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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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Histone signature at the MAGE-A1 promoter as examined by chromatin immunoprecipitation. DNA was derived from unstimulated (basal) MCF-7 cells, 5-aza-CdR- and/or TSA-stimulated MCF-7 cells and MCF-7 cells transfected with the expression plasmid encoding for BORIS. DNA-bound histones were immunoprecipitated by antibodies specific for methylated and acetylated histones, and amplified in a real-time PCR by a primer pair specific for the MAGE-A1 promoter. The background of the non-specific IgG immunoprecipitation was subtracted from the calculated ratio between the data derived from the histone-specific immunoprecipitation and input DNA. H3K9, Lysine 9 of histone H3; H4K8, Lysine 8 of histone H4; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; ac, acetylated; me, monomethylated; me2, dimethylated; me3, trimethylated. The significant p-values are shown.
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Fig4: Histone signature at the MAGE-A1 promoter as examined by chromatin immunoprecipitation. DNA was derived from unstimulated (basal) MCF-7 cells, 5-aza-CdR- and/or TSA-stimulated MCF-7 cells and MCF-7 cells transfected with the expression plasmid encoding for BORIS. DNA-bound histones were immunoprecipitated by antibodies specific for methylated and acetylated histones, and amplified in a real-time PCR by a primer pair specific for the MAGE-A1 promoter. The background of the non-specific IgG immunoprecipitation was subtracted from the calculated ratio between the data derived from the histone-specific immunoprecipitation and input DNA. H3K9, Lysine 9 of histone H3; H4K8, Lysine 8 of histone H4; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; ac, acetylated; me, monomethylated; me2, dimethylated; me3, trimethylated. The significant p-values are shown.

Mentions: Since BORIS may demethylate the MAGE-A1 promoter, we also analyzed its impact on the modifications of histones bound at the MAGE-A1 promoters. To investigate the changes in the histone signature of MAGE-A1 promoter, it was compared in basal MCF-7 cells (no expression of MAGE-A1, Table 1) to the signature in MCF-7 cells stimulated by 5-aza-CdR with/without TSA or transfected with the expression plasmid encoding for BORIS. For these analyses we used antibodies specific for acetylated histones H3K9 and H4K8, and for methylated histones H3K4, H3K9 and H4K20. We performed immunoblot analyses and documented specific recognition of histone modifications by these specific antibodies. The histone modifications could not be determined in the micrometastatic BCM1 cells because of their slow cell growth and high cell death caused by 5-aza-CdR and TSA. Upon treatment of MCF-7 cells with TSA, an enrichment of H3K9ac could be observed, indicating the function of TSA as histone deacetylase inhibitor (p = 0.001). While DNA demethylation by 5-aza-CdR had no or a minor effect on the histone modifications, 5-aza-CdR and TSA were able to enrich H3K9ac, H4K8ac, H3K9me, and H3K4me2 (p = 0.0001). Based on their low levels, the relative changes in the histone modifications of H3K4me3, H4K20me, H4K20me2 and H4K20me3 could not be evaluated, but did not seem to be significant (Figure 4). Due to the nature of the experimental procedures of ChIP, we could not sort transfected cells from untransfected cells by FACS analyses. Therefore, the predominant occurrence of untransfected cells in the transfection assay (10% of transfection efficiency) may be the reason, that we could not observe any alterations in the histone modifications mediated by transfected BORIS (Figure 4).Figure 4


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)

Histone signature at the MAGE-A1 promoter as examined by chromatin immunoprecipitation. DNA was derived from unstimulated (basal) MCF-7 cells, 5-aza-CdR- and/or TSA-stimulated MCF-7 cells and MCF-7 cells transfected with the expression plasmid encoding for BORIS. DNA-bound histones were immunoprecipitated by antibodies specific for methylated and acetylated histones, and amplified in a real-time PCR by a primer pair specific for the MAGE-A1 promoter. The background of the non-specific IgG immunoprecipitation was subtracted from the calculated ratio between the data derived from the histone-specific immunoprecipitation and input DNA. H3K9, Lysine 9 of histone H3; H4K8, Lysine 8 of histone H4; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; ac, acetylated; me, monomethylated; me2, dimethylated; me3, trimethylated. The significant p-values are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig4: Histone signature at the MAGE-A1 promoter as examined by chromatin immunoprecipitation. DNA was derived from unstimulated (basal) MCF-7 cells, 5-aza-CdR- and/or TSA-stimulated MCF-7 cells and MCF-7 cells transfected with the expression plasmid encoding for BORIS. DNA-bound histones were immunoprecipitated by antibodies specific for methylated and acetylated histones, and amplified in a real-time PCR by a primer pair specific for the MAGE-A1 promoter. The background of the non-specific IgG immunoprecipitation was subtracted from the calculated ratio between the data derived from the histone-specific immunoprecipitation and input DNA. H3K9, Lysine 9 of histone H3; H4K8, Lysine 8 of histone H4; H3K4, Lysine 4 of histone H3; H4K20, Lysine 20 of histone H4; ac, acetylated; me, monomethylated; me2, dimethylated; me3, trimethylated. The significant p-values are shown.
Mentions: Since BORIS may demethylate the MAGE-A1 promoter, we also analyzed its impact on the modifications of histones bound at the MAGE-A1 promoters. To investigate the changes in the histone signature of MAGE-A1 promoter, it was compared in basal MCF-7 cells (no expression of MAGE-A1, Table 1) to the signature in MCF-7 cells stimulated by 5-aza-CdR with/without TSA or transfected with the expression plasmid encoding for BORIS. For these analyses we used antibodies specific for acetylated histones H3K9 and H4K8, and for methylated histones H3K4, H3K9 and H4K20. We performed immunoblot analyses and documented specific recognition of histone modifications by these specific antibodies. The histone modifications could not be determined in the micrometastatic BCM1 cells because of their slow cell growth and high cell death caused by 5-aza-CdR and TSA. Upon treatment of MCF-7 cells with TSA, an enrichment of H3K9ac could be observed, indicating the function of TSA as histone deacetylase inhibitor (p = 0.001). While DNA demethylation by 5-aza-CdR had no or a minor effect on the histone modifications, 5-aza-CdR and TSA were able to enrich H3K9ac, H4K8ac, H3K9me, and H3K4me2 (p = 0.0001). Based on their low levels, the relative changes in the histone modifications of H3K4me3, H4K20me, H4K20me2 and H4K20me3 could not be evaluated, but did not seem to be significant (Figure 4). Due to the nature of the experimental procedures of ChIP, we could not sort transfected cells from untransfected cells by FACS analyses. Therefore, the predominant occurrence of untransfected cells in the transfection assay (10% of transfection efficiency) may be the reason, that we could not observe any alterations in the histone modifications mediated by transfected BORIS (Figure 4).Figure 4

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