**1. Introduction**

The placenta is a specialized organ playing a crucial role in the health of the fetus and mother during pregnancy, regulating the exchange of nutrients, gases, hormones and waste [1]. Inefficient transfer of materials from the mother to the fetus is frequently associated with adverse health outcomes for the fetus [2]. The placenta also plays a protective role, regulating the immune response of cells and preventing toxic elements from reaching the fetus [3,4]. Placental insufficiency, as well as many other placental abnormalities, have been attributed to misregulated gene expression [5,6]. Despite its importance, many aspects of placental development and the genetic mechanisms involved in its function remain unknown.

To elucidate these mechanisms, many researchers utilize the mouse model, which has contributed greatly to our understanding of the development of the placenta. Similar to humans, rodents have a single disc-shaped, hemochorial placenta [7] to sustain the fetus during development. In the human placenta, trophoblast cells invade deeply into the myometrium, and partially replace the endothelial wall of maternal spiral arteries, thereby channeling maternal blood into the intervillous spaces. Trophoblast lining the villi can then exchange factors between the maternal blood and fetal vessels running through the villous core [1]. Similarly, in the mouse, trophoblast cells replace the endothelial wall of maternal spiral arteries in the near decidua, channeling maternal blood through trophoblast-lined canals to spaces in the labyrinth, which are surrounded by trophoblast that exchange material between the maternal blood and the adjacent fetal vessels [8]. Labyrinth formation begins around embryonic day (e) 8.5 with the fusing of the chorion and allantois. Subsequently, the chorion begins to fold and form branches that become the trophoblast-lined maternal blood spaces, while allantoic offshoots form the interdigitating fetal vessels [9]. Thus, coordinated interactions between trophoblast and endothelial cells, with trophoblasts assuming key endothelial properties, are critical for placental development and function.

Regulation of these complex interactions requires transcriptional machinery to interact with the appropriate regulatory elements and cofactors in a spatiotemporal manner. A common cause of genetic misregulation is disruption of cis-regulatory elements, specifically in enhancers [10,11]. Despite the crucial role enhancers play in gene regulation, there remains a large deficit in our understanding of the gene-enhancer regulatory networks that are important for placental function [12], including trophoblast-endothelial cell interactions.

A previous study compared the enhancer landscape before (e7.5) and after (e9.5) chorioallantoic fusion in the mouse placenta, identifying an e7.5-specific cell migration network of transcription factors (TFs) and enhancers [13]. The goal of the present study is to identify e9.5-specific TFs and enhancers critical to placental development. In order to identify novel TFs that could be important for regulating processes in the mouse placenta after chorioallantoic fusion, we utilized publicly available H3k27ac ChIP-seq data, defining putative active enhancers in the e7.5 and e9.5 placenta, as well as RNA-seq data generated at the same timepoints [13]. We identified several highly expressed TFs that were upregulated in the e9.5 placenta. One of these TFs, pleiomorphic adenoma gene-like 1 (PLAGL1), has a motif highly enriched in e9.5-specific enhancers compared to e7.5-specific enhancers. Overexpression of *PLAGL1* has been associated with transient neonatal diabetes mellitus [14], while reduced expression has been found in female intrauterine growth restricted placentas, suggesting it plays an important role [15].

To further understand the functions of PLAGL1, we carried out additional experiments in mouse placenta and a human trophoblast cell line, HTR-8/SVneo, due to the high expression of *Plagl1* in both systems. By combining computational predictions with experimental validations, we were able to identify novel functions for PLAGL1 in two models frequently used to study human placenta, which could contribute to insights into placental development and certain pregnancy disorders.
