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Differential regulation of MAGE-A1 promoter activity by BORIS and Sp1, both interacting with the TATA binding protein.

Schwarzenbach H, Eichelser C, Steinbach B, Tadewaldt J, Pantel K, Lobanenkov V, Loukinov D - BMC Cancer (2014)

Bottom Line: Our findings show that BORIS and Sp1 have opposite effects on the regulation of MAGE-A1 gene expression.This differential regulation may be explained by direct protein-protein interaction of both factors or by interaction of MAGE-A1 promoter with BORIS alternatively spliced isoforms with different sequence specificity.We also show here that ectopic expression of BORIS can activate transcription from its own locus, inducing all its splice variants.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinistraße 52, Hamburg 20246, Germany. hschwarz@uke.uni-hamburg.de.

ABSTRACT

Background: As cancer-testis MAGE-A antigens are targets for tumor immunotherapy, it is important to study the regulation of their expression in cancers. This regulation appears to be rather complex and at the moment controversial. Although it is generally accepted that MAGE-A expression is controlled by epigenetics, the exact mechanisms of that control remain poorly understood.

Methods: We analyzed the interplay of another cancer-testis gene, BORIS, and the transcription factors Ets-1 and Sp1 in the regulation of MAGE-A1 gene expression performing luciferase assays, quantitative real-time PCR, sodium bisulfite sequencing, chromatin immunoprecipitation assays and pull down experiments.

Results: We detected that ectopically expressed BORIS could activate and demethylate both endogenous and methylated reporter MAGE-A1 promoter in MCF-7 and micrometastatic BCM1 cancer cell lines. Overexpression of Ets-1 could not further upregulate the promoter activity mediated by BORIS. Surprisingly, in co-transfection experiments we observed that Sp1 partly repressed the BORIS-mediated stimulation, while addition of Ets-1 expression plasmid abrogated the Sp1 mediated repression of MAGE-A1 promoter. Both BORIS and Sp1 interacted with the TATA binding protein (hTBP) suggesting the possibility of a competitive mechanism of action between BORIS and Sp1.

Conclusions: Our findings show that BORIS and Sp1 have opposite effects on the regulation of MAGE-A1 gene expression. This differential regulation may be explained by direct protein-protein interaction of both factors or by interaction of MAGE-A1 promoter with BORIS alternatively spliced isoforms with different sequence specificity. We also show here that ectopic expression of BORIS can activate transcription from its own locus, inducing all its splice variants.

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MAGE-A1 promoter activity in basal and transfected cancer cells. Schematic view of the MAGE-A1 promoter fragment (-81/-185). The binding sites for Ets-1, Sp1 and basal transcription complex are indicated by grey boxes. The start site is indicated by an arrow. The vertical lines with the numbers mark the cytosine in the CpG dinucleotides (A). Luciferase activity of the HpaII-methylated plasmid containing the MAGE-A1 promoter fragment (-77/+183) in BCM1 cells which were transiently co-transfected with expression plasmids encoding for BORIS, Ets-1 and Sp1. The basal MAGE-A1 promoter activity was set to 100%. The activities derived from the reference plasmid encoding for the Renilla Luciferase were used to normalize the variability in transfection efficiency. The significant p-values are shown (B). Endogenous mRNA expression of MAGE-A1 in MCF-7 and BCM1 cells basal or transfected with expression plasmids encoding for BORIS, Ets-1 and Sp1 as determined by RT-PCR and gel electrophoresis. The housekeeping gene β-Actin was selected as an internal control due to the lack of influence of any stimulation involved (C).
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Fig5: MAGE-A1 promoter activity in basal and transfected cancer cells. Schematic view of the MAGE-A1 promoter fragment (-81/-185). The binding sites for Ets-1, Sp1 and basal transcription complex are indicated by grey boxes. The start site is indicated by an arrow. The vertical lines with the numbers mark the cytosine in the CpG dinucleotides (A). Luciferase activity of the HpaII-methylated plasmid containing the MAGE-A1 promoter fragment (-77/+183) in BCM1 cells which were transiently co-transfected with expression plasmids encoding for BORIS, Ets-1 and Sp1. The basal MAGE-A1 promoter activity was set to 100%. The activities derived from the reference plasmid encoding for the Renilla Luciferase were used to normalize the variability in transfection efficiency. The significant p-values are shown (B). Endogenous mRNA expression of MAGE-A1 in MCF-7 and BCM1 cells basal or transfected with expression plasmids encoding for BORIS, Ets-1 and Sp1 as determined by RT-PCR and gel electrophoresis. The housekeeping gene β-Actin was selected as an internal control due to the lack of influence of any stimulation involved (C).

Mentions: In order to functionally investigate the impact of BORIS on promoter settings, we examined the influence of BORIS on the activity of the methylated MAGE-A1 promoter in the context of transcription factors Ets-1 and Sp1. We transiently co-transfected methylated reporter plasmids pGL2/MAGE-A1 (-77/+183) containing the BORIS binding site located downstream of the start site (Figure 5A), and expression plasmids encoding for BORIS, Ets-1 or Sp1 into BCM1 cells. As expected, aberrantly expressed transcription factors Ets-1 and Sp1 had no effect on methylated MAGE-A1 [11]. However, transfected BORIS was able to activate the methylated promoter in these cells (p = 0.0001). Overexpression of Ets-1 could not further upregulate the promoter activity mediated by BORIS, as shown by several repetitions. Surprisingly, co-transfection with an expression plasmid encoding for Sp1 partly repressed the stimulatory effect mediated by BORIS (p = 0.001), whereas the addition of expression plasmid encoding for Ets-1 abrogated this repression (Figure 5B).


Differential regulation of MAGE-A1 promoter activity by BORIS and Sp1, both interacting with the TATA binding protein.

Schwarzenbach H, Eichelser C, Steinbach B, Tadewaldt J, Pantel K, Lobanenkov V, Loukinov D - BMC Cancer (2014)

MAGE-A1 promoter activity in basal and transfected cancer cells. Schematic view of the MAGE-A1 promoter fragment (-81/-185). The binding sites for Ets-1, Sp1 and basal transcription complex are indicated by grey boxes. The start site is indicated by an arrow. The vertical lines with the numbers mark the cytosine in the CpG dinucleotides (A). Luciferase activity of the HpaII-methylated plasmid containing the MAGE-A1 promoter fragment (-77/+183) in BCM1 cells which were transiently co-transfected with expression plasmids encoding for BORIS, Ets-1 and Sp1. The basal MAGE-A1 promoter activity was set to 100%. The activities derived from the reference plasmid encoding for the Renilla Luciferase were used to normalize the variability in transfection efficiency. The significant p-values are shown (B). Endogenous mRNA expression of MAGE-A1 in MCF-7 and BCM1 cells basal or transfected with expression plasmids encoding for BORIS, Ets-1 and Sp1 as determined by RT-PCR and gel electrophoresis. The housekeeping gene β-Actin was selected as an internal control due to the lack of influence of any stimulation involved (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230356&req=5

Fig5: MAGE-A1 promoter activity in basal and transfected cancer cells. Schematic view of the MAGE-A1 promoter fragment (-81/-185). The binding sites for Ets-1, Sp1 and basal transcription complex are indicated by grey boxes. The start site is indicated by an arrow. The vertical lines with the numbers mark the cytosine in the CpG dinucleotides (A). Luciferase activity of the HpaII-methylated plasmid containing the MAGE-A1 promoter fragment (-77/+183) in BCM1 cells which were transiently co-transfected with expression plasmids encoding for BORIS, Ets-1 and Sp1. The basal MAGE-A1 promoter activity was set to 100%. The activities derived from the reference plasmid encoding for the Renilla Luciferase were used to normalize the variability in transfection efficiency. The significant p-values are shown (B). Endogenous mRNA expression of MAGE-A1 in MCF-7 and BCM1 cells basal or transfected with expression plasmids encoding for BORIS, Ets-1 and Sp1 as determined by RT-PCR and gel electrophoresis. The housekeeping gene β-Actin was selected as an internal control due to the lack of influence of any stimulation involved (C).
Mentions: In order to functionally investigate the impact of BORIS on promoter settings, we examined the influence of BORIS on the activity of the methylated MAGE-A1 promoter in the context of transcription factors Ets-1 and Sp1. We transiently co-transfected methylated reporter plasmids pGL2/MAGE-A1 (-77/+183) containing the BORIS binding site located downstream of the start site (Figure 5A), and expression plasmids encoding for BORIS, Ets-1 or Sp1 into BCM1 cells. As expected, aberrantly expressed transcription factors Ets-1 and Sp1 had no effect on methylated MAGE-A1 [11]. However, transfected BORIS was able to activate the methylated promoter in these cells (p = 0.0001). Overexpression of Ets-1 could not further upregulate the promoter activity mediated by BORIS, as shown by several repetitions. Surprisingly, co-transfection with an expression plasmid encoding for Sp1 partly repressed the stimulatory effect mediated by BORIS (p = 0.001), whereas the addition of expression plasmid encoding for Ets-1 abrogated this repression (Figure 5B).

Bottom Line: Our findings show that BORIS and Sp1 have opposite effects on the regulation of MAGE-A1 gene expression.This differential regulation may be explained by direct protein-protein interaction of both factors or by interaction of MAGE-A1 promoter with BORIS alternatively spliced isoforms with different sequence specificity.We also show here that ectopic expression of BORIS can activate transcription from its own locus, inducing all its splice variants.

View Article: PubMed Central - PubMed

Affiliation: Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Martinistraße 52, Hamburg 20246, Germany. hschwarz@uke.uni-hamburg.de.

ABSTRACT

Background: As cancer-testis MAGE-A antigens are targets for tumor immunotherapy, it is important to study the regulation of their expression in cancers. This regulation appears to be rather complex and at the moment controversial. Although it is generally accepted that MAGE-A expression is controlled by epigenetics, the exact mechanisms of that control remain poorly understood.

Methods: We analyzed the interplay of another cancer-testis gene, BORIS, and the transcription factors Ets-1 and Sp1 in the regulation of MAGE-A1 gene expression performing luciferase assays, quantitative real-time PCR, sodium bisulfite sequencing, chromatin immunoprecipitation assays and pull down experiments.

Results: We detected that ectopically expressed BORIS could activate and demethylate both endogenous and methylated reporter MAGE-A1 promoter in MCF-7 and micrometastatic BCM1 cancer cell lines. Overexpression of Ets-1 could not further upregulate the promoter activity mediated by BORIS. Surprisingly, in co-transfection experiments we observed that Sp1 partly repressed the BORIS-mediated stimulation, while addition of Ets-1 expression plasmid abrogated the Sp1 mediated repression of MAGE-A1 promoter. Both BORIS and Sp1 interacted with the TATA binding protein (hTBP) suggesting the possibility of a competitive mechanism of action between BORIS and Sp1.

Conclusions: Our findings show that BORIS and Sp1 have opposite effects on the regulation of MAGE-A1 gene expression. This differential regulation may be explained by direct protein-protein interaction of both factors or by interaction of MAGE-A1 promoter with BORIS alternatively spliced isoforms with different sequence specificity. We also show here that ectopic expression of BORIS can activate transcription from its own locus, inducing all its splice variants.

Show MeSH
Related in: MedlinePlus