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

Telugu BP, Walker AM, Green JA - BMC Genomics (2009)

Bottom Line: The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order.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.These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the boPAG gene family.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA. telugub@missouri.edu

ABSTRACT

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.

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Electrophoretic mobility shift assays demonstrating that the putative ETS site and the repeated elements in the boPAG-2 promoter are capable of binding proteins in trophoblast nuclear extracts. A. Competition of ETS-2 binding activity (20 μg protein) with cold ETS-2 probe. Nuclear extracts were incubated with 1 μL of 50 pmol probe, in the absence or presence of the indicated molar excess of cold probe (indicated along the top). B. The ETS-2 complex composition was examined by depleting ETS-2 with an antibody specific to ETS-2. Preincubation of the ETS antibody with the nuclear extracts followed by binding reaction with the probe resulted in specific dissociation of the complex. Control: normal rabbit serum. C and D. Competition assays indicating specificity of association of, as yet unknown, TFs capable of binding to the unique bovine tandem repeats, BR1(C) and BR2 (D). Lane 1: labeled probe and nuclear extract; Lane 2: same as lane 1 except for addition of a 50-fold molar excess of unlabeled double-stranded oligonucleotide; Lane 3: 250-fold molar excess of unlabeled probe; Lane 4: 500-fold molar excess.
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Figure 8: Electrophoretic mobility shift assays demonstrating that the putative ETS site and the repeated elements in the boPAG-2 promoter are capable of binding proteins in trophoblast nuclear extracts. A. Competition of ETS-2 binding activity (20 μg protein) with cold ETS-2 probe. Nuclear extracts were incubated with 1 μL of 50 pmol probe, in the absence or presence of the indicated molar excess of cold probe (indicated along the top). B. The ETS-2 complex composition was examined by depleting ETS-2 with an antibody specific to ETS-2. Preincubation of the ETS antibody with the nuclear extracts followed by binding reaction with the probe resulted in specific dissociation of the complex. Control: normal rabbit serum. C and D. Competition assays indicating specificity of association of, as yet unknown, TFs capable of binding to the unique bovine tandem repeats, BR1(C) and BR2 (D). Lane 1: labeled probe and nuclear extract; Lane 2: same as lane 1 except for addition of a 50-fold molar excess of unlabeled double-stranded oligonucleotide; Lane 3: 250-fold molar excess of unlabeled probe; Lane 4: 500-fold molar excess.

Mentions: Since boPAG-2 was the most abundant transcript observed in the bovine genome, we set out to study its promoter in some detail. ETS-2 is a key TF involved in the regulation of numerous placenta-specific genes, such as interferon-tau (IFNT) [61] and the human chorionic gonadotropin (hCG) beta subunit [62]. As mentioned previously, an ETS-2 site is present in all boPAG promoters (Figure 5), including boPAG-2, and may be critical to its transcriptional regulation. Competition and super shift assays (Figure 8A, and 8B) were performed with 32P-labeled oligonucleotides representing the putative ETS site from -226 to -229 (Figure 5). We utilized nuclear extracts from JAr human choriocarcinoma cells for this experiment, since nuclear extracts from bovine placental samples couldn't be obtained. EMSA's with nuclear extracts from JAr cells, which constitutively express ETS-2, indicated the presence of a protein(s) capable of specific association with the oligonucleotide probe. The complex could be competed away by excess unlabeled probe and could be decreased by the addition of an anti-ETS antibody. Likewise, the unique bovine tandem repeats (BR-1 and -2) which were reported previously and were found to be conserved across most of the PAGs [60] were also investigated by EMSAs to determine if proteins present in human JAr cells are capable of binding to these repeats. A specific complex was identified that could be competed away with an excess of non radiolabeled specific competitor (Figure 8C and 8D) implying that these repeats could possibly bind to endogenous TFs in placenta. Although, the experiments were conducted with cells of chorionic or placental origins from human, we anticipate that the observed results would also hold true with bovine placental samples.


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

Telugu BP, Walker AM, Green JA - BMC Genomics (2009)

Electrophoretic mobility shift assays demonstrating that the putative ETS site and the repeated elements in the boPAG-2 promoter are capable of binding proteins in trophoblast nuclear extracts. A. Competition of ETS-2 binding activity (20 μg protein) with cold ETS-2 probe. Nuclear extracts were incubated with 1 μL of 50 pmol probe, in the absence or presence of the indicated molar excess of cold probe (indicated along the top). B. The ETS-2 complex composition was examined by depleting ETS-2 with an antibody specific to ETS-2. Preincubation of the ETS antibody with the nuclear extracts followed by binding reaction with the probe resulted in specific dissociation of the complex. Control: normal rabbit serum. C and D. Competition assays indicating specificity of association of, as yet unknown, TFs capable of binding to the unique bovine tandem repeats, BR1(C) and BR2 (D). Lane 1: labeled probe and nuclear extract; Lane 2: same as lane 1 except for addition of a 50-fold molar excess of unlabeled double-stranded oligonucleotide; Lane 3: 250-fold molar excess of unlabeled probe; Lane 4: 500-fold molar excess.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2682831&req=5

Figure 8: Electrophoretic mobility shift assays demonstrating that the putative ETS site and the repeated elements in the boPAG-2 promoter are capable of binding proteins in trophoblast nuclear extracts. A. Competition of ETS-2 binding activity (20 μg protein) with cold ETS-2 probe. Nuclear extracts were incubated with 1 μL of 50 pmol probe, in the absence or presence of the indicated molar excess of cold probe (indicated along the top). B. The ETS-2 complex composition was examined by depleting ETS-2 with an antibody specific to ETS-2. Preincubation of the ETS antibody with the nuclear extracts followed by binding reaction with the probe resulted in specific dissociation of the complex. Control: normal rabbit serum. C and D. Competition assays indicating specificity of association of, as yet unknown, TFs capable of binding to the unique bovine tandem repeats, BR1(C) and BR2 (D). Lane 1: labeled probe and nuclear extract; Lane 2: same as lane 1 except for addition of a 50-fold molar excess of unlabeled double-stranded oligonucleotide; Lane 3: 250-fold molar excess of unlabeled probe; Lane 4: 500-fold molar excess.
Mentions: Since boPAG-2 was the most abundant transcript observed in the bovine genome, we set out to study its promoter in some detail. ETS-2 is a key TF involved in the regulation of numerous placenta-specific genes, such as interferon-tau (IFNT) [61] and the human chorionic gonadotropin (hCG) beta subunit [62]. As mentioned previously, an ETS-2 site is present in all boPAG promoters (Figure 5), including boPAG-2, and may be critical to its transcriptional regulation. Competition and super shift assays (Figure 8A, and 8B) were performed with 32P-labeled oligonucleotides representing the putative ETS site from -226 to -229 (Figure 5). We utilized nuclear extracts from JAr human choriocarcinoma cells for this experiment, since nuclear extracts from bovine placental samples couldn't be obtained. EMSA's with nuclear extracts from JAr cells, which constitutively express ETS-2, indicated the presence of a protein(s) capable of specific association with the oligonucleotide probe. The complex could be competed away by excess unlabeled probe and could be decreased by the addition of an anti-ETS antibody. Likewise, the unique bovine tandem repeats (BR-1 and -2) which were reported previously and were found to be conserved across most of the PAGs [60] were also investigated by EMSAs to determine if proteins present in human JAr cells are capable of binding to these repeats. A specific complex was identified that could be competed away with an excess of non radiolabeled specific competitor (Figure 8C and 8D) implying that these repeats could possibly bind to endogenous TFs in placenta. Although, the experiments were conducted with cells of chorionic or placental origins from human, we anticipate that the observed results would also hold true with bovine placental samples.

Bottom Line: The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the Artiodactyla order.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.These experiments mark the crucial first step in discerning the complex transcriptional regulation operating within the boPAG gene family.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Animal Sciences, University of Missouri, Columbia, MO 65211, USA. telugub@missouri.edu

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus