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Originally published In Press as doi:10.1074/jbc.M310353200 on October 15, 2003

J. Biol. Chem., Vol. 279, Issue 1, 556-561, January 2, 2004
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Distinct RE-1 Silencing Transcription Factor-containing Complexes Interact with Different Target Genes*

Nikolai D. Belyaev{ddagger}, Ian C. Wood{ddagger}, Alexander W. Bruce{ddagger}§, Miyoko Street{ddagger}, Jean-Baptiste Trinh{ddagger}, and Noel J. Buckley{ddagger}||

From the {ddagger}School of Biochemistry and Molecular Biology and School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom

Received for publication, September 17, 2003 , and in revised form, October 13, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Establishment of neuronal identity requires co-ordinated expression of specific batteries of genes. These programs of gene expression are executed by activation of neuron-specific genes in neuronal cells and their repression in non-neuronal cells. Such co-ordinate regulation requires that individual activators and repressors regulate transcription from specific subsets of their potential target genes, yet we know little of the mechanisms that underlie this selective process. The RE-1 silencing transcription factor (REST) is a repressor that is proposed to silence transcription of numerous neuron-specific genes in non-neuronal cells via recruitment of two independent histone deacetylase (HDAC)-containing co-repressor complexes. However, in vivo, REST appears to be an obligate silencer for only a minority of RE-1-bearing genes. Here we examine the interaction of REST, Co-REST, Sin3A, HDAC1, and HDAC2 with two archetypical endogenous target genes, the M4 muscarinic receptor and the sodium type II channel (NaV1.2) genes. We find that these genes are present in distinct chromosomal domains. The NaV1.2 gene is actively transcribed but repressed by REST independently of histone deacetylation or DNA methylation and does not co-localize with epigenetic markers of silence, including dimethylation of H3K9 and HP1. In contrast, the M4 gene is maintained in a silent state independently of REST and co-localizes with dimethylated H3K9 and HP1{alpha} and HP1{gamma}, characteristic of silenced or senescent euchromatic DNA. This contrasts with the coordinate REST-dependent regulation of this locus reported previously. Taken together, we infer that distinct repressor complexes and mechanisms are operative at particular loci even in cell lines derived from a common embryological origin.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Transcriptional repression is a principal means by which developmental programs of gene expression are established. This is manifestly clear in the differentiation of the vertebrate nervous system where repressive mechanisms are employed at all stages of neural development including neurogenesis (1), specification of regional identity (2, 3), and terminal differentiation (3, 4). An emerging theme in transcriptional regulation is the diversity of co-repressor platforms that may be recruited by individual repressors. This diversity has the potential to expand the repertoire of transcriptional regulation directed by individual repressors and may be especially pertinent to phenotypically complex tissues exemplified by the vertebrate nervous system. In this study, we explore this notion using the RE-1 silencing transcription factor (REST)1 (4) also known as the neuron-restrictive silencer factor (5). This transcription factor has been proposed to play an important role in establishing and maintaining expression of neuron-specific genes. This multizinc finger protein interacts with a 23-bp RE-1 cis-element, also known as the neuron-restrictive silencer element. The RE-1 was originally identified as a silencer element in the 5'-flanking regions of the SCG10 and NaV1.2 channel genes (6, 7) and was proposed to be responsible for restricting expression of these genes to the nervous system. Since then, REST has been shown to repress (and, in some cases, activate) expression of at least 30 genes (8), including those encoding the SCG10 (7), NaV1.2 (6), M4 muscarinic receptor (9), synapsin I (10), L1 CAM (11), ChAT (12), {beta}2 nicotinic receptor (13), and GluR2 receptor (14).

In situ hybridization studies have shown that REST is expressed in most non-neural tissues throughout embryonic development and into adulthood. In contrast, in the developing nervous system, REST is transiently expressed by neuroepithelial cells but appears to be extinguished upon terminal differentiation (4, 5). All of these early observations were consistent with the role of REST as a global silencer of neuron-specific gene expression outside of the nervous system. However, several observations are inconsistent with this notion. First, mice lacking REST do not show widespread ectopic expression of all RE-1-containing genes but do show numerous malformations in both epithelial and mesenchymal tissue by E9.5 and die at E11.5 (16). Of several RE-1-bearing genes, including neuronal class III tubulin, SCG10, L1, synapsin, calbindin, and middle neurofilament (NF-M), only {beta}III tubulin was found to be de-repressed. Analysis was complicated by the fact that many RE-1-bearing genes were not expressed in the wild type at the point of embryonic lethality in the knockout mouse. Thus it was possible that continued silence in the homozygous knockout was due to absence of appropriate transcriptional activators. Infection of chick myotome with a viral vector carrying a dominative negative REST construct led to de-repression of two further RE-1-bearing genes, NgCAM and SCG10, each in a specific manner. However, no de-repression of middle neurofilament was observed (16). Although usually associated with repression, recruitment of REST can also lead to activation of gene expression. This can be seen from the repression of NaV1.2 expression in Xenopus neural stripes upon injection of a dominant-negative REST constructs into two-cell stage embryos (17). We have also shown that REST acts as a repressor but not silencer of neuronal gene expression (18). These observations can be placed in a wider context when it is considered that mining of the human genome sequence reveals the full repertoire of RE-1-bearing genes in the human genome to be in excess of 800 (44).2 Taken together, these observations all indicate that, in vivo, RE-1-bearing genes do not show a uniform response to REST. How does this selectivity arise?

The ability to interact with and act upon a select subset of target genes must underpin the transcriptional programs driven by any individual transcription factor in any one cell at any one time, but little is known of how this selectivity is achieved. In principle, selectivity could be achieved at several distinct levels, including access of the transcription factor to its DNA recognition element, recruitment of co-factors and ancillary transcription factors, or response of the general transcription machinery. Several studies have examined the mechanism of REST-mediated repression. An N-terminal repression domain of REST interacts with a Sin3-containing co-repressor complex (19–21), whereas the C-terminal recruits a distinct co-repressor complex that contains Co-REST (22) and a high mobility group-containing component, BRAF35 (23) and BAF57, a component of the hSWI/SNF chromatin-remodeling complex (24). Both co-repressor complexes contain histone deacetylases HDAC1 and HDAC2 that are required for repression of transcription. In the present study we have used two prototypical RE-1 target genes, the M4 muscarinic receptor gene and the NaV1.2 gene, to examine the mechanism of REST-mediated repression. Using chromatin immunoprecipitation we show that these two targets differ in their ability to interact with REST, Sin3, Co-REST, and HDACs and that this difference is reflected in the epigenetic markers present at the chromatin of the target genes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Transient Transfections—JTC-19 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, streptomycin (10 g/liter), and penicillin (10 g/liter). TSA treatments were carried out by supplementing growth media with 100 nM TSA for 24 h.

Transfections—All transfections were carried out using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. Adenoviral delivery was affected by using high titer stocks (1011-1012 plaque-forming units/ml) at a dilution of 1:1,000 to 1:10,000. Infection was close to 100% as adjudged by green fluorescent protein fluorescence. Cells were harvested 48 h after transfection or infection.

Chromatin Immunoprecipitation—Chromatin immunoprecipitations were performed according to established protocols (25, 26), with minor modifications. Cells were fixed with 1% formaldehyde for 8–10 min, after which fixation was stopped by addition of glycine to a final concentration of 125 mM. Cells were washed two times with phosphate-buffered saline, and cell pellets were suspended in IP lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride) and disrupted by extensive shaking with acid-washed glass beads. Cell extracts were removed from the glass beads by puncturing the bottoms of the Eppendorf tubes containing both beads and extracts, and the extract was rapidly spun into a fresh Eppendorf tube. Sonication of the cell extract was performed four times at 15-s pulses with microprobe at 40–50% output and 70% duty cycle. To minimize foaming of the solution, sonications were performed in glass tubes in a total extract volume not less than 2 ml. Under these conditions DNA fragments with an average size of 500–700 bp were obtained. Sonicated extracts were precleared with blocked protein G-Sepharose and incubated by overnight rotation with 1 µg of primary antibodies at 4 °C followed by 1 h with Protein G-Sepharose. Beads were spun at 10,000 x g for 30 s and washed sequentially with increasing concentrations of salts and nonionic detergents. Finally, beads were eluted with 1% SDS in 0.1 M NaHCO3. Both unbound and bound fractions were de-cross-linked by incubation at 65 °C for 6–8 h. De-cross-linked samples were treated with RNase and Proteinase K, and DNA was purified by phenol-chloroform extraction and amplified using Taq polymerase. A rabbit antisera was generated against REST using C-terminal peptide as immunogen. Anti-HDAC1, anti-HDAC2, anti-Sin3A, and anti-HA were all obtained from Santa Cruz Biotechnology, and anti-Co-REST antibody was a kind gift from Gail Mandel (Stony Brook, NY). Anti-HP1{alpha} and anti-HP1{gamma} were obtained from Serotec and Upstate, respectively.

RNA Analysis—RNA was extracted from cells using TRI reagent (Sigma) according to the manufacturer's instructions. RNA was treated with RQ DNase (Promega) prior to reverse transcription with H-MMLV-RT (Invitrogen). cDNA was amplified using Taq polymerase (Promega).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
REST, Sin3A, Co-REST, and HDAC1/2 Are Present at the RE-1s of Both the NaV1.2 and M4 Genes on Transiently Transfected Templates—RT/PCR analysis showed that the REST-expressing cell line, JTC-19, does not express the M4 gene but do express low, but clearly detectable levels of NaV1.2 (see Fig. 4 below). We first sought to verify that reporter genes, containing M4 and NaV1.2 RE-1s, were repressed by REST in JTC-19 cells. For this purpose, reporter genes containing RE-1 elements derived from the human M4 and NaV1.2 genes were cloned upstream of the minimal NaV1.2 promoter. Fig. 1 shows that luciferase activity is 2- to 3-fold lower in reporter gene constructs containing an RE-1 than in the empty vector. Furthermore, TSA relieved repression to a level similar to that seen with the minimal promoter and induced a higher incremental increase in luciferase activity in RE-1-containing reporter genes than those lacking a RE-1. This is a similar fold sensitivity as reported in other RE-1 reporter gene assays (19, 20). Hence REST interacts with the RE-1 of M4 and NaV1.2 reporter genes and represses transcription in a TSA-sensitive manner for both promoters. Interestingly, the NaV1.2 RE-1 was more effective at repressing transcription from the minimal NaV1.2 promoter than was the M4 RE-1. These data mirror earlier results obtained using Neuro2A cells (19). Furthermore, these data show that both promoters are capable of interacting with the general transcription apparatus in these cells and that the silence of the endogenous M4 promoter is not due to an inability to interact with components of the transcriptional machinery.



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  		FIG. 4.
TSA does not induce expression of either the M4 or NaV1.2 genes. JTC19 cells were treated with 100 nM TSA for 24 h, and RNA was harvested, digested with RQ DNase, and reverse-transcribed as described under "Experimental Procedures." cDNA was amplified using PCR and primer pairs specific for the M4 and NaV1.2 transcripts. Controls were provided by omitting reverse transcriptase (-RT).

 



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  		FIG. 1.
The M4 and NaV1.2 promoters are regulated by TSA in transient transfection assays. JTC19 cells were transfected with M4 and NaV1.2 luciferase reporter gene constructs and treated with 100 nM TSA for 24 h. M4-RE-1 and NaV1.2RE-1 contain the RE-1 elements of the M4 and NaV1.2 genes cloned upstream of the minimal NaV1.2 promoter. Values represent the average of three independent transfections, each of which was carried out in triplicate and normalized to cotransfected cytomegalovirus-Renilla. Results are expressed as -fold activity of the minimal NaV1.2 promoter, and error bars represent S.E.

 
Next, we used chromatin immunoprecipitation to see if REST was recruited to the RE-1 elements of the reporter genes. We distinguished signal specifically amplified from transient templates by using primer pairs derived from the pGL3 reporter vector. Fig. 2 shows that REST is recruited to the RE-1 of both M4 and NaV1.2 transient-transfected templates. Control immunoprecipitations were carried out by using an antibody directed against the hemagglutinin (HA) epitope.



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  		FIG. 2.
The transiently transfected M4 and NaV1.2 promoters both interact with REST, Sin3A, and Co-REST. JTC19 cells were transfected with the same reporter constructs as used in Fig. 1. Cells were cross-linked and immunoprecipitated using antibodies against REST, Sin3A, HDAC1/2, Co-REST, and HA as described under "Experimental Procedures." Precipitated DNA was then amplified using PCR. Primers were designed to anneal to vector sites flanking the RE-1 sites to specifically amplify sequences derived only from the transfected template. "input" represents signal derived using input chromatin as template.

 
Having established the presence of REST at the RE-1 of the transient template, we next sought to establish which members of the co-repressor apparatus were also present. Accordingly, a further series of ChIPs was carried out using antisera against Sin3A, Co-REST, HDAC1, and HDAC2. Fig. 2 shows that Sin3A, Co-REST, and HDAC1 and -2 are all recruited to both M4- and NaV1.2 RE-1-containing reporter genes, whereas at the empty vector, REST, Sin3A, and Co-REST were all absent and HDAC1 and HDAC2 were either absent or present at only very low levels. Hence, REST, Sin3A, Co-REST, and HDAC containing co-repressor platforms are all present at the RE-1 of transiently transfected templates containing the M4 and NaV1.2 promoters. The resolution of ChIP assays is clearly limited by the shear size of the DNA but, because the only difference between the reporter genes is the presence or absence of an RE-1, then it can be clearly concluded that Sin3A, Co-REST, HDAC1, and HDAC2 are all recruited by REST and not by adjacent sequence. The presence of HDAC at RE-1-containing reporter genes is entirely consistent with ability of TSA to de-repress the NaV1.2- and M4 RE-1-containing reporter genes. Furthermore, these data show that both RE-1s are capable of interacting with a complex or complexes that contains REST, Sin3A, Co-REST, HDAC1, and HDAC2.

REST Is Present at the Endogenous NaV1.2 RE-1 but Not at the Endogenous M4 RE-1—Next, we carried out a further series of chromatin immunoprecipitation assays to assess the interaction of REST with the RE-1 of the endogenous M4 and NaV1.2 genes. Cross-linking and immunoprecipitations were carried out in an identical manner to those used for assessing REST interaction with transient reporter templates, but PCR was carried out using primer pairs flanking the RE-1 of the chromosomal genes. In contrast to results obtained with the transiently transfected templates, these experiments showed a marked difference between the interaction of REST with the M4 gene and with the NaV1.2 gene. Whereas REST is present at the RE-1 of the NaV1.2 gene, it was not detectable at the RE-1 of the M4 gene (Fig. 3). Failure to detect a bound transcription factor by ChIP may mean that the factor needs ancillary co-factors to allow binding or that the epitope is occluded by bound co-factors. We consider that both of these explanations are unlikely, because the same sequence can be immunoprecipitated when present as a transient template in the same cells (see above). Furthermore, use of REST antibodies directed against a different epitope gave identical results (18). All of these observations strongly suggest that REST is absent from the endogenous M4 locus.



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  		FIG. 3.
The chromosomal M4 and NaV1.2 promoters differentially interact with REST, Sin3A, Co-REST, and HDAC1/2. JTC19 cells were cross-linked and immunoprecipitated using antibodies against REST, Sin3A, HDAC1, HDAC2 Co-REST, HP1{alpha}, HP1{gamma}, dimethylated H3K9, and HA as described under "Experimental Procedures." Precipitated DNA was then amplified using PCR. Primers were designed to amplify regions flanking the chromosomal RE-1 sites of the NaV1.2 and M4 genes. "input" represents signal derived using input chromatin as template. All image blocks were processed identically during construction to ensure valid comparison of band intensities.

 
Sin3A and Co-REST Are Present at the Endogenous NaV1.2 RE-1 but Not at the M4 RE-1—Having shown that transiently transfected reporter genes and their chromosomal counterparts differ in their ability to bind REST, we then investigated whether there were any further differences with respect to their ability to recruit co-repressors. Again we used ChIP to detect the presence of Sin3A, Co-REST, and HDAC1/2 at these two chromosomal loci. At the M4 locus where REST is not detected, all co-repressor molecules were also not detected. However, at the NaV1.2 locus, where REST was present, Sin3A and Co-REST were also present, but surprisingly, HDAC1 and HDAC2 were absent (Fig. 3). Because HDACs were absent from both chromosomal loci, these results led us to propose that expression of both the endogenous NaV1.2 and M4 genes would be insensitive to attenuation of HDAC activity by TSA challenge. This indeed was the case. RT-PCR analysis showed that expression of neither gene was altered by 24-h treatment with 100 nM TSA (Fig. 4). This is in marked contrast to the observed activation of the corresponding reporter gene by the same treatment (see above) and demonstrates a clear difference between the co-repressor platforms recruited to transient and chromosomal templates of these two genes.

REST Mediates Repression of the Endogenous NaV1.2 Gene in an HDAC-independent Manner—The presence of REST and absence of either HDAC1 and HDAC2 at the NaV1.2RE-1 combined with the lack of effect of TSA on NaV1.2 expression led us to investigate whether REST was required for NaV1.2 repression. Accordingly we infected JTC-19 cells with an adenoviral construct carrying a dominant-negative REST construct consisting of the REST DNA binding domain (AdVREST:DBD). 48 h later, RNA was harvested and analyzed. As can be seen from Fig. 5, infection led to activation of NaV1.2 expression but M4 expression was undetectable either in the absence or presence of TSA. Infection with empty adenovirus (AdV) had no effect. Clearly REST does repress NaV1.2 expression but in a manner that does not require histone deacetylase activity.



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  		FIG. 5.
The endogenous NaV1.2 but not the M4 gene is induced by dominant-negative REST. JTC-19 cells were infected with either AdEASY (AdV) or AdEASY carrying the DNA binding domain of REST (AdVREST:DBD). RNA was harvested 48 later and subjected to RTPCR analysis as described. At the point of cell harvest, 100% of cells appeared infected as adjudged by green fluorescent protein fluorescence. Primer pairs derived from the coding region of glyceraldehyde-3-phosphate dehydrogenase, M4, and NaV1.2 were used to amplify cDNA. Note that fewer amplification cycles were used than in the experiment in Fig. 4 to ensure that the enhanced signal seen in Ad-VREST:DBD-treated cells was not saturating.

 
The Endogenous M4 but Not the NaV1.2 Gene Is Associated with Heterochromatic Markers—Because the M4 gene appeared to be silenced in an REST-independent manner, and the NaV1.2 gene appeared to be active but repressed in an REST-dependent manner, we used ChIP to examine both genes for the presence of epigenetic makers of silenced chromatin, namely dimethylated K9H3 and HP1{alpha} and HP1{gamma} (27). As can be seen in Fig. 3, H3K9 is dimethylated and HP1{alpha} and HP1{gamma} are recruited at the M4 gene but not the NaV1.2 gene supporting the view that only the M4 gene is present in silent chromatin, whereas the NaV1.2 gene is present in open chromatin but is nevertheless repressed by REST


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Many studies have examined interaction of REST with its potential target genes using a combination of DNA-protein interaction assays (such as electromobility shift assays) and by monitoring expression of transiently transfected reporter genes (6, 7, 9, 10, 12, 14, 19, 28–33). Such studies invariably show that all bone fide RE-1s can interact with REST and that this recruitment results in repression or silencing of the reporter gene. However, it is probably more parsimonious to regard such studies as providing insight into the potential of a cis regulatory element to interact with its cognate transcription factor. Both techniques take advantage of DNA templates that are either devoid of nucleosomes or have a chromatin conformation that differs from that of their chromosomal counterparts. This difference in transcription factor accessibility between transient and chromosomal templates is well known and can be clearly seen in studies of the mouse mammary-tumor virus promoter where transiently transfected templates containing the mouse mammary-tumor virus promoter are assembled on unphased nucleosomes, and transactivation by the glucocorticoid receptor is independent of the human Brahma-related gene chromatin-remodeling complex (34). In contrast, the chromosomal template is packaged into a nucleosomal array and glucocorticoid receptor transactivation does require remodeling by the human Brahma-related gene complex. Similarly, the nuclear factor 1/CCAAT transcription factor is constitutively bound at the transient template, but binding to the chromosomal template is hormone-dependent (35). In other words, the chromatin conformation of an endogenous locus is a determining factor in specifying which target genes interact with their cognate transcription factor in vivo.

Transiently Transfected Templates and Endogenous Target Genes Recruit Different REST Complexes—Against this background, we examined interaction of REST with its endogenous chromosomal target genes using chromatin immunoprecipitation. When this is done, a different picture emerges of REST action than that seen using transiently transfected templates. Although both the M4 and NaV1.2 genes are widely recognized as REST targets (3, 6, 7, 9, 19, 31), our results show that only the NaV1.2 RE-1 is occupied by REST in vivo in JTC-19 cells. Surprisingly, when HDAC activity is inhibited by TSA, neither of these two genes is induced, despite the presence of REST at the NaV1.2 RE-1. Yet reporter genes containing the promoters and RE-1s of both of these genes can drive transcription when expressed as transient-transfected templates and RE-1-dependent repression of reporter gene expression is TSA-sensitive. This latter observation clearly shows that the lack of induction of the endogenous chromosomal genes by TSA is not attributable to absence of appropriate activators or the inability to detect HDACs at REST occupied RE-1s. The presence of REST, HDAC1 and -2, Sin3A, and Co-REST at the RE-1 of the M4 transient templates shows that REST is perfectly capable of recruiting the co-repressor apparatus to the M4 RE-1 and that the absence of the equivalent complex at the endogenous gene probably reflects a difference in chromatin conformation between the chromosomal gene and the corresponding transiently transfected template. Whereas, the absence of effect of TSA on endogenous M4 expression was consistent with the absence of REST at the RE-1 of the M4 promoter, the inability of TSA to induce NaV1.2 expression was more surprising, given that REST is bound and known to be capable of recruiting two HDAC1/2 containing co-repressor complexes (3, 19–23, 36, 37). The presence of REST at the NaV1.2 RE-1 gene coupled with the absence of any effect of TSA suggested that, despite the presence of REST, other members of the co-repressor apparatus were either absent or non-functional. ChIPs using antibodies against Sin3A, Co-REST, HDAC1, and HDAC2 showed all co-repressor molecules were absent from the RE-1 of the M4 gene, whereas, in contrast both Co-REST and Sin3A were present at the NaV1.2 RE-1 but HDAC1 and HDAC2 were absent. Hence, the lack of effect of TSA on endogenous NaV1.2 expression was clearly consistent with an absence of any recruited deacetylase activity, despite the presence of both corepressors, Sin3A and Co-REST.

Distinct HDAC Requirement by REST at Different Promoters— REST-dependent repression has been shown to be TSA-sensitive at a number of transiently transfected reporter genes, including the M4 (19), NaV1.2 (3, 19, 21), GluR2 (21), CRH (38), and ANP (28). Antibody microinjection studies have also shown that REST repression of transiently transfected reporter genes in Rat-1 fibroblasts requires N-CoR, Sin3A/B, and HDAC2 (39). However, an increase in activity of endogenous RE-1-bearing genes in response to TSA has been reported in only a minority of studies (3, 20, 28). In the case of the ANP gene, although it was not directly demonstrated that REST was actually present at the endogenous ANP gene, it was nevertheless shown that TSA increased activity and acetylation of both the endogenous ANP gene and RE-1-bearing reporter genes (28). By itself, TSA data must be treated with caution, because TSA can effect global acetylation patterns of core histones and non-histone targets (40–43), and consequently, its effects may not be solely attributable to targeted deacetylation by REST. Interestingly, one recent study reported that TSA relieved repression of the silent endogenous NaV1.2 in rat L6 cells and ChIP assays showed the RE-1 of NaV1.2 to be occupied by REST, Co-REST, and HDAC2 (3). However, this same group later reported that the silent NaV1.2 in Rat-1 fibroblasts recruited only REST and Co-REST but no HDACs, and accordingly expression of NaV1.2 was TSA-insensitive (44). This raises the possibility that repressor and co-repressor recruitment by REST varies according to both promoter and cell type (see below). Indeed this is likely to be the case, because not all RE-1-bearing genes are silenced in all REST-expressing cell types. In fact, this cell selectivity can be seen even with transient templates. An earlier study from our laboratory showed that REST-mediated repression of both M4 and NaV1.2 reporter genes was TSA-sensitive in 3T3 cells, but only repression of the M4 reporter was TSA-sensitive in Neuro2A cells (19). A further disparity between HDAC recruitment and TSA sensitivity was revealed in a study of the effects of TSA on histone acetylation at the GluR2 promoter in C6 glioma cells. In this case, TSA increased H3 and H4 acetylation, whereas, in cortical neurons, basal levels of both H3 and H4 acetylation were high and TSA produced no incremental increase in histone acetylation (21). Thus, HDAC recruitment and HDAC requirement do not always go hand in hand.

Co-repressor Recruitment Varies According to Cell Type— The data we present here show marked differences with those of Lunyak et al. (44) who reported that, in Rat-1 fibroblasts, the M4 and NaV1.2 genes were present within and flanked a 10-centimorgan silenced domain of chromosome 3 that contained the NaChIII, GAD1, and HoxD9 genes. This region had characteristics of silenced chromatin, including DNA methylation and presence of MeCP2 and HP1. Furthermore, they showed that this silencing was dependent upon REST, Co-REST, and DNA methylation but independent of histone deacetylation and proposed that the entire locus was silenced by REST recruited at the RE-1 sites of the M4, NaV1.2, and GAD1 genes. However, inspection of a later release of the rat genome (eNSEMBL 15.2.1; release of July 1, 2003) shows that, in fact, this 23-Mbp locus contains somewhat more than five genes and that neither the NaChIII nor HoxD9 are present within this location. In fact, HoxD9 is present within the HoxD cluster on chromosome 11, whereas NaChIII actually lies outside of the silent locus that the authors describe. Furthermore, NeuroD, which the authors claimed to lie outside of the locus and to be regulated independently, actually lies within the locus. Here we show that in rat JTC-19 fibroblasts, the same two genes are present in distinct chromosomal domains. On the one hand, the NaV1.2 is actively transcribed but repressed by REST independently of histone deacetylation or DNA methylation and does not colocalize with either HP1{alpha}, HP1{gamma}, or dimethylated H3K9. On the other hand, the M4 gene is maintained in a silent state independently of REST, is unaffected by inhibition of histone deacetylase or DNA methylation (data not shown), and colocalizes with markers of silent or senescent (27) DNA, including dimethylation of H3K9 and HP1{alpha} and HP1{gamma} (Fig. 6). This leads to the intriguing notion that different transcriptional mechanisms are operative on a particular locus even in cell lines derived from a common embryological origin. Seemingly, in Rat-1 fibroblasts, the NaV1.2 and M4 genes appear to be silenced in a manner that is seeded or maintained, at least partially, by REST and Co-REST, whereas in JTC-19 fibroblasts, these two genes are not co-ordinately regulated and do not co-habit a common chromatin domain. Furthermore, it would appear that REST is capable of acting as a silencer of the NaV1.2 in Rat-1 fibroblasts and as a repressor of the same gene in JTC-19 cells and that this difference is reflected in recruitment of distinct co-repressor complexes. We do not know at present the factors that determine this differential regulation among closely related cell types.



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  		FIG. 6.
RE-1 sites recruit a range of REST-containing repressor complexes. A, the recruitment of a complete REST/Sin3A/Co-REST/HDAC1/HDAC2 repressor complex to the RE-1 of a reporter gene and the subsequent de-repression by TSA. B, shows the absence of any REST-containing repressor complex at the silent endogenous M4 gene. H3K9 is dimethylated and HP1 is present. No repression is seen and no induction is seen upon TSA inhibition of HDAC activity. Note that the position of the RE-1 is not meant to infer that it is nucleosomal; it is indicated purely for graphic purposes. C, similarly shows the recruitment of a REST/Sin3A/Co-REST complex that contains no HDAC1 or HDAC2 to the RE-1 of the endogenous NaV1.2 gene. H3K9 is not methylated, and HP1{alpha} and HP1{gamma} are absent. Repression occurs independently of HDAC activity, and accordingly, TSA does not relieve repression.

 
Although co-repressor complexes are often regarded as stable, fixed entities, several studies have shown that HDAC association with co-repressor complexes is labile and subject to regulation. One recent study has shown that hyperphosphorylation of HDAC1 and -2 leads to their dissociation from Sin3 during mitosis (45). In another study, the DNA binding domain of myocyte enhancer factor-2 was shown to interact with the class II HDACs, HDAC4 and HDAC5, in the absence of growth factors, but upon activation by calmodulin-dependent protein kinase by mitogen challenge, HDAC dissociated with concomitant unmasking of the myocyte enhancer factor-2 transactivation domain (46). Retinoblastoma protein (Rb) forms a complex with HDAC and SWI/SNF to inhibit transcription of the cyclin E gene, but phosphorylation of Rb by cyclinD/cdk4 leads to release of HDACs and relief of repression of cyclin E transcription (47). Interestingly. In this latter study Rb/SWI/SNF was shown to be sufficient to maintain inhibition of cyclin A and cdc2 in the absence of associated HDAC activity (15, 47) indicating that different target genes require different co-repressor platforms. Recruitment of multiple co-repressor and co-activator platforms by individual transcription factors is an increasingly recognized phenomenon. Its importance lies in the potential increase in the repertoire of responses mediated by a single transcription factor and further underscores the need to examine interaction of transcription factors and their co-factors at potential target genes in vivo. Taken together, we have shown that REST is capable of recruiting at least two co-repressor complexes, one containing and one lacking HDAC activity. Furthermore, the nature of the interaction of REST with its endogenous target genes is governed by its ability to recognize its recognition site and by its recruitment of components of the co-repressor apparatus and their subsequent relationship with modified histones and associated proteins. These interactions are complex and vary among the same genetic loci even within cells of common embryological origin. We envisage that this spectrum of interactions is not dictated by the particular promoter type per se but that REST target gene profiles will probably vary according to cell type and developmental stage.


   FOOTNOTES
 
* This work was supported by grants from the Wellcome Trust and the Biotechnology and Biological Sciences and Research Council (BBSRC). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ A BBSRC Ph.D. student. Back

|| To whom correspondence should be addressed. Tel.: 44-113-343-3020; Fax: 44-113-343-3167; E-mail: n.j.buckley{at}leeds.ac.uk.

1 The abbreviations used are: REST, RE-1 silencing transcription factor; E, embryonic day; HDAC, histone deacetylase; TSA, trichostatin A; HA, hemagglutinin; ChIP, chromosomal immunoprecipitation; DBD, DNA binding domain; AdV, adenovirus; RT, reverse transcriptase. Back

2 A. W. Bruce, I. J. Donaldson, I. C. Wood, S. A. Yerbury, A. Kel, M. Chapman, M. I. Sadowski, B. Cöttgens, and N. J. Buckley, manuscript submitted. Back



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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