{'fraction_non_alphanumeric': 0.029208450978483107, 'fraction_numerical': 0.0169165611917048, 'mean_word_length': 4.996934754050503, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Interplay between REST and nucleolin transcription factors: a key mechanism in the overexpression of genes upon increased phosphorylation

Non-malignant cells can be transformed via the activation of kinases that control degradation of neural-restrictive silencer factor (REST). Here, we identify a mechanism that contributes to the activation of genes, expression of which is controlled by responsive elements containing overlapping binding sites for REST and nucleolin. We demonstrate that both phosphorylated and non-phosphorylated nucleolin-bound DNA; however, only phosphorylated nucleolin successfully competed with either full-length REST or a REST-derived DNA-binding peptide, REST68, for binding to the overlapping binding sites. We show that this interplay between the two transcription factors regulates the activation of cell survival and immunomodulatory genes in tumors and non-malignant cells with activated protein kinase C, which is accompanied with alterations in cell proliferation and apoptosis. We propose a model for the regulation of these genes, which brings a new insight into the molecular mechanisms that control cellular transformation driven by activation of protein kinases.


INTRODUCTION
A number of studies demonstrated the central role that protein kinases play in transformation of non-malignant cells. Human mammary epithelial cell can be transformed through activation of phosphatidylinositol 3-kinase (PI3K) (1). PI3K also regulates cellular growth (2) and prevents apoptosis (3). Protein kinase Cn (PKCn) has been implicated as a mediator of epidermal growth factor receptor signaling in certain cell types. PKCn activation can transform non-malignant cells (4,5) resulting in cancer cell growth, survival and metastasis (6)(7)(8).
The neural-restrictive silencer factor (REST), originally described as a transcriptional repressor of neuronal gene expression (9,10), has recently emerged as a tumor suppressor capable of transforming epithelial cells when mutated (11), or subsequent to degradation of REST by b-TRCP ligase upon activation of cellular phosphorylation (12). This latter process could be triggered downstream of PI3K and PKC, which contribute to cellular transformation (1,5).
Recently, there has been an increased interest in using the host immune system to treat patients with malignancies. However, a recurring problem is that tumor cells often escape from immune control by overexpressing immunosuppressive molecules, such as membrane-bound complement regulators (mCReg), or proteins that directly impair immune responses to tumors (13,14). In many tumors, mCReg expression is much greater than on normal surrounding tissue (15)(16)(17)(18). The role of CD59, the only mCReg inhibitor of the cytolytic membrane attack complex of complement system, in enhancing tumor survival and growth in vivo was clearly demonstrated (19), prompting the search for an efficient strategy for CD59 suppression in tumors. We identified REST as an important regulator of the transcriptional machinery of the cd59 gene. Guided by the central role of REST in cellular transformation, we designed a peptide, REST68, derived from the DNA-binding domain of REST (18), which inhibited the expression of CD59 in tumors expressing a mutated REST which lacks DNA-binding activity.
Here, we identify nucleolin (NCL) as a key player in the modulation of expression of CD59. We elucidate the interplay between REST and NCL, transcription factors that bind to overlapping sites within the promoters of genes essential for cell survival and tumor growth. This interplay causes the overexpression of the above genes, exemplified by cd59 and mcl1, in tumors and in non-malignant cells where PKC is activated. We propose a model for the regulation of genes with overlapping REST/NCL binding sites in their promoters allowing better understanding of the molecular machinery involved in cellular transformation driven by activation of protein kinases.

Cell lines and treatments
Human neuroblastoma cell lines IMR32 and Kelly, the human colon carcinoma cell line Caco2, the malignant melanoma cell line G361 and the Chinese hamster ovary (CHO) cells (all from the European Collection of Animal Cell Cultures; ECACC) were maintained in RPMI 1640 with 10% heat-inactivated fetal calf serum (FCS), supplemented with glutamine, penicillin and streptomycin (Invitrogen). CHO cells were transfected with expression plasmids containing the full-length cDNAs for either human NCL or human REST (OriGene) using jetPEI reagent (Autogen Bioclear UK Ltd.). In some experiments, the above cells were treated for 24 or 48 h with 10 mM GF109203X, an inhibitor of PKC family kinases (Merck Chemicals Ltd.).
Normal human dermal fibroblasts (HDF; Millipore) were maintained in basal HDF medium with supplement as recommended by the supplier. In some studies, HDF cells were treated with 4 mM PDBu (Merck) over a period of 48 h prior to and after transformation with lentiviral particles to ensure maintenance of high protein kinase C (PKC) activity. ReNcell VM human neural progenitor cells (Millipore) were grown and differentiated as described previously (20).
Preparation of nuclear extracts followed by pulldown of proteins and mass spectrometry identification (21,22), western blotting (23,24), chromatin immunoprecipitations (ChIPs) (Magna ChIP A kit; Millipore), quantitative reverse transcription-PCR and qPCR (primers used are given in Supplementary Table  S1) and flow cytometry (25)(26)(27), were performed as described previously. Expression of REST was detected with rabbit polyclonal anti-REST antibody raised against amino acids 1-290 of the protein (H-290; Santa Cruz Biotechnology, Inc.). This antibody recognizes both the full-length and truncated REST. REST68, NCL and phosphorylated NCL were detected with mouse monoclonal anti-His (Millipore), anti-NCL (Millipore) and anti-NCL-phosphorylated (Thr76/Thr84) (BioLegend). Phosphorylation of REST was determined by anti-phosphoserine antibody (clone 4A4; Millipore). To control for the amount of sample loaded, blots were stained with polyclonal anti-b-actin antibody (Cambridge Bioscience Ltd).
Electrophoretic mobility shift assay DIG-labeled sense and antisense strands of the 35-bp responsive element (RE) within the cd59 promoter were purchased (Sigma-Aldrich). Oligonucleotides (100 pmol each) were mixed in equimolar amounts and annealed (18). The annealed DNA probe (10 pmol per reaction) was incubated with 10 mg of nuclear protein extracts from CHO cells or CHO transfected with either NCL-or REST-expressing plasmids. In some reactions, a 50-fold excess of non-labeled 35 bp RE was included as a control for specificity. To identify the presence of NCL and REST in DNA-protein complexes, antibodies against these two proteins were used. DNA was separated (28) and detected with anti-DIG-AP Fab fragments (Roche Diagnostics). For the NCL/REST displacement experiments, the same amount of protein was used per reaction (10 mg); however, only 0.2 pmol of the labeled probe was added to ensure that no unbound DNA would remain after the first incubation with the nuclear extract.
Subcloning of REST and preparation of reporter constructs with mutated RE from the cd59 promoter To enable generation of Kelly cells expressing the fulllength REST, we cut out the REST-coding sequence from the OriGene plasmid using BamHI followed by subsequent ligations with NotI-BamHI adaptor (Supplementary  Table  S1) and NotI-digested pDR2DEF1a expression plasmid. Clones with the correct orientation (confirmed by sequencing) were used for transfection into Kelly cells. Transfected cells were selected in medium containing hygromycin B (InvivoGen).
Cd59 promoter fragment, 2140-bp long, was previously cloned into pEGFP-1 vector (Clontech), generating a reporter system for studying the role of promoter sequences in expression of the cd59 gene (18). To study the interplay between REST and NCL transcription factors, we generated two additional reporter constructs in which the wild-type RE was mutated in such a manner that it still binds REST but not NCL (mutCD59) or does not bind either of the two transcription factors (replaced by an irrelevant 35-bp sequence; irr35 bp). To generate these two constructs, we used the previously prepared pEGFP-1 vector with the wild type cd59 promoter as a template in overlapping PCR mutagenesis with oligonucleotides given in Supplementary Table S1. Sequences for the both constructs were designed based on the previously published consensus binding sequences for REST (29) and Sp1/NCL (30,31). Constructs were transfected into different cell types (Effectene reagent; Qiagen) and in some experiments transfected cells were selected by G418 (Merck).

RNA interference
NCL was knocked down in IMR32 and Kelly cells by transfecting the cells with pKD-Nucleolin-v1 plasmid (Millipore) expressing siRNA specific for the NCL mRNA. pKD-NegCon-v1 (Millipore) was used as a negative control. REST was knocked down in G361 and HDF cells by transfecting the cells with plasmid expressing siRNA specific for the REST mRNA (18) and transfected cells were selected by puromycin (10 ng/ml). Transfections were performed using Effectene reagent (Qiagen) following the supplier's recommendations. IMR32 and Kelly cells with knocked down REST expression were generated previously (18) using the same plasmids as for the G361 and HDF cells. The effect of the NCL and REST knockdown was assessed by qPCR 48 h after the transfection.
To achieve a higher efficiency of the NCL knockdown in G361 and HDF cells, a lentiviral approach was used. Constructs were engineered based on the pRRLSIN.cPPT.PGK-GFP.WPRE lentiviral vector (Addgene), which expresses GFP. The h1 promoter together with the siRNA sequence from the pKD-Nucleolin-v1 plasmid was excised using SpeI and XhoI restriction enzymes and the residual fragment ligated into pRRLSIN.cPPT.PGK-GFP.WPRE, which contains the same unique restriction sites, using T4 ligase. The control siRNA sequence from pKD-NegCon-v1in was inserted in a similar manner. To enable the detection of REST and NCL binding to certain promoters using relatively low number of cells, fragments from promoter sequences of the cd59, mcl1 and adcy5 genes that contain REST/NCL binding sites (Supplementary  Table S1 and Supplementary Figure S2) were inserted into both plasmids. The cd59 fragment was inserted into the NruI restriction site, the mcl1 fragment into a BamHI site and the adcy5 promoter fragment into a KpnI site. Oligonucleotides for these three promoter sequences (Biomers.net GmbH) were designed such that after annealing, they formed cloning-ready ends. Restriction enzymes, T4 ligase and NEB 5-alpha competent Escherichia coli were purchased from New England Biolabs. The correctness of all constructs was confirmed by sequencing. Lentiviral particles were prepared using MISSION Lentiviral Packaging Mix (Sigma-Aldrich) and stored at -80 o C until use.

Cell viability, apoptosis and cell proliferation assays
Cells transfected with either construct expressing NCL-specific siRNA or a control plasmid were treated with PDBu activator or the same volume of dimethyl sulfoxide (DMSO) as described above. The percentage of viable and apoptotic cells was determined by staining with BD Cell Viability and PE Annexin V Apoptosis Detection Kits (BD Pharmingen), respectively, following the manufacturer's recommendations. The effect of each treatment on cellular proliferation was assessed using CyQUANT Direct Cell Proliferation Assay Kit (Invitrogen).

Statistical analysis
The data are expressed as mean ± SEM and were analyzed for statistical significance by the two-tailed Student's t-test to compare two paired groups of data. P < 0.05 was considered to be statistically significant.

NCL is involved in the regulation of CD59 expression
Recently, we identified a 35-bp RE within the cd59 promoter containing the REST binding site, which is involved in the overexpression of CD59 in neuroblastoma (18). Here, we elucidate the molecular mechanism resulting in the overexpression of CD59 in tumors by identifying transcription factors that bind to the RE. In our study we used Kelly and IMR32 cells, human neuroblastoma lines expressing high and low amounts of CD59, respectively, and primary neurons differentiated from human neural progenitor cells (REN), which have a negligible CD59 expression ( Figure 1A). Nuclear extracts from all three cell types were generated and proteins that bind to the 35-bp RE from the cd59 promoter were precipitated ( Figure 1B). In each precipitation, we observed three bands (marked by arrows); however, the highest molecular weight band was enriched in preparations from Kelly cells in comparison to those from IMR32 and REN cells. Using mass spectrometry, we unequivocally identified this protein as NCL.
We next investigated whether NCL was involved in the regulation of CD59 expression in living cells. Using RNAi, NCL was knocked down in IMR32 and Kelly cells with similar efficiency (75% decreased expression of NCL at the RNA level) ( Figure 1C). CD59 expression was decreased by 80% following knockdown in Kelly cells, but remained unaffected in IMR32 cells. A likely reason for this result is that because IMR32 cells express full-length REST binding of NCL to the RE is blocked, whereas in Kelly cells which express the truncated REST isoform the RE is unoccupied and NCL can bind. It is also possible that the failure of NCL knockdown to affect CD59 expression in IMR32 cells is a consequence of NCL phosphorylation status. NCL is a heavily phosphorylated protein substrate for PKCn and p34 cdc2 kinases (32,33). To test this, western blots were performed ( Figure 1D), which showed that IMR32 and Kelly cells have similar levels of expression of NCL; however, phosphorylation of NCL is much lower in IMR32 cells as compared to Kelly cells.

Phosphorylation of NCL plays a major role in competition with REST for binding to DNA
To test the importance of NCL phosphorylation in its binding to the DNA consensus sequence overlapping with the REST binding site, we first determined the effect of different inhibitors of p34 cdc2 and PKCn kinases on NCL phosphorylation (Supplementary Figure S1A). The most remarkable inhibition was achieved by GF109203X (GF), an inhibitor of PKC. Similar effects were observed with LY294002, an inhibitor of PI3K. We chose to use GF in our further experiments. We detected low levels of binding of NCL to the cd59 promoter in IMR32 cells, while antibody against REST enriched the chromatin fraction in the RE by 4-fold compared to the input control ( Figure 2A). Treatment of IMR32 cells with GF led to a loss of NCL phosphorylation and abolished its binding to the cd59 promoter. In concert with this, REST binding to the promoter was increased 34-fold compared to binding in untreated cells. These events resulted in a 4-fold decrease in CD59 expression in IMR32 cells. To demonstrate interplay between REST and NCL for their binding to the cd59 promoter, we performed ChIP with chromatin fragments from IMR32 cells in which REST was knocked down. This resulted in a considerable decrease in REST binding to the promoter and an 8-fold increase in NCL binding augmenting expression of CD59 by 60%. To determine if the detected binding occurred within the 35-bp RE of the cd59 promoter, we designed a plasmid in which the RE from the cd59 promoter was replaced with an irrelevant 35-bp sequence (irr35 bp) that does not bind REST and NCL (Supplementary Figure S2A). ChIP with chromatin fragments from IMR32 and Kelly cells transfected with plasmids containing either the wild-type promoter or the irr35-bp sequence (Supplementary Figure S2B) showed that this substitution abolished binding of both transcription factors. This clearly demonstrated that the binding to the cd59 promoter we detect in our ChIP experiments  requires presence of the 35-bp RE. Importantly, this experiment also showed that binding of REST and NCL to the wild-type cd59 promoter is not a function of potential cis elements flanking the 35-bp RE.
Western blots showed that the GF treatment of IMR32 cells (Figure 2A) increased REST content compared to non-treated cells, which supports the previous finding that phosphorylation at the C-terminus of REST is a signal for its degradation (12). Considering this, we determined if the GF treatment contributes to changes in the phosphorylation status of REST (Supplementary Figure S1B). We performed western blots using equal amounts of immunoprecipitated REST and monoclonal antibody against phosphoserine, a posttranslational modification shown to be responsible for REST degradation (12). These experiments showed that treatment of IMR32 cells with GF decreased phosphorylation in REST. However, in Kelly cells we did not detect significant phosphorylation of the truncated REST expressed by these cells. To show that this lack of phosphorylation in REST is due to the expression of truncated isoform, we carried out similar experiments with lysates from Kelly cells transfected with plasmids expressing the full-length REST. The full-length REST was heavily phosphorylated and GF treatment led to marked decrease in phosphorylation of this protein.
Taken together, the above experiments suggest that serine phosphorylation occurs to a great extent within the full-length REST and that GF inhibitor can inhibit this phosphorylation resulting in accumulation of this transcriptional suppressor. Therefore, the observed decrease in NCL binding to the RE (Figure 2A) might be, at least in part, a result of the increased amount of REST protein. To address this issue, we carried out similar experiments but with Kelly cells (Figure 2B), which express only truncated REST that cannot be degraded in response to phosphorylation (12). ChIP analyses did not detect any difference in NCL binding to the 35-bp RE, suggesting that in the absence of full-length REST, the phosphorylation status of NCL is not important for its interaction with DNA. The lack of regulatory role of the truncated REST in expression of CD59 was confirmed by ChIP with chromatin fragments from Kelly cells with knocked down REST, which did not alter binding of NCL to the RE. As a consequence, expression of CD59 was unaffected by the GF treatment and REST knockdown (qPCR data). To address if GF did not alter NCL binding in Kelly cells due to the lack of full-length REST, we transfected Kelly cells with plasmids expressing the full-length REST and analyzed these cells for binding of REST and NCL to the 35-bp RE ( Figure 2C). In the presence of full-length REST, which bound to the RE, NCL binding was decreased 5-fold compared to untransfected Kelly cells ( Figure 2B). GF treatment abolished NCL binding and increased the number of REST molecules bound to the RE, which resulted in the inhibition of CD59 expression.
Next, we took advantage of a previously generated Kelly cell line transfected with a plasmid that expresses REST68, a peptide comprising the DNA-binding zinc finger domains 6-8 of REST (18). REST68 binds DNA, but does not contain the phosphorylation signal for degradation, thus, its expression should not be affected by GF. This expectation was confirmed by western blotting ( Figure 2D). Although the expression of REST68 was unaltered by GF, ChIP analysis showed 5-fold less binding of NCL to the 35-bp RE in GF-treated cells compared to non-treated ones. The GF treatment resulted in the replacement of NCL by REST68 and an 80% decrease in the expression of CD59. These data clearly show that the phosphorylation status of NCL is of major importance for the interplay between REST and NCL when they bind to overlapping binding sites.
To elucidate if the REST/NCL interplay is a competition for binding to the overlapping binding sites within the 3-bp RE or co-recruitment, we carried out re-ChIP with chromatin fragments from untreated Kelly cells or transfected with REST expressing plasmid, incubated with GF inhibitor or both together ( Figure 3A). When anti-NCL was used as first antibody, we did not detect any REST binding to the 35-bp RE with anti-REST used in the second immunoprecipitation. When anti-REST was the primary antibody, we detected binding of REST to the 35-bp RE in Kelly cells transfected with REST expression construct with or without GF treatment. However, no binding of NCL was found in any of the cells. These data unambiguously showed that REST and NCL compete for binding to overlapping binding sites and do not co-recruit.
We further addressed the importance of phosphorylation status of NCL for its competition with REST for binding to overlapping binding sites by performing a series of electrophoretic mobility shift assays (EMSAs; Figure 3B and C). CHO cells were transfected with expression plasmids containing the full-length cDNAs for human REST and NCL. We used cells from evolutionary diverse specie to be able to perform competition assays for binding of human REST and NCL to DNA and to avoid complications that may occur by adding extracts that contain both proteins. Cells were grown and treated with GF. Prepared nuclear extracts were analyzed for expression and phosphorylation status of REST and NCL ( Figure 3B). The antibody against REST recognized only the human protein and not the endogenous hamster protein; GF treatment slightly increased the expression of human REST. BLAST search shows that NCL is highly conserved, and the antibody against human NCL also recognized hamster NCL. However, transfection of CHO cells with human NCL-expressing vector markedly increased the amount of protein detected by the antibody, confirming that human NCL was expressed. We performed densitometry analysis of phospho-NCL band intensities for untreated and GF-treated CHO cells. Similar analysis was carried out for CHO cells transfected with plasmids expressing either REST or NCL ( Figure 3B, right). We obtained similar ratios for all three cell lines, suggesting that phosphorylation of both hamster and human NCL is similarly inhibited by the GF treatment. EMSA of the 35-bp RE with nuclear extracts from untransfected CHO cells, untreated or treated with GF, showed only a single band, which was shown by supershift Either anti-NCL or anti-REST was used as first antibody, followed by second ChIP with anti-NCL, anti-REST and non-immune rabbit IgG as background control. Binding was quantified by qPCR and was set as 1 for the input control of each treatment. Columns, results from three independent experiments; bars, SEM. (B) Nuclear extracts from non-treated CHO cells or cells transfected with either a REST or NCL expression plasmid were analyzed by western blots (left) for expression of REST, NCL and phosphorylated NCL using antihuman antibodies. Similar analysis was performed for the above cells, after they were treated 24 h with 10-mM GF inhibitor. Ratios between band intensities for phospho-NCL without and with GF treatment were calculated for each cell line using densitometry analysis (right). Columns, results from three independent experiments; bars, SEM. (C) EMSA with the 35-bp RE labeled with DIG (10 pmol/reaction), incubated with 10 mg of nuclear protein extracts from cells described in (B) in the presence of 0.1 mg/ml poly(deoxyinosinicdeoxycytidylic acid) and 1 mg/ml salmon sperm DNA. Antibodies against either REST or NCL were added in some reactions (+) to test for presence of these two proteins in the observed complexes. The complexes were separated in a 2.5% agarose gel for 2 h at 100 V, transferred onto a nylon membrane and detected by anti-DIG-AP Fab fragments. (D) EMSA demonstrating the NCL/ REST competition for binding to the 35-bp RE. Labeled probe (0.2 pmol/reaction; no unbound DNA remains after the incubation) was incubated with nuclear protein extracts (10 mg/reaction) from CHO cells expressing either human NCL or REST, treated or untreated with GF. Complexes obtained after incubation of the 35-bp RE with CHO cells expressing human REST and treated with GF were subjected to competition with nuclear extracts from CHO cells expressing human NCL with or without GF treatment. with antibody to contain NCL ( Figure 3C). Extracts from cells expressing human REST gave two bands. Supershift with anti-NCL and anti-REST showed that the complex with the higher electrophoretic mobility contained NCL, while the slower migrating band contained REST. In untreated cells, both bands had comparable intensity; however, when the transfected cells were treated with GF, the intensity of the NCL complex was markedly reduced ( Figure 3C).
Once the distinct binding pattern of each nuclear extract to the 35-bp RE was established, we explored whether NCL could replace REST in preformed complexes ( Figure 3D). First, we optimized the concentration of the 35-bp RE to ensure that there was no residual unbound DNA after the initial formation of complexes by incubating the DNA with nuclear extracts from CHO cells expressing human REST. We finally added nuclear extracts from CHO cells expressing human NCL, with or without GF, to the preformed complexes. The majority of the REST-DNA complex was replaced with NCL-DNA when extracts without GF were added ( Figure 3D; lanes 3 and 4); however, when the NCL was not phosphorylated ( Figure 3D; lane 5), this shift was not observed. This experiment confirms the critical role of NCL phosphorylation in the REST/NCL competition for binding to overlapping binding sites.

Interplay between REST and NCL is a key mechanism in gene activation by PKC
Considering the high number of genes regulated by REST [binding sites mapped to 1946 locations (34)], we reasoned that REST/NCL interplay might be involved in the regulation of many genes in which the two transcription factors have overlapping binding sites. This issue is of particular importance to tumor biology considering that REST is a major player in cellular transformation upon increased phosphorylation (12). We first studied REST/NCL interplay in G361 melanoma cells, a tumor type that originates from neural crest like neuroblastoma, however, unlike the neuroblastoma cells express the full-length DNA-binding form of REST (18,35). G361 cells were infected with lentiviral particles containing plasmids that express GFP and either siRNA for knocking down NCL (NCL-RNAi) or a control sequence (cntr-RNAi). Both plasmids also contained fragments from the promoters of cd59, mcl1 and adcy5, each including a REST binding site. NCL was recently shown to bind to Sp1 binding sequences in DNA (31) and here we demonstrated that the Sp1 binding sites are required for binding of NCL to the 35-bp RE (Supplementary Figure S2A and B). The REST binding site overlaps with a NCL binding sequence in the mcl1 and cd59, but not in the adcy5 promoter fragments (Supplementary Figure S2C). An average multiplicity of infection (MOI) of around 15-16 was achieved (Supplementary Figure S3), which allowed performing the subsequent analyses with a relatively low number of cells ($7 Â 10 6 ) sorted by expression of GFP (Figure 4). The knockdown caused decreased expression of NCL at both the RNA (by 90%) and protein level. This reduced expression led to an 8-fold lower binding of NCL to mcl1 and cd59 promoters ( Figure 4B). Depletion of the NCL from these promoters allowed increased binding of REST (3.5-to 4-fold) and a 60% decrease in the expression of the cd59 and mcl1 genes ( Figure 4C). Primers for the assessment of Mcl1 expression were designed to detect only the longer splice variant, which stimulates cell survival. No binding of NCL to the adcy5 promoter was detected, and we did not observe any alteration in the binding of REST or in the expression level of the adcy5 gene following the NCL knockdown.
These data show that the interplay between REST and NCL is a key mechanism in tumors for the regulation of the expression of genes with overlapping binding sites in their promoters for these two transcription factors. The data also show that the REST/NCL interplay controls the expression of genes involved in cell survival (i.e. mcl1; similar results for bcl2l1, not shown) and modulation of immune responses and tumor growth (cd59). Thus, our next aim was to determine if this interplay is involved in gene overexpression occurring during cellular transformation by PKC (5). Primary HDFs were infected at a MOI of 12 (Supplementary Figure S3), with either NCL-RNAi or cntr-RNAi plasmids. In some experiments, HDFs were treated for 48 h with an activator of PKC, PDBu, prior to infection. Western blots showed that the PDBu Figure 5. REST/NCL interplay is involved in gene overexpression occurring during cellular transformation by PKC. (A) HDF cells, treated with either the PDBu activator of PKC or DMSO as a control, were infected with lentiviral particles expressing either siRNA specific for NCL or a control siRNA. Infected cells were separated by flow sorting, and the effect of this knockdown and/or PKC activation on the expression of NCL, phosphorylated NCL and REST were examined by western blots. Equal amounts of nuclear protein extracts were loaded in each lane. (B) The effect of NCL knockdown and/or PDBu treatment on REST and NCL binding to the promoters of cd59, mcl1 and adcy5 was determined by ChIP with anti-REST and anti-NCL antibodies, respectively. Binding was quantified by qPCR and was set as 1 for the input control of each treatment. Immunoprecipitation with non-immune rabbit IgG was carried out as a control for the assay background. Columns, results from three independent experiments; bars, SEM. (C) Alterations in expression of NCL, CD59, Mcl1 and ADCY5 as a result of the NCL knockdown and/or PKC activation were quantified by qPCR. Expression in control cells was set as 100%. Columns, results from three independent experiments; bars, SEM. treatment increased the phosphorylation of NCL in HDF cells, which, in general, do not contain many phosphorylated molecules ( Figure 5A). Notably, PDBu treatment resulted in a lower amount of REST at the protein level, most likely a result of increased protein degradation (12). Knocking down NCL expression by 85% ( Figure 5C) resulted in a 60% decrease in its binding to the cd59 and mcl1 promoters ( Figure 5B). PDBu treatment resulted in 25-fold more NCL binding to the promoters of cd59 and mcl1, a consequence of both the increased phosphorylation of NCL and the degradation of REST. Indeed, knockdown of NCL in PDBu-treated HDF cells caused a considerable decrease in the binding of this protein to the promoters and increased binding of REST. However, REST binding was still lower than in the control HDF cells, likely a consequence of REST degradation following PDBu treatment. Critically, knocking down NCL expression in PDBu-treated HDF cells resulted in a 7-fold reduction of CD59 and Mcl1 mRNA expression compared to PDBu-treated cells infected with the control virus ( Figure 5C). This result unambiguously showed that the interplay between REST and NCL controls the activation of a set of key genes in non-malignant cells upon activation of PKC, a process which is implicated in cellular transformation.
It is well-known that NCL has numerous functions including that of a histone chaperone (36). To clarify whether NCL exerts its main effect on CD59 expression by binding to the 35-bp RE, we generated EGFP-reporter constructs in which expression of EGFP was controlled by the wild-type cd59 promoter or a mutated one in which the 35-bp RE still binds REST but not NCL (mutCD59) (Supplementary Figure S2A). G361 malignant melanoma cells transfected with the mutated or the wild-type cd59 promoter reporter constructs ( Figure 6) demonstrated the need of NCL binding sites within the 35-bp RE for the increased gene expression.

Interplay between REST and NCL regulates cellular proliferation and apoptosis
Considering that the interplay between REST and NCL regulates expression of genes involved in cell survival and apoptosis, we next decided to test the effect of the NCL knockdown alone or together with REST knockdown on major cellular processes, resulting in tumor growth. Knockdown efficiency of REST in G361 and HDF cells was similar to that obtained for NCL (around 85%; Figures 4C, 5C and 7A). Double knockdown of REST and NCL restored expression of CD59 to levels similar to these in corresponding controls ( Figure 7A). Unfortunately, we could not perform clonal growth in soft agar, because this assay requires a relatively long culturing of cells (1-2 weeks), while an efficient knockdown of the NCL expression results in cell death within several days. Viability of G361 and HDF cells remained unaffected within first 48 h after different treatments ( Figure 7B). Therefore, we assessed the percentage of apoptotic cells in G361 malignant melanoma and HDF non-malignant cells as a result of NCL knockdown or REST/NCL double knockdown ( Figure 7C) 48 h after the treatments. Knockdown of NCL increased the number of annexin V positive G361 cells by 3.5-fold, while for the non-malignant HDF cells this increase was around 1.3-fold only. To address if the phosphorylation status of NCL is important for the observed lack of significant alteration in percentage of apoptotic HDF cells, we treated cells with PDBu prior to the NCL knockdown. Two days after that we found that the NCL knockdown resulted in $4-fold increase in percentage of annexin V positive cells, which well correlated with the results obtained for the G361 cells. However, double knockdown of REST and NCL in these cell lines took the percentage of apoptotic cells back to levels similar to these in control cells transfected with plasmid expressing cntr-siRNA. The increased percentage of apoptotic cells because of NCL knockdown correlated with a slower proliferation rate of the G361 tumor cells and the PDBu-treated HDF cells ( Figure 7D). However, no significant slow down in proliferation of HDF cells was observed as a result of the NCL knockdown. To demonstrate further that the interplay between REST and NCL regulates cell proliferation, we carried out double knockdown of REST and NCL in the above cell lines, which restored proliferation rates to those characteristic for the control cells. Figure 6. NCL exerts its effect on CD59 expression by binding to the 35-bp RE. Two EGFP-reporter constructs were generated: in one of them expression of EGFP is controlled by the wild-type cd59 promoter (wtCD59 promoter); in the other construct, the 35-bp RE was mutated in such a way that it still binds REST but not NCL (mutCD59 promoter) (Supplementary Figure S2A). Expression of EGFP in G361 transfected with each of these two constructs was determined by flow cytometry. Promoterless pEGFP-1 vector was used as a negative control. Promoter of the constitutively active elongation factor alpha 1 (EFa1) was introduced in the pEGFP-1 and used as a positive control. Columns, results from three independent experiments; bars, SEM.

DISCUSSION
REST is a transcriptional repressor that regulates numerous genes, notably those involved in neuronal differentiation and cellular transformation (12,37). The essential role of this repressor in cellular transformation was found to be controlled via the phosphorylation of a conserved degron at the C-terminus of the protein, initiating its degradation by SCF bTRCP ligase (12). In this study, we have identified a novel mechanism that contributes to the regulation of a large number of genes controlled by REST, which have overlapping binding sites for REST and a second important transcription factor, NCL, which requires Sp1 binding sites for its direct interaction with DNA (31). A number of key genes, including those encoding proteins regulating immune responses and tumor growth (e.g. cd59) (18) and cell survival (mcl1, bcl2l1), belong to this group of REST/NCL-regulated genes (Supplementary Figure S2). It has been demonstrated that activation of PI3K and PKCn protein kinases can transform non-malignant cells (1,5). The increased kinase activity not only leads to the degradation of REST (12), but also phosphorylates NCL (5). Phosphorylation of NCL allows displacement of the remaining transcriptional suppressor from the promoter, which further increases the expression of the gene (Figures 2 and 3). Our experiments with knocked down NCL (Figures 4 and 5) unambiguously showed that the lack of NCL results in increased binding of REST to gene promoters and reduced activation of REST/NCL controlled genes. The overall effect of this inhibited expression of genes involved in cell survival was that cells slowed down their proliferation and became more susceptible to apoptosis (Figure 7). These effects on proliferation and apoptosis were however reversed by simultaneous knockdown of both REST and NCL.
The above results, together with our previously published data, enabled us to propose a model for the upregulation of expression of genes with overlapping REST/NCL binding sites in their promoters upon activation of protein kinases (Figure 8). In non-malignant cells, REST is expressed at a relatively high level, and NCL phosphorylation is moderate compared to transformed cells. Thus, REST is bound to the gene promoters. However, upon activation of kinases like PKCn, p34 cdc2 and PI3K, which occurs in cellular transformation (1,5,38), NCL is phosphorylated and the half-life of REST decreases as a consequence of its increased degradation (12). These events cause decreased binding of REST and increased binding of NCL to the overlapping binding sites in the gene promoter ( Figure 8A). Treatment of such tumors with the REST68 peptide, degradation of which is not affected by phosphorylation, suppresses expression of the gene by competing with phosphorylated nucleolin. However, if cells are treated further with inhibitors of the protein kinases that use NCL as a substrate, the amount of endogenous REST increases which together with the REST68 peptide and inhibited phosphorylation of NCL results in additional inhibition of gene expression.
Many tumors do not express the full-length REST ( Figure 8B), either due to an insertion that introduces a stop codon and results in the expression of non-DNA-binding truncated REST (39,40), or by deletion of the gene during tumorigenesis (11). In the absence of functional REST, both phosphorylated and nonphosphorylated NCL bind to the gene promoter and activates expression via formation of complexes with other transcription factors (i.e. c-Jun/NCL) (31). Treatment of tumors with kinase inhibitors will therefore not significantly affect expression of the genes controlled by the REST/NCL interplay ( Figure 2B, Supplementary Figure S4). However, treatment of such tumors with a peptide that binds the REST binding site (i.e. REST68) will inhibit the expression of the target genes by competing with phosphorylated NCL for binding to the overlapping binding sites. Inhibition of NCL phosphorylation will further enhance the binding of REST68 to the promoter In malignant cells, protein kinases such as PI3 and PKC are activated resulting in degradation of the full-length REST that binds to a responsive element within the promoter of the gene and in phosphorylation of NCL, which together leads to a displacement of the remaining REST from the gene promoter and high expression levels. Treatment of such tumors with the REST68 peptide (degradation is not affected by phosphorylation) suppresses expression of the gene by competing with phosphorylated NCL. However, if cells are treated further with inhibitors of NCL phosphorylation, NCL will no longer be in competition for binding and gene expression will be suppressed further. (B) The model is similar to the one shown in (A); however, tumor types like colon carcinoma, neuroblastoma and small-cell lung carcinoma lack REST that can bind to DNA and suppress gene expression. In these tumors, both phosphorylated and non-phosphorylated NCL can bind to the gene promoter and activate expression. REST68 treatment prevents non-phosphorylated NCL from binding to the REST/NCL overlapping binding sites, partially decreasing the expression. Hence, further treatment with kinase inhibitors does not allow for competition between REST68 and NCL, resulting in even stronger inhibition of the controlled gene.
by taking the NCL out of the competition with this peptide, leading to further suppression of the genes with overlapping REST/NCL-binding sites.
Our data demonstrate a new mechanism involving interplay between two transcription factors and phosphorylation status that play a major role in the regulation of expression of genes involved in tumor growth and survival. The current study provides a model, which allows a better understanding of the molecular machinery involved in cellular transformation driven by activation of protein kinases and enables a better design of adjuvant treatments for achieving efficient suppression of survival and immunomodulatory genes controlled by the REST/ NCL interplay.