Functional characterization of cis- and trans-regulatory elements involved in expression of phospholipid hydroperoxide glutathione peroxidase.
Phospholipid hydroperoxide glutathione peroxidase (phGPx) is a member of the seleno glutathione peroxidase family that is comprised of five selenoproteins capable of reducing hydroperoxy lipids to the corresponding alcohols. The enzyme has been implicated in antioxidative defense, but its high expression level in testicular tissue suggests a more specific function during sperm maturation. The phGPx is encoded for by a joint sperm nucleus/phGPx gene (sn/phGPx) and can be expressed as a mitochondrial or cytosolic isoform. Although sn/phGPx genes have been cloned from various mammalian species expression regulation of the enzyme has not been studied in detail. We investigated the 5'-flanking region of the murine sn/phGPx gene and observed basic promoter activity in a 200 bp region localized immediately upstream of the translational initiation site of the cytosolic isoform (3'-ATG). DNase protection assays indicated the presence of five distinct protein-binding regions and electrophoretic mobility shift assays and supershift experiments revealed binding of stimulating protein 1 (SP1), nuclear factor Y (NF-Y) and members of the SMAD family. Site-directed mutagenesis of the consensus binding sequences abolished in vitro transcription factor binding. Expression of reporter genes was most effectively impaired when SP1/SP3 and NF-Y binding site-deficient constructs were tested. Chromatin immunoprecipitation suggested the in vivo relevance of these transcription factors. Our data indicate that the basic phGPx promoter constitutes a 200 bp oligonucleotide, which is localized immediately upstream of the 3'-ATG and involves functional SP1/SP3, NF-Y and SMAD binding sites. The corresponding trans-regulatory proteins may contribute to differential expression regulation of the mitochondrial and cytosolic phGPx isoforms.
Ufer
Christoph
C
Institute of Biochemistry, Humboldt University Medical School Charité, Monbijoustrasse 2, 10117 Berlin, Germany.
Borchert
Astrid
A
Kuhn
Hartmut
H
D024506:5' Flanking Region; D000818:Animals; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000378:metabolism; D002460:Cell Line; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D005979:Glutathione Peroxidase / Q000235:genetics*; D006801:Humans; D051379:Mice; D009154:Mutation; D023282:Nuclease Protection Assays; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D011401:Promoter Regions, Genetic; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism; D024363:Transcription Initiation Site; D015533:Transcriptional Activation
D023081:CCAAT-Binding Factor; D016329:Sp1 Transcription Factor; D000080662:Phospholipid Hydroperoxide Glutathione Peroxidase; D005979:Glutathione Peroxidase