Novel activator protein-2alpha splice-variants function as transactivators of the ovine placental lactogen gene.
Activator protein-2 (AP-2) has been implicated as a transactivator of the human and ovine placental lactogen (oPL) genes. Transcriptional enhancement through an AP-2 cis-acting element has been described for other genes expressed in the placenta, but the AP-2 isoform enhancing expression is species dependent. Transactivation of the oPL minimal promoter (-124 bp to +16 bp) by AP-2 was confirmed by mutational analysis in transiently transfected human choriocarcinoma cells (BeWo). AP-2alpha was localized in ovine chorionic epithelial cells by immunohistochemistry and a 3-kb transcript was identified by Northern hybridization. Four nearly full-length AP-2 cDNAs were isolated from an ovine placenta cDNA library. Nucleotide sequencing these cDNAs revealed that the AP-2 mRNA expressed in the ovine placenta shares identity with human AP-2alpha, but variations in the predicted N-terminus were observed, and three unique AP-2alpha splice-variants were identified. Expression of AP-2alpha variants in HepG2 cells, devoid of endogenous AP-2, indicates that enhancement through the AP-2 element in the oPL gene minimal promoter was variant dependent. RNA transcripts for all of the ovine AP-2alpha splice-variants were confirmed in ovine placenta by RT-PCR, and homologs for two variants were found in human placenta. However, only one AP-2alpha transcript, which shares identity to Xenopus AP-2alpha, was expressed in BeWo cells. Immunoblot analysis confirmed AP-2alpha variants in ovine chorionic binucleate cell nuclear extracts, one of which migrates similar to the AP-2alpha variant identified in BeWo cell nuclear extracts. These data indicate the presence of new mammalian AP-2alpha splice-variants that augment transactivation of the oPL gene in ovine chorionic binucleate cells.
Limesand
S W
SW
Animal Reproduction and Biotechnology Laboratory, Department of Physiology, Colorado State University, Fort Collins, CO 80523-1683, USA.
Anthony
R V
RV
D017398:Alternative Splicing; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002822:Choriocarcinoma / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D051379:Mice; D008957:Models, Genetic; D008969:Molecular Sequence Data; D010928:Placental Lactogen / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D020033:Protein Isoforms; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D012756:Sheep; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D014407:Tumor Cells, Cultured; D014981:Xenopus; D029867:Xenopus Proteins
D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D020033:Protein Isoforms; C493673:TFAP2A protein, human; C493668:Tfap2a protein, mouse; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D029867:Xenopus Proteins; C493675:tfap2a protein, Xenopus; D010928:Placental Lactogen