Several genes encoding proteins critical to the neuronal phenotype, such as the brain type II sodium channel gene, are expressed to high levels only in neurons. This cell specificity is due, in part, to long-term repression in nonneural cells mediated by the repressor protein REST͞NRSF (RE1 silencing transcription factor͞neural-restrictive silencing factor). We show here that CoREST, a newly identified human protein, functions as a corepressor for REST. A single zinc finger motif in REST is required for A large number of genes encoding neuronal phenotypic traits, 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 involves the DNA-binding protein REST͞ NRSF (RE1 silencing transcription factor͞neural-restrictive silencing factor) (1-4), which serves to block expression of its target genes in nonneural tissues. Such maintained gene repression is 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, see ref. 5).One REST target gene essential for neuronal physiology is 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 cotransfected into neuronal cells along with a type II sodium channel reporter, the expression of the reporter gene is 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 distinct repressor domains have been identified and characterized in REST (6, 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 domain is sufficient to repress type II sodium channel reporter genes when expressed as a Gal4 fusion protein (6). The C-terminal repressor domain contains a C 2 H 2 class zinc finger beginning approximately 40 aa upstream of the stop codon. Deleting this domain, or introducing a point mutation critical to the zinc finger motif, abolishes repressor activity (6). Because zinc finger motifs often mediate proteinprotein interactions, we proposed that REST might function in conjunction with other nuclear factors or corepressors.In this study, we find that repression of the type II sodium channel promoter by REST requires a newly identified protein, CoREST, which fulfills the criteria for a bona fide corepressor. CoREST is a repressor; mutations that disrupt CoREST...

The voltage-gated ionic currents of two rodent prostatic cancer cell lines were investigated using the whole-cell patch clamp technique. The highly metastatic Mat-Ly-Lu cells expressed a transient, inward Na ÷ current (blocked by 600 nM tetrodotoxin), which was not found in any of the weakly metastatic AT-2 cells. Although both cell lines expressed a sustained, outward K ÷ current, this occurred at a significantly higher density in the AT-2 than in the Mat-Ly-Lu cells. Incubation of the MatLy-Lu cell line with 600 nM tetrodotoxin significantly reduced the invasive capacity of the cells in vitro. Under identical conditions, tetrodotoxin had no effect on the invasiveness of the AT-2 cells.

(1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9873-9878). Here we show that the co-repressor mSin3A also interacts with REST. The REST-mSin3A association involves the NH 2 -terminal repressor domain of REST and the paired amphipathic helix 2 domain of mSin3A. REST forms complexes with endogenous mSin3A in mammalian cells, and both mSin3A and CoREST interact with REST in intact mammalian cells. REST repression is blocked in yeast lacking Sin3 and rescued in its presence. In mammalian cells, repression by REST is reduced when binding to mSin3A is inhibited. In mouse embryos, the distribution of mSin3A and REST transcripts is largely coincident. The pattern of CoREST gene expression is more restricted, suggesting that mSin3A is required constitutively for REST repression, whereas CoREST is recruited for more specialized repressor functions.Many genes essential for neuronal functioning, including the brain type II voltage-dependent sodium channel, neuronal growth factors, and neurotransmitter receptors, are repressed in non-neuronal cells by the transcriptional repressor, REST/ NRSF (2-5). Removal of the REST/NRSF binding site (RE1/ NRSE) from the regulatory region of transgenes (6) or expression of a dominant negative form of REST/NRSF in vivo (5) results in aberrant expression of the target genes in non-neural tissues. Deletion of the mouse REST/NRSF gene by homologous recombination results in embryonic lethality (5). Because of its importance in establishing and maintaining the expression pattern of a large number of genes required for neuronal functioning, it was of interest to identify the molecules involved in the repressor mechanism.Previous studies identified two distinct domains in the NH 2 and COOH termini of REST that were both necessary and sufficient to repress brain type II sodium channel reporter genes in transient transfection assays (7) and showed that repression by each of these two domains required distinct titratable factors (8). Recently, repression from the COOH-terminal domain was determined to be mediated by the co-repressor, CoREST (1). We sought to determine whether, in addition to CoREST, mSin3A, a co-repressor for several regulated repressor complexes, might also be involved in REST repression. We found that mSin3A is indeed a functional co-repressor for REST. mSin3A interacts with REST in vitro, in yeast and in intact mammalian cells, and interestingly, the binding site maps to the NH 2 -terminal repressor domain in REST. Furthermore, experiments both in yeast and mammalian cells showed that mSin3A is involved in repressor function. In vivo, the expression patterns of the co-repressors mSin3A and CoREST are distinct. Specifically, in early embryogenesis CoREST exhibits a much more restricted pattern of expression compared with REST and mSin3A, suggesting that the composition of the REST repressor complex during development is dynamic. EXPERIMENTAL PROCEDURES Plasmid ConstructionsYeast Two-hybrid Constructs-LexA-mSin3A was obtained by cloning full-length mSin3A into pBTM116 by standard PCR 1...

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Voltage-gated K+ currents expressed in two rat prostate cancer ("Dunning") cell lines of markedly different metastatic ability were characterised using electrophysiological, pharmacological and molecular approaches. Whole-cell patch-clamp recordings showed that both strongly metastatic MAT-LyLu and weakly metastatic AT-2 cell lines possessed outward (delayed-rectifier type) K+ currents, which activated at around -40 mV. From the parameters measured, several characteristics of the two cell lines were similar. However, a number of statistically significant differences were noted for MAT-LyLu versus the AT-2 cells as follows: (1) current densities were smaller; (2) the slope factor for channel activation was smaller; (3) the voltage at which current was half-inactivated, and the slope factor for channel inactivation were greater; (4) the time constants for current decay at -20 and 0 mV were smaller; and (5) the residual peak current was larger following 60 s of repetitive voltage pulses for stimulation frequencies in the range 0.05-0.2 Hz. On the other hand, the K+ currents in both cell lines showed similar pharmacological profiles. Thus, the currents were blocked by 4-aminopyridine, tetraethylammonium, verapamil, margatoxin, and charybdotoxin, with highly similar IC(50)s for given blockers. The electrophysiological and pharmacological data taken together suggested expression of voltage-gated K+ channels of the Kv1 family, expression of the Kv1.3 subunit being predominant. Western blot and RT-PCR tests both confirmed that the cells indeed expressed Kv1.3 and to a lesser extent Kv1.4 and Kv1.6 channel alpha-subunits. In view of the similarity of channel expression in the two cell lines, voltage-gated K+ channel activity may not be a primary determinant of metastatic potential in the rat model of prostate cancer, but the possible contribution of K+ channel activity to the metastatic process is discussed.