Characterization of the bovine pregnancy-associated glycoprotein gene family - analysis of gene sequences, regulatory regions within the promoter and expression of selected genes

Background: The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order. In cattle, the PAG gene family is comprised of at least 22 transcribed genes, as well as some variants. Phylogenetic analyses have shown that the PAG family segregates into 'ancient' and 'modern' groupings. Along with sequence differences between family members, there are clear distinctions in their spatio-temporal distribution and in their relative level of expression. In this report, 1) we performed an in silico analysis of the bovine genome to further characterize the PAG gene family, 2) we scrutinized proximal promoter sequences of the PAG genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3) we determined relative transcript abundance of selected PAGs during pregnancy and, 4) we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, bovine (bo) PAG-2. Results: From our analysis of the bovine genome, we identified 18 distinct PAG genes and 14 pseudogenes. We observed that the first 500 base pairs upstream of the translational start site contained multiple regions that are conserved among all boPAGs. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern boPAG grouping, but not the ancient boPAGs. We gathered evidence by means of Q-PCR and screening of EST databases to show that boPAG-2 is the most abundant of all boPAG transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the boPAG-2 gene. Conclusion: PAGs represent a relatively large gene family in the bovine genome. The proximal promoter regions of these genes display differences in putative TF binding sites, likely contributing to observed differences in spatial and temporal expression. We also discovered that boPAG-2 is the most abundant of all boPAG transcripts and provided evidence for the role of ETS and DDVL TFs in its regulation. These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the boPAG gene family.