Limits...
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.

Show MeSH

Related in: MedlinePlus

Comparison of the MAGE-A1 mRNA expression in 5-aza-CdR- and/or TSA-stimulated MCF-7 and BCM1 cells with the expression in BORIS-transfected cells. RT-PCR products of MAGE-A1 mRNA expression prior and after stimulation of MCF-7 (A) and BCM1 cells (B) with the demethylating agent 5-aza-CdR and/or the histone deacetylase inhibitor TSA or after transient transfection of these cells with an expression plasmid encoding for BORIS were separated on an agarose gel. The bar chart shows the relative changes in mRNA expression levels of MAGE-A1 in MCF-7 cells by quantitative real-time PCR. The significant p-values are shown (C). H2O lane serves as a negative control. The housekeeping gene β-Actin was selected as an internal control due to the lack of influence of any stimulation involved.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Comparison of the MAGE-A1 mRNA expression in 5-aza-CdR- and/or TSA-stimulated MCF-7 and BCM1 cells with the expression in BORIS-transfected cells. RT-PCR products of MAGE-A1 mRNA expression prior and after stimulation of MCF-7 (A) and BCM1 cells (B) with the demethylating agent 5-aza-CdR and/or the histone deacetylase inhibitor TSA or after transient transfection of these cells with an expression plasmid encoding for BORIS were separated on an agarose gel. The bar chart shows the relative changes in mRNA expression levels of MAGE-A1 in MCF-7 cells by quantitative real-time PCR. The significant p-values are shown (C). H2O lane serves as a negative control. The housekeeping gene β-Actin was selected as an internal control due to the lack of influence of any stimulation involved.

Mentions: We previously demonstrated that the demethylating agent 5-aza-CdR and the histone deacetylase inhibitor TSA synergistically upregulate MAGE-A1 expression in cell lines derived from different cancer types [6]. Moreover, Vatolin et al. reported that conditionally expressed BORIS induces expression of a series of CTA genes, including MAGE-A1 gene [14], but converse data have also been reported demonstrating that stable expression of BORIS in melanoma cell lines did not induce expression of MAGE-A1 [20]. In order to examine whether BORIS is actually able to activate the MAGE-A1 promoter and to which extent, we compared its influence with the stimulatory effect of 5-aza-CdR and/or TSA on MAGE-A1 transcription in cancer cell line settings. For our current investigations, we chose 3 breast cancer cell lines: MDA-MB-468, MCF-7 and BCM1 because of their different levels of MAGE-A1 and BORIS transcripts. As shown in Table 1 and measured by quantitative real-time PCR, MDA-MB-468 cells express relatively high levels of MAGE-A1 [2^(ΔCt) 19.33] and BORIS mRNA [2^(ΔCt) 48.78], whereas MCF-7 cells do not (or negligibly) express MAGE-A1 mRNA [2^(ΔCt) 2.00] and express low levels of BORIS [2^(ΔCt) 6.92 with a high standard deviation]. In the micrometastatic cell line BCM1, the expression of both genes is opposite: no levels of MAGE-A1 [2^(ΔCt) 1.07] and high levels of BORIS [2^(ΔCt) 24.39]. We transiently transfected expression plasmid encoding BORIS into both cell lines, with negligible transcript levels of MAGE-A1, and quantified endogenous MAGE-A1 mRNA by RT (reverse transcription)-PCR and gel electrophoresis. As depicted in Figure 1, BORIS was able to stimulate or induce the expression of MAGE-A1 in MCF-7 cells (Figure 1A) and BCM1 (Figure 1B) cells. In both cell lines, the BORIS-mediated stimulation was much weaker than the stimulatory effect by both agents (5-aza-CdR and/or TSA, Figure 1). Performing real-time PCR, we found that 5-aza-CdR (p = 0.0001), TSA (p = 0.001), 5-aza-CdR plus TSA (p = 0.0001) and BORIS (p = 0.04) stimulated the RNA expression 30-, 18-, 60- and 7-fold, respectively, in MCF-7 cells (Figure 1C). This ostensibly weaker activation by transfected BORIS may be partly due to the fact that transfection efficiency is usually much lower and about 10% (as deduced from FACS analyses and shown later), but 5-aza-CdR and TSA treatment can affect 100% of cells taken into experiment.Table 1


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)

Comparison of the MAGE-A1 mRNA expression in 5-aza-CdR- and/or TSA-stimulated MCF-7 and BCM1 cells with the expression in BORIS-transfected cells. RT-PCR products of MAGE-A1 mRNA expression prior and after stimulation of MCF-7 (A) and BCM1 cells (B) with the demethylating agent 5-aza-CdR and/or the histone deacetylase inhibitor TSA or after transient transfection of these cells with an expression plasmid encoding for BORIS were separated on an agarose gel. The bar chart shows the relative changes in mRNA expression levels of MAGE-A1 in MCF-7 cells by quantitative real-time PCR. The significant p-values are shown (C). H2O lane serves as a negative control. The housekeeping gene β-Actin was selected as an internal control due to the lack of influence of any stimulation involved.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Comparison of the MAGE-A1 mRNA expression in 5-aza-CdR- and/or TSA-stimulated MCF-7 and BCM1 cells with the expression in BORIS-transfected cells. RT-PCR products of MAGE-A1 mRNA expression prior and after stimulation of MCF-7 (A) and BCM1 cells (B) with the demethylating agent 5-aza-CdR and/or the histone deacetylase inhibitor TSA or after transient transfection of these cells with an expression plasmid encoding for BORIS were separated on an agarose gel. The bar chart shows the relative changes in mRNA expression levels of MAGE-A1 in MCF-7 cells by quantitative real-time PCR. The significant p-values are shown (C). H2O lane serves as a negative control. The housekeeping gene β-Actin was selected as an internal control due to the lack of influence of any stimulation involved.
Mentions: We previously demonstrated that the demethylating agent 5-aza-CdR and the histone deacetylase inhibitor TSA synergistically upregulate MAGE-A1 expression in cell lines derived from different cancer types [6]. Moreover, Vatolin et al. reported that conditionally expressed BORIS induces expression of a series of CTA genes, including MAGE-A1 gene [14], but converse data have also been reported demonstrating that stable expression of BORIS in melanoma cell lines did not induce expression of MAGE-A1 [20]. In order to examine whether BORIS is actually able to activate the MAGE-A1 promoter and to which extent, we compared its influence with the stimulatory effect of 5-aza-CdR and/or TSA on MAGE-A1 transcription in cancer cell line settings. For our current investigations, we chose 3 breast cancer cell lines: MDA-MB-468, MCF-7 and BCM1 because of their different levels of MAGE-A1 and BORIS transcripts. As shown in Table 1 and measured by quantitative real-time PCR, MDA-MB-468 cells express relatively high levels of MAGE-A1 [2^(ΔCt) 19.33] and BORIS mRNA [2^(ΔCt) 48.78], whereas MCF-7 cells do not (or negligibly) express MAGE-A1 mRNA [2^(ΔCt) 2.00] and express low levels of BORIS [2^(ΔCt) 6.92 with a high standard deviation]. In the micrometastatic cell line BCM1, the expression of both genes is opposite: no levels of MAGE-A1 [2^(ΔCt) 1.07] and high levels of BORIS [2^(ΔCt) 24.39]. We transiently transfected expression plasmid encoding BORIS into both cell lines, with negligible transcript levels of MAGE-A1, and quantified endogenous MAGE-A1 mRNA by RT (reverse transcription)-PCR and gel electrophoresis. As depicted in Figure 1, BORIS was able to stimulate or induce the expression of MAGE-A1 in MCF-7 cells (Figure 1A) and BCM1 (Figure 1B) cells. In both cell lines, the BORIS-mediated stimulation was much weaker than the stimulatory effect by both agents (5-aza-CdR and/or TSA, Figure 1). Performing real-time PCR, we found that 5-aza-CdR (p = 0.0001), TSA (p = 0.001), 5-aza-CdR plus TSA (p = 0.0001) and BORIS (p = 0.04) stimulated the RNA expression 30-, 18-, 60- and 7-fold, respectively, in MCF-7 cells (Figure 1C). This ostensibly weaker activation by transfected BORIS may be partly due to the fact that transfection efficiency is usually much lower and about 10% (as deduced from FACS analyses and shown later), but 5-aza-CdR and TSA treatment can affect 100% of cells taken into experiment.Table 1

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