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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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BORIS-specific shRNA knocks down BORIS and decreases MAGE-A1 gene expression. MDA-MB-468 (A, B, C) and MCF-7 (D, E) cells were transiently transfected with expression plasmid containing BORIS-specific shRNA and control plasmid encoding for a scramble shRNA. In contrast to MDA-MB-468 cells with their high levels of endogenous MAGE-A1 and BORIS mRNA levels, MCF-7 cells showing no expression of MAGE-A1 were additionally cotransfected with the expression plasmid containing the BORIS sequence. After a 48 hour transfection, mRNA levels were measured by PCR. Changes in mRNA expression levels of BORIS (A) and MAGE-A1 (B) by quantitative real-time PCR and MAGE-A1 by gel electrophoresis (C) in MDA-MB-468 cells. Real-time PCR derived changes in mRNA expression levels of BORIS (D) and MAGE-A1 (E) in MCF-7 cells. The significant p-values are shown.
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Fig3: BORIS-specific shRNA knocks down BORIS and decreases MAGE-A1 gene expression. MDA-MB-468 (A, B, C) and MCF-7 (D, E) cells were transiently transfected with expression plasmid containing BORIS-specific shRNA and control plasmid encoding for a scramble shRNA. In contrast to MDA-MB-468 cells with their high levels of endogenous MAGE-A1 and BORIS mRNA levels, MCF-7 cells showing no expression of MAGE-A1 were additionally cotransfected with the expression plasmid containing the BORIS sequence. After a 48 hour transfection, mRNA levels were measured by PCR. Changes in mRNA expression levels of BORIS (A) and MAGE-A1 (B) by quantitative real-time PCR and MAGE-A1 by gel electrophoresis (C) in MDA-MB-468 cells. Real-time PCR derived changes in mRNA expression levels of BORIS (D) and MAGE-A1 (E) in MCF-7 cells. The significant p-values are shown.

Mentions: We knocked down the high expression of endogenous BORIS in MDA-MB-468 cells by a BORIS specific shRNA cassette. The transfection with a plasmid encoding for a scramble shRNA served as a control. At 48 or 72 hour post-transfection, we quantified the changes in the BORIS and MAGE-A1 mRNA levels by quantitative real-time PCR and RT-PCR/gel electrophoresis. As measured by real-time PCR, BORIS-specific shRNA reduced the endogenous BORIS mRNA expression from 100% down to 20% in basal MDA-MB-468 cells (p = 0.0001) and, documenting more the specificity of the experiment, from 75% down to 40% in MDA-MB-468 cells transfected with the control plasmid encoding for scramble shRNA (Figure 3A, p = 0.008). As shown by quantitative real-time PCR (Figure 3B, p < 0.05) and on an agarose gel (Figure 3C), the BORIS-specific shRNA (with and without scramble shRNA) downregulated the basal endogenous MAGE-A1 expression approximately 30%. We also carried out these knock-down experiments in MCF-7 cells that were additionally transfected with an expression plasmid encoding for BORIS. Therefore, we co-transfected MCF-7 cells with an expression plasmid encoding for BORIS, to upregulate MAGE-A1 expression in this cell line. BORIS-specific shRNA reduced the BORIS mRNA expression nearly completely in presence and absence of scramble shRNA (Figure 3D, p = 0.0001). Likewise, the downregulation of MAGE-A1 expression by BORIS-specific shRNA was more prominent in MCF-7 cells than in MDA-MB-468 cells. As measured by quantitative real time PCR, BORIS-specific shRNA reduced the MAGE-A1 expression down to 10% in presence and absence of scramble shRNA (Figure 3E, p = 0.0001). This stronger downregulation of BORIS and MAGE-A1 in MCF-7 cells is caused by the overexpression of BORIS in these cells, whereas the analyses in MDA-MB-468 were carried with endogenous BORIS.Figure 2


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)

BORIS-specific shRNA knocks down BORIS and decreases MAGE-A1 gene expression. MDA-MB-468 (A, B, C) and MCF-7 (D, E) cells were transiently transfected with expression plasmid containing BORIS-specific shRNA and control plasmid encoding for a scramble shRNA. In contrast to MDA-MB-468 cells with their high levels of endogenous MAGE-A1 and BORIS mRNA levels, MCF-7 cells showing no expression of MAGE-A1 were additionally cotransfected with the expression plasmid containing the BORIS sequence. After a 48 hour transfection, mRNA levels were measured by PCR. Changes in mRNA expression levels of BORIS (A) and MAGE-A1 (B) by quantitative real-time PCR and MAGE-A1 by gel electrophoresis (C) in MDA-MB-468 cells. Real-time PCR derived changes in mRNA expression levels of BORIS (D) and MAGE-A1 (E) in MCF-7 cells. The significant p-values are shown.
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Fig3: BORIS-specific shRNA knocks down BORIS and decreases MAGE-A1 gene expression. MDA-MB-468 (A, B, C) and MCF-7 (D, E) cells were transiently transfected with expression plasmid containing BORIS-specific shRNA and control plasmid encoding for a scramble shRNA. In contrast to MDA-MB-468 cells with their high levels of endogenous MAGE-A1 and BORIS mRNA levels, MCF-7 cells showing no expression of MAGE-A1 were additionally cotransfected with the expression plasmid containing the BORIS sequence. After a 48 hour transfection, mRNA levels were measured by PCR. Changes in mRNA expression levels of BORIS (A) and MAGE-A1 (B) by quantitative real-time PCR and MAGE-A1 by gel electrophoresis (C) in MDA-MB-468 cells. Real-time PCR derived changes in mRNA expression levels of BORIS (D) and MAGE-A1 (E) in MCF-7 cells. The significant p-values are shown.
Mentions: We knocked down the high expression of endogenous BORIS in MDA-MB-468 cells by a BORIS specific shRNA cassette. The transfection with a plasmid encoding for a scramble shRNA served as a control. At 48 or 72 hour post-transfection, we quantified the changes in the BORIS and MAGE-A1 mRNA levels by quantitative real-time PCR and RT-PCR/gel electrophoresis. As measured by real-time PCR, BORIS-specific shRNA reduced the endogenous BORIS mRNA expression from 100% down to 20% in basal MDA-MB-468 cells (p = 0.0001) and, documenting more the specificity of the experiment, from 75% down to 40% in MDA-MB-468 cells transfected with the control plasmid encoding for scramble shRNA (Figure 3A, p = 0.008). As shown by quantitative real-time PCR (Figure 3B, p < 0.05) and on an agarose gel (Figure 3C), the BORIS-specific shRNA (with and without scramble shRNA) downregulated the basal endogenous MAGE-A1 expression approximately 30%. We also carried out these knock-down experiments in MCF-7 cells that were additionally transfected with an expression plasmid encoding for BORIS. Therefore, we co-transfected MCF-7 cells with an expression plasmid encoding for BORIS, to upregulate MAGE-A1 expression in this cell line. BORIS-specific shRNA reduced the BORIS mRNA expression nearly completely in presence and absence of scramble shRNA (Figure 3D, p = 0.0001). Likewise, the downregulation of MAGE-A1 expression by BORIS-specific shRNA was more prominent in MCF-7 cells than in MDA-MB-468 cells. As measured by quantitative real time PCR, BORIS-specific shRNA reduced the MAGE-A1 expression down to 10% in presence and absence of scramble shRNA (Figure 3E, p = 0.0001). This stronger downregulation of BORIS and MAGE-A1 in MCF-7 cells is caused by the overexpression of BORIS in these cells, whereas the analyses in MDA-MB-468 were carried with endogenous BORIS.Figure 2

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