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Vol. 16, Issue 12, 5630-5638, December 2005
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* School of Biochemistry and Microbiology, University of Leeds, Leeds LS2 9JT, United Kingdom;
School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom; and
School of Biology, University of Leeds, Leeds LS2 9JT, United Kingdom
Submitted July 28, 2005;
Revised September 15, 2005;
Accepted September 20, 2005
Monitoring Editor: Marianne Bronner-Fraser
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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REST interacts with RE1 sites and recruits corepressors including mSin3A (Huang et al., 1999
, Roopra et al., 2000) and CoREST (Andrés et al., 1999, Ballas et al., 2001), which in turn, recruit multiple cofactors and transcriptional regulators including histone deacetylases 1 and 2, G9a histone methyltransferase (Roopra et al., 2004), H3 lysine 4 demethylase LSD1 (Shi et al., 2004), and chromatin remodeling machinery, including BRG1, BAF57, and BAF170 (Battaglioli et al., 2002) and BRAF35 (Hakimi et al., 2002). There are over 1300 RE1 sites in the human and murine genomes (Bruce et al., 2004; www.bioinformatics.leeds.ac.uk/group/online/RE1db/re1db_home.htm) and the majority encode ion channels, neurotransmitters, growth factors and hormones, and factors involved in axonal guidance and vesicle trafficking, and molecules involved in maintenance of the cytoskeleton and extracellular matrix (Schoenherr et al., 1996; Bruce et al., 2004). Because many of these target genes are expressed by postmitotic neurons, the role of REST was originally perceived as that of silencer of neuron-specific genes in nonneuronal cells (Chong et al., 1995; Schoenherr and Anderson, 1995). However, several observations have tempered this view. First, REST is expressed in many adult postmitotic neurons, most notably those of the hippocampus, where both REST and its target genes are modulated in the adult hippocampus in response to ischemic or epileptic insults (Palm et al., 1998; Calderone et al., 2003). Second, we have shown that, in most neural and nonneural cell lines, REST principally acts as a repressor of active gene transcription rather than as a silencer (Wood et al., 2003; Belyaev et al., 2004; Bruce et al., 2004). Third, REST has been shown to be sufficient to drive neuronal differentiation of adult neural stem cells (Kuwabara et al., 2004; Su et al., 2004) and even myoblasts (Watanabe et al., 2004). Fourth, REST is present at all stages of neural development from the blastocyst, through embryonic development in most tissues including the neuroepithelium. Finally, REST–/– mice are embryonic lethal around E10 and do not show widespread precocious expression of REST target genes and introduction of a dominant-negative REST construct into chick myotome caused up-regulation of some, but not all, REST target genes (Chen et al., 1998), whereas constitutive expression of REST down-regulated some target genes and caused axon pathfinding errors (Paquette et al., 2000). Taken together, these observations suggest that REST plays distinct roles in embryonic and adult cells and that this complexity might be reflected in the range of target genes that REST interacts with at different developmental stages.
In this study, we directly investigate this hypothesis by using a novel chromatin immunoprecipitation (ChIP) cloning strategy based on selective amplification of ChIP (SACHI) to identify REST target gene interactions in ES cells, hippocampal NS cell line, MHP36 (Kershaw et al., 1994) and mature hippocampus, all of which represent distinct stages of neuronal development. Using this technique, we identify 81 novel REST targets and further, we show that REST occupies a unique subset of target genes in each cell type.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-mercaptoethanol (Invitrogen, Carlsbad, CA). Neural stem cells, MHP36 (a gift from Prof. J. Price, King's College London), were plated out in 10-cm dishes precoated with 10 µl/ml fibronectin and cultured in DMEM/F12 medium in the presence of 47 µg/ml gentamicin and 200 µg/ml geneticin (Invitrogen). The medium was supplemented with 0.03% bovine serum albumin (BSA) Pathocyte (ICN, Costa Mesa, CA), 94 µg/ml human apo-transferrin, 15 µg/ml human putrescine dihydrochloride, 4.7 µg/ml bovine pancreatic insulin, 380 ng/ml L-thyroxine, 317 ng/ml tri-iodo-L-thyronine, 58 ng/ml progesterone, 1.9 mM L-glutamine, 38 ng/ml sodium selenite, 9.4 U/ml heparin sodium salt (all from Sigma), 10 ng/ml human bFGF, and 10 ng/ml murine interferon (PeproTech, Rocky Hill, NJ). Hippocampal tissues were collected by gross dissection from F1 mice (FVB/N x C57BL6) aged between 16 and 20 d. RNA was extracted using Tri reagent (Sigma) from cells or tissues. RNA was treated with DNAse (Ambion, Austin, TX) and cDNA was made from 2 µg RNA using MMLV (H-point mutant) reverse transcriptase and oligo dT and random hexamers as primers. cDNA was then purified through spin columns (Qiagen, Santa Clarita, CA) using ultra centrifugation and eluted in 50 µl of elution buffer.
Neuronal Differentiation
MH-1 ES cells were differentiated to NS cells and subsequently to mature neurons using monolayer culture in N2B27 medium (Ying et al., 2003). ES cells were plated onto 0.1% gelatin-coated cover slips in 24-well plates at a density of 1 x 104/cm2 in ES cell medium. The next day, the ES cell medium was replaced with N2B27 medium, which is a 1:1 mixture of DMEM/F12 medium supplemented with N2 and Neurobasal medium supplemented with B27 (all from Invitrogen). N2 consisted of 25 µg/ml insulin, 100 µg/ml apotransferrin, 6 ng/ml progesterone, 16 µg/ml putrescine, 30 nM sodium selenite, and 50 µg/ml BSA fraction V. Medium was changed every 2 d. Cells were processed for immunocytochemistry at day 0 (prior differentiation) and days 6, 9, and 18 after differentiation.
Immunostaining
Mice (C57BL6) were anesthetized with intraperitoneal Sagatal (60 mg/kg) and transcardially perfused with 4% paraformaldehyde (in 0.1 M phosphate buffer PB; pH 7.4). The brains were removed and postfixed in the same solution for 2 h at 4°C. Transverse sections (50 µm) were then cut on a vibrating microtome (Leica, Milton Keynes, United Kingdom) and collected into phosphate-buffered saline (PBS; pH 7.2). After 10-min washes three times in PBS, the sections were incubated with rabbit anti-REST primary antibody P18 (Santa Cruz Biotechnology, Santa Cruz, CA) for 12–18 h at 4°C. After 10-min washes three times in PBS, sections were incubated with a Cy3-conjugated anti-rabbit secondary antibody (1:1000 in PBS; Jackson ImmunoResearch, Luton, United Kingdom) for 
4 h at room temperature. These sections were then washed 10 min three times in PBS, air-dried onto glass slides, and mounted under a coverslip using VectaMount mounting medium (Vector Laboratories, Peterborough, United Kingdom). The sections were viewed under a Nikon E600 microscope (Melville, NY) equipped with epifluorescence and images obtained via an integrating analogue CCD camera (JVC KYF 55B) attached to an Acquis image capture system (Synoptics Ltd, Cambridge, United Kingdom). The fluorescent images were converted to greyscale, inverted and adjusted for brightness/contrast/intensity and color balance using CorelDraw 11 (Corel Systems, Ottawa, Ontario, Canada).
ES cells, derived NS cells and neurons, and MHP36 cells were fixed in 3.5% paraformaldehyde for 20 min and stained with REST antibody (1: 300; Santa Cruz Biotechnology) alone or in combination with Sox1 (1:1000; Chemicon), Tuj1 (1:200; COVANCE, Princeton, NJ) and NeuN (1:500; Chemicon) antibodies overnight at 4°C.
Chromatin Immunoprecipitation
Cells (1 x 108 cells) or tissues (minced, 100 mg wet weight) were fixed for 10 min in 0.37% or 15 min in 1% formaldehyde, respectively. The fixation reaction was quenched with 0.125 M glycine. Nuclei were isolated by incubation on ice for 10 min in cell lysis buffer (10 mM Tris-HCl, pH 8.0, 10 mM NaCl, 0.2% NP40, 10 mM sodium butyrate, and protease inhibitors) and lysed in nuclei lysis buffer (50 mM Tris-HCl, pH 8.1, 10 mM EDTA, 1% SDS, 10 mM sodium butyrate, and protease inhibitors) for 10 min on ice. The lysate was then sonicated to shear the chromatin, cell debris was removed by centrifugation, and IP dilution buffer was added (20 mM Tris-HCl, pH 8.1, 150 mM NaCl, 2 mM EDTA, 1% Triton-X-100, 0.01% SDS, 10 mM sodium butyrate, and proteinase inhibitors). Chromatin was precleared for 3 h at 4°C with 200 µl of protein G-Sepharose blocked with 5% BSA. Whole cell extract was divided into aliquots: 270 µl kept as input chromatin and equal aliquots were incubated separately with anti-REST (P-18) and anti-HA (as a negative control) antibodies (Santa Cruz Biotechnology). Immune complexes were collected by rotation at 4°C for 3 h with 100 µl blocked protein G-Sepharose. Beads were washed twice with wash buffer 1 (20 mM Tris-HCl, pH 8.1, 50 mM NaCl, 2 mM EDTA, 1% Triton-X-100, and 0.1% SDS), once with wash buffer 2 (10 mM Tris-HCl, pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid), and twice with TE buffer. Immune complexes were eluted with elution buffer (100 mM NaHCO3, 1% SDS) and cross-links were reversed by incubation at 65°C for 6 h in the presence of 5 µg RNase A and 0.3 M NaCl. Proteinase K, 90 µg, was added and samples were incubated overnight at 45°C. DNA was purified using phenol:chloroform extraction and ethanol precipitation. DNA from input sample was resuspended in 200 µl water, and DNA was pulled down by REST and HA antibodies in 100 µl water. Two-microliter aliquots were analyzed in real time PCR using primers designed adjacent to the RE1 regions.
ChIP Cloning
A cloning protocol was devised to specifically amplify RE1 containing sequences from the DNA output of anti-REST immunoprecipitated DNA. The principal of this procedure was to enrich target sequences by a nested PCR strategy using primers corresponding to the RE1 sequence and to end-ligated adaptors. The detailed procedure is summarized in Figure 1. After immunoprecipitation of fixed, sonicated chromatin but before elution of DNA from protein G-Sepharose, the sheared DNA ends were blunt-ended using mung bean nuclease, followed by adding phosphate to the 5'-ends of DNA using T4 polynucleotide kinase (BioLabs, St. Paul, MN). ChIP DNA was ligated with a specific adaptor [5'-CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT-3']. The RE1-containing fragments were amplified from the enriched ChIP DNA by two rounds of PCR with primer pairs. First-round PCR used an RE1 degenerate primer 5'-[TT(C)AGA(C)G(A)CCA(T)A(T)GGACAG-3'] and adaptor primer A [CTAATACGACTCACTATAGGGC-3']. PCR cycling parameters were 95°C x 5 min; 94°C x 10 s, 52°C x 20 min, 72°C x 2 min, for 10 cycles; 94°C x 10 s, 56°C x 20 min, 72°C xx 2 min, for 25 cycles. Second-round PCR used the RE1 degenerate primer and a nested adaptor primer B [5'-TCGAGCGGCCGCCCGGGCAGGT-3']. Cycling conditions were 95°C x 5 min; 94°C x 10 s, 58°C x 20 min, 72°C x 2 min, for 35 cycles. Second-round PCR-products were size selected at 200–800 base pairs and cloned into the pGEM-easy vector. Seven hundred clones from each cell line were isolated and sequenced. We found that the crucial factor for designing an effective RE1 degenerate primer was to contain no degeneracy within the first six base pairs of the 3'-end.
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). Nuclear protein extract from U373 expressing the REST DNA-binding domain was mixed with a 32P-labeled NaV1.2 consensus RE1 probe and 100-fold molar excess unlabeled 24–28-base pair oligonucleotides, derived from cloned REST target genes, were used to compete the gel shift.
Quantitative Real-time PCR
Primers were designed using the primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_slow.cgi) and "mfold" (http://www.bioinfo.rpi.edu/applications/mfold/old/dna) programs to obtain amplicons using the following parameters: Tm between 58 and 62°C, devoid of secondary structure at Tm 60°C and amplicon size of 50–150 base pairs. ChIP primers were derived from sequence within 500 base pairs of the RE1. Real-time PCR was carried out using SYBR green containing Supermix (Bio-Rad, Hercules, CA) on a Bio-Rad iCycler. All reactions were performed in duplicate. PCR cycle parameters were 95°C for 3 min, followed by 45 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 30 s. At the end of the program, the temperature was reduced to 50°C and then gradually increased by 1°C for 10 s up to 96°C, to produce a melt curve. Gene expression was normalized to cyclophilin using the formula 1/2
Ct, where Ct = average unknown gene threshold cycle-average cyclophilin threshold cycle. The expression levels of cyclophilin in ES, MHP cells, and hippocampus showed no significant differences (Supplementary Figure 1).
Data Analysis
The sequences of clones obtained from ChIP cloning were blast-searched against Ensembl mouse database (http://www.ensembl.org/Multi/blastview?Species=Mus_musculus). Homologous sequences were investigated to identify the RE1 consensus: 5'-TY(TC)AGM(AC)R(AG)CCN(ACGT)NRGMCAG-3' (Bruce et al., 2004). Sequences were classified according to 0, 1, 2, 3, or 4 base pairs mismatch to the consensus RE1. All fragments showing no deviation from the consensus RE1 were then annotated according to the closest genes.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Our results suggest that, during ES cell differentiation, the expression profile of REST declines as neuronal differentiation proceeds.
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). Accordingly we examined REST expression in an embryonic hippocampal NS cell line (MHP36 cells) and in hippocampal neurons. In MHP36 cells, nuclear REST was evident (Figure 3A). MHP36 cells are homogenous with regard to both the expression of REST and the progenitor marker, nestin (unpublished data). Furthermore, in hippocampus, REST was restricted to neuronal cell bodies throughout the pyramidal cell layer and the granular layer of the dentate gyrus (Figure 3B), a distribution that is consistent with the REST mRNA expression profile (Kuwabara et al., 2004). These results are different from those reported in early cortical neurons (Ballas et al., 2005). This is a key difference because it illustrates heterogeneity of REST expression within both progenitors and within differentiated postmitotic neurons. Accordingly, we used immunocytochemistry to demonstrate that REST was expressed in HM-1 ES cells, ES-derived NS cells, MHP36 NS cells, and in neurons of the mature hippocampus.
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Identification of REST Target Genes in Embryonic Stem Cells, Neural Stem Cells, and Hippocampus
Ninety-three independent clones containing consensus RE1s were isolated from ES, NS, and hippocampus using our ChIP cloning procedure. Eighty-nine of these sequences were proximal to a known or predicted transcriptional unit. Sixty RE1 sites were located within intragenic regions, of which 50% were located in the first intron. Another 25 (28%) and 7 (7.8%) RE1s were located in the 5'flanking and 3'flanking regions, respectively (Tables 1 and 2). Four genes comprising L1cam, Syt2, GEF1, and ENSMUSG00000041339 were adjacent to, or contained, two RE1 sites. Thirty-eight genes (42%) were annotated in the Ensembl database (Supplementary Table 1) and were classified into six groups according to their biological functions (Table 1), whereas the remaining 51 genes (58%) corresponded to novel, predicted transcriptional units (Table 2). Of the annotated genes, 24 (65%) are expressed specifically or selectively in the nervous system (Table 1), including those concerned with neurite outgrowth, axonal guidance, vesicle trafficking, signaling, and transcription, consonant with the role of REST in regulating neuronal gene expression. The fact that more than 85% of these genes have not previously been shown to be direct targets of REST is testament to the power of this ChIP cloning protocol to reveal novel target genes.
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Validation of Bona Fide REST Target Genes
Validation of REST target genes isolated from ChIP cloning was carried out using conventional ChIP analysis. Thirty-three genes were randomly selected for testing from the isolated 89 genes and included 23 annotated genes and 10 predicted genes. Thirty-two genes (97%) were occupied by REST and showed greater than 1.5-fold enrichments in ChIP assays, in at least one of the three cell types (Figure 5). Four genes comprising Resp18 and ENSMUSG00000038457 in NS cells, Zfm1 in ES cells, and NM_173446
[GenBank]
in NS cells and hippocampus exhibited markedly high enrichments of 18-, 20-, 46-, 36-, and 12.6-fold, respectively.
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The results showed that the equivalent RE1 sites were differentially bound by REST in ES, NS cells, and hippocampus. RE1 sites associated with Ntrk3, ENMUSG000000 41339/17k, Olfr1336, ENSMUSESTG00000031508, CREBBP/EP300 inhibitory protein 2, Accn1, Arc, Atp2b2, and Zfm1 genes were bound by REST in ES and NS cells, but not in hippocampus, whereas the RE1 sites associated with Drd2, ENSMUSG00000028804, Syt2, ENSMUSG00000015719, and Kirrel3 genes were bound by REST in ES and hippocampus, but not in NS cells. Conversely, RE1 sites associated with NM_175461 [GenBank] , Apba2, and NM_173446 [GenBank] genes were bound by REST in NS cells and hippocampus, but not in ES cells. Furthermore, some RE1 sites were bound by REST in only one cell type including Pkd1l3 and Mbp genes in ES cells, the Cacna2d3 gene in NS cells, and Kcnh1 and Cspg3 genes in hippocampus. Only one RE1 site, which was located 30 kb upstream from the coding region of Cspg4 gene, failed to show any occupancy by REST in any cell type. Collectively, these data show that each cell type bears a unique REST occupancy signature.
Gene Expression Profile of the REST Differentially Targeted Genes
To examine the relationship between REST occupancy and expression of the corresponding gene, we carried out real-time quantitative PCR to determine the expression profile of REST target genes in each cell type. Of the 33 verified REST target genes, 20 were randomly selected to determine their expression pattern. One striking difference was evident in the contrast of the expression profile seen in ES and NS cells compared with that seen in the hippocampus. Although most genes were either silent or expressed at relatively low level in both ES and NS cells, with one exception, all genes were expressed at relatively high levels in hippocampus (Table 3). In ES cells, only the Olfr1336 was silent. In NS cells, 10 of the 20 genes were silent, whereas CREBBP/EP300 inhibitory protein 2, Kirrel3, and ENSMUSG00000051719 genes were expressed at relatively high levels. In hippocampus, only the olfactory receptor gene, Olfr1336, was silent. All other genes, including the five unannotated predicted genes were expressed at high levels relative to ES and NS cells.
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Most of these target genes are reported to be specifically or selectively expressed in neurons. Exceptions include CREBBP/EP300 inhibitory protein 2 and Zfm1, which are widely expressed and Kirrel3, which is expressed in both neural and stromal cells. The fact that REST and so many target genes are coexpressed in the hippocampus, further underscores the principle role of REST in the adult CNS as a regulator, but not a silencer of neuronal gene expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), ChIP Display (Barski and Frenkel, 2004), SACO (Impey et al., 2004), GMAT (Roh et al., 2005), and STAGE (Kim et al., 2005). All of these methodologies combine ChIP with SAGE, differential display or microarray to reduce the overwhelming contribution of nonspecific products to ChIP pulldown. Their success rate in identifying validated target genes ranged from 60 to 85%. In this study, we have developed a novel strategy that uses a selective amplification of ChIP (SACHI) to identify cell and stage specific interactions of REST with its target genes with a success rate up to 97%. Among the isolated REST target genes, Crhr2, Cdk5r2, Syt2, Syt7, Kcnh1, Cspg3, L1cam, and Ntrk3 have been identified in our previous study (Bruce et al., 2004). We have used this new strategy to identify 81 additional REST target genes from ES, NS, and hippocampus. This new protocol could also be used to isolate the target genes of other transcription factors as long as the length of their DNA binding sites exceeds five base pairs.
REST Target Genes and Neuronal Function
Many REST target genes involved in regulating core aspects of neuronal phenotype such as vesicular transport and release, signaling, and neurite outgrowth. Several known REST target genes are involved in neurite outgrowth and/or axonal guidance through regulation of intercellular and cell matrix interactions and of the intracellular cytoskeleton and include L1cam, Cdh4, Adam23, Catnd2, Ppp2r2c, and Unc5d (Gerhardt et al., 2000; Kim et al., 2002, Strack, 2002; Nishimura et al., 2003; Frappé et al., 2004; Goldsmith et al., 2004; Liu et al., 2004). This list can now be expanded by the addition of a further four REST target genes, Cspg3, Shank2, Extl3, and Arc, all of which are involved in regulation of cell adhesion or modulation of the cytoskeleton (Fujimoto et al., 2004; Osman et al., 2004; Qualmann et al., 2004). This cohort of target genes lends mechanistic insight into the axon pathfinding errors resulting from constitutive expression of REST (Paquette et al., 2000). Two new targets regulating vesicular trafficking and release include Rab4a and Apba2. These add to the known REST targets, Snap25, Syt2, Syt7, synapsin 1, and synaptophysin and illustrate the ability of REST to regulate every step of neurosecretion from trafficking, through docking to fusion and release.
REST Interacts with the Chromatin of Transcriptionally Active Genes in Differentiated Neurons
Our results show that REST occupies distinct subsets of target genes in ES cells, NS cells and hippocampus. A total of 89 genes were identified in this study, 38 of which are annotated, whereas 51 have no annotation and represent hypothetical genes. Of the 38 annotated REST target genes 24 (65%) encode gene products that are specifically or selectively expressed in neural tissue. Most target genes are transcribed at high levels in the hippocampus compared with ES and NS cells.
All target genes selected for expression analysis were transcribed in hippocampus, with the exception of Olfr1336. Most of the RE1 sites of these genes are occupied by REST. How does REST act principally as a repressor and become an activator in hippocampus? A previous study showed that NRSE dsRNA (RE1 dsRNA) appeared in the early stage of neurogenesis and in hippocampus. During the differentiation of NS cell to neurons, NRSE dsRNA triggered gene expression of neuron-specific genes through interaction with REST transcriptional machinery, by which REST was converted from a repressor to an activator (Kuwabara et al., 2004). If this is the case, why are not all the REST target genes occupied by REST? For example, five genes comprising Atp2b2, Cacna2d3, Ntrk3, Zfm1, and CREBBP/EP300 inhibitory protein 2 are actively transcribed but not occupied by REST in hippocampus. This indicates that existence of NRSE dsRNA cannot explain all facets of REST interaction with its target genes. Equally clear, is that REST cannot operate purely as an "on/off" switch whereby REST acts as a silencer.
REST Interaction with Target Genes in Stem Cells
In contrast to the hippocampus, the majority of target genes are silent in MHP36 neural stem cells and most are occupied by REST, irrespective of their transcriptional status. The only unoccupied sites lie within the Cspg3, Kcnh1, Drd2, and Kirrel 3 genes. The profile of REST occupancy in ES cells is very similar to that of MHP36 cells but unlike MHP36 cells, most REST target genes are transcribed in ES cells, albeit at low levels compared with the hippocampus. As with MHP36 cells, Cspg3, and Kcnh1 are both silent and occupied by REST. This low level of transcription likely reflects a transcriptionally poised state whereby the chromatin is an open configuration before full transcriptional activation upon differentiation (Schubeler et al., 2000; Ragoczy et al., 2003). In general, this study shows that many REST target genes are either silent or weakly expressed in ES and MHP36 cells but are strongly expressed in hippocampus. This may imply that REST is acting to silence or strongly repress these genes in both multipotent and pluripotent cells and then switches to become a regulator in postmitotic differentiated neurons.
A recent study reported that REST was present in ES cells but upon retinoic acid differentiation into neurons, REST was rapidly down-regulated in NS cells and was absent from both cultured cortical progenitors and cortical neurons (Ballas et al., 2005). However, in our study, REST continues to be expressed in NS cells, embryonic hippocampal stem cells, and ES cell-derived NS cells. In vivo, REST is expressed widely throughout the ventricular neuroepithelium (Schoenherr et al., 1995) and is present in many adult cortical neurons (Palm et al., 1998). In the hippocampus, most neurons of the pyramidal and granular layers express REST. In fact, REST levels and REST target gene levels are dynamically regulated in response to ischemic or epileptic insults (Palm et al., 1998; Calderone et al., 2003). Here, we have used the MHP36 neural stem cell line, derived from embryonic mouse hippocampus and which is capable of differentiating into neurons and glia both in vitro and in vivo (Mellodew et al., 2004; Hugnot et al., 2001). As such it represents a homogenous, tractable system to examine REST interactions with its target genes at the neural stem cell stage. In contrast to the study of Ballas et al. (2005) on cortical progenitors and neurons, we find REST is expressed in both hippocampal stem cells and neurons. This difference probably indicates diversity of REST actions in different populations of CNS progenitors and neurons but may also reflect differences in developmental stage.
In summary, we have used a novel ChIP cloning protocol (SACHI) to identify 89 REST target genes. We show that the REST occupies a distinct set of target genes in ES cells, NS cells and hippocampal neurons and that occupancy is not simply related to expression levels. The results suggest that the REST-directed mechanism in the gene expression of its target genes extends beyond the simple "on/off" model and the mechanism may be compounded by combinatorial regulation from other transcription factors or corepressors.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: RE1, repressor element 1; NRSE, neuron-restrictive silencer element; REST, repressor element 1 silencing transcription factor; NRSF, neuron-restrictive silencing factor; ES, embryonic stem cell; NS, neural stem cell; EMSA, electrophoretic mobility shift assay.
The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org). 
These authors contributed equally to this work.
Address correspondence to: Yuh-Man Sun (y.sun{at}bmb.leeds.ac.uk).
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