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

Upregulation of alternatively spliced BORIS transcripts in MCF-7 and BCM1 cells as determined by quantitative real time PCR. Changes in mRNA expression levels of BORIS isoforms sf2 to sf6 in MCF-7 (A) and micrometastatic breast cancer BCM1 cells (B), which were basal or transiently transfected with the expression plasmid containing the BORIS (sf1) sequence. The significant p-values are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig6: Upregulation of alternatively spliced BORIS transcripts in MCF-7 and BCM1 cells as determined by quantitative real time PCR. Changes in mRNA expression levels of BORIS isoforms sf2 to sf6 in MCF-7 (A) and micrometastatic breast cancer BCM1 cells (B), which were basal or transiently transfected with the expression plasmid containing the BORIS (sf1) sequence. The significant p-values are shown.

Mentions: The differential regulation of MAGE-A1 gene expression could be explained by a separate site in MAGE-A1 promoter that could be recognized by a BORIS alternatively spliced isoform with different sequence specificity. For this reason, we compared mRNA patterns of alternatively spliced BORIS isoforms in basal MCF-7 and BCM1 cells with analogous cells transfected with BORIS (the first cloned, original form B0). As we recently described [17], BORIS isoforms could not be specifically discriminated by quantitative RT-PCR because most isoforms share sequence similarities, making it impossible to design primers and probes that would detect each BORIS isoform as a separate species. Therefore, we operationally divided the 23 isoforms into six subfamilies (sf1 to sf6) based on their unique 3’ terminal sequences, which were used to design 6 Taqman probes for quantitative real-time PCR [17]. With the exception of sf1, which detects the original BORIS form (B0) that was transfected into the cell lines, we measured the relative units of BORIS isoforms sf2 to sf6 in basal and BORIS-transfected MCF-7 and BCM1 cells. As shown in Figure 6, BORIS stimulated substantially its isoforms sf3 to sf6 in MCF-7 cells which do not express endogenous BORIS (Figure 6A, p = 0.0001), whereas BORIS only stimulated weakly sf2 and sf3 in BCM1 cells which express high levels of endogenous BORIS (Figure 6B).Figure 6


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)

Upregulation of alternatively spliced BORIS transcripts in MCF-7 and BCM1 cells as determined by quantitative real time PCR. Changes in mRNA expression levels of BORIS isoforms sf2 to sf6 in MCF-7 (A) and micrometastatic breast cancer BCM1 cells (B), which were basal or transiently transfected with the expression plasmid containing the BORIS (sf1) sequence. 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

Fig6: Upregulation of alternatively spliced BORIS transcripts in MCF-7 and BCM1 cells as determined by quantitative real time PCR. Changes in mRNA expression levels of BORIS isoforms sf2 to sf6 in MCF-7 (A) and micrometastatic breast cancer BCM1 cells (B), which were basal or transiently transfected with the expression plasmid containing the BORIS (sf1) sequence. The significant p-values are shown.
Mentions: The differential regulation of MAGE-A1 gene expression could be explained by a separate site in MAGE-A1 promoter that could be recognized by a BORIS alternatively spliced isoform with different sequence specificity. For this reason, we compared mRNA patterns of alternatively spliced BORIS isoforms in basal MCF-7 and BCM1 cells with analogous cells transfected with BORIS (the first cloned, original form B0). As we recently described [17], BORIS isoforms could not be specifically discriminated by quantitative RT-PCR because most isoforms share sequence similarities, making it impossible to design primers and probes that would detect each BORIS isoform as a separate species. Therefore, we operationally divided the 23 isoforms into six subfamilies (sf1 to sf6) based on their unique 3’ terminal sequences, which were used to design 6 Taqman probes for quantitative real-time PCR [17]. With the exception of sf1, which detects the original BORIS form (B0) that was transfected into the cell lines, we measured the relative units of BORIS isoforms sf2 to sf6 in basal and BORIS-transfected MCF-7 and BCM1 cells. As shown in Figure 6, BORIS stimulated substantially its isoforms sf3 to sf6 in MCF-7 cells which do not express endogenous BORIS (Figure 6A, p = 0.0001), whereas BORIS only stimulated weakly sf2 and sf3 in BCM1 cells which express high levels of endogenous BORIS (Figure 6B).Figure 6

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