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.

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...

Histone demethylase LSD1 regulates transcription by demethylating Lys 4 of histone H3. The crystal structure of the enzyme in complex with CoREST and a substrate-like peptide inhibitor highlights an intricate network of interactions and a folded conformation of the bound peptide. The core of the peptide structure is formed by Arg 2 , Gln 5 , and Ser 10 , which are engaged in specific intramolecular H-bonds. Several charged side chains on the surface of the substrate-binding pocket establish electrostatic interactions with the peptide. The three-dimensional structure predicts that methylated Lys 4 binds in a solvent inaccessible position in front of the flavin cofactor. This geometry is fully consistent with the demethylation reaction being catalyzed through a flavin-mediated oxidation of the substrate amino-methyl group. These features dictate the exquisite substrate specificity of LSD1 and provide a structural framework to explain the fine tuning of its catalytic activity and the active role of CoREST in substrate recognition.Lysine methylation is among the most well characterized histone modifications, and its existence has been known since the early days of chromatin research (1, 2). This type of epigenetic mark provides a huge potential for functional responses in that it can occur in different forms (mono-, di-, and tri-methylation) and on different histone sites, each having a specific physiological meaning. Histone methylation has been long thought to be a low turnover epigenetic mark, but the recent discovery of histone demethylases (3, 4) has challenged this view by demonstrating that histone lysine methylation can be actively and dynamically regulated. Two classes of histone demethylases have been uncovered; the enzymes of the JmjC family use iron as cofactor, whereas lysine-specific demethylase 1 (LSD1) 4 employs FAD as the prosthetic group (5).LSD1 catalyzes the oxidative demethylation of mono-and dimethyl Lys 4 of histone H3, generating hydrogen peroxide and formaldehyde (3, 4). The enzyme is implicated as a key component of distinct co-activator and co-repressor complexes in a surprisingly wide range of cellular processes where it participates in the dynamic transition of transcriptional programs (6). Its catalytic activity is finely tuned by the epigenetic marks present on the H3 N-terminal tail (7) and by other protein partners, such as CoREST, that form a stable complex with the enzyme (8, 9). The three-dimensional structure of LSD1 in its native state (10, 11) and in complex with the LSD1-binding domain of CoREST (12) have revealed that the catalytic center is located in the core of the enzyme main body. A protruding tower domain consisting of two remarkably long helices forms the docking site for the co-repressor protein (Fig. 1A).Here, we describe the structural analysis of LSD1-CoREST bound to a 21-amino acid H3 peptide in which pLys 4 ("p" is for peptide) is mutated to Met. The structural analysis illuminates the molecular properties that enable LSD1 to function as a key transcriptional reg...