PHGPx (phospholipid hydroperoxide glutathione peroxidase) is a selenoprotein present in at least three isoforms in testis: cytosolic, mitochondrial and nuclear. All of these derive from the same gene and are structurally related with the exception of the snPHGPx (sperm nucleus-specific form), which differs from the others due to the presence of an arginine-rich N-terminus. It has been demonstrated recently that this N-terminus is encoded by an alter- native exon located in the first intron of the PHGPx gene. The expression of snPHGPx has been attributed either to an alternative pre-mRNA splicing or to the presence of a distinct promoter re- gion. Nevertheless, the exact molecular mechanism by which the expression of snPHGPx occurs has not been demonstrated so far. Preliminary sequence analysis of the region located upstream of the alternative exon revealed some potential DNA-binding sites, one of which is specific to the binding of CREM(cAMP-response elementmodulator) transcription factors.By using electrophoretic mobility-shift assays, we demonstrated that both nuclear protein extract from highly purified rat spermatid cells and recombinant CREM-τ protein can specifically bind to this element. Further- more, we cloned a 1059 bp comprising the intron and the alter- native exon for snPHGPx in the pCAT®3 reporter vector. By transient transfection experiments, we demonstrated that the ex- pression of the transcription factor CREM-τ can induce the activ- ation of the reporter gene in NIH-3T3 cell line. These results were confirmed by chromatin immunoprecipitation experiments per- formed on highly purified rat spermatid cells.On the basis of these results, we demonstrate that snPHGPx expression is mediated by the transcription factor CREM-τ , which acts as a cis-acting element localized in the first intron of the PHGPx gene.

cAMP-response element modulator-tau activates a distinct promoter element for the expression of phospholipid hydroperoxide/sperm nucleus glutathione peroxidase gene

TRAMER, FEDERICA;VETERE, AMEDEO;MARSICH, ELEONORA;
2004-01-01

Abstract

PHGPx (phospholipid hydroperoxide glutathione peroxidase) is a selenoprotein present in at least three isoforms in testis: cytosolic, mitochondrial and nuclear. All of these derive from the same gene and are structurally related with the exception of the snPHGPx (sperm nucleus-specific form), which differs from the others due to the presence of an arginine-rich N-terminus. It has been demonstrated recently that this N-terminus is encoded by an alter- native exon located in the first intron of the PHGPx gene. The expression of snPHGPx has been attributed either to an alternative pre-mRNA splicing or to the presence of a distinct promoter re- gion. Nevertheless, the exact molecular mechanism by which the expression of snPHGPx occurs has not been demonstrated so far. Preliminary sequence analysis of the region located upstream of the alternative exon revealed some potential DNA-binding sites, one of which is specific to the binding of CREM(cAMP-response elementmodulator) transcription factors.By using electrophoretic mobility-shift assays, we demonstrated that both nuclear protein extract from highly purified rat spermatid cells and recombinant CREM-τ protein can specifically bind to this element. Further- more, we cloned a 1059 bp comprising the intron and the alter- native exon for snPHGPx in the pCAT®3 reporter vector. By transient transfection experiments, we demonstrated that the ex- pression of the transcription factor CREM-τ can induce the activ- ation of the reporter gene in NIH-3T3 cell line. These results were confirmed by chromatin immunoprecipitation experiments per- formed on highly purified rat spermatid cells.On the basis of these results, we demonstrate that snPHGPx expression is mediated by the transcription factor CREM-τ , which acts as a cis-acting element localized in the first intron of the PHGPx gene.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/1698502
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