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Interplay between REST and nucleolin transcription factors: a key mechanism in the overexpression of genes upon increased phosphorylation.

Tediose T, Kolev M, Sivasankar B, Brennan P, Morgan BP, Donev R - Nucleic Acids Res. (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK.

ABSTRACT
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.

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Interplay between REST and NCL regulates expression of genes with overlapping REST/NCL-binding sites in melanoma tumors. (A) G361 cells were infected with lentiviral particles expressing either siRNA specific for NCL or a control siRNA. Infected cells (expressing GFP) were separated by flow sorting, and the effect of this knockdown on the expression of NCL, phosphorylated NCL, REST and β-actin were examined by western blots. Equal amounts of nuclear protein extracts were loaded in each lane. (B) The effect of NCL knockdown 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 were quantified by qPCR. Expression in control cells was set as 100%. Columns, results from three independent experiments; bars, SEM.
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Figure 4: Interplay between REST and NCL regulates expression of genes with overlapping REST/NCL-binding sites in melanoma tumors. (A) G361 cells were infected with lentiviral particles expressing either siRNA specific for NCL or a control siRNA. Infected cells (expressing GFP) were separated by flow sorting, and the effect of this knockdown on the expression of NCL, phosphorylated NCL, REST and β-actin were examined by western blots. Equal amounts of nuclear protein extracts were loaded in each lane. (B) The effect of NCL knockdown 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 were quantified by qPCR. Expression in control cells was set as 100%. Columns, results from three independent experiments; bars, SEM.

Mentions: 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 × 106) 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.Figure 4.


Interplay between REST and nucleolin transcription factors: a key mechanism in the overexpression of genes upon increased phosphorylation.

Tediose T, Kolev M, Sivasankar B, Brennan P, Morgan BP, Donev R - Nucleic Acids Res. (2010)

Interplay between REST and NCL regulates expression of genes with overlapping REST/NCL-binding sites in melanoma tumors. (A) G361 cells were infected with lentiviral particles expressing either siRNA specific for NCL or a control siRNA. Infected cells (expressing GFP) were separated by flow sorting, and the effect of this knockdown on the expression of NCL, phosphorylated NCL, REST and β-actin were examined by western blots. Equal amounts of nuclear protein extracts were loaded in each lane. (B) The effect of NCL knockdown 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 were quantified by qPCR. Expression in control cells was set as 100%. Columns, results from three independent experiments; bars, SEM.
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Figure 4: Interplay between REST and NCL regulates expression of genes with overlapping REST/NCL-binding sites in melanoma tumors. (A) G361 cells were infected with lentiviral particles expressing either siRNA specific for NCL or a control siRNA. Infected cells (expressing GFP) were separated by flow sorting, and the effect of this knockdown on the expression of NCL, phosphorylated NCL, REST and β-actin were examined by western blots. Equal amounts of nuclear protein extracts were loaded in each lane. (B) The effect of NCL knockdown 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 were quantified by qPCR. Expression in control cells was set as 100%. Columns, results from three independent experiments; bars, SEM.
Mentions: 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 × 106) 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.Figure 4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Infection, Immunity and Biochemistry, School of Medicine, Cardiff University, Cardiff CF14 4XN, UK.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus