Life Science 74(2004) 2213-2225 Minireview REST-NRSF

MinireviewRegulation of the cholinergic gene locus by the repressorelement-1silencing transcription factor/neuron restrictivesilencer factor (REST/NRSF)Masahito Shimojo,Louis B.Hersh *Department of Molecular and Cellular Biochemistry,University of Kentucky,Chandler Medical Center,800Rose Street,Lexington,KY 40536-0298,USAReceived 17July 2003;accepted 21August 2003AbstractThe cholinergic gene locus is comprised of two genes,the choline acetyltransferase gene and the vesicular acetylcholine transporter gene.The vesicular acetylcholine transporter gene is located within the first intron of the choline acetyltransferase gene.This arrangement permits coordinate regulation of the locus.Protein kinase A regulates expression of the cholinergic gene locus in PC12cells.This regulation was found to be dependent on the presence of a 21-bp DNA sequence known as the repressor element-1(RE-1)/neuron-restrictive silencer element (NRSE).Repressor element-1silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF),which binds to the RE-1/NRSE,is a zinc finger containing transcriptional repressor that blocks the expression of many neuronal RE-1/NRSE containing genes in nonneuronal cells.However,REST/NRSF expression has also been observed in neurons as well as the PC12cell line used in these studies.REST/NRSF truncated isoforms were expressed in neuronal cells,suggesting they also function in regulating neuronal gene expression.A study of REST4,one of the REST/NRSF isoforms,suggests that it regulates transcription of the cholinergic gene locus by blocking the repressor activity of REST/NRSF.Protein kinase A regulation of the cholinergic gene locus in PC12cells can thus be attributed,at least in part,to increased synthesis of REST4,which in turn derepresses the repressor activity of REST/NRSF.D 2004Elsevier Inc.All rights reserved.Keywords:Gene transcription;Gene regulation;Cholinergic gene;Transcriptional repression0024-3205/$-see front matter D 2004Elsevier Inc.All rights reserved.doi:10.1016/j.lfs.2003.08.045*Corresponding author.Tel.:+1-859-323-5549;fax:+1-859-323-1727.E-mail address:lhersh@ (L.B.Hersh)./locate/lifescieLife Sciences 74(2004)2213–2225IntroductionThe ability of a cell to regulate its phenotype is dependent upon its ability to carefully and precisely control which genes are expressed and which are not.In the case of neurons there are many genes whose expression is necessary to produce a neuronal phenotype,but whose expression outside the central nervous system is not needed and likely to be detrimental.Recent evidence suggests that one mechanism utilized to prevent expression of neuronal genes outside the central nervous system is that of gene repression.This review focuses on the regulation of one neuron specific gene locus,the cholinergic gene locus,by transcriptional repression.In addition to describing the regulation of the cholinergic gene locus,the complexity of this system,its application to other neuronal genes,and the many questions yet to be answered are noted.Cholinergic neurotransmission in the central nervous system is a key process dependent on the coexpression of proteins involved in the synthesis,storage,and release of the neurotransmitter acetylcholine (ACh).ACh plays an important role in fundamental brain processes such as memory,learning,and sleep (Karczmar,1976;Bartus et al.,1982;Aigner and Mishkin,1986).ACh is used in a cyclic process involving the uptake of choline by a high-affinity uptake system (Jope,1979),the formation of ACh through acetylation of choline catalyzed by the enzyme choline acetyltransferase (ChAT)(EC 2.3.1.6)and the transport of ACh into synaptic vesicles by the vesicular ACh transporter (V AChT).Upon stimulation,synaptic vesicles fuse with the plasma membrane and release ACh,which can then be hydrolyzed by acetylcholinesterase to produce free choline,thereby completing the cycle.The cholinergic gene locus is comprised of both the choline acetyltransferase gene as well as the vesicular acetylcholine transporter gene,the latter being located within the first intron of the ChAT gene.ChAT is primarily if not exclusively a cytosolic protein whereas V AChT has 12transmembrane domains and is integrated in the membrane of synaptic vesicles.In the CNS,ChAT and V AChT are both required for cholinergic neurotransmission.In some neurodegenerative disorders,i.e.Alzheimer’s disease,amyotrophic lateral sclerosis,and schizophrenia,there is a dysfunction of central cholinergic neurons involving a loss of,or abnormality in,basal forebrain ChAT activity (Davies and Maloney,1976;Coyle et al.,1983;Price,1986;Wu and Hersh,1994).Genomic structure and transcription of the cholinergic gene locusThe genes for mouse (Misawa et al.,1992;Pu et al.,1993),rat (Brice et al.,1989;Ishii et al.,1990),and human ChAT (Kong et al.,1989;Hersh et al.,1993)have been isolated and shown to contain three 5V -noncoding exons;exon 1,referred to as the R exon;exon 2,referred to as the N exon;and exon 3,referred to as the M exon (Misawa et al.,1992;Kengaku et al.,1993),Fig.1.The alternative splicing of these three exons results in multiple 5V -mRNA species,which are transcribed from three different promoters upstream of the R,N,and M exons,Fig.1.In the mammalian gene,the M exon resides f 5kb downstream from the R exon and transcription from its promoter appears to represent the major promoter used for generating ChAT mRNA.The V AChT gene in mammalian species (Bejanin et al.,1994;Erickson et al.,1994)as well as C.elegans (Alfonso et al.,1993)was found to lie uniquely within the first intron of the cholinergic gene locus between the first (R)and second (N)non-coding exons.This gene arrangement is conserved across such diverse species such as the nematode,Drosophila ,and mammals.M.Shimojo,L.B.Hersh /Life Sciences 74(2004)2213–22252214There are also multiple mRNA species found for V AChT that differ in their5V-untranslated sequences (Bejanin et al.,1994;Cervini et al.,1995).These mRNA isoforms appear to be generated from either a transcriptional start site upstream of the R exon,which may also be used for ChAT,or from sites within the V AChT exon(Bejanin et al.,1992;Misawa et al.,1992;Kengaku et al.,1993;Alfonso et al.,1994; Cervini et al.,1995),Fig.1.The human ChAT gene(and thus the cholinergic gene locus)has been mapped to chromosome10,region10q11.2–10q22.2(Cervini et al.,1991).Coordinate regulation of ChAT and V AChT gene expressionThe organization of the cholinergic gene locus suggested the possibility of coordinate regulation of the ChAT and V AChT genes at the transcriptional level(Berrard et al.,1995;Berse and Blusztajn,1995;Erickson et al.,1994;Shimojo et al.,1998).Prior to the discovery of the gene structure within the cholinergic gene locus,several studies reported that ChAT activity was regulated by growth factors (Hefti et al.,1986;Gould and Butcher,1989;Saadat et al.,1989;Knusel et al.,1991)and cytokines (Kamegai et al.,1990).Using a PC12mutant cell line,in which protein kinase A (PKA)activity is dramatically reduced by the expression of a defective PKA regulatory subunit,Inoue et al.(1995)reported that PKA could regulate expression of the ChAT gene at the transcriptional level.Transient transfection analysis using a human genomic reporter gene revealed that PKA acts through a site on the human ChAT gene upstream of the R exon.Lonnerberg et al.(1996)reported that this region of the rat ChAT gene contains both enhancer and silencer elements.Misawa et al.(1993)reported that dbcAMP increased ChAT mRNA and activity as well as transcription from the R,N,and M exons,suggesting that the cholinergic gene locus might be regulated by a PKA dependent pathway.Although a number of studies had reported regulation of the ChAT gene (see above),several reports emerged suggesting that the expression of ChAT and V AChT might be regulated coordinately in the CNS (Erickson et al.,1994;Berrard et al.,1995;Berse and Blusztajn,1995;Misawa et al.,1995).Berse and Blusztajn (1995)studied the expression of the ChAT and V AChT genes in a murine septal cell line,SN56,and found that ciliary neurotrophic factor (CNF),retinoic acid,leukemia inhibitory factor (LIF),and dbcAMP increased the mRNA levels for both ChAT and V AChT,although the effects appeared quantitatively different for the two genes.Studies on the regulation of ChAT and V AChT gene transcription in PC12cells using selective inhibitors and two protein kinase A (PKA)mutant PC12cell lines suggested that PKA signaling pathways involving PKAII coordinately regulate both the ChAT and V AChT genes at the transcriptional level (Shimojo et al.,1998).Transcriptional regulation of cholinergic gene locus by REST/NRSFStudies from a number of laboratories suggest that the transcriptional regulation of the ChAT and V AChT genes involves multiple cell specific regulatory elements.The region of the cholinergic gene locus upstream of the R-type promoter contains a consensus repressor element-1(RE-1)/neuron-restrictive silencer element (NRSE)sequence and a cholinergic specific enhancer that presumably operate in a cooperative manner to control cholinergic gene expression in the CNS,Fig.1.The RE-1/NRSE is a 21-basepair sequence originally isolated from the promoters of the type II sodium channel (Maue et al.,1990;Mori et al.,1992;Kraner et al.,1992)and SCG10(Mori et al.,1990,1992),and is implicated in silencing the cholinergic gene locus in non-neuronal cells (Lonnerberg et al.,1996).This sequence is found in a large number of neuron-specific genes including the genes for the N-methyl-D-aspartate receptor (NMDAR)(Bai et al.,1998),synapsin I (Schoch et al.,1996),the m4muscarinic acetylcholine receptor (Wood et al.,1996)and BDNF (Timmusk et al.,1999)as well as choline acetyltransferase (Lonnerberg et al.,1996).The transcription factor that binds to the RE-1/NRSE sequence was cloned independently by two groups;one naming it the RE-1silencing transcription factor or REST (Chong et al.,1995),the other naming it the neuron-restrictive silencing factor or NRSF (Schoenherr and Anderson,1995).The RE-1/NRSE sequence,through its binding of REST/NRSF,leads to the repression of the expression of neuron-specific genes in nonneuronal cells (Chong et al.,1995;Schoenherr and Anderson,1995).REST/NRSF is a member of the Gli-Kruppel family of transcription zinc-finger proteins being a 210-kDa glycoprotein containing nine zinc finger domains (Fig.2).M.Shimojo,L.B.Hersh /Life Sciences 74(2004)2213–22252216It was shown that REST/NRSF acts as a silencer of neuron-specific gene expression in nonneuronal tissues and in undifferentiated neuronal progenitor cells(Chong et al.,1995;Schoenherr and Anderson, 1995).The absence of the RE-1/NRSE sequence(Kallunki et al.,1997)or expression of a dominant negative form of REST/NRSF can produce expression of neuronal genes in both nonneuronal tissues and central nervous system progenitors(Chen et al.,1998).A mouse model in which the REST/NRSF gene was deleted,although embryonic lethal,showed aberrant expression of SCG10and tubulin h III in nonneuronal tissues(Chen et al.,1998).Although it was thought that expression of REST/NRSF was limited to non-neuronal cells where neuron-specific genes are repressed,recent evidence suggests that REST/NRSF may control the expression of neuron-specific genes containing an RE-1/NRSE element in neurons(Kallunki et al.,1997;Palm et al.,1998;Timmusk et al.,1999).To date five REST/NRSF isoforms arising from alternative splicing of the REST/NRSF pre-mRNA have been reported(Palm et al.,1998)and referred to as REST1through REST5.Two of these REST isoforms,REST4and REST5,are expressed specifically in mature neurons of adult brain,albeit at low levels.REST4and REST5contain an insertion of either16nucleotides or28nucleotides respectively,in the region of the gene encoding a spacer between zinc fingers5and6,Fig.2.These insertions in REST4 and REST5,as well as similar insertions in REST2and REST3lead to truncated isoforms that contain only 5of the9zinc finger domains found in REST/NRSF.REST1differs in that it contains only4zinc finger domains,Fig.2.Interestingly,REST4formation is induced by neuronal activity produced by kainate-induced seizure(Palm et al.,1998).REST4exhibits weak binding to the RE-1/NRSE element(Lee et al., 2000),and thus cannot directly regulate neuronal gene expression in RE-1/NRSE containing genes.As noted above the level of transcription of the cholinergic gene locus is greatly reduced in two PC12 cell lines;A123.7(Correll et al.,1989;Ginty et al.,1991)and A126.1B2(Van Buskirk et al.,1985)that have reduced protein kinase A ing reporter gene analysis it was found that the RE-1/NRSEelement was required for repression of the gene in the protein kinase A deficient PC12cells.Introduction of protein kinase A into the PKA deficient cells restored expression from the cholinergic gene locus,that is ChAT and V AChT mRNAs were now detectable.The finding that the concentration of REST/NRSF was essentially the same in both wild type and protein kinase A deficient PC12cells suggests that protein kinase A does not affect the expression levels of REST/NRSF.REST/NRSF in nuclear extracts from the protein kinase A deficient cells bound to the cholinergic RE-1/NRSE element as determined by electrophoretic mobility shift assays.On the other hand nuclear extracts derived from wild type PC12cells exhibited barely detectable binding to the RE-1/NRSE sequence,despite the fact that both nuclear extracts contained the same levels of REST/NRSF immunoreactive protein.The finding of active REST/NRSF in the protein kinase A deficient PC12cells suggests that transcription of the cholinergic locus was repressed in these cells by REST/NRSF,thus accounting for the absence of ChAT and V AChT mRNAs.However,the reason for the absence of repression of the cholinergic locus by REST/NRSF in wild type PC12cells was unclear.The possibility that protein kinase A phosphorylated REST/NRSF and generated an inactivate repressor was considered.However,purified protein kinase A catalytic subunit did not phosphorylate recombinant REST/NRSF.Wild type,but not protein kinase A deficient,PC12cells expressed REST4,the neuron-specific isoform of REST/NRSF described above and in Fig.2.It was shown that REST4forms a heterodimer with REST/NRSF.This heterodimer does not bind to the RE-1/NRSE element from the cholinergic gene locus.Thus even though REST/NRSF is expressed in wild type PC12cells,the presence of REST4inhibits the silencing of the cholinergic gene by blocking the repressor activity of REST/NRSF through heterodimer formation.Treatment of PC12cells with forskolin,which increases intracellular cAMP levels by activating adenylate cyclase,induced REST4expression within 4h and ChAT mRNA expression within 6to 12h.These kinetics are consistent with cAMP activating protein kinase A,which in turn leads to an increase in REST4levels,followed by derepression of the cholinergic locus and increased levels of ChAT mRNA.Thus it appears that protein kinase A regulation of the cholinergic gene locus occurs by PKA increasing the splicing of REST/NRSF pre-mRNA to produce REST4.REST4in turn blocks the repressor activity of REST/NRSF leading to increased gene transcription.Palm et al.(1998)found that a truncated form of REST/NRSF,REST2-5trunc ,also acted as a transcriptional repressor of a REST/NRSF containing reporter gene in Neuro-2A cells,a cell line that does not contain detectable REST/NRSF.On the other hand relatively weak repression of the REST/NRSF containing reporter gene was seen in C6cells,which contain relatively high levels of endogenous REST/NRSF.It was concluded that truncated forms of REST/NRSF produce repression independent of the activity of REST/NRSF and that the repressor activity of the truncated forms resides in an N-terminal repressor domain.It was further suggested that the repressor activity of the REST/NRSF truncated forms could be attributed to the formation of a complex with essential components of the transcriptional machinery.Regulation of REST/NRSF function by its REST4isoformThe finding that REST4can block REST/NRSF repressor activity,coupled with REST4expression in neuronal cells,can account for the presence of REST/NRSF in neurons without neuronal gene expression being repressed.Palm et al.(1998)reported the presence of REST/NRSF in adult neurons,M.Shimojo,L.B.Hersh /Life Sciences 74(2004)2213–22252218albeit at rather low levels relative to its expression in non-neuronal cells.It was suggested that the inability of REST/NRSF to repress gene expression in neuronal cells could be explained by its low concentration.However,as noted above,it appears that the relative levels of REST/NRSF and REST4 determine the extent of transcriptional repression.Although REST/NRSF levels appear to be constant in PC12cells,the levels of REST4can be regulated by cAMP through protein kinase A activity(Shimojo et al.,1999).In the PC12cell line we studied,the cholinergic gene,although transcriptionally active,was not maximally active since there was an excess of REST/NRSF relative to REST4.Thus modulation of transcriptional repression occurs through modulation of REST4,not REST/NRSF.This mechanism, rather than cAMP acting on a cAMP response element within the cholinergic locus,can explain at leasta Fig.3.Schematic showing the possible mechanisms for the de-repression activity of REST4.A.The formation of heterodimers between REST4and REST/NRSF prevents the binding of REST/NRSF to the cholinergic RE-1/NRSE and leads to de-repression of the cholinergic gene locus.Zinc finger domains are represented by numbered black circles.B.One of two repressor domains is located at the N-terminal domain and binds Sin3and histone deacetylase(HDAC).The other,located at the C-terminal domain is the CoREST binding site.All of the REST truncated forms retain the Sin3/HDAC binding site,but not the CoREST binding site.The truncated REST isoforms could form complexes with Sin3/cofactor complexes,resulting in the supression of available Sin3.C.Regulation of RNA splicing.The various truncated forms of REST shown in Fig.2are produced by RNA splicing as illustrated.Exons are shown as boxes and introns are shown as lines.The ORF of each transcript is indicated as a black box.The neuron-specific exon(N)located between exons V and VI has a stop codon thus when inserted produces truncated proteins.The relative expression levels for REST/NRSF and the splice variant REST4is illustrated for neurons and non-neuronal cells.M.Shimojo,L.B.Hersh/Life Sciences74(2004)2213–22252219part of the effects of cAMP on cholinergic gene expression in PC12cells.Whether this mechanism can be extended to the cholinergic gene locus in vivo remains to be determined.Supporting this hypothesis is the report of Tabuchi et al.(2002)who found that REST4affects the activity-dependent activation of the BDNF gene promoter I (BDNFp-I)in cultured rat cortical neurons.As we observed in PC12cells,REST4antagonized silencing of the BDNF gene by REST/NRSF,suggesting a role for REST4in preventing the neuron-specific BDNF gene from being transcriptionally repressed by REST/NRSF.In this case REST4activity varied in response to a variety of neuronal stimuli.Recently Magin et al.(2002)reported that humanized REST4had no discernable effect in NS20Y neuroblastoma cells.REST4did not impair transcriptional repression of the human synapsin I promoter by REST nor did it have a direct effect on the synapsin I promoter.The reason for this discrepancy with our studies and those of Tabuchi et al.(2002)remain unclear,but could be related to the cell type studied.As Tabuchi et al.(2002)suggested,the mechanism of regulation of gene transcription by REST/NRSF isoforms is complex in neurons.There are several mechanisms whereby REST isoforms can function to regulate transcription in the neuron.As shown in Fig.3A ,one mechanism is by heterodimer formation between REST/NRSF and REST4.Since REST4and other REST isoforms retain the N-terminal repressor domain,but do not bind efficiently to DNA,an alternative,or possibly additional mechanism,is that REST isoforms regulate gene expression by competing for binding of the transcriptional repressor complex made up of Sin3and HDAC as illustrated in Fig.3B (Palm et al.,1998;Roopra et al.,2001).The mRNA levels of REST/NRSF and REST isoforms are determined by the extent of alternative splicing of REST/NRSF pre-mRNA (Fig.3C).Our studies suggest that protein kinase A regulates REST/NRSF pre-mRNA splicing.The ability of a cell signaling pathway to regulate neuronal gene transcription through the REST/NRSF -REST isoform system provides a mechanism for a neuron to regulate its plasticity in response to neuronal activity,neuronal stimuli,or neuronal development.Role of zinc finger domains of REST/NRSFREST/NRSF contains nine Cys 2-His 2type zinc finger domains,the first of these is near the N-terminus and is followed by a cluster of seven zinc finger domains,with the last zinc finger domain being near the C-terminus of the molecule,Fig.2.REST4,being a truncated form of REST/NRSF contains only zinc finger domains 1–5,Fig.2.It binds weakly to the RE-1/NRSE with the strength of binding dependent on the number of zinc finger domains present (Lee et al.,2000).These findings suggest that zinc finger domains 2–5are required for DNA binding,while zinc finger domains 6–8contribute to the strength of the binding interaction (Shimojo et al.,2001;Chong et al.,1995).In order to study the effect of zinc finger conformation on REST/NRSF activity,mutational analysis was conducted in which zinc finger domains 6–8were studied.Conversion of a Cys to Arg within a zinc finger domain was shown to disrupt the conformation and function of the Cys 2-His 2type zinc finger domain (Tapia-Ramirez et al.,1997).The results of analyzing zinc finger mutations in REST/NRSF suggest that a combination of three zinc finger domains (zinc finger domains 6–8)contribute to DNA binding,however two adjacent zinc fingers,either zinc finger 6plus7or zinc finger 7plus 8,are required to produce maximal DNA binding.Analysis of the REST4sequence reveals that it lacks the putative nuclear localization signal found in the C-terminal region of REST/NRSF (Chong et al.,1995;Grimes et al.,2000).However,REST4isM.Shimojo,L.B.Hersh /Life Sciences 74(2004)2213–22252220M.Shimojo,L.B.Hersh/Life Sciences74(2004)2213–22252221efficiently targeted to the nucleus.Thus the translocation of REST4and REST/NRSF to the nucleus appears to be regulated by different mechanisms.Either REST/NRSF contains an additional N-terminally located nuclear localization signal,or a different nuclear localization signal in REST4is functional.Deletion mutagenesis indicated that the nuclear targeting signal of REST4resides within the C-terminal region containing zinc finger domains2–5(Lee et al.,2000;Shimojo et al.,2001).Fusion protein analysis showed that zinc finger domains2through5,but not2through4,could target h-galactosidase to the nucleus(Shimojo et al.,2001).Mutation of zinc finger domain5of REST4did not prevent its nuclear localization.However,the REST isoform REST1,which is similar to REST4but lacks zinc finger domain5,was not targeted to the nucleus.These data indicate the importance of the region around zinc finger5and suggest that there is a nuclear targeting signal,other than the zinc finger domain,located within the region preceding and going through zinc finger domain5.An alternative explanation is that deletion of the region around zinc finger5changes the structure of REST4such that a critical conformation needed for nuclear targeting is disrupted.To further study whether the zinc finger domain structure is important for nuclear targeting,REST4 with combinations of Cys to Arg point mutations in the zinc finger domains was fused to GFP.None of the combinations of point mutations altered the localization of REST4from nuclear to cytoplasmic.These data suggest that a functional structure of the zinc finger domains is not required to target REST4to the nucleus.However,certain combinations of zinc finger domain mutations produced abnormal nuclear localization.In these cases,REST4-GFP constructs appeared to localize to the edge of the nucleus,as if they were attached to the nuclear envelope(Shimojo et al.,2001).These data are consistent with previous findings that suggest that zinc finger domains not only function in DNA recognition(Wolfe et al.,2000), but can also be involved in protein-protein interactions(Lee et al.,1993;Milne and Segall,1993). REST/NRSF Repressor DomainsREST/NRSF is a modular protein that represses neuronal gene expression via two distinct repressor domains.Repression through an amino terminal domain is mediated by a Sin3-histone deacetylase (HDAC)complex,which affects chromosomal structure by promoting histone deacetylation(Tapia-Ramirez et al.,1997;Grimes et al.,2000;Huang et al.,1999;Roopra et al.,2001;Naruse et al.,1999). The other repressor domain is found in the carboxyl-terminal domain and binds a novel protein, CoREST and HDAC(Ballas et al.,2001;Andres et al.,1999).In situ hybridization studies of CoREST and Sin3A expression as well as REST/NRSF expression suggest that Sin3is expressed more ubiquitously during early embryogenesis(embryonic day8.5),but at latter stages of embryogenesis (day11.5)CoREST and Sin3A are both expressed fairly ubiquitously throughout the embryo.Evidence has recently been obtained that gene silencing by REST through these domains involves both histone deacetylation and ATP-dependent chromatin remodeling(Battaglioli et al.,2002).The future for RESTIt is evident that the regulation of neuronal gene expression by REST/NRSF and its truncated isoforms is complex and far from understood.As noted above REST mediated gene repression involves two distinct repressor domains,one binding Sin3A and the other binding CoREST.Why two distinctrepressor domains are present in REST/NRSF is puzzling.It is unclear whether both domains are utilized simultaneously or represent different modes of gene regulation that are utilized differentially.The need for a complex system whereby REST4modulates REST/NRSF activity (Fig.3)is also not clear,and whether REST4and the other REST isoforms have additional functions yet to be elucidated is an intriguing question.To date a function has only been attributed to REST4,a function for REST1,REST2,REST3,and REST5remain to be described.It has been shown that a signaling molecule,cAMP,acting through protein kinase A and REST/NRSF pre-mRNA splicing,can regulate cholinergic gene expression in PC12cells.Is this a general mechanism in all neurons and does cAMP and other signaling pathways regulate transcription of neuronal genes in response to cellular stimuli?On the surface it would appear that protein kinase A,through it increase in REST4,would lead to derepression of all REST/NRSF responsive genes.Whether this is the case or whether there are additional mechanisms that permit only a subset of REST/NRSF responsive genes to be affected by REST4remains to be established.Clearly there are many unanswered questions and much to be done to understand how the REST/NRSF system regulates the cholinergic gene locus as well as a host of other neuron specific genes.AcknowledgementsThis work was supported in part by grants AG46734and AG05893from the National Institutes of Health.ReferencesAigner,T.G.,Mishkin,M.,1986.The effects of physostigmine and scopolamine on recognition memory in monkeys.Behav-ioral and Neural Biology 45(1),81–87.Alfonso,A.,Grundahl,K.,Duerr,J.S.,Han,H.-P.,Rand,J.B.,1993.The Caenorhabditis elegans unc-17gene:a putative vesicular acetylcholine transporter.Science 261(5121),617–619.Alfonso,A.,Grundahl,K.,McManus,J.R.,Asbury,J.M.,Rand,J.B.,1994.Alternative splicing leads to two cholinergic proteins in Caenorhabditis elegans.Journal of Molecular Biology 241(4),627–630.Andres,M.E.,Burger,C.,Peral-Rubio,M.J.,Battaglioli,E.,Anderson,M.E.,Grimes,J.,Dallman,J.,Ballas,N.,Mandel,G.,1999.CoREST:a functional corepressor required for regulation of neural-specific gene expression.Proceedings of the National Academy of Sciences,USA 96(17),9873–9878.Bai,G.,Norton,D.D.,Prenger,M.S.,Kusiak,J.W.,1998.Single-stranded DNA-binding proteins and neuron-restrictive silencer factor participate in cell-specific transcriptional control of the NMDAR1gene.Journal of Biological Chemistry 273(2),1086–1091.Ballas,N.,Battaglioli,E.,Atouf,F.,Andres,M.E.,Chenoweth,J.,Anderson,M.E.,Burger,C.,Moniwa,M.,Davie,J.R.,Bowers,W.J.,Federoff,H.J.,Rose,D.W.,Rosenfeld,M.G.,Brehm,P.,Mandel,G.,2001.Regulation of neuronal traits by a novel transcriptional complex.Neuron 31(3),353–365.Bartus,R.T.,Dean,R.L.III,Beer,B.,Lippa,A.S.,,1982.The cholinergic hypothesis of geriatric memory dysfunction.Science 217(4558),408–414.Battaglioli,E.,Andres,M.E.,Rose,D.W.,Chenoweth,J.G.,Rosenfeld,M.G.,Anderson,M.E.,Mandel,G.,2002.REST repression of neuronal genes requires components of the hSWI.SNF complex.J.Biol.Chem.277(43),41038–41045.Bejanin,S.,Habert,E.,Berrard,S.,Edwards,J.B.,Loeffler,J.P.,Mallet,J.,1992.Promoter elements of the rat choline acetyltransferase gene allowing nerve growth factor inducibility in transfected primary cultured cells.Journal of Neuro-chemistry 58(4),1580–1583.M.Shimojo,L.B.Hersh /Life Sciences 74(2004)2213–22252222。