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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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Related in: MedlinePlus

Changes in expression profile is dependent on Sp1 binding to DNA.Kinetic of expression levels of five differentially expressed genes (Neogenin, Slc39a8, Gelsolin, Cxcl4, Jun) following doxycycline removal in Baf-3-Sp1 clone 1 (left panel). At the indicated times, cells were harvested and mRNA levels were measured by real-time PCR and normalized to HPRT mRNA levels. Results show the ratio of the mRNA levels measured in the absence of doxycycline relative to mRNA levels measured in the presence of doxycycline at each time point. Results show the mean ± sd of at least 2 independent experiments. Expression levels in Baf-3 cells transduced with either control, Sp1 or Sp1Zn2,3 retroviruses purified by magnetic selection with anti-CD2 antibody 30 hrs post-infection (right panel). Results show the mean ± sd of at least 2 independent experiments.
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pone-0007035-g003: Changes in expression profile is dependent on Sp1 binding to DNA.Kinetic of expression levels of five differentially expressed genes (Neogenin, Slc39a8, Gelsolin, Cxcl4, Jun) following doxycycline removal in Baf-3-Sp1 clone 1 (left panel). At the indicated times, cells were harvested and mRNA levels were measured by real-time PCR and normalized to HPRT mRNA levels. Results show the ratio of the mRNA levels measured in the absence of doxycycline relative to mRNA levels measured in the presence of doxycycline at each time point. Results show the mean ± sd of at least 2 independent experiments. Expression levels in Baf-3 cells transduced with either control, Sp1 or Sp1Zn2,3 retroviruses purified by magnetic selection with anti-CD2 antibody 30 hrs post-infection (right panel). Results show the mean ± sd of at least 2 independent experiments.

Mentions: To have a better insight into the molecular perturbations induced by Sp1 overexpression, we performed a genome-wide expression profiling to identify set of genes that are affected by overexpression of Sp1. Expression profiling was conducted comparing Baf-3-Sp1 clone 1 grown in the presence of doxycycline for 28 hrs as control and the same clone grown 28 hrs without doxycycline. This time corresponds to a point where cells already expressed high levels of Sp1 protein without any detectable cell death (Figure 1A). Three independent experiments were performed and analysed using the Affymetrix genechip Mouse genome 430 2.0 arrays (data deposited in ArrayExpress http://www.ebi.ac.uk/microarray-as/ae, accession number E-MEXP-1702). Sp1 overexpression was associated with a substantial modification of transcription profiles (Figure S4). Differential probesets expression following Sp1 overexpression relative to control was determined using statistical analysis in GeneSpring: 1294 genes with unique gene symbol identifiers were found to be differentially expressed with a fold change of expression of 1.3. Among them, the expression of 766 genes was increased whereas the expression of 528 genes was decreased. With a two-fold change of expression, 172 genes with unique gene symbol identifiers were found to be differentially expressed (162 up-regulated genes, 10 down-regulated genes). Table 1 shows the most up-regulated and down-regulated genes. Sp1 overexpression has an effect on the expression of many genes involved in metabolism, ubiquitination and transcription but also many genes with unknown functions. The kinetic of expression of five of the most up-regulated genes (Neo1, Cxcl4, Gsn, Slc39a8, and Jun) was precisely monitored using quantitative real-time PCR (Figure 3, left panels). Their up-regulation starts to be detected 16 hrs following doxycycline removal and is also observed in Baf-3 cells transduced with wild-type Sp1 but not Sp1Zn2,3 indicating that the binding of Sp1 to DNA is necessary for their induction (Figure 3, right panels).


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)

Changes in expression profile is dependent on Sp1 binding to DNA.Kinetic of expression levels of five differentially expressed genes (Neogenin, Slc39a8, Gelsolin, Cxcl4, Jun) following doxycycline removal in Baf-3-Sp1 clone 1 (left panel). At the indicated times, cells were harvested and mRNA levels were measured by real-time PCR and normalized to HPRT mRNA levels. Results show the ratio of the mRNA levels measured in the absence of doxycycline relative to mRNA levels measured in the presence of doxycycline at each time point. Results show the mean ± sd of at least 2 independent experiments. Expression levels in Baf-3 cells transduced with either control, Sp1 or Sp1Zn2,3 retroviruses purified by magnetic selection with anti-CD2 antibody 30 hrs post-infection (right panel). Results show the mean ± sd of at least 2 independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2737146&req=5

pone-0007035-g003: Changes in expression profile is dependent on Sp1 binding to DNA.Kinetic of expression levels of five differentially expressed genes (Neogenin, Slc39a8, Gelsolin, Cxcl4, Jun) following doxycycline removal in Baf-3-Sp1 clone 1 (left panel). At the indicated times, cells were harvested and mRNA levels were measured by real-time PCR and normalized to HPRT mRNA levels. Results show the ratio of the mRNA levels measured in the absence of doxycycline relative to mRNA levels measured in the presence of doxycycline at each time point. Results show the mean ± sd of at least 2 independent experiments. Expression levels in Baf-3 cells transduced with either control, Sp1 or Sp1Zn2,3 retroviruses purified by magnetic selection with anti-CD2 antibody 30 hrs post-infection (right panel). Results show the mean ± sd of at least 2 independent experiments.
Mentions: To have a better insight into the molecular perturbations induced by Sp1 overexpression, we performed a genome-wide expression profiling to identify set of genes that are affected by overexpression of Sp1. Expression profiling was conducted comparing Baf-3-Sp1 clone 1 grown in the presence of doxycycline for 28 hrs as control and the same clone grown 28 hrs without doxycycline. This time corresponds to a point where cells already expressed high levels of Sp1 protein without any detectable cell death (Figure 1A). Three independent experiments were performed and analysed using the Affymetrix genechip Mouse genome 430 2.0 arrays (data deposited in ArrayExpress http://www.ebi.ac.uk/microarray-as/ae, accession number E-MEXP-1702). Sp1 overexpression was associated with a substantial modification of transcription profiles (Figure S4). Differential probesets expression following Sp1 overexpression relative to control was determined using statistical analysis in GeneSpring: 1294 genes with unique gene symbol identifiers were found to be differentially expressed with a fold change of expression of 1.3. Among them, the expression of 766 genes was increased whereas the expression of 528 genes was decreased. With a two-fold change of expression, 172 genes with unique gene symbol identifiers were found to be differentially expressed (162 up-regulated genes, 10 down-regulated genes). Table 1 shows the most up-regulated and down-regulated genes. Sp1 overexpression has an effect on the expression of many genes involved in metabolism, ubiquitination and transcription but also many genes with unknown functions. The kinetic of expression of five of the most up-regulated genes (Neo1, Cxcl4, Gsn, Slc39a8, and Jun) was precisely monitored using quantitative real-time PCR (Figure 3, left panels). Their up-regulation starts to be detected 16 hrs following doxycycline removal and is also observed in Baf-3 cells transduced with wild-type Sp1 but not Sp1Zn2,3 indicating that the binding of Sp1 to DNA is necessary for their induction (Figure 3, right panels).

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