The selective pressures operating on the ORFs of various boPAG genes have been analyzed systematically in prior publications 2932. The availability of full length gene sequences has made it possible to extend similar types of analyses to the PAG promoter regions. Two different lengths of promoter sequence were chosen for comparison [1000 bp as well as 500 bp proximal to the TSS] between several ancient boPAGs (boPAG-2, 8, 10, 11 and 12) and some representative modern boPAGs (boPAG-1, 3, 4, 5, 6, 7, 15, 18, 19, 20 and 21) to simplify the analysis. The nucleotide sequences were aligned by using CLUSTALW in the MEGA4 software suite. All the deletions and gaps arising from the alignment were eliminated by using the pairwise deletion option. The aligned boPAG sequences were subjected to pairwise comparisons in MEGA4 by using the Maximum Composite Likelihood method with 1000 bootstrap replicates to calculate the p-distance (number of differences/total length of sequence analyzed).

In order to understand the type of evolutionary pressures operating on the promoter regions, we plotted the inferred p-distances obtained from the promoter analysis against the proportion of synonymous changes per synonymous site (dS) estimated for the corresponding boPAG ORFs. The underlying assumption for this approach was that, dS within the ORFs would approximately reflect the rate of nucleotide change in the locus in the absence of selection. In other words, if the p-distance of the promoter equals dS of the corresponding exons of the gene (p-distance/dS = 1), then the boPAG promoter is accumulating substitutions in this region at a rate that corresponds to that expected, based on normal mutation rates. A value >1 would indicate that nucleotide changes are occurring faster than would be predicted and a value <1 would suggest stringent purifying selection, with fewer substitutions being tolerated and hence retained.

To explain for the apparent disparities in evolutionary pressures operating on the non-coding proximal promoter sequences of the boPAG genes, 1000 bp upstream of the translational start codon (ATG) were aligned with CLUSTALW. Within this alignment, insertions of TE, identified by the repeat masker program, were visually detected and mapped to the boPAG promoter sequences.

DiAlign TF, a component of the comprehensive promoter analysis software, Genomatix GEMS launcher http://www.genomatix.de/products/GEMSLauncher/47, was used to align and search for putative transcription factor (TF) binding sites within the proximal promoter regions of select PAGs. Approximately 1000 bp upstream of the TSS (proximal promoter) of eight boPAGs [4 ancient (boPAG-2, -8, -11, and -12), and 4 modern (boPAG-3, -5, -15, and -18)] that were recognized by the GEMS database were used in the analysis. The following parameters were selected for performing the analysis: Matrix library 7.0 was used as the default library to match the TF binding sites, and 'all' the matrix groups from 'embryo' tissue type were selected as a reference. Input sequences were aligned and regions closely matching known TF-binding sites that were conserved in more than 50% of the input sequences (4 out of 8) were mapped. The output from the analysis was modified and presented in multiple sequence alignment with artificial shading to facilitate easier comprehension.

In order to estimate, how differences within the boPAG promoters reflect in vivo expression differences, relative levels of transcription were determined based on the representation of each gene in common bovine EST databases. Known boPAG cDNAs were each queried by BLASTN in the NCBI bovine EST database. ESTs that exceeded 98% in identity in at least 350 bp of query nucleotide sequence were considered to be a positive match with a particular PAG.

It was noted from the analysis of the proximal promoters and EST frequencies, that there were some distinct differences in both the TF-binding sites within the regulatory regions and the EST frequencies of the boPAGs, particularly among the ancient boPAG members. Such differences in putative regulatory elements were even observed between two closely related ancient boPAG members (boPAG-2 and -12). In order to determine if these minor differences in the purported promoter elements can influence the relative expression of the boPAGs, quantitative Real-time PCR (Q-PCR) was performed to monitor relative transcript abundance of the ancient PAGs in placental RNA harvested from different stages of pregnancy.

RNA was extracted from placental cotyledons at various stages of pregnancy (days 45, 60, 75, 90, 140, 170, 220 and 280) by using STAT-60 RNA extraction reagent (IsoTex diagnostics, TX, USA). Each gestational stage was represented by two different animals. The extracted RNA preparations were treated with amplification grade DNAse I (Invitrogen, CA, USA) at room temperature according to the manufacturer's recommendations. The DNA-free RNA samples were quantified and analyzed for quality (260/280) and agarose gel electrophoresis. Two micrograms of high quality RNA from each sample were reverse transcribed by using an oligo-dT primer and SuperScript III-reverse transcriptase (Invitrogen, CA, USA) at 50°C for 1 hr.

Oligonucleotides for Q-PCR were designed to span exons of each boPAG to prevent unwarranted amplification of any trace carry-over contamination from the genomic DNA. Oligonucleotides were also designed for a control gene in cattle, YWHAG (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide). Power SYBR^® Green PCR master mix (Applied Biosystems, CA, USA) reagent and the Applied Biosystems ABI Prism 7500 Real-Time PCR system were employed for the Q-PCR. The reaction conditions for the Q-PCR were optimized by determining the amplification efficiency, as well as the dynamic range for each primer set, according to methods described by the manufacturer. Following the preliminary evaluation, the optimum oligonucleotide sets were selected (Table 2). The Q-PCR for each candidate gene was performed with two biological replicates and duplicate technical replicates. The cycling conditions were: pre-heating for: 50°C for 2 min (1 cycle); followed by a pre-run to activate the polymerase at 95°C for 10 min (1 cycle) followed by 40 cycles of 95°C for 15 sec, 65°C for 30 sec and 75°C for 1 min, with the data being acquired in the 75°C window. The data was analyzed by the ABI-PRISM 7500 sequence detection system software and the results from the analysis were graphed.

It was noted that the regulatory regions of the boPAGs do not share any conserved sequences with other genes whose expression is restricted to trophoblast (data not shown). This analysis sought to improve the understanding of the proximal promoters of boPAGs and identify any conserved elements within the family members. In order to better understand the selective pressures operating on the promoters, the observed p-distance of the promoters were plotted against the rate at which synonymous substitutions are occurring (dS) within the nucleotide sequences of each corresponding ORF. There were two principal assumptions within this analysis; these were that (1) dS of the exons of each analyzed gene pair was under neutral selection and would reflect the normal mutation rate for this chromosomal location, and (2) if the calculated p-distance within the promoter is equal to the dS of the exons, then the promoter is mutating at a rate that is expected for this location. If the observed ratio is above one, it was considered positive selection for nucleotide substitutions and if below one, it was purifying selection.

The analysis was performed with two variable lengths of promoter sequence. When the p-distance v. the ORF for the proximal 1000 bp was mapped, all of the boPAGs were undergoing neutral to purifying selection (Figure 3A and 3B), with the exception of boPAG-10 and -6, which had ratios of more than one (Figure 3A). These promoters seemed to have accumulated more mutations than would have been predicted by molecular clocks. The analysis, when confined to the first 500 bp, generated similar results except that both boPAG-6 and -10 showed a ratio close to neutrality (Figure 3B). Overall, the boPAG promoters are being conserved, particularly in the first 500 bp upstream of the TSS (Figure 3B) implying that critical regulatory elements responsible for trophoblast expression may be positioned within this region.

To account for observed differences within the proximal promoter elements, the 1000 bp upstream of each TSS was assessed for the presence of repeat element insertions. The sequences of the promoters were aligned and the position and types of TE insertions were identified and mapped (Figure 4). Among all the boPAG promoters analyzed there were no TE insertions within the proximal 600 bp region with the exception of boPAG-10 which had a SINE (MIRb) insertion at -317 bp corresponding to -390 bp in multiple sequence alignment (TSS being base pair position +1) (Figure 4). An interesting observation was that the type of TEs detected in distinct boPAG promoters differed between modern and ancient boPAGs. In boPAG-10 (an ancient PAG) for instance, there was a long SINE-element insertion from -524 to -1066 bp (-631 to > -1250 bp in alignment) (Figure 4). The corresponding region was occupied by DNA element Charlie-8 in all modern boPAGs and an additional LINE element (L2) in boPAG-4, -5, -7, and -15 (Figure 4). In the ancient boPAGs there was a ~200 bp DNA MER-108 element upstream of -750 bp that was conserved in all the ancient boPAGs, with the exception of boPAG-10. Therefore, the two groups of the boPAG promoters deviated in the types of TEs that were inserted in their upstream regulatory regions, which also accounts for the large deviations in p-distances between the modern and ancient boPAG promoters (Figure 3A). Similarly, a lengthy SINE insertion was identified in the boPAG-10 promoter that was not found in any other boPAG promoters. The boPAG-10 promoter diverged considerably from the remainder of the boPAG promoters. The functional significance of these inserted TEs is not known, but a potential role for these elements in influencing the expression of boPAGs could not be ruled out.

Based on previous reports, the boPAGs are known to exhibit differences in both their spatial and temporal expression patterns 13141528. The availability of the full-length promoter sequences provided an opportunity to study putative regulatory elements that could potentially explain the observed differences in the temporal and spatial expression patterns.

For this analysis, the first 1000 bp upstream of the TSS of various boPAGs was examined by using the DiAlign TF program of Genomatix-GEMS launcher. Among the aligned boPAG-promoter sequences, there were regions that were conserved in both the ancient and modern PAGs and, therefore, may contribute to trophoblast-specific expression. However, there were also a number of isolated conserved regions corresponding to consensus sequences for TF binding that were specific for ancient or modern boPAGs suggesting that the divergence of such elements could be responsible for the observed differences in the spatial distribution of the two boPAG groups. Examples of such regions within the first 350 bp of the TSS were boxed and listed in the Figure 5. Based on this analysis, conserved putative TF binding sites are highly prevalent in modern boPAGs. For example, there are predicted binding sites for these TFs: HOXC13 at position -109 to -125, RPOA (DTYPEPA) at -111 to -132, a FREAC17 at -124 to -141, FREAC2 at -149 to -166, LEF1 at -182 to -199 and -246 to -262, EN1 at -207 to -224 and SKN1 at -322 (TSS is +1). In addition, an atypical ETS site was conserved in all boPAGs and is located at position -227 bp to -230. Besides these sites, there were two tandem repeats (TTTCTCCA) 11 bp apart at positions -284 and -302 bp, respectively. Of these two repeats, the distal repeat was predicted to be recognized by DDVL (drosophila dorsal ventral factor) a homolog of vertebrate c-Rel TF. These repeats were conserved in most of the boPAGs and were referred to as 'bovine repeats' (BR); the presence of these repeats has been reported previously 60.

In order to verify if apparent differences observed in the promoter sequence might be associated with the relative levels of transcription of various genes, the bovine EST database was searched to define the relative distribution of various boPAG transcripts. Of all the boPAGs that were investigated, boPAG-2 had the highest occurrence, with 92 ESTs represented in the database (Figure 6). The next most abundant member was boPAG-11 with 46 ESTs (Figure 6). Of the modern boPAGs that were assessed, boPAG-1 had the highest number of EST matches with 28, followed by boPAG-17 with 25 matches (Figure 6).

As described above, boPAG-2 was an extremely abundant transcript. Therefore, follow-up experiments were performed to study the relative expression of boPAG-2 in comparison to its closest relative, boPAG-12, and to the other ancient bovine PAGs. Real time quantitative PCR of boPAG -2, -8, -10, -11, and -12 were performed and message abundance was assessed relative to an endogenous control transcript, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide(YWHAG). The source of RNA was obtained from placental cotyledons harvested at different stages of pregnancy, between d 45 and term. The relative amount of message for each target gene was graphed (Figure 7). BoPAG-2 was the highly abundant transcript relative to other ancient PAGs, while its most closely related family member, boPAG-12, was the least abundant under identical reaction conditions (Figure 7). Relative transcript abundance of boPAG-2 ranged from 186–1745 times greater than the control transcript, YWHAG, depending on the stage of pregnancy. In contrast, boPAG-12 message was much closer to that of YWHAG; its relative abundance varied from 0.16 to 2.21 that of the YWHAG transcript. The relative transcript abundance of boPAG-8 ranged from 0.5 to 14.83, boPAG-10 from 0.4 – 38.6 and boPAG-11 ranged from 0.9 to 21.4 times YWHAG-expression. Regardless of the stage of pregnancy that was examined, the transcript abundance of boPAG-2 was at least a 100 times greater than boPAG -12 and, when compared to other ancient PAGs, boPAG-2 message was at least 5 times greater (Figure 7). Finally, the relative profiles of each PAG transcript were distinct and they did not parallel one another. One interesting observation in particular, was that the relative temporal expression profiles of boPAG-8 and -10 were essentially opposite to one another. While the relative abundance of boPAG-8 was higher on d45 and was relatively stable across all other stages of pregnancy, boPAG-10 on the contrary had relatively low level of expression on d45 and had its highest level of expression at term.

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.