Transcription Factor PLAGL1 Is Associated with Angiogenic Gene Expression in the Placenta

Abstract

During pregnancy, the placenta is important for transporting nutrients and waste between the maternal and fetal blood supply, secreting hormones, and serving as a protective barrier. To better understand placental development, we must understand how placental gene expression is regulated. We used RNA-seq data and ChIP-seq data for the enhancer associated mark, H3k27ac, to study gene regulation in the mouse placenta at embryonic day (e) 9.5, when the placenta is developing a complex network of blood vessels. We identified several upregulated transcription factors with enriched binding sites in e9.5-specific enhancers. The most enriched transcription factor, PLAGL1 had a predicted motif in 233 regions that were significantly associated with vasculature development and response to insulin stimulus genes. We then performed several experiments using mouse placenta and a human trophoblast cell line to understand the role of PLAGL1 in placental development. In the mouse placenta, Plagl1 is expressed in endothelial cells of the labyrinth layer and is differentially expressed in placentas from mice with gestational diabetes compared to placentas from control mice in a sex-specific manner. In human trophoblast cells, siRNA knockdown significantly decreased expression of genes associated with placental vasculature development terms. In a tube assay, decreased PLAGL1 expression led to reduced cord formation. These results suggest that Plagl1 regulates overlapping gene networks in placental trophoblast and endothelial cells, and may play a critical role in placental development in normal and complicated pregnancies.

Adaptive structural and functional evolution of the placenta protects fetal growth in high elevation deer mice

“…For example, in the junctional zone/decidua, we found associations between fetal growth and the expression of fetal hemoglobins Hba-x and Hbb-y (oxygen transport), Plin2 (trophoblast cell survival under hypoxia (50)), and F3 (clotting, pre-eclampsia risk (51, 52)). In the labyrinth zone, we found a priori genes associated with outcomes of interest that are involved in diverse processes including nutrient transport (i.e., Slc32a2 , Slc38a1 , Fabp4 ; (53, 54)), vascular growth and function (i.e., Edn1 and Plagl1 ; (55–57)), and trophoblast cell differentiation or survival (i.e., Pappa2 , Prkcz , Furin , and Mmp28 ; (58–63)). The two most prominent candidates from human literature, PRKAA1 and EDNRA (38), were not significant for multiple factors of interest in deer mice.…”

Mapping cis-regulatory elements in the midgestation mouse placenta

“…Interestingly, fetal sex has been shown to impact neurolation 83 , as well as placental development and gene expression, both in normal development and following in utero environment changes 84 . For example, sex-specific changes in placental gene regulation has been seen in response to maternal diets 85 – 87 and stress hormones 88 , 89 . Therefore, it is possible that the regulatory landscape of the placenta differs when it is obtained from male versus female embryos, which is not addressed in our study.…”

Identifying novel regulators of placental development using time series transcriptomic data and network analyses

“…To investigate this further, we performed gene knockdown and migration assays for four candidate genes from four different networks in the HTR-8/SVneo cell line, an established model for studying TB migration [72]- [74]. From the lists of hub genes and their directly connected nodes (Supplementary table S7), we obtained genes that met the following criteria: having expression levels > 5 TPM in the mouse placenta transcriptome data we generated, having expression levels > 5 FPKM (fragments per kilobase of transcript per million of mapped reads) in human TB cell lines [75] and having expression levels > 20 TPM in HTR-8/SVneo cell line [20] (Supplementary Table S7). From this list, we selected four genes: Mtdh and Siah2 (from the e7.5_1_STRING and e7.5_2_GENIE3 network, respectively), Hnrnpk (from the e8.5_2_GENIE3), and Ncor2 (from the e9.5_2_GENIE3), none of which have been studied in TB migration function.…”

“…siRNA knockdown -HTR-8/SVneo cells were transfected with two different siRNA for each target gene knockdown (KD). Cells were split at 80% confluency, and siRNA transfection was performed in 6-well plates; 150,000 cells/well were seeded [20]. After 24 hours, cells were transfected with 30nM siRNA using RNAiMax 3000 (Thermofisher, 13778150).…”