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

**Rebekah R. Starks 1,2, Rabab Abu Alhasan <sup>1</sup> , Haninder Kaur <sup>1</sup> , Kathleen A. Pennington <sup>3</sup> , Laura C. Schulz <sup>4</sup> and Geetu Tuteja 1,2,\***


Received: 7 October 2020; Accepted: 2 November 2020; Published: 6 November 2020

**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.

**Keywords:** RNA-seq; ChIP-seq; enhancers; transcription factors; placenta; PLAGL1; gestational diabetes; tube formation; blood vessel development
