The transcriptional repressor, REST, helps restrict neuronal traits to neurons by blocking their expression in nonneuronal cells. To examine the repercussions of REST expression in neurons, we generated a neuronal cell line that expresses REST conditionally. REST expression inhibited differentiation by nerve growth factor, suppressing both sodium current and neurite growth. A novel corepressor complex, CoREST/HDAC2, was shown to be required for REST repression. In the presence of REST, the CoREST/HDAC2 complex occupied the native Nav1.2 sodium channel gene in chromatin. In neuronal cells that lack REST and express sodium channels, the corepressor complex was not present on the gene. Collectively, these studies define a novel HDAC complex that is recruited by the C-terminal repressor domain of REST to actively repress genes essential to the neuronal phenotype.

(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...

Pancreatic beta cells (insulin-producing cells) and neuronal cells share a large number of similarities. Here, we investigate whether the same mechanisms could control the expression of neuronal genes in both neurons and insulin-producing cells. For that purpose, we tested the role of the transcriptional repressor neuron-restrictive silencing factor/repressor element silencing transciption factor (NRSF/REST) in the expression of a battery of neuronal genes in insulin-producing cells. NRSF/REST is a negative regulator of the neuronal fate. It is known to silence neuronal-specific genes in non-neuronal cells. We demonstrate that, as in the case of the neuronal pheochromocytoma cell line PC12, mRNA coding for NRSF/REST is absent from the insulinoma cell line INS-1 and from three other insulin-and glucagon-producing cell lines. NRSF/REST activity is also absent from insulin-producing cell lines. Transient expression of REST in insulin-producing cell lines is sufficient to silence a reporter gene containing a NRSF/ REST binding site, demonstrating the role of NRSF/ REST in the expression of neuronal markers in insulinproducing cells. Finally, by searching for the expression of NRSF/REST-regulated genes in insulin-producing cells, we increased the list of the genes expressed in both neurons and insulin-producing cells.It is now well established that, in spite of different embryological origins, beta and neuronal cells share a large number of similarities. Indeed, molecules such as glutamic acid decarboxylase (1), tyrosine hydroxylase (2), dopamine ␤-hydroxylase (3), type II voltage-dependent sodium channel (4), glutamate receptor (5), neurofilament proteins (6, 7), receptors for neurotrophins (8, 9), and thyrotropin-releasing hormone (10, 11) have been shown to be expressed in both beta and neuronal cells.The regulation of the expression of neuronal markers in beta cells at the molecular level is not understood. Theoretically, the expression of the same gene in two different cell types can be explained by either the presence in both cell types of specific transcriptional activators or the absence of specific transcriptional repressors. At least three specific transcriptional activators, Islet-1 (12), Pax-6 (13), and Beta2 (14), are expressed in both beta and neuronal cells. To our knowledge, there are no data demonstrating the role of transcriptional repressors in the expression of neuronal molecules by beta cells.Recently, progress has been made on the mechanisms underlying the specific expression of genes in neurons. It has indeed been demonstrated that a large battery of neuron-specific genes was regulated by one silencer protein. This repressor, named NRSF/REST, 1 is present in non-neuronal cells and absent from neuronal cells (15, 16). It was cloned according to its ability to bind a 24-bp cis-element necessary and sufficient for silencing neuron-specific genes such as the type II voltage-dependent sodium channel gene and the SCG10 gene, another neuron-specific gene. This 24-bp cis-element, which represents a bin...