o c i ical and help ul discussions. We also hank Da id
Kennedy o excellen echnical expe ise and D . Co inna Be ge o
he an i-REST an ibody. This wo k was suppo ed by Na ional Ins i-
u es o Heal h G an NS22518 o G.M. and by ellowships om he
Na ional Mul iple Scle osis Socie y, he Human F on ie Science
P og am, and he Spanish Go e nmen o J.J.T.-A., B.J.L.E., and
M.J.P.-R., espec i ely.
1. Mandel, G. (1992) J. Memb. Biol. 125, 193–205.
2. Noda, M., Ikeda, T., Suzuki, H., Takeshima, H., Takahashi, T.,
Kuno, M. & Numa, S. (1986) Na u e (London) 322, 826–828.
3. Auld, V. J., Goldin, A. L., K a e, D. S., Ma shall, J., Dunn, J. M.
Ca e all., W. A., Les e , H. A., Da idson, N. & Dunn, R. J.
(1988) Neu on 1, 449–461.
4. Beckh, S., Noda, M., Lubbe , H. & Numa, S. (1989) EMBO J. 8,
3611–3616.
5. Wes enb oek, R. E., Me ick, D. K. & Ca e all, W. A. (1989)
Neu on 3, 695–704.
6. Maue, R. A., K ane , S. D., Goodman, R. H. & Mandel, G.
(1990) Neu on 4, 223–231.
7. K ane , S. D., Chong, J. A., Tsay, H.-H. & Mandel, G. (1992)
Neu on 9, 37–44.
8. Schoenhe , C. J., Paque e, A. J. & Ande son, D. J. (1996) P oc.
Na l. Acad. Sci. USA 93, 9881–9886.
9. Mo i,N.,S ein,R.,Sigmund,O.&Ande son,D. J.(1990)Neu on
4, 588–594.
10. Mo i, N., Schoenhe , C., Vandenbe gh, D. J. & Ande son, D. J.
(1992) Neu on 9, 45–54.
11. Thiel, G., G eenga d, P. & Su¨dho , T. C. (1991) P oc. Na l. Acad.
Sci. USA 88, 3431–3435.
12. Bessis, A., Salmon, A.-M., Zoli, M., LeNo e e, N., Piccio o, M.
& Changeux, J.-P. (1995) J. Neu osci. 69, 807–819.
13. Mieda, M., Haga, T. & Sa en, D. W. (1996) J. Biol. Chem. 271,
5177–5182.
14. Wood, I. C., Roop a, A. & Buckley, N. J. (1996) J. Biol. Chem.
271, 14221–14225.
15. Kallunki, P., Jenkinson, S., Edelman, G. M. & Jones, F. S. (1995)
J. Biol. Chem. 270, 21291–21298.
16. Chong, J. A., Tapia-Ramı´ ez, J., Kim, S., Toledo-A al, J. J.,
Zheng, Y., Bou os, M. C., Al shulle , Y. M., F ohman, M. A.,
K ane , S. D. & Mandel, G. (1995) Cell 80, 949–957.
17. Schoenhe , C. J. & Ande sen, D. J. (1995) Science 267, 1360–
1363.
18. Shi, Y., Se o, E., Chang, L.-S. & Shenk, T. (1991) Cell 67,
377–388.
19. Toledo-A al, J., B ehm, P., Halegoua, S. & Mandel, G. (1995)
Neu on 14, 607–611.
20. Wigle , M., Pellice , A., Sil e s ein, S., Axel, R., U laub, G. &
Chasin, L. (1979) P oc. Na l. Acad. Sci. USA 76, 1373–1376.
21. Johnson, A. D. (1995) Cell 81, 655–658.
22. Lich , J. D., G ossel, J., Figge, J. & Hansen, U. M. (1990) Na u e
(London) 346, 76–79.
23. Jaynes, J. B. & O’Fa ell, P. H. (1991) EMBO J. 10, 1427–1433.
24. Han, K. & Manley, J. L. (1993) EMBO J. 12, 2723–2733.
25. Wi zgall, R., O’Lea y, E., Lea , A., O
¨naldi, D. & Bon en e, J. V.
(1994) P oc. Na l. Acad. Sci. USA 91, 4514–4518.
26. Ma golin, J. F., F iedman, J. R., Meye , W. K.-H., Vissing, H.,
Thiesen, H.-J. & Rausche , F. J., III (1994) P oc. Na l. Acad. Sci.
USA 91, 4509–4513.
27. Scholl, T., S e ens, M. B., Manhan a, S. & S ominge , J. L.
(1996) J. Immunol. 156, 1448–1457.
28. Klug, A. & Schwabe, W. R. (1995) FASEB J. 9, 597–658.
29. Ha, I., Robe s, S., Maldonado, E., Sun, X., Kim, L.-U., G een,
M. & Reinbe g, D. (1993) Genes De . 7, 1021–1032.
30. Lee, J.-S., Gal in, K. M. & Shi, Y. (1993) P oc. Na l. Acad. Sci.
USA 90, 6145–6149.
31. Se o, E., Lewis, B. & Shenk, T. (1993) Na u e (London) 365,
462–464.
32. Geisbe g, J. V., Lee, W. S., Be k, A. J. & Riccia di, R. P. (1994)
P oc. Na l. Acad. Sci. USA 91, 2488–2492.
33. Zhou, Q., Ged ich, R. W. & Engel, D. A. (1995) J. Vi ol. 69,
4323–4330.
34. Me ika, M. & O kin, S. H. (1995) Mol. Cell. Biol. 15, 2437–2447.
35. Bushmeye , S., Pa k, K. & A chison, M. L. (1995) J. Biol. Chem.
270, 30213–30220.
36. Choo, Y. & Klug, A. (1993) Nucleic Acids Res. 21, 3334–3346.
37. Chen, J. D. & E ans, R. M. (1995) Na u e (London) 377, 454–
457.
38. Ho¨ lein, A. J., Na¨a¨ , A. M., Heinzel, T., To chia, J., Gloss, B.,
Ku okawa, R., Ryan, A., Kamei, Y., So¨de s o¨m, M., Glass, C. K.
& Rosen eld, M. G. (1995) Na u e (London) 377, 397–403.
39. Aye , D. E., Law ence, Q. A. & Eisenman, R. N. (1995) Cell 80,
767–776.
40. Kelehe , C. A., Redd, M. J., Schul z, J., Ca lson, M. & Johnson,
A. D. (1992) Cell 68, 709–719.
41. Russo, M. W., Se e son, B. R. & Milb and , J. (1995) P oc. Na l.
Acad. Sci. USA 92, 6873–6877.
42. Pa oush, Z., Finley, R. L., J ., Kidd, T., Wainw igh , S. M.,
Ingham, P. W., B en , R. & Ish-Ho owicz, D. (1994) Cell 79,
805–815.
43. Fishe , A. L., Ohsako, S. & Caudy, M. (1996) Mol. Cell. Biol. 16,
2670–2677.
1182 Biochemis y: Tapia-Ramı´ ez e al. P oc. Na l. Acad. Sci. USA 94 (1997)