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

<p>Abstract</p> <p>Background</p> <p>The Pregnancy-associated glycoproteins (PAGs) belong to a large family of aspartic peptidases expressed exclusively in the placenta of species in the <it>Artiodactyla </it>order. In cattle, the <it>PAG </it>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 <it>in silico </it>analysis of the bovine genome to further characterize the <it>PAG </it>gene family, 2) we scrutinized proximal promoter sequences of the <it>PAG </it>genes to evaluate the evolution pressures operating on them and to identify putative regulatory regions, 3) we determined relative transcript abundance of selected <it>PAGs </it>during pregnancy and, 4) we performed preliminary characterization of the putative regulatory elements for one of the candidate PAGs, <it>bovine </it>(<it>bo</it>) <it>PAG-2</it>.</p> <p>Results</p> <p>From our analysis of the bovine genome, we identified 18 distinct <it>PAG </it>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 <it>boPAGs</it>. However, a preponderance of conserved regions, that harbor recognition sites for putative transcriptional factors (TFs), were found to be unique to the modern <it>boPAG </it>grouping, but not the ancient <it>boPAGs</it>. We gathered evidence by means of Q-PCR and screening of EST databases to show that <it>boPAG-2 </it>is the most abundant of all <it>boPAG </it>transcripts. Finally, we provided preliminary evidence for the role of ETS- and DDVL-related TFs in the regulation of the <it>boPAG-2 </it>gene.</p> <p>Conclusion</p> <p><it>PAGs </it>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 <it>boPAG-2 </it>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 <it>boPAG </it>gene family.</p

SMARCAD1 Contributes to the Regulation of Naive Pluripotency by Interacting with Histone Citrullination

Histone citrullination regulates diverse cellular processes. Here, we report that SMARCAD1 preferentially associates with H3 arginine 26 citrullination (H3R26Cit) peptides present on arrays composed of 384 histone peptides harboring distinct post-transcriptional modifications. Among ten histone modifications assayed by ChIP-seq, H3R26Cit exhibited the most extensive genomewide co-localization with SMARCAD1 binding. Increased Smarcad1 expression correlated with naive pluripotency in pre-implantation embryos. In the presence of LIF, Smarcad1 knockdown (KD) embryonic stem cells lost naive state phenotypes but remained pluripotent, as suggested by morphology, gene expression, histone modifications, alkaline phosphatase activity, energy metabolism, embryoid bodies, teratoma, and chimeras. The majority of H3R26Cit ChIP-seq peaks occupied by SMARCAD1 were associated with increased levels of H3K9me3 in Smarcad1 KD cells. Inhibition of H3Cit induced H3K9me3 at the overlapping regions of H3R26Cit peaks and SMARCAD1 peaks. These data suggest a model in which SMARCAD1 regulates naive pluripotency by interacting with H3R26Cit and suppressing heterochromatin formation