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nature neuroscience • volume 2 no 10 • october 1999 867

Defining the mechanisms controlling neuronal gene expression iskey to understanding the phenotypic differences between neu-rons and glia and among different populations of neurons, aswell as how the brain responds to insults or learns. The repres-sor element-1 (RE1, also known as the neuronal restrictivesilencer element or NRSE) and its cognate RE1-silencing tran-scription factor REST (or neuronal-restrictive silencer factor,NRSF) have been identified as a negative regulatory system thatcontrols the expression of many neuron-specific genes1–9. RESTis dominantly expressed in nonneuronal tissues and in neuronalprecursors; in nonneuronal cells, binding of REST to its silencercauses a two- to tenfold reduction of transcription for genes withan RE1 element3–5. However, REST mRNA is expressed in neu-rons in specific brain regions during development10, suggestingthat RE1/REST may also regulate the expression of a subset ofgenes in neurons8. Occupancy of the RE1 silencer by REST thusprovides a means for regulating the expression of several essentialgenes in the central nervous system.

The molecular mechanisms of gene silencing mediated bythe RE1/REST system have been elusive. Evidence for a modelof transcriptional repression involves repressor proteins bind-ing a corepressor (Sin3A or Sin3B), which, in turn, recruitshistone deacetylase to nucleosomes11–13. This mechanism wasoriginally invoked to explain transcriptional repression ofhypermethylated regions of the genome14, but also applies tocertain transcriptional repressors such as retinoblastoma (RB)protein15 and MAD16. The chromatin stabilization resultingfrom histone deacetylation is thought to mask nearby pro-moters from the transcription apparatus17–20. Thus, it is becom-ing clear that the acetylation state of histone tail lysines innucleosomes can regulate transcription of many eukaryoticgenes21–23. Based on genetic evidence, REST can interact withyeast Sin3 (N.J. Buckley and A. Roopra, Soc. Neurosci. Abstr.24, 1566, 1998). Here we demonstrate that REST represses RE1-containing genes in nonneuronal cells by recruiting histone

deacetylase activity to their promoter regions, suggesting thata mechanism involving chromatin remodeling regulates theexpression of a neuronal phenotype.

RESULTSRegulation of RE1-containing genes by acetylationThe GluR2 subunit controls numerous properties of AMPA-type glutamate receptors, including Ca2+ permeability, singlechannel conductance and rectification24. Expression of theGluR2 gene is limited to neurons in the CNS25,26. A functionalRE1-like silencer element exists in the proximal GluR2 pro-moter, which, when deleted, increases promoter activity approx-imately threefold in astrocytes and C6 glioma cells but does notaffect neurons6. Another RE1-containing gene with neuronallyrestricted expression is the type II sodium-channel gene1,2. Todetermine whether transcriptional suppression of GluR2 andtype II Na+-channel genes in nonneuronal cells may involvedeacetylation, we investigated the effect of a specific inhibitor ofhistone deacetylase, trichostatin A (TSA)27, on the expressionof the endogenous GluR2 and Na+ II genes. C6 glioma cells weretreated with 300 nM TSA for 16 h, and RT-PCR was used tocompare the levels of mRNAs encoding GluR2, Na+ II andGAPDH (as control) in treated and untreated cultures. Endoge-nous GluR2 mRNA expression was increased in TSA-treatedC6 cells with no effect on the expression of GAPDH (Fig. 1a,top panel; compare lanes 7 and 8 with lanes 6 and 9). A similarinduction of Na+ II gene expression was produced by 300 nMTSA in C6 glioma cells (Fig. 1a, bottom panel).

These results demonstrate that the expression of two RE1-con-taining genes is enhanced in C6 glioma cells by a histone deacety-lase inhibitor. We have previously shown that the proximal GluR2promoter is approximately 30-fold more active in cultured cor-tical neurons than in astrocytes or C6 glioma cells, when assessedby transient expression of promoter-luciferase constructs6. TSAcaused a 26 ± 9-fold increase in GluR2 promoter activity in C6

Transcriptional repression by REST:recruitment of Sin3A and histonedeacetylase to neuronal genes

Yunfei Huang, Scott J. Myers and Raymond Dingledine

Department of Pharmacology and Biochemistry, Cell and Developmental Biology Graduate Program, Emory University School of Medicine,Atlanta, Georgia 30322, USA

Correspondence should be addressed to R.D. ([email protected])

Many genes whose expression is restricted to neurons in the brain contain a silencer element(RE1/NRSE) that limits transcription in nonneuronal cells by binding the transcription factor REST(also named NRSF or XBR). Although two independent domains of REST are known to confer repres-sion, the mechanisms of transcriptional repression by REST remain obscure. We provide multiplelines of evidence that the N-terminal domain of REST represses transcription of the GluR2 and type IIsodium-channel genes by recruiting the corepressor Sin3A and histone deacetylase (HDAC) to thepromoter region in nonneuronal cells. These results identify a general mechanism for controlling theneuronal expression pattern of a specific set of genes via the RE1 silencer element.

© 1999 Nature America Inc. • http://neurosci.nature.com©

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868 nature neuroscience • volume 2 no 10 • october 1999

glioma cells but had no effect on GluR2 promoter activity in cul-tured cortical neurons (Fig. 1b). Thus TSA essentially eliminat-ed the neuronal selectivity of the GluR2 promoter.

The RE1 silencer is a TSA-sensitive regulatory elementTo identify the TSA-sensitive element(s) in the GluR2 promoter,we tested a series of 14 GluR2-promoter constructs that contain20–25-base internal deletions6. We found that deletion of the RE1-like silencer element from the GluR2 promoter reduced the TSApotentiation by 74 ± 2% in C6 glioma cells without measurableeffect on promoter activity in cultured cortical neurons (Fig. 2a,construct B). Similar-size deletions flanking the silencer were with-out effect. Thus, the majority of the TSA effect on GluR2 pro-moter activity in C6 glioma can be accounted for by the RE1-likesilencer element in the proximal promoter. The GluR2 promot-er core region, containing Sp1 and NRF-1 elements6, also con-tributes to the TSA effect, but not in a cell-specific manner, asdeleting these elements had similar effects in C6 cells or primarycortical neurons (Fig. 2a; constructs G, H and I). Reduced poten-tiation by TSA in the silencer-deletion construct in C6 cells result-ed from derepression of GluR2 promoter activity in the absence ofTSA, rather than reduced promoter activity in TSA. Indeed,reporter activity in the presence of TSA was similar to that of thewild-type promoter (Fig. 2b). This result would be expected ifTSA prevented repression mediated by the GluR2 silencer, andfurther supports the conclusion that the RE1 silencer is a TSA-sensitive element. In contrast, promoter activity of the constructslacking Sp1, NRF1 or the bridge spacer element was reduced by

TSA in neurons (Fig. 2b). Although the mechanism underlyingthis effect is unknown, it is possible that histone deacetylase func-tionally interacts with28 or directly modifies these transcriptionfactors29–31, therefore regulating their transcription activities.

A previous study indicated that TSA treatment changes theexpression of only 2% of 340 human genes examined by differ-ential display32. Indeed, when tested over its effective range of con-centrations for enhancing GluR2-promoter activity in C6 gliomacells, TSA had minimal effects on the SV40 and CMV promoters,which are not neural selective (Fig. 3a). TSA also enhanced GluR2promoter activity in cultured astrocytes (not shown). Sodiumbutyrate (1 mM), another histone-deacetylase inhibitor33, alsoincreased GluR2-promoter activity in C6 glioma cells (not shown).The biphasic concentration–response relationship found for TSA(Fig. 3a) is probably due to pleiotropic effects of this drug at highconcentrations, such as induction of histone-deacetylase expres-sion itself34. TSA also enhanced the basal promoter activity of thetype II Na+-channel gene by 9.4-fold. When the RE1 silencer wasdeleted, sensitivity of the Na+ II promoter to TSA was reduced by55% (Fig. 3b). These data indicate that transcriptional repressionof both GluR2 and type II Na+-channel promoters in C6 gliomacells involves a TSA-sensitive mechanism that seems to be medi-ated through the RE1 silencer.

We next investigated whether TSA could prevent transcrip-tional repression mediated by REST itself. REST is known to haveat least two independently acting repressor domains, one each atthe extreme N and C termini of the protein35,36. These domainsare sufficient, when tethered to a promoter through a Gal4 DNA

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Fig. 2. Deletion scan of the proximal GluR2 promoter identifies two TSA-sensitive regions. (a) A series of GluR2-promoter constructs with internal dele-tions in the context of R2(–302/+320)luc were transfected into C6 gliomas and primary neuronal cultures; nomenclature as published6. Cultures were pre-treated with 100 nM TSA for 6 h before transfection, then harvested 16 h aftertransfection for luciferase activity assays. The fold effect of TSA onGluR2-promoter activity, from the untreated controls, was determined for each construct and normalized to the fold TSA effect on the wild-type GluR2promoter (WT defined as 100%; opensquare). The entire deletion series wastransfected in parallel into five (C6 glioma)and four (primary neurons) culture prepa-rations. The mean ± s.e. were plotted;note log scale on y-axis. *p < 0.05 from theWT control by ANOVA and post-hocDunnett’s test. (b) The raw data of Turnerlight units was averaged from 5–6 indepen-dent experiments and presented as mean ±s.e. Open bars, control; black bars, samplestreated with 100 nM TSA.

Fig. 1. Trichostatin A derepresses the GluR2 geneby a silencer-dependent mechanism. (a) EndogenousGluR2 and type II Na+-channel expression in C6glioma cells. C6 glioma cells were treated with 300nM TSA for 16 h. Total RNA was harvested and usedin RT-PCR reactions to amplify GluR2, GAPDH andthe type II Na+ channel genes. Results from two inde-pendent TSA treatments are shown for GluR2(upper panel), and three independent TSA treat-ments for the Na+ II mRNA amplifications (lowerpanel). (b) TSA derepresses GluR2 promoter activityin C6 glioma cells but not cortical neurons. Cultureswere transfected with a 622 bp GluR2 promoter,treated with 100 nM TSA and harvested 15–18 hourslater. In 6 experiments, TSA produced a 26 ± 9-fold

increase of GluR2 promoter activity from 454 ± 272 Turner light units (TLU) per well in C6 gliomas. In contrast, after TSA treatment, GluR2 pro-moter activity was unchanged in neurons (0.79 ± 0.53-fold of control from a basal expression of 10,500 ± 3,900 TLU per well, n = 5).

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binding domain, to reduce transcription35,36. We asked whethereither of these domains contributes to the RE1-dependent, TSA-sensitive component of repression observed in C6 glioma cells.To minimize effects due to the action of endogenous REST ontransfected promoters, or exogenous REST on other cellular genes,we replaced the RE1 silencer element in the GluR2 promoter withfour contiguous Gal4-targeting sequences. This promoter–reporterconstruct was then coexpressed in C6 glioma cells with plasmidsencoding the yeast Gal4 DNA-binding domain fused to three dif-ferent REST domains, each driven by the CMV promoter. Thisexperiment showed that N-terminal REST (residues 1–152) andC-terminal REST (953–1097), both of which possess functionalrepressor activity36, strongly repressed GluR2-promoter activityin C6 glioma cells, whereas the inactive N-terminal fragment ofREST (62–152) was without measurable effect (Fig. 4a). Notably,luciferase-reporter levels were reduced 3.8-fold by co-expressionwith the active N-terminal REST domain and 2.5-fold with theactive C-terminal domain. TSA relieved promoter repression inall of the cotransfections (Fig. 4b), but importantly, TSA treat-ment was more effective for the N-terminal REST (1–152), com-pared to the C-terminal REST (953–1097) or inactive N-terminal(62–152) REST fragments. These results suggest that the repressoractivity of the N-terminal, but not C-terminal, domain of REST inC6 glioma cells depends largely on the ability of this domain torecruit a TSA-sensitive complex.

Association of REST with Sin3A and HDAC1Next, we asked whether REST associates with histone deacety-lase 1 (HDAC1) and its corepressor, Sin3A, in intact cells. Myc-REST was co-expressed with flag-tagged HDAC1 in C6 glioma

cells. After one day, cells were homogenized, and aliquots wereimmunoprecipitated with an anti-myc antibody and western-blotted to detect HDAC1 or endogenous Sin3A. This experimentdemonstrated that HDAC, Sin3A and REST all resided in thesame complex (Fig. 5a). Further, endogenous HDAC1 could beco-immunoprecipitated by an anti-myc antibody when C6glioma cells were transfected with myc-tagged full-length REST(Fig. 5b). To determine whether endogenous REST interactedwith Sin3A and HDAC1 in vivo, C6 glioma cells were lysed andimmunoprecipitated with mouse monoclonal antibody againstendogenous REST. Immunoblots with anti-Sin3A and anti-HDAC1 showed the presence of endogenous Sin3A and HDAC1in the immunoprecipitate (Fig. 5c). These observations indicatethat both the corepressor, Sin3A, and histone deacetylase 1 forma complex with REST in C6 glioma cells.

The TSA-sensitive repression mediated by the N-terminaldomain of REST (Fig. 4b) suggests that this domain may interactwith Sin3A. Accordingly, myc-tagged REST fragments (residues1–152, 62–152 and 953–1097) were transfected into C6 gliomacells, which were harvested and immunoprecipitated with ananti-myc antibody. Western blots demonstrated that the tran-scriptionally active N-terminal REST fragment (1–152) pulleddown Sin3A, whereas the TSA-insensitive C-terminal repressorfragment (953–1097) did not (Fig. 5d). This finding supportsthe observation that TSA specifically relieves transcriptionalrepression mediated by the N-terminal but not the C-terminalREST domain. When normalized to the levels of transfected pro-teins precipitated from the cell lysate, the truncated 62–152 frag-ment of the N-terminal domain bound only 11% as much Sin3Aas the 1–152 fragment (Fig. 5d). This weak interaction may have

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Fig. 3. Selectivity of trichostatin A effect. (a) Concentration–responserelationships for TSA in C6 gliomas transfected with luciferase con-structs (0.75 µg per well) driven by the three promoters as indicated.Cells were pretreated with TSA for 6 h, then transfected and harvested12 h later. Values shown are triplicates from the following levels ofexpression in the absence of TSA: 129 ± 2.6 TLU per well for GluR2,10,400 ± 55 TLU per well for SV40 and 38,800 ± 420 TLU per well forCMV. (b) C6 glioma cells were transfected with a luciferase reporterdriven by the type II sodium channel promoter, containing or lacking theRE1 silencer as indicated, and the effect of 100 nM TSA treatment wasmeasured. Values are from triplicate wells representative of threeexperiments.

Fig. 4. The N-terminal repressor domain of REST is TSA-sensitive. C6glioma cells were transfected with the GluR2-luciferase construct modi-fied to replace the RE1 silencer element with four contiguous Gal4 DNAbinding sites. Cultures were cotransfected with one of three plasmidsencoding a Gal4-REST fusion protein (either residues 1–152, 62–152 or953–1097) or with pcDNA3 as control. Cells were pretreated with 100nM TSA for 6 h and harvested 15 h later for measurement of luciferaseactivity. (a) Values shown are mean ± s.e. of five experiments, each trans-fection done in triplicate wells. Luciferase activity resulting from cotrans-fection of the Gal4/GluR2 reporter gene and the empty pcDNA3 plasmidvector in the absence of TSA was set to 100% (58.8 ± 10.9 TLU per well).In the absence of TSA, the N-terminal (1–152) and C-terminal(953–1097) REST constructs, but not the truncated N-terminal domain(62–152), repressed GluR2 promoter activity. *p < 0.05 from thepcDNA3 cotransfection control by ANOVA. (b) Effect of 100 nM TSAon C6 cotransfections, shown as fold increase in promoter activity fromthe respective untreated control. *p < 0.05 from all other treatmentgroups by ANOVA and post-hoc Bonferroni test.

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been insufficient to be detected in the functional assay of Fig. 4b;alternatively, the extreme N terminus of REST (residues 1–61)may be required for repressor complex activity.

Acetylation of histones bound to GLUR2 promoterIn many genes, transcriptional activity is enhanced by acetylationof lysines in the tails of histone proteins that are physically associ-ated with their promoter regions21, a process that is regulated byhistone deacetylase. We therefore asked whether induction ofGluR2 expression by TSA was accompanied by a change in acety-lation of histones near the GluR2 promoter. A chromatin immuno-precipitation (CHIP) assay was used to measure the levels ofacetylated H3 and H4 histones physically associated with theendogenous GluR2 promoter. After chromosomal DNA wassheared to 0.2–3 kb, antibodies directed against acetylated H3 andH4 histone proteins were used to immunoprecipitate chromatin.PCR with GluR2-specific primers was then used to monitor theamount of GluR2 gene in the immunoprecipitates. The CHIP assayshowed that treatment of C6 glioma cultures with 300 nM TSAdramatically increased the acetylated form of histone H4 associ-ated with the endogenous GluR2 promoter region (Fig. 6a) andmoderately increased the acetylated form of histone H3. In con-trast, for cortical neurons in primary culture, the GluR2 promot-er was associated with a large amount of acetylated H3 and H4even under basal conditions, and TSA treatment had minimaleffects (Fig. 6b). These data, taken together with those of Fig. 1,further imply that the endogenous GluR2 promoter is sensitive tochanges in the acetylation state of histones in C6 glioma, but not inneurons, and supports the conclusion that a RE1/REST interac-tion represses promoter activity by recruiting histone deacetylase tothe GluR2 promoter.

DISCUSSIONThe RE1 element and its cognate transcription factor, REST, rep-resent a negative regulatory system that restricts neuronal geneexpression in nonneuronal tissues, neuronal precursor cells andcertain differentiated neurons3,4,7,8. Our data suggest that RE1and the N-terminal repressor domain of REST cooperate torecruit a Sin3A/HDAC1 repressor complex into the immediatevicinity of the promoter. Repression by the C-terminal domainmay occur by a different mechanism37,38. A novel corepressor,CoREST, interacts with the C-terminal domain38. As transcrip-tional repression mediated by the C-terminal domain was notrelieved by TSA (Fig. 4b), CoREST may repress transcription by

a mechanism independent of histone deacetylase activity, or byrecruiting different deacetylases containing repression complex-es that are less sensitive to TSA39,40. Six mammalian histonedeacetylases have been reported that differ in their histone sub-strate specificity and form different corepressor complexes41–43.Because RE1 silencer elements reside in many genes that have aneuron-selective expression pattern, the identification of a mech-anism of promoter repression by REST is an important steptoward understanding how neuronal and nonneuronal pheno-type is controlled through regulation of RE1-containing genes.

Although recruitment by RE1/REST of a Sin3A/HDAC-con-taining complex can lead to the repression of neural-specific genesin nonneuronal tissues, a number of issues remain to fully under-stand the basis for neuron-selective expression of the GluR2 gene.First, our results do not indicate whether the relevant deacetylasetargets associated with the GluR2 and type II Na+-channel genesare histone proteins themselves or instead are transcriptional acti-vators. For example, an erythroid-specific member of the Sp1 fam-

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Fig. 5. Co-immunoprecipitation of REST, Sin3A and HDAC1. (a) C6glioma cells were transfected with flag-tagged HDAC1 with or withoutmyc-REST, as indicated. One day later, cells were harvested and immuno-precipitated with anti-myc antibody. The immunoprecipitates were sepa-rated on an SDS acrylamide gel, blotted to nitrocellulose and probed withflag antibody (1:1000). Then, after stripping, blots were reprobed with anantibody to endogenous Sin3A (1:800). (b) C6 glioma cells were trans-fected with either myc-tagged REST or pcDNA3 vector, harvested 36 hlater, and immunoprecipitated with anti-myc as indicated. The immunopre-cipitates were separated by polyacrylamide gel electrophoresis (PAGE) andprobed with anti-HDAC1 antibody (1:100). (c) C6 glioma-cell lysate wasimmunoprecipitated with monoclonal anti-REST (1:20 dilution). Theimmunoprecipitates were separated on an SDS gel and blotted with anti-Sin3A (1:1000) and anti-HDAC1 (1:100). (d) C6 glioma cells were trans-fected with either myc-tagged REST fragment (1–152, 62–152 and953–1097) constructs or pcDNA3 vector. Lysates were immunoprecipi-tated with anti-myc as indicated. The immunoprecipitates were separatedby PAGE and probed with anti-Sin3A antibody (1:1000).

Fig. 6. TSA increased the association of acetylated histone H3 and H4with GluR2 promoter in C6 glioma but not cortical neurons. C6 gliomacells (a) or primary cortical neurons (b) were treated with 300 nM ofTSA for 17 h. Chromatin immunoprecipitation samples were amplifiedby PCR using primers from the core region of the GluR2 promoter. Atitration of genomic DNA isolated from C6 glioma cells with 32 cyclesof PCR indicated that the intensities of bands are proportional to theamount of input DNA. Each individual input represents 0.0075% of totalgenomic DNA samples before chromatin immunprecipitation. Thisexperiment was repeated three times with similar results.

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ily of transcriptional activators is known to be acetylated29 and it ispossible that transcription factors could be differentially regulatedby acetylation in neurons versus C6 glioma cells29–31. Second, thefinding that TSA could stimulate the GluR2 and type II Na+-chan-nel promoters in the absence of the RE1 silencer or Gal4 targetingsequence (Figs. 2a and 3b) makes it likely that additional deacety-lase-related transcriptional mechanisms operate on these pro-moters. A core region of the GluR2 promoter and its transcriptionfactors may contribute to TSA-mediated transcriptional derepres-sion (Fig. 2a). This possibility is supported by the finding that Sp1can negatively regulate gene expression by recruiting HDAC1through its C-terminal domain28. Alternatively, the differentialDNA methylation of the CpG island that occurs in the GluR2 pro-moter6 may be an additional determinant of neuronal selectivityby recruiting histone deacetylase6,14,45,46 . Third, as described above,the two repressor domains of REST recruit different corepressorcomplexes, Sin3A and CoREST. It is plausible that in different celltypes, REST may recruit other complexes to modify promoteractivity8, for example, the NuRD protein complex42.

Finally, it is interesting that certain neuronally expressed splicevariants of REST, including those that lack the C-terminal repres-sor domain, are upregulated following kainate-induced seizures10.It will be important to determine whether REST-mediateddeacetylation of histones or transcription factors is involved inthe downregulation of GluR2 following seizures47–50.

METHODSCulture and transfections. The culture of C6 glioma cells and rat corticalneurons, the preparation of GluR2–luciferase constructs ([–302/+320]luc),type II Na+-channel constructs and the luciferase assay were as described6.The transfection protocol was similar to a described protocol6, except thatLipofectamine Plus (Gibco) was used and a total of 0.75 µg DNA was usedin transfection. For cotransfections, 0.7 µg of reporter constructs and 0.05µg of Gal4/REST constructs were as described36. Trichostatin A was dis-solved in ethanol and stored at –20ºC. When needed, cultures were pre-treated for 3–6 h with TSA before transfection, and TSA was maintained inthe culture medium until harvest. Similar results were obtained when TSApretreatment was omitted. TSA does not increase transfection efficiencyin C6 glioma cells (data not shown). The Gal4x4 target sequence was gen-erated by annealing and Klenow filling a pair of oligonucleotides(5 ′ -GGTATGCATAGGCCTCGGAGGACAGTCCTCCGCGGAG-GACAGTCCTCCGCCTAGGCCTCGGAG-3′, 5′-CCAATGCATCGGCG-GACTGTCCTCCGCGGAGGACTGTCCTCCGAGGCCTAGGCGGAG-3′.The resulting Gal4x4 fragment was restricted by NsiI and inserted into aprepared GluR2 promoter.

RT-PCR. C6 glioma cells were treated for 16 h with 300 nM TSA and totalRNA was isolated using TRIzol Reagent (Gibco). For RT-PCR, 1 or 2 µgtotal RNA was reverse transcribed with Superscript II (Stratagene) primedwith oligo-dT. A 1% aliquot of the RT reaction was taken for GluR2 andGAPDH PCR reactions (96°C for 30 s, 50–55°C for 30 s, 72°C for 60 s;30–35 cycles) using specific primers to produce ∼ 500 and ∼ 250-bp prod-ucts, respectively. A 10% aliquot of the RT reaction was used to amplifythe type II Na+ channel gene. Serial dilutions of plasmids encoding GluR2and GAPDH were also amplified to ensure that PCR reactions had not‘plateaued’. PCR reactions were conducted with multiple RNA prepara-tions from independent TSA treatments of C6 glioma cells. The experi-ments shown in Fig. 1a were repeated at least four times with similar results.

Immunoprecipitations. For the experiment shown in Fig. 5a, C6 gliomacells were transfected with myc-tagged REST (4 µg per 100-mm dish)and/or flag-tagged HDAC1 (4 µg per 100-mm dish) as indicated, eachunder control of a CMV promoter, and cells were harvested 20–24 h later.Control dishes were transfected with pcDNA3. Cells were homogenizedin buffer containing Boehringer’s protease-inhibitor cocktail plus PMSF(Sigma). An aliquot equivalent to 1% of a dish of a 10,000 × g supernatespin was applied directly to the gel to assess the expression level of

HDAC1 and Sin3A ('cell lysate' in Fig. 5a and d). Monoclonal anti-mycantibody (1:20) was used for immunoprecipitation at 4°C overnight.Aliquots of the immunoprecipitate equivalent to 25% of a dish were sep-arated on a 10% SDS-acrylamide gel. Bands were electroblotted to anitrocellulose membrane, rinsed in PBS and blocked with 5% milk inPBS + 0.05% Tween 20 for 1 h at room temperature; then a mouse mon-oclonal anti-flag antibody (1:1000, Sigma) was added for an overnightincubation at 4ºC. The membrane was then stripped and reblotted withanti-Sin3A antibody (1:800, Santa Cruz). For the experiment shown inFig. 5b, C6 glioma cells were transfected with myc-tagged REST orpcDNA3 as control (1.5 µg per well for 6-well plate). After 36 hours incu-bation, cells were rinsed in ice-cold PBS twice, lysed in 150 mM NaCl,50 mM TrisCl pH 7.5 and 0.2% NP40 plus proteinase inhibitors at pH7.5 on ice and centrifuged at 10,000 × g for 10 min. The supernate wascleaned with protein G beads, then REST was immunoprecipitated witha monoclonal anti-myc antibody (1:20). An aliquot was separated on a4–12% MOPS gel and immunoblotted with a polyclonal antibody againstHDAC1 (1:100). For the experiment in Fig. 5c, C6 glioma cells from two10-cm dishes were homogenized in 3 ml ice-cold buffer (150 mM NaCland 10 mM Tris at pH 8, with proteinase inhibitors). Then, NP40 wasadded to each lysate to a final concentration of 0.1%, and the lysate wasincubated at 4°C for 10 min with shaking. The lysate was immunopre-cipitated with a monoclonal antibody (1:20) against the N-terminaldomain of REST7 at 4°C overnight, and resulting pellets were separatedon a 7.5% SDS-acrylamide gel. The membrane was then blotted withanti-Sin3A antibody (1:1000, Santa Cruz) and anti-HDAC1(1:100 SantaCruz). For the experiment shown in Fig. 5d, C6 glioma cells in 60-mmdishes were transfected with 2 µg of plasmids encoding myc-tagged RESTfragments (1–152, 62–152, 953–1097), each under control of a CMVpromoter. Control dishes were transfected with pcDNA3 as indicated.Cells were harvested 44 h later. After the lysate was precleared with pro-tein G beads at 4°C for 1 h, anti-myc antibody (1:20) was added forovernight incubation at 4°C. Immunoprecipitated samples were sepa-rated on a 8–16% tris/glycine gradient gel and immunoblotted with anti-Sin3A as above.

CHIP assays. C6 glioma cells and primary cortical neurons were treatedwith 300 nM TSA for 17 h. The chromatin immunoprecipitation assaywas performed following the protocol provided in anti-acetyl H3 andanti-acetyl H4 CHIP assay kits (Upstate Biotech, Rochester). The celllysates were sonicated by a 60 Sonic-Dismembrator (Fisher Scientific)at 75% of maximium power for 10 s on ice, repeated 4 times. The sizes ofsonicated genomic DNA were 0.2–3 kb. After chromatin immunopre-cipitation, DNA samples were purified by gel exclusion (P6, BioRAD)equilibrated with Tris-HCl (pH 8.0). 5% of the DNA collected from each100-mm culture dish was used as PCR template. PCR primers amplifieda GluR2 genomic DNA fragment in the core region of GluR2 promoterfrom –43 to +183 relative to the transcription start site6. Genomic DNA(0, 5, 10 or 25 ng) was also amplified by 32 cycles to verify that the PCRreaction was not saturated. PCR products were electrophoresed on 1.5%agarose gels, ethidium-bromide stained and imaged with a StratageneEagleEyeTM imaging system.

ACKNOWLEDGEMENTSWe thank Rick Kahn for the mouse anti-myc antibody, David Anderson for the

mouse anti-REST antibody, Stuart Schrieber for the HDAC constructs, Gerald

Thiel for the myc-REST and Gal4-REST constructs, Gail Mandel for the type II

sodium-channel promoter and Nancy F. Ciliax for neuronal cultures. We thank

Jerry Boss, John Lucchesi and Steve Warren for comments on an early version of

the manuscript. Supported by NIH grant NS36604 (R.D.) and an NRSA (S.J.M.).

RECEIVED 12 JULY; ACCEPTED 24 AUGUST 1999

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