Proc. Natl. Acad. Sci. USA Vol. 96, pp. 9873–9878, August 1999 Neurobiology CoREST: A functional corepressor required for regulation of neural-specific gene expression MARI´A E. ANDRE´S*†,CORINNA BURGER†‡,MARI´A J. PERAL-RUBIO†§,ELENA BATTAGLIOLI*, MARY E. ANDERSON*, ࿣ JULIA GRIMES*, JULIA DALLMAN*, NURIT BALLAS*¶, AND GAIL MANDEL* *Howard Hughes Medical Institute and Department of Neurobiology and Behavior, and ¶Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794 Communicated by William J. Lennarz, State University of New York, Stony Brook, NY, June 25, 1999 (received for review April 30, 1999) ABSTRACT Several genes encoding proteins critical to Two distinct repressor domains have been identified and the neuronal phenotype, such as the brain type II sodium characterized in REST (6, 7). These domains are located in the channel gene, are expressed to high levels only in neurons. amino and carboxyl termini of the protein. Both domains are This cell specificity is due, in part, to long-term repression in required for full repression in the context of the intact mole- nonneural cells mediated by the repressor protein cule, but each domain is sufficient to repress type II sodium REST͞NRSF (RE1 silencing transcription factor͞neural- channel reporter genes when expressed as a Gal4 fusion restrictive silencing factor). We show here that CoREST, a protein (6). The C-terminal repressor domain contains a C2H2 newly identified human protein, functions as a corepressor for class zinc finger beginning approximately 40 aa upstream of the REST. A single zinc finger motif in REST is required for stop codon. Deleting this domain, or introducing a point CoREST interaction. Mutations of the motif that disrupt mutation critical to the zinc finger motif, abolishes repressor binding also abrogate repression. When fused to a Gal4 activity (6). Because zinc finger motifs often mediate protein– DNA-binding domain, CoREST functions as a repressor. protein interactions, we proposed that REST might function in CoREST is present in cell lines that express REST, and the conjunction with other nuclear factors or corepressors. proteins are found in the same immunocomplex. CoREST In this study, we find that repression of the type II sodium ͞ ͞ ͞ contains two SANT (SW13 ADA2 NCoR TFIIIB B) do- channel promoter by REST requires a newly identified pro- mains, a structural feature of the nuclear receptor and tein, CoREST, which fulfills the criteria for a bona fide silencing mediator for retinoid and thyroid human receptors corepressor. CoREST is a repressor; mutations that disrupt (SMRT)-extended corepressors that mediate inducible repres- CoREST’s binding to REST also interfere with REST repres- sion by steroid hormone receptors. Together, REST and sion. Endogenous REST and CoREST proteins form a com- CoREST mediate repression of the type II sodium channel ͞ plex in cells that do not express the type II sodium channel promoter in nonneural cells, and the REST CoREST com- gene. plex may mediate long-term repression essential to mainte- nance of cell identity. MATERIALS AND METHODS A large number of genes encoding neuronal phenotypic traits, Two-Hybrid Screening. The REST cDNA used for bait including ion channels, neurotransmitters, synaptic proteins, (LexA-C-REST) contains amino acids 525-1097 of REST. G. and cell-adhesion molecules, are expressed only in neurons. Hannon (Cold Spring Harbor Laboratory, Cold Spring Har- One mechanism important in establishing and maintaining this bor, NY) provided the HeLa cell cDNA library. Library ͞ neural specificity involves the DNA-binding protein REST screens were carried out, as described previously (8, 9), by ͞ NRSF (RE1 silencing transcription factor neural-restrictive using HIS3 and LacZ reporters and the L40 strain provided by silencing factor) (1–4), which serves to block expression of its R. Sternglanz (State University of New York at Stony Brook). target genes in nonneural tissues. Such maintained gene Specificity of the interaction was examined by a mating assay repression is in contrast to the more dynamic repression between the positive L40 transformants and an AMR-70 strain mechanism that regulates inducible gene expression in re- expressing REST-related proteins [LexA-N-REST, amino ac- sponse to steroid hormone receptors, one of the best-studied ids 1–525; LexA-C-RESTM1, amino acids 525-1097 (Cys-1062 3 mammalian repressor mechanisms (for review, see ref. 5). Arg); LexA-C3-REST, amino acids 1013–1097] and several One REST target gene essential for neuronal physiology is LexA fusion proteins not related to REST. All cDNAs were that encoding the brain type II voltage-dependent sodium characterized by sequence analysis. channel. This ion channel is required for the propagation of Library Screening. A ␭ Zap HeLa cDNA library (Strat- fast electrical signals in neurons, in the form of neuronal agene) was screened with a cDNA probe representing a 5Ј impulses, and is not expressed in nonneural tissues. As is true for other REST target genes, there is a reciprocal relationship between expression of the type II sodium channel gene and Abbreviations: REST, RE1 silencing transcription factor; NRSF, neural-restrictive silencing factor; SMRT, silencing mediator for ret- expression of REST. Additionally, when a REST expression inoid and thyroid hormone receptor; SMRTe, SMRT-extended; plasmid is cotransfected into neuronal cells along with a type NCoR, nuclear receptor corepressor; SANT, SW13͞ADA2͞NCoR͞ II sodium channel reporter, the expression of the reporter gene TFIIIB; GST, glutathione S-transferase; DBD, DNA-binding domain; is reduced dramatically (1). This result indicates either that CAT, chloramphenicol acetyltransferase. REST alone is sufficient to repress its target genes or that Data deposition: The sequence reported in this paper has been deposited in the GenBank database (accession no. AF155595). REST accessory factors are present in neuronal cells despite †M.E.A., C.B., and M.J.P-R. contributed equally to this work. the absence of REST. ‡Present address: Department of Molecular Genetics and Microbiol- ogy and Gene Therapy Center, University of Florida, Gainesville, FL The publication costs of this article were defrayed in part by page charge 32610-0266. §Present address: Department of Physiology, University of Seville, payment. This article must therefore be hereby marked ‘‘advertisement’’ in 41012 Seville, Spain. accordance with 18 U.S.C. §1734 solely to indicate this fact. ࿣To whom reprint requests should be addressed. E-mail: gmandel@ PNAS is available online at www.pnas.org. brain.neurobio.sunysb.edu. 9873 Downloaded by guest on September 28, 2021 9874 Neurobiology: Andre´s et al. Proc. Natl. Acad. Sci. USA 96 (1999) fragment of KIAA0071 (10). Positive clones were excised from macia). PCR and subsequent cloning into the vector pCGN the phagemid according to the manufacturer’s instructions (provided by J. Trimmer, State University of New York at (Stratagene) and characterized by chain-termination sequence Stony Brook) created an epitope-tagged version of the zinc analysis (Amersham Pharmacia) by using an ABI 300 auto- finger domain of the C terminus of REST (HA-C3, amino mated sequencer (Applied Biosystems). One clone extended acids 1009–1097). LexA-N-REST (amino acids 1–525) was the published sequence of KIAA0071 by 101 aa and contains created by a three-way ligation among pBTM116 cut with 227 nt of 5Ј untranslated region. This cDNA (termed CoR5b) BamHI and SalI-blunt, a BamHI-ClaI fragment of pCBY was joined to KIAA0071 to generate full-length CoREST REST2, and a ClaI-HincII fragment of REST. LexA-C-REST (described below). (amino acids 525-1097) was created by cloning a HindII-PstI Plasmid Constructions. A CoREST expression vector (pc- fragment of NH7 (1) into pBTM116 cut with BamHI-blunt and DNACoREST) was constructed by inserting an EcoRI-BstXI PstI. LexA-C-RESTM1 (amino acids 525-1097, Cys1062 3 fragment from Cor5b and a BstXI-XbaI fragment from Arg) was created by a three-way ligation of an EaeI-PstI KIAA0071 into pcDNA1.1amp (Invitrogen). Cloning a fragment from C3M1 (6) plus a HindII-PstI fragment of NH7 BsaBI͞EcoRI-blunted fragment of CoREST cDNA into (1) into pBTM116 cut with BamHI and PstI. LexA-C3-REST pcDNA3.1A (Invitrogen) linearized with EcoRV created an (amino acids 1013–1097) was generated by cloning a PCR epitope-tagged version of CoREST (CoREST myc). Cloning fragment containing amino acids 1013–1097 into pBTM116 cut CoREST cDNA into vector pSG424 (6) created with BamHI and PstI. The REST cDNAs lacking the N- Gal4CoREST. The glutathione S-transferase (GST) CoREST terminal REST domain (REST⌬N; REEX 8) lacking the vector used in the pulldown assays contains amino acids C-terminal zinc finger domain (REST⌬C; REEX9) or con- 109–293 of CoREST. The CoREST sequences were placed taining a point mutation in the zinc finger motif (RESTM1; in-frame with GST in the vector pGEX-3X (Amersham Phar- Cys-1062 3 Arg) have been described previously (6). Gal4C3 FIG. 1. Characterization of CoREST. (a) Predicted amino acid sequence of human CoREST (hCoREST) (accession no. AF155595). The underlined amino acids represent the protein fragment isolated in the yeast two-hybrid screen. The two SANT domains (15) are shaded. (b) Alignment of the two repeated SANT domains in CoREST and NCoR. For comparison, the SANT consensus and predicted structure are shown below. h, hydrophobic residues; ϩ and Ϫ, positively and negatively charged residues. Amino acids conforming to the SANT consensus are in boldface. Additional homologies between CoREST and NCoR are shaded. (c) Alignment of hCoREST (h) with a similar Drosophila protein (d) (accession no. AA392295). Conserved amino acids are shaded. Boldface type indicates the SANT domain. (d) Western blot of nuclear extracts from HEK293 cells transfected with pcDNA or with a myc-tagged CoREST cDNA. The probes were a polyclonal CoREST antibody (␣CoREST) and a monoclonal myc antibody (␣myc). The positions of migration of endogenous CoREST (66 kDa) and myc-CoREST (70 kDa) are indicated.