A Single Zinc Finger Motif in the Silencing Factor Rest Represses the Neural-Specific Type II Sodium Channel Promoter.

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. 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