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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 and Sp1 interact with the TATA binding protein(hTBP). The methionine labeled “prey” fusion proteins Sp1, hTBP and the carboxyl terminus of hTBP (hTBP-C) were incubated with various resin-bound “bait” proteins as indicated below the polyacrylamide gels. The protein complexes were collected by precipitation and separated by gel electrophoresis. The weak band in the TNT-mix (negative control) is unspecific and serves as a background level (A). Schematic view of the detected protein-protein interactions. N, amino terminus; C, carboxyl terminus (B).
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Fig7: BORIS and Sp1 interact with the TATA binding protein(hTBP). The methionine labeled “prey” fusion proteins Sp1, hTBP and the carboxyl terminus of hTBP (hTBP-C) were incubated with various resin-bound “bait” proteins as indicated below the polyacrylamide gels. The protein complexes were collected by precipitation and separated by gel electrophoresis. The weak band in the TNT-mix (negative control) is unspecific and serves as a background level (A). Schematic view of the detected protein-protein interactions. N, amino terminus; C, carboxyl terminus (B).

Mentions: In our recent publication, we have shown the cooperative and competitive interplay of transcription factors (Ets-1, Sp1) and epigenetic factors (BORIS, MBD1, MBD2a) to activate or repress the MAGE-A1 promoter by transient transfection assays [11]. To determine if these proteins, together with BORIS, can form secondary complexes with each other in the absence of DNA and to understand better the mechanism underlying the regulation of MAGE-A1 expression, we carried out in vitro protein-protein interaction assays. Each of the proteins was either a resin-bound “bait” fusion protein or a [35S]-L-methionine labeled “prey” protein. As shown by the separation of the bound proteins on polyacrylamide gels and previously reported [28, 29], the transcription factor Sp1 efficiently interacted with Ets-1 and the amino-terminus of the human TATA binding protein (hTBP), the general transcription factor of the basal transcription complex. We show for the first time, that Sp1 also interacted with BORIS and MBD1v1, which up- and downregulate MAGE-A1 promoter activity, respectively, but this protein-protein interaction was much weaker than its interaction with Ets-1 and hTBP amino-terminus. Conversely, hTBP interacted with MBD2b, BORIS, Ets-1 and Sp1 (Figure 7A). For the interaction with MBD2b and BORIS the evolutionarily conserved carboxyl (C) terminus of hTBP was necessary and sufficient. At present, it is unclear whether these interactions with hTBP play a functional role in the competitive transcriptional regulation of Sp1 and BORIS. However, this observation is supported by our ChIP-seq data on several cancer cell lines – BORIS sites are frequently overlapped with hTBP [17]. For a better overview, Figure 7B schematically summarizes the protein-protein interactions.Figure 7


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 and Sp1 interact with the TATA binding protein(hTBP). The methionine labeled “prey” fusion proteins Sp1, hTBP and the carboxyl terminus of hTBP (hTBP-C) were incubated with various resin-bound “bait” proteins as indicated below the polyacrylamide gels. The protein complexes were collected by precipitation and separated by gel electrophoresis. The weak band in the TNT-mix (negative control) is unspecific and serves as a background level (A). Schematic view of the detected protein-protein interactions. N, amino terminus; C, carboxyl terminus (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig7: BORIS and Sp1 interact with the TATA binding protein(hTBP). The methionine labeled “prey” fusion proteins Sp1, hTBP and the carboxyl terminus of hTBP (hTBP-C) were incubated with various resin-bound “bait” proteins as indicated below the polyacrylamide gels. The protein complexes were collected by precipitation and separated by gel electrophoresis. The weak band in the TNT-mix (negative control) is unspecific and serves as a background level (A). Schematic view of the detected protein-protein interactions. N, amino terminus; C, carboxyl terminus (B).
Mentions: In our recent publication, we have shown the cooperative and competitive interplay of transcription factors (Ets-1, Sp1) and epigenetic factors (BORIS, MBD1, MBD2a) to activate or repress the MAGE-A1 promoter by transient transfection assays [11]. To determine if these proteins, together with BORIS, can form secondary complexes with each other in the absence of DNA and to understand better the mechanism underlying the regulation of MAGE-A1 expression, we carried out in vitro protein-protein interaction assays. Each of the proteins was either a resin-bound “bait” fusion protein or a [35S]-L-methionine labeled “prey” protein. As shown by the separation of the bound proteins on polyacrylamide gels and previously reported [28, 29], the transcription factor Sp1 efficiently interacted with Ets-1 and the amino-terminus of the human TATA binding protein (hTBP), the general transcription factor of the basal transcription complex. We show for the first time, that Sp1 also interacted with BORIS and MBD1v1, which up- and downregulate MAGE-A1 promoter activity, respectively, but this protein-protein interaction was much weaker than its interaction with Ets-1 and hTBP amino-terminus. Conversely, hTBP interacted with MBD2b, BORIS, Ets-1 and Sp1 (Figure 7A). For the interaction with MBD2b and BORIS the evolutionarily conserved carboxyl (C) terminus of hTBP was necessary and sufficient. At present, it is unclear whether these interactions with hTBP play a functional role in the competitive transcriptional regulation of Sp1 and BORIS. However, this observation is supported by our ChIP-seq data on several cancer cell lines – BORIS sites are frequently overlapped with hTBP [17]. For a better overview, Figure 7B schematically summarizes the protein-protein interactions.Figure 7

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