Corepressor-Dependent Silencing of Chromosomal Regions Encoding Neuronal Genes Victoria V. Lunyak, 1 Robert Burgess, 1 * Gratien G. Prefontaine, 1 Charles Nelson, 1 Sing-Hoi Sze, 2 Josh Chenoweth, 3 Phillip Schwartz, 4 Pavel A. Pevzner, 2 Christopher Glass, 5 Gail Mandel, 3 Michael G. Rosenfeld 1 † Themolecularmechanismsbywhichcentralnervoussystem–specificgenesare expressed only in the nervous system and repressed in other tissues remain a central issue in developmental and regulatory biology. Here, we report that the zinc-finger gene-specific repressor element RE-1 silencing transcription factor/ neuronal restricted silencing factor (REST/NRSF) can mediate extra- neuronal restriction by imposing either active repression via histone deacety- lase recruitment or long-term gene silencing using a distinct functional com- plex. Silencing of neuronal-specific genes requires the recruitment of an as- sociated corepressor, CoREST, that serves as a functional molecular beacon for the recruitment of molecular machinery that imposes silencing across a chro- mosomal interval, including transcriptional units that do not themselves con- tain REST/NRSF response elements. Specific strategies mediating gene repression and gene silencing are required to generate cell-type diversity and promote inheritable cell-type identity [reviewed in (1)]. For ex- ample, the transcriptional repression of neu- ronal-specific genes is necessary to maintain functions unique to nonneuronal systems. Al- though the precise mechanisms responsible for this tissue-specific transcriptional inacti- vation remain unclear, it has been shown that repressor element RE-1 silencing transcrip- tion factor/neuronal restricted silencing factor (REST/NRSF) is a negative regulator that restricts expression of neuronal genes to neu- rons in a variety of genetic contexts (2–4 ). About 35 neuronal target genes have been identified for REST/NRSF [reviewed in (4 )]. REST/NRSF is a 116-kD protein that con- tains a DNA binding domain with eight zinc fingers and two repressor domains (4–6 ) and binds to a 21– to 23– base pair (bp) conserved DNA response element, RE-1/NRSE (2–4 ). It has been shown that REST/NRSF can me- diate repression, in part, through the associ- ation of its NH 2 -terminal repression domain with the mSin3/histone deacethylase 1,2 (HDAC1,2) complex and with the nuclear receptor corepressor (N-CoR) participating in the context of certain genes (7, 8). The REST/ NRSF COOH-terminal repression domain as- sociates with at least one other factor, the transcriptional corepressor CoREST, charac- terized by two SWI3, ADA2, N-Cor, TFIIIB (SANT) domains (9), that may serve as a platform protein for assembly of specialized repressor machinery (10–12) (fig. S1). HDAC-dependent repression of the neuron-specific gene SCG10. REST/NRSF alternatively recruits mSin3A/HDAC1,2 (7, 8) or CoREST complexes (10–12). To investigate the molecular mechanisms involved in REST/ NRSF-mediated gene repression and corepres- sor complexes, we studied one of the most well-characterized neuronal-specific genes, NaCh type II/Nav1.2, and compared its regula- tion to that of SCG10 (5–8). In a chromatin immunoprecipitation assay (ChIP) (8, 13) from Rat-1 fibroblasts, REST/NRSF and CoREST were highly recruited to the NaCh II promoter, whereas N-CoR was not (Fig. 1A). HDAC1, HDAC3, and HDAC2 were detected in small quantities or not at all in some experiments (14 ). In contrast, REST/NRSF was present on the SCG10 gene promoter with HDAC2, HDAC3, and N-CoR (6, 8, 14 ). Transfection of a construct that encodes the REST/NRSF DNA binding domain (REST DBD ) harboring dele- tions of the defined NH 2 - and COOH-terminal repressor domains (6, 13), and hence a potential dominant negative, resulted in the specific de- repression of both the SCG10 and NaCh II genes (Fig. 1B). Thus, the binding of REST functions in both establishing and maintaining repression (2–4 ). Overexpression of the REST/NRSF inter- action domain of CoREST (CoREST RID )(6 ) served as a dominant negative in the Rat-1 cells and resulted in the specific derepression of the NaCh II gene (Fig. 1C); in contrast, there was no effect on repression of the SCG10 gene (Fig. 1C). Because CoREST can form a biochemical complex with HDAC1/2 (10–12), we investigated the functional im- portance of HDACs by treating Rat-1 cells with an HDAC inhibitor, trichostatin A (TSA) (300 nM) (7 ). When exponentially proliferating Rat-1 cells were incubated in the presence of 300 nM TSA, ectopic activation of the SCG10 gene was observed, with the maximum level of expression activity 8 hours after treatment (Fig. 1D). In contrast, even after a 48-hour treatment with TSA no detect- able activation of the NaCh II gene was observed (Fig. 1D). These data indicate that CoREST is selectively required to maintain NaCh II but not SCG10 gene repression. CpG methylation is required for silenc- ing NaCh II gene transcription. Because TSA failed to reduce NaCh II gene repression and because DNA methylation is a widely used strategy in gene silencing (15), we examined the CpG methylation status of the NaCh II gene in Rat-1 cells. Within the genome, from 60 to 90% of the cytosine methylation occurs at CpG dinucleotides (15–17 ). With the use of the so- dium bisulfite genomic-modification sequenc- ing approach (13), we found that the NaCh II promoter region exhibited a sparse pattern of CpG methylation (C m pG), with three sites (– 447, –259, and +45) preferentially methyl- ated, whereas the CpGs further along the 3' end of the gene exhibited a more robust methylated CpG pattern (Fig. 1E) (14 ). Treatment of Rat-1 cells with 5'-aza-cytidine (5AzaC) for a pro- longed period of time (up to 72 hours) to re- verse DNA methylation reduced specific CpG methylation in the NaCh II gene promoter (Fig. 1F and fig. S2) and caused derepression of the NaCh II but not the SCG-10 gene (Fig. 1G). These data suggest that the NaCh II gene might be silenced in a C m pG-dependent manner. Among the many proteins that bind to methylated DNA, MeCP2 characteristically binds to single, symmetrical C m pG pairs in any sequence context (18–20) and has been functionally linked to gene silencing (21–24 ). Because it is also robustly expressed in Rat-1 cells (fig. S3), we investigated the possible participation of MeCP2 in NaCh II gene re- pression. ChIPs were performed from Rat-1 cells using a MeCP2-specific immunoglobu- lin G (IgG) (Fig. 2A) and primers from the REST-binding element in the promoters as well as from the 3'-coding regions of SCG10 and NaCh II genes. MeCP2 is present in both the promoter and exon and intron regions of 1 Howard Hughes Medical Institute (HHMI), 2 Depart- ment of Computer Science and Engineering, School of Medicine, University of California, San Diego, 9500 Gilman Drive, Room 345, La Jolla, CA 92093–0648, USA. 3 Howard Hughes Medical Institute, Department of Neurobiology, State University of New York, Stony Brook, NY 11794, USA. 4 Affinity BioReagents, Incor- porated, 14818 West 6th Avenue, Suite 10A, Golden, CO 80401, USA. 5 Department of Cellular and Molec- ular Medicine, School of Medicine, University of Cal- ifornia, San Diego, La Jolla, CA 92093, USA. *Present address: Beckman Institute for Biomedical Research, Department of Functional Genomics, Te- mecula, CA 92590, USA. †To whom correspondence should be addressed. E- mail: mrosenfeld@ucsd.edu R ESEARCH A RTICLES www.sciencemag.org SCIENCE VOL 298 29 NOVEMBER 2002 1747