The Co-repressor mSin3A Is a Functional Component of the REST-CoREST Repressor Complex

Abstract

(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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