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Overexpression of transcription factor Sp1 leads to gene expression perturbations and cell cycle inhibition.

Deniaud E, Baguet J, Chalard R, Blanquier B, Brinza L, Meunier J, Michallet MC, Laugraud A, Ah-Soon C, Wierinckx A, Castellazzi M, Lachuer J, Gautier C, Marvel J, Leverrier Y - PLoS ONE (2009)

Bottom Line: These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms.The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis.This study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Inserm, U851, Lyon, France.

ABSTRACT

Background: The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression.

Methodology and principal findings: We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis.

Conclusion: This study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms.

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Deregulation of the expression of wild-type but not truncated cytoplasmic Sp1 induces apoptosis.(A) Baf-3-Sp1 clone 1 and (B) Baf-3-Sp1 clone 2 were grown in presence of doxycycline (+ dox) or in absence of doxycycline for the indicated time (- dox). Analysis of Sp1 and actin protein levels by Western Blot (left panel). Viability measured by flow cytometry after staining with propidium iodide (right panel). (C) Inducible clone Baf-3-tSp1 was grown with (+ dox) or without (-dox) doxycycline for the indicated time. Analysis of Sp1 and actin protein levels by Western Blot (left panel). tSp1: truncated Sp1. Cells were costained for DNA (blue) and Sp1 (red) and analysed by confocal microscopy (middle panel). Scale bar 10 µm. Viability measured by flow cytometry after staining with propidium iodide (right panel). Results show the mean ± sd of 3 independent experiments.
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pone-0007035-g001: Deregulation of the expression of wild-type but not truncated cytoplasmic Sp1 induces apoptosis.(A) Baf-3-Sp1 clone 1 and (B) Baf-3-Sp1 clone 2 were grown in presence of doxycycline (+ dox) or in absence of doxycycline for the indicated time (- dox). Analysis of Sp1 and actin protein levels by Western Blot (left panel). Viability measured by flow cytometry after staining with propidium iodide (right panel). (C) Inducible clone Baf-3-tSp1 was grown with (+ dox) or without (-dox) doxycycline for the indicated time. Analysis of Sp1 and actin protein levels by Western Blot (left panel). tSp1: truncated Sp1. Cells were costained for DNA (blue) and Sp1 (red) and analysed by confocal microscopy (middle panel). Scale bar 10 µm. Viability measured by flow cytometry after staining with propidium iodide (right panel). Results show the mean ± sd of 3 independent experiments.

Mentions: The murine IL-3-dependent Baf-3 cell line was maintained in DMEM (Gibco) containing 6% FBS (Pan Biotech GmbH) and 5% IL-3 as described [20]. Drosophila Schneider's SL2 cells were cultured in Drosophila medium (Invitrogen) containing 10% FBS at 28°C [21]. Baf-3 clones expressing inducible Sp1 and GFP were obtained as described previously [18]. To repress ectopic Sp1 expression, cells were grown in presence of doxycyline (30 ng/ml, Sigma). To induce Sp1 and GFP expression, cells were washed 3 times and cultured without doxycyline. Among the inducible clones generated, one expressed a truncated form of Sp1 (tSp1, Figure 1C) due to the integration into the genome of a retrovirus coding for a truncated Sp1 (data not shown). tSp1 is composed of the first 418 amino acids and is devoid of DNA binding domain and nuclear localisation sequences (Figure S1).


Overexpression of transcription factor Sp1 leads to gene expression perturbations and cell cycle inhibition.

Deniaud E, Baguet J, Chalard R, Blanquier B, Brinza L, Meunier J, Michallet MC, Laugraud A, Ah-Soon C, Wierinckx A, Castellazzi M, Lachuer J, Gautier C, Marvel J, Leverrier Y - PLoS ONE (2009)

Deregulation of the expression of wild-type but not truncated cytoplasmic Sp1 induces apoptosis.(A) Baf-3-Sp1 clone 1 and (B) Baf-3-Sp1 clone 2 were grown in presence of doxycycline (+ dox) or in absence of doxycycline for the indicated time (- dox). Analysis of Sp1 and actin protein levels by Western Blot (left panel). Viability measured by flow cytometry after staining with propidium iodide (right panel). (C) Inducible clone Baf-3-tSp1 was grown with (+ dox) or without (-dox) doxycycline for the indicated time. Analysis of Sp1 and actin protein levels by Western Blot (left panel). tSp1: truncated Sp1. Cells were costained for DNA (blue) and Sp1 (red) and analysed by confocal microscopy (middle panel). Scale bar 10 µm. Viability measured by flow cytometry after staining with propidium iodide (right panel). Results show the mean ± sd of 3 independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2737146&req=5

pone-0007035-g001: Deregulation of the expression of wild-type but not truncated cytoplasmic Sp1 induces apoptosis.(A) Baf-3-Sp1 clone 1 and (B) Baf-3-Sp1 clone 2 were grown in presence of doxycycline (+ dox) or in absence of doxycycline for the indicated time (- dox). Analysis of Sp1 and actin protein levels by Western Blot (left panel). Viability measured by flow cytometry after staining with propidium iodide (right panel). (C) Inducible clone Baf-3-tSp1 was grown with (+ dox) or without (-dox) doxycycline for the indicated time. Analysis of Sp1 and actin protein levels by Western Blot (left panel). tSp1: truncated Sp1. Cells were costained for DNA (blue) and Sp1 (red) and analysed by confocal microscopy (middle panel). Scale bar 10 µm. Viability measured by flow cytometry after staining with propidium iodide (right panel). Results show the mean ± sd of 3 independent experiments.
Mentions: The murine IL-3-dependent Baf-3 cell line was maintained in DMEM (Gibco) containing 6% FBS (Pan Biotech GmbH) and 5% IL-3 as described [20]. Drosophila Schneider's SL2 cells were cultured in Drosophila medium (Invitrogen) containing 10% FBS at 28°C [21]. Baf-3 clones expressing inducible Sp1 and GFP were obtained as described previously [18]. To repress ectopic Sp1 expression, cells were grown in presence of doxycyline (30 ng/ml, Sigma). To induce Sp1 and GFP expression, cells were washed 3 times and cultured without doxycyline. Among the inducible clones generated, one expressed a truncated form of Sp1 (tSp1, Figure 1C) due to the integration into the genome of a retrovirus coding for a truncated Sp1 (data not shown). tSp1 is composed of the first 418 amino acids and is devoid of DNA binding domain and nuclear localisation sequences (Figure S1).

Bottom Line: These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms.The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis.This study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Inserm, U851, Lyon, France.

ABSTRACT

Background: The ubiquitous transcription factor Sp1 regulates the expression of a vast number of genes involved in many cellular functions ranging from differentiation to proliferation and apoptosis. Sp1 expression levels show a dramatic increase during transformation and this could play a critical role for tumour development or maintenance. Although Sp1 deregulation might be beneficial for tumour cells, its overexpression induces apoptosis of untransformed cells. Here we further characterised the functional and transcriptional responses of untransformed cells following Sp1 overexpression.

Methodology and principal findings: We made use of wild-type and DNA-binding-deficient Sp1 to demonstrate that the induction of apoptosis by Sp1 is dependent on its capacity to bind DNA. Genome-wide expression profiling identified genes involved in cancer, cell death and cell cycle as being enriched among differentially expressed genes following Sp1 overexpression. In silico search to determine the presence of Sp1 binding sites in the promoter region of modulated genes was conducted. Genes that contained Sp1 binding sites in their promoters were enriched among down-regulated genes. The endogenous sp1 gene is one of the most down-regulated suggesting a negative feedback loop induced by overexpressed Sp1. In contrast, genes containing Sp1 binding sites in their promoters were not enriched among up-regulated genes. These results suggest that the transcriptional response involves both direct Sp1-driven transcription and indirect mechanisms. Finally, we show that Sp1 overexpression led to a modified expression of G1/S transition regulatory genes such as the down-regulation of cyclin D2 and the up-regulation of cyclin G2 and cdkn2c/p18 expression. The biological significance of these modifications was confirmed by showing that the cells accumulated in the G1 phase of the cell cycle before the onset of apoptosis.

Conclusion: This study shows that the binding to DNA of overexpressed Sp1 induces an inhibition of cell cycle progression that precedes apoptosis and a transcriptional response targeting genes containing Sp1 binding sites in their promoter or not suggesting both direct Sp1-driven transcription and indirect mechanisms.

Show MeSH
Related in: MedlinePlus