cAMP-response element modulator-tau activates a distinct promoter element for the expression of the phospholipid hydroperoxide/sperm nucleus glutathione peroxidase gene.
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 alternative 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 region. 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 element modulator) transcription factors. By using electrophoretic mobility-shift assays, we demonstrated that both nuclear protein extract from highly purified rat spermatid cells and recombinant CREM-tau protein can specifically bind to this element. Furthermore, we cloned a 1059 bp comprising the intron and the alternative exon for snPHGPx in the pCAT3 reporter vector. By transient transfection experiments, we demonstrated that the expression of the transcription factor CREM-tau can induce the activation of the reporter gene in NIH-3T3 cell line. These results were confirmed by chromatin immunoprecipitation experiments performed on highly purified rat spermatid cells. On the basis of these results, we demonstrate that snPHGPx expression is mediated by the transcription factor CREM-tau, which acts as a cis-acting element localized in the first intron of the PHGPx gene.
Tramer
Federica
F
Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Via L. Giorgieri 1, 34127 Trieste, Italy.
Vetere
Amedeo
A
Martinelli
Monica
M
Paroni
Federico
F
Marsich
Eleonora
E
Boitani
Carla
C
Sandri
Gabriella
G
Panfili
Enrico
E
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D051786:Cyclic AMP Response Element Modulator; D004268:DNA-Binding Proteins / Q000502:physiology*; D024202:Electrophoretic Mobility Shift Assay; D015971:Gene Expression Regulation, Enzymologic; D005979:Glutathione Peroxidase / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007438:Introns; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D021141:Nucleic Acid Amplification Techniques; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D051381:Rats; D017208:Rats, Wistar; D020218:Response Elements / Q000502:physiology*; D013094:Spermatozoa / Q000201:enzymology* / Q000648:ultrastructure; D015534:Trans-Activators / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*
D004268:DNA-Binding Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D051786:Cyclic AMP Response Element Modulator; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase