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Chromatin remodeling enzymes, such as SWI/SNF, use the hydrolysis of ATP to power the movement of nucleosomes with respect to DNA. BRG1, one of the ATPases of the SWI/SNF complex can be recruited by both activators and repressors, though the precise role of BRG1 in mechanisms of repression has thus far remained unclear. One transcription factor that recruits BRG1 as a corepressor is the Repressor Element 1-Silencing Transcription factor (REST). Here we address for the first time the mechanism of BRG1 activity in gene repression. We show that BRG1 enhances REST mediated repression at some REST target genes by increasing the interaction of REST with the local chromatin at its binding sites. Furthermore, REST-chromatin interactions, mediated by BRG1, are enhanced following an increase in histone acetylation in a manner dependent on the BRG1 bromodomain. Our data suggest that BRG1 facilitates REST repression by increasing the interaction between REST and chromatin. Such a mechanism may be applicable to other transcriptional repressors that utilise BRG1.
Repression of genes often involves the establishment and maintenance of chromatin in a structure that prevents transcription. ATP-dependent chromatin remodeling activities have more typically been associated with transcriptional activators but more recent evidence has implied their role in transcriptional repression. Many of these chromatin modifying enzymes do not bind DNA specifically and whilst it is possible for chromatin to be modified in a global manner (1), more often the enzymatic activity is targeted to appropriate genes by site-specific transcription factors via complexes containing multiple proteins.
The Repressor Element 1-Silencing Transcription factor (REST), also known as Neuron Restrictive Silencing Factor (NRSF), is a 116 kDa C2H2 Krüppel-type transcription factor that acts as a repressor of numerous genes, many of which are involved in neuronal function (2,3). However, this protein has varied and complex functions that extend into neurogenesis (4-6), cardiogenesis (7) and oncogenesis (8,9). REST binds to the 21 bp Repressor Element 1 (RE1), also known as Neuron Restrictive Silencing Element (NRSE), present in the regulatory regions of its target genes. REST was first identified on account of its binding to a negative-acting DNA regulatory element in the 5' regions of the genes encoding two proteins that are important for neuronal function, the voltage-dependent sodium channel type II (NaV1.2, encoded by SCN2A2) and the growth-associated protein, superior cervical ganglion 10 (SCG10, encoded by STMN2) (2,3). Recently, we have identified putative RE1s in more than 1000 loci within the human genome and these sequences are responsible for the negative regulation of genes that code for proteins involved in many cellular functions (10,11).
REST has been shown to interact with a number of proteins, many of which are required for its repressor function, including histone deacetylases HDAC1 and HDAC2 (12-14), SWI/SNF components BAF57, BAF170, and BRG1 (15), a H3 K4 histone demethylase LSD1 (16) and the H3 K9 histone methyltransferase G9a (17). Whereas many of these proteins have known repressor functions, BRG1 is more commonly associated with transcriptional activation. However, some studies have suggested that BRG1 is required for repression. For example, in Rb-E2F-(18) and p65-mediated repression (19) and in the repression of the c-fos (20), CAD (21) and TRPO (22) genes (18, 21, 22). BRG1 has also been found in corepressor complexes associated with the transcriptional repressor REST (15) and tumour cell lines lacking BRG1 activity show increased expression of some REST-repressed genes (23). Despite the evidence of a role for BRG1 in transcriptional repression, the mechanisms underlying this repression have not been identified. Here we show that the chromatin remodeling activity of BRG1 is required for efficient REST occupancy at some RE1 sites within chromatin, suggesting that BRG1 acts to facilitate REST binding to promoters. This BRG1 facilitated occupancy is dependent on chromatin remodelling activity and is enhanced by increased histone acetylation via the BRG1 bromodomain. This is the first description of a mechanism by which a chromatin remodeling enzyme facilitates repression by a transcription factor and is likely to be applicable to other transcriptional repressors that utilise BRG1.
HEK293 cells were cultured at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium (PAA Laboratories.), supplemented with 10% (v/v) foetal calf serum, 2 mM L-glutamine, 6 g/L penicillin, 10 g/L streptomycin and 1% non-essential amino acids.
Nuclear protein extracts from HEK293 cells were prepared essentially as described by Andrews and Faller (24). Protein concentrations were determined by Bradford assay and 20 μg of each extract was boiled for 10 minutes in 2x SDS loading buffer (100 mM Tris-HCl pH 6.8, 4% SDS, 200 mM DTT, 20% glycerol, 0.2% bromophenol blue) and cooled on ice. Denatured protein samples were electrophoresed through an SDS/6% (w/v) polyacrylamide gel. Proteins were transferred to a HybondC Extra membrane (Amersham) and the membrane screened with 1:1000 anti-BRG1 (25) in 5% (w/v) milk powder in PBS Tween 20 0.1% (v/v), followed by 1:2000 secondary HRP-conjugated anti-rabbit antibody (Santa-Cruz). Bands were visualised using an ECL plus detection kit (Amersham) and the membrane was exposed to x-ray film (Kodak). To control for equal protein loading, membranes were stripped in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris HCl pH 6.7 for 30 minutes at 50°C, washed in PBS 0.1% Tween 20, and re-probed with an anti-mouse TAF1 antibody (Upstate).
Cells were washed twice with PBS, resuspended in extraction buffer (10mM Tris-HCl pH 8.0, 0.15 M NaCl, 5mM EDTA, 0.5% Triton X-100) and passed through a 23G needle 10 times. Extracts were centrifuged for 5 minutes at 13,000 rpm at 4°C and equal aliquots of the supernatant were incubated with a sham antibody (pre-immune serum) or an anti-REST antibody (26) overnight at 4°C. Antibody complexes were precipitated with protein G-sepharose and washed twice with extraction buffer. Proteins were eluted in SDS loading buffer along with 10% input and subjected to Western blotting with an anti-BRG1 antibody (Abcam).
the FLAG epitope-tagged BRG1 DN construct was excised from pBS.CehBRG1K798R (a kind gift from Dr. Tony Imbalzano, University of Massachusetts) using ClaI and SpeI and ligated into the pCS2+ vector, digested with ClaI and XbaI.
HEK293 cells were transfected with pAdTrack.RESTDN (REST DN), pCS2+.BRG1K798R (BRG1 DN), pcDNA4.BRG1Δbromo (BRG1 deltabromo) (27) or without DNA for mock transfection, with Lipofectamine and Plus reagent (Invitrogen), according to manufacturer's protocol. TSA treated cells were incubated in media, including 100 nM trichostatin A (TSA) or an equivalent volume of methanol (vehicle control), at 37°C in 5% CO2 for 48 hours, following transfection and TSA treatment, before harvesting for reverse transcription-PCR (RT-PCR) and chromatin immunoprecipitation (ChIP).
Cells were harvested in 1 ml Tri-reagent (Sigma) and RNA purified as per manufacturer's instructions and resuspended in 50 μl TE, pH 8.0. Genomic DNA was removed by incubation with 1 μl DNase (2 U) and 5 μl of 10x buffer (Ambion) for 30 minutes at 37°C. 5 μg RNA in 50 μl was reverse transcribed with 2.5 μl of random primers (1.25 μg) and 2.5 μl of oligo (dT) (1.25 μg), 20 μl 5x buffer, 20 μl 8 mM dNTPs, 2.5 μl RNasin (100 U) and 2.5 μl Murine Moloney Leukaemia virus (M-MLV) H (-) reverse transcriptase (500 U) at 37°C for 60 minutes. cDNA samples were purified using a PCR purification kit (Qiagen) and of the resulting 100 μl cDNA, 2 μl were used in a 20 μl real time PCR reaction in duplicate (BioRad iCycler MyiQ). Non-reverse transcribed RNA samples were included in the PCR to control for genomic DNA contamination. Primers were designed to coding regions of genes and expression levels were divided by those of the housekeeping gene cyclophilin, to normalise for starting levels of DNA. Primer sequences: CHRM4 sense 5'-GGCAGTTTGTGGTGGGTAAG-3', antisense 5'-GCAGGTAGAAGGCAGCAATG-3'; Cyclophilin sense 5'-ACCCCACCGTGTTCTTCGAC-3', antisense 5'-TGGACTTGCCACCAGTGCCA-3', L1CAM sense 5'-AGGAAGGGGAGTCAGTGGTT-3', antisense 5'-GTCCTGCTTGATGTGCAAGA-3'; REST sense 5'-ACTTTGTCCTTACTCAAGTTCTCAG-3', antisense 5'-ATGGCGGGTTACTTCATGTT-3'; SCN2A2 sense 5'-TGTGGTGGTCATTCTCTCCA-3', antisense 5'-GGATCACTCGGAACAGGGTA-3'; SNAP25 sense 5'-TACACAGAATCGCCAGATCG-3', antisense 5'-ACCACTTCCCAGCATCTTTG-3'; STMN2 sense 5'-TGAAGTCGTTTCTCCCCAAC-3', antisense 5'-TCACAGCTTGCTCACAATGA-3'.
Chromatin was immunoprecipitated from ten 10 cm dishes of HEK293 cells (approximately 1 × 108 cells) as follows. Cells were washed in PBS and cross-linked with formaldehyde at a final concentration of 1% in PBS for 10 minutes at room temperature. Fixation was quenched by addition of glycine to a final concentration of 125 mM and the cells harvested and centrifuged for 5 minutes at 13,000 rpm at 4°C. The chromatin was fragmented into approximately 500 base pair lengths by sonication (Bandelin), 8 × 30 second pulses, on ice, at 30% power, duty cycle 7. The chromatin was pre-cleared with 5% BSA-blocked protein G-Sepharose and 270 μl input chromatin was removed at this point and stored at -20°C. Samples of chromatin were incubated at 4°C overnight with 5 μg of primary antibodies or control IgG or 10 μl rabbit serum antibodies or normal rabbit serum. Protein G-Sepharose beads were incubated with the chromatin-antibody complexes for 3 hours and the chromatin-antibody-bead complexes were collected by centrifugation. The beads were washed twice in wash buffer 1 (20 mM Tris-HCl, pH 8.1, 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once in wash buffer 2 (10 mM Tris-HCl, pH 8.1, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid) and twice in 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA before the chromatin-antibody complexes were eluted with 1% SDS in 100 mM NaHCO3. The immunoprecipitated samples, along with the inputs, were decross-linked by incubation at 65°C for 6 hours. Decross-linked samples were treated with 0.5 μl of 10 mg/ml RNase (0.35 U) and 9 μl of 25 mg/ml Proteinase K (6.75 U), and DNA was purified by phenol-chloroform extraction and resuspended in 100 μl water.
Anti-REST antibody (P18) was obtained from Santa Cruz Biotechnology, anti-BRG1 serum (25) and anti-acetyl H4 was obtained from Upstate. Primers were designed proximal to RE1s and real time PCR was performed on the samples in duplicate. The fold enrichment of the IP was calculated by dividing the starting quantities of DNA in the immunoprecipitated samples by that of the control antibody (IgG for affinity purified antibodies or normal rabbit serum for crude sera antibody preparations). Primers to the CHRM4 coding region (non-RE1 region) were used as a negative control. Primer sequences: CHRM4 RE1 sense 5'-GGCCTGTAACCCCAAATTC-3', antisense 5'-GGGGAGGGTCTTGAGTTGTT-3'; L1CAM RE1 sense 5'-TCCTCACCTTCTCCCTGTTC-3', antisense 5'-TACCCAACGTCCTGGCTATC-3'; SCN2A2 RE1 sense 5'-GGCTGATCTGGGGACTTTTA-3', antisense 5'-AGGTCTTAAACGGGTCTGAAA-3'; SNAP25 RE1 sense 5'-ACCAAATTGTCTCCCTGAGA-3', antisense 5'-GGAGGGGAACAGGAAATTGT-3'; STMN2 RE1 sense 5'-CTGTTCCAGTCCAGTAGCATC-3', antisense 5'-AACCTCATGGACATTTTGCTG-3'.
To determine the role of BRG1 in REST repression we used HEK293 cells, which express functional REST protein (28) and BRG1 (Figure 1a lane 1). First we examined the recruitment of BRG1 to known RE1 sites. Chromatin from HEK293 cells was precipitated with the anti-BRG1 antibody or IgG and the presence of RE1 sites from five well characterized REST regulated genes (2,3,10,29,30) was quantified by real time PCR. BRG1 was enriched at each of the five RE1s under study but not at a control sequence in HEK293 cells (Figure 1b). BRG1 and REST are present in a single complex, as shown by the co-immunoprecipitation of the two proteins in HEK293 cells (Figure 1c). To determine whether the BRG1 recruitment to RE1s is mediated by REST, a dominant negative REST construct (REST DN) was transfected into HEK293. REST DN contains only the DNA binding domain of REST, without the N- and C-terminal repression domains and inhibits REST function (31,32). Expression of REST DN resulted in a specific reduction in the recruitment of BRG1 at each of the five RE1s under study but had no effect on the control sequence (Figure 1d). Together, this data suggested that BRG1 is recruited to each of these RE1s and that this interaction occurs via the N- or C-terminus of REST (Figure 1d). This observation is consistent with earlier studies that have reported interactions between BRG1 and mSin3A (21,33) and BRG1 and CoREST (15).
Having shown that BRG1 is recruited to RE1 sites, we wanted to determine whether BRG1 is required for REST-mediated repression. To do this we utilised BRG1 dominant negative, which contains a point mutation in the ATPase domain (K798R) (34). Accordingly, we transfected HEK293 cells with REST DN or BRG1 DN and examined the effect on the expression of the five RE1 containing genes previously examined. Expression of either REST DN or BRG1 DN resulted in increased expression of each of the five transcripts (Figures 2a and 2b). We considered the possibility that BRG1 DN may affect expression of RE1 containing genes indirectly by decreasing endogenous REST expression levels. Thus, we examined the expression level of REST in response to BRG1 DN. REST levels were not changed in response to BRG1 DN (Figure 2b). Taken together with the demonstration that BRG1 DN was recruited to each of the RE1 sequences (Figure 2c), this suggests that BRG1 plays a direct role in facilitating REST repression.
Stable recruitment of MyoD to the myogenin promoter during muscle differentiation requires BRG1 chromatin remodeling activity (35). Thus we hypothesised that the chromatin remodeling activity of BRG1 may also be important for efficient occupancy of REST at RE1 sites within chromatin. To test if BRG1 chromatin remodeling activity affects the ability of REST to bind RE1s we examined the effect of BRG1 DN on REST recruitment to RE1 sites. Expression of BRG1 DN resulted in a significant decrease in REST recruitment at the highly enriched SNAP25 and L1CAM RE1 sequences (Figure 2d). Recruitment at the moderately enriched CHRM4 RE1 was also reduced (though this reduction did not achieve statistical significance) whilst we could not detect any change in REST occupancy at the STMN2 and SCN2A2 RE1 sequences (Figure 2d). Although the BRG1 DN appeared to have no effect on REST recruitment at the STMN2 and SCN2A2 RE1 sequences, both of these sites are only modestly enriched in control cells and given the inherent variation with ChIP we cannot be certain whether the BRG1 DN has had no effect at these sites or whether an effect is lost within the experimental variation. Nevertheless, our data showing that REST occupancy at the SNAP25 and L1CAM RE1 sequences is significantly reduced in the presence of BRG1 DN suggests that BRG1 chromatin remodeling activity is indeed important for REST recruitment to some, if not all, RE1s.
In addition to containing an ATP-dependent nucleosome remodeling activity, BRG1 also contains a bromodomain (36). Bromodomains are present in many chromatin-associated proteins and have been shown to be acetyl lysine-binding motifs (37,38). In S. cerevisiae the bromodomain of Swi2/Snf2, the yeast homologue of BRG1, binds chromatin following histone acetylation by SAGA or NuA4 (39). In fact, the bromodomain of Swi2/Snf2 is important for anchoring the SWI/SNF complex to acetylated promoters (40) and increasing histone H4 K8 acetylation increases Swi2/Snf2 recruitment (41). If BRG1 remodeling activity is involved in the stable recruitment of REST to RE1 sites, then an increase in H4 K8 acetylation levels should result in increased REST recruitment. To test this idea we assessed REST occupancy consequent to inhibition of HDAC activity with TSA. These experiments showed that TSA treatment resulted in an increase in both acetylation of H4 and in REST occupancy at RE1 sequences (Figures 3a and 3b). Inhibition of HDAC activity with TSA results in global hyperacetylation of histones and a consequent increase in 'open' chomatin. Accordingly, any increase in REST recruitment may be the result of such an open chromatin configuration rather than being due to an increase in BRG1 activity. To determine if the increased REST recruitment was mediated by BRG1, we examined the effect of TSA on REST recruitment in cells expressing BRG1 DN. Expression of BRG1 DN had resulted in reduced REST recruitment at the SNAP25 RE1 sequence (Figure 2d) and while TSA had no effect on REST expression (Figure 3c), it did result in increases in acetylation of H4 (Figure 3a) and a 5 fold increase in the level of REST recruitment to the SNAP25 RE1 (Figure 3b). However, in the presence of BRG1 DN, TSA had no effect on the levels of REST recruitment (Figure 3d), indicating that the acetylation-dependent recruitment of REST requires BRG1 remodeling activity. Because the bromodomain of Swi2/Snf2 has been shown to be important for binding acetylated histones, we wanted to determine whether the bromodomain of BRG1 was required for the acetylation-dependent increased REST occupancy. To do this, we utilised a BRG1 mutant lacking the bromodomain (BRG1 deltabromo) (27). As for the BRG1 ATPase point mutant, BRG1 deltabromo was expressed in HEK293 in the presence of TSA or a vehicle control and subjected to ChIP. Expression of BRG1 deltabromo abrogated the TSA-induced increase in REST recruitment, suggesting that the bromodomain of BRG1, as well as the ATPase domain, is crucial in mediating this effect (Figure 3d). Together our data show that BRG1 chromatin remodelling activity facilitates REST mediated repression by increasing REST occupancy. Furthermore this increased occupancy is influenced by the level of histone acetylation in a manner dependent on the BRG1 bromodomain. Thus acetylation levels at REST regulated promoters will influence the level of REST repression.
BRG1 has previously been shown to affect transcription in both a positive and negative manner. Over-expression of BRG1 DN in B22 cells led to increased and decreased gene expression (35) and in yeast and mammals, the SWI/SNF complex has been shown to interact with both positive-acting factors, e.g. SAGA-Gcn5, (42) and p300/CBP (43) and negative-acting factors, e.g. HDAC1 and Sin3-Rpd3 (33). Battaglioli et al. have shown that BRG1 and its associated factors BAF57 and BAF170 interact with the corepressor CoREST and that BRG1 is required for REST-mediated repression (15). Furthermore, decreased BRG1 function in cancer cell lines has been linked with an increase in expression of REST regulated genes (23). The results presented here show that BRG1 is recruited to RE1s by REST (Figures 1b and 1d) and that inhibition of BRG1 nucleosomal remodeling activity with the BRG1 DN resulted in a reduction in REST enrichment at some RE1s and a derepression of REST target genes (Figures 2b and d). These results suggest that BRG1 activity increases the stability of REST-RE1 interactions in chromatin. BRG1 dependent transcription factor binding has been reported for transcriptional activators such as MyoD, which can bind to the myogenin promoter with weak affinity, but recruitment of BRG1 and its chromatin remodeling activity by MyoD is required to permit the stable binding of MyoD to the promoter along with its cofactors (35). However, enhancement of repressor binding by BRG1 has not been previously demonstrated.
Many complexes that contain chromatin remodeling activity also contain histone acetylases and/or deacetylases and it would seem that the two activities are linked both in the repression and activation of genes. BRG1 binds chromatin via acetyl lysines (39-41) and inhibition of HDAC activity with TSA resulted in an increase in both histone acetylation and in recruitment of REST to RE1s (Figures 3a, 3b and 3d). Since DNA can be made inaccessible to binding proteins by nucleosomes, the role of BRG1 in REST-mediated repression may therefore be to allow REST to gain better access to its chromatin targets by remodeling the local chromatin environment. It may be the case that only high levels of REST recruitment (e.g. SNAP25, L1CAM) are observed at those RE1s that utilise BRG1 activity, whereas lower REST recruitment is observed at RE1s that recruit BRG1 but whose activity has little effect (e.g. STMN2, SCN2A2). Interestingly, despite the fact that only a small change in REST recruitment at the STMN2 RE1 was observed following expression of the BRG1 DN, it resulted in an 8 fold increase in STMN2 expression. Furthermore, expression of REST DN caused only a 2 fold increase in STMN2 expression. This could potentially be due to a role for BRG1 in repression via a mechanism that does not involve affecting REST recruitment. This may be due to the presence of a second transcriptional repressor which also interacts with BRG1 or a secondary effect of BRG1 DN on the expression of another transcription factor that regulates STMN2.
The binding of REST to DNA is highly specific. Its only known binding site is the 21 bp RE1 and yet, the presence of this sequence in a gene is not sufficient to result in REST binding in vivo. For example, REST was recruited to the RE1s of the expressed L1CAM and SNAP25 genes but not to the RE1s of the silent BDNF, STMN2 and SYN1 genes in the human glioma cell line U373 (10). The presence of acetylated lysines at particular genes may explain the cell specific recruitment of REST at different RE1 sites. Similarly, this may explain the distinct profiles of REST recruitment observed in different cell types. Different complements of genes were regulated by REST in three different developmental stages in embryonic stem cells, neural stem cells and hippocampal neurons (44). Additionally, both BRG1 null mice (45) and REST knockout mice (31) are embryonic lethal. The fact that changes in both REST (46) and BRG1 activity affect neural stem cell differentiation and gliogenesis (47) are consistent with the notion that BRG1 activity is important for maintaining appropriate REST function.
Our data shows that REST occupancy is also influenced by the level of histone acetylation in a manner dependent on BRG1. This data predicts that the level of REST repression at any particular gene will be modified by local levels of histone acetylation and provides one potential explanation for different levels of REST repression seen at different target genes (10,26,32). Despite the fact that BRG1 is thought to be involved in the repression of genes by different transcription factors (reviewed in (48)), the precise role of BRG1 in this repression had not previously been identified. The mechanism proposed here in which BRG1 remodels chromatin to allow increased interaction of repressors with their binding sites is likely to be applicable to BRG1 mediated repression by other transcription factors.
The abbreviations used are:
The BRG1 DN-encoding plasmid, pBSCehBRG1K798R, was a generous gift from Dr. Tony Imbalzano (University of Massachusetts). This work was supported by the Wellcome Trust. L.O. was a Wellcome Trust PhD Student.