The REST cDNA utilized for bait (LexA-C-REST) contains amino acids 525-1097 of REST. silencing mediator for retinoid and thyroid human receptors (SMRT)-extended corepressors that mediate inducible repression by steroid hormone receptors. Together, REST and CoREST mediate repression of the type II sodium channel promoter in nonneural cells, and the REST/CoREST complex may mediate long-term repression essential to maintenance of cell identity. A large number of genes encoding neuronal phenotypic characteristics, including ion channels, neurotransmitters, synaptic proteins, and cell-adhesion molecules, are expressed only in neurons. One mechanism important in establishing and maintaining this neural specificity entails the DNA-binding protein REST/NRSF (RE1 silencing transcription factor/neural-restrictive silencing factor) (1C4), which serves to block expression of its target genes in nonneural tissues. Such managed gene repression is usually in contrast to the more dynamic repression mechanism that regulates inducible gene expression in response to steroid hormone receptors, one of the best-studied mammalian repressor mechanisms (for review, observe ref. 5). One REST target gene essential for neuronal physiology is usually that encoding the brain type II voltage-dependent sodium channel. This ion channel is required for the propagation of fast electrical signals in neurons, in the form of neuronal 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 expression of REST. Additionally, when a REST expression plasmid is usually cotransfected into neuronal cells along with a type II sodium channel reporter, the expression of the reporter gene is usually reduced dramatically (1). This result indicates either that REST alone is sufficient to repress its target genes or that REST accessory factors are present in neuronal cells despite the absence of REST. Two unique repressor domains have been recognized and characterized in REST (6, TOK-8801 7). These domains are located in the amino and carboxyl termini of the protein. Both domains are required for full repression in the context of the intact molecule, but each TOK-8801 domain name is sufficient to repress type II sodium channel reporter genes when expressed as a Gal4 fusion protein (6). The C-terminal repressor domain name contains a C2H2 class zinc finger beginning approximately 40 aa upstream of the quit codon. Deleting this domain name, or introducing a point mutation crucial to the zinc finger motif, abolishes repressor activity (6). Because zinc finger motifs often mediate proteinCprotein interactions, we proposed that TOK-8801 REST might function in conjunction with other nuclear factors or corepressors. In this study, we find that repression of the type Mouse monoclonal to RBP4 II sodium channel promoter by REST requires a newly recognized protein, CoREST, which fulfills the criteria for a bona fide corepressor. CoREST is usually a repressor; mutations that disrupt CoRESTs binding to REST also interfere with REST repression. Endogenous REST and CoREST proteins form a complex in cells that do not express the type II sodium channel gene. MATERIALS AND METHODS Two-Hybrid Screening. The REST cDNA utilized for bait (LexA-C-REST) contains amino acids 525-1097 of REST. G. Hannon (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) provided the HeLa cell cDNA library. Library screens were carried out, as described previously (8, 9), by using HIS3 and LacZ reporters and the L40 strain provided by R. Sternglanz (State University of New York at Stony Brook). Specificity of the conversation was examined by a mating assay between the positive L40 transformants and an AMR-70 strain expressing REST-related proteins [LexA-N-REST, amino acids 1C525; LexA-C-RESTM1, amino acids 525-1097 (Cys-1062 Arg); LexA-C3-REST, amino acids 1013C1097] and several LexA fusion proteins not related to REST. All cDNAs were characterized by sequence analysis. Library Screening. A Zap HeLa cDNA library (Stratagene) was screened with a cDNA probe representing a 5 fragment of KIAA0071 (10). Positive clones were excised from your phagemid according to the manufacturers instructions (Stratagene) and characterized by chain-termination sequence analysis (Amersham Pharmacia) by using an ABI 300 TOK-8801 automated sequencer (Applied Biosystems). One clone extended the published sequence of KIAA0071 by 101 aa and contains 227 nt of 5 untranslated region. This cDNA (termed CoR5b) was joined to KIAA0071 to generate full-length CoREST (explained below). Plasmid Constructions. A CoREST expression vector (pcDNACoREST) was constructed by inserting an protein (d) (accession no. “type”:”entrez-nucleotide”,”attrs”:”text”:”AA392295″,”term_id”:”13766491″AA392295). Conserved amino acids are shaded. Boldface type indicates the SANT domain name. (by coimmunoprecipitation analysis. Whole-cell lysates prepared from L6 skeletal muscle mass cells were immunoprecipitated with the indicated antibodies and then subjected to Western blotting. The membrane was probed with REST antibody (1). The arrow depicts migration of REST protein..