*2.1. E9.5-Specific Genes and Enhancers Are Associated with Vasculature Development*

RNA-seq and H3k27ac ChIP-seq were previously carried out on mouse placenta at e7.5 and e9.5 to identify genes upregulated and enhancers specific to the e7.5 timepoint [13]. However, the genes upregulated at e9.5, as well as the enhancers more active at this timepoint were not thoroughly investigated. We first analyzed the 583 genes that were highly expressed and upregulated at e9.5 (fragments per kilobase of transcript per million of mapped reads (FPKM) ≥ 10, fold ≥ 2, and FDR ≤ 0.05). As expected, we see higher e9.5 expression of genes related to processes important in the e9.5 placenta including, *Apoa1* (lipid metabolism [16]); *Notch1*, *Col1a1*, and *Dlc1* (blood vessel development [17–19]); *Gcm1* (hormone production [20]); *Igfbp2*, *Pappa2*, *Ada* (fetal growth and development [21–23]); and *Syna*

(trophoblast differentiation [24]) (Figure 1a). We then used the Genomic Regions Enrichment of Annotations Tool (GREAT) [25] to identify the biological processes associated with the highly expressed and upregulated e9.5 genes using the single nearest gene option. In general, we found that terms related to metabolism and vasculature development are more enriched in genes upregulated at e9.5 than genes upregulated at e7.5 (Figure 1b). *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 3 of 21 associated with the highly expressed and upregulated e9.5 genes using the single nearest gene option. In general, we found that terms related to metabolism and vasculature development are more enriched in genes upregulated at e9.5 than genes upregulated at e7.5 (Figure 1b).

**Figure 1.** Genes and enhancers enriched in the placenta at e9.5 are associated with vasculature development. (**a**) Examples of genes significantly upregulated at e9.5 (FPKM 10, fold 2, and FDR ≤ 0.05) that are also associated with placental functions. (**b**) Top 10 GO biological process terms, according to GREAT, using the hypergeometric fold (≥2), FDR (≤0.05), and gene association (≥5 associated genes) cutoffs. Genes significantly upregulated at e9.5 are related to lipid metabolism, and vasculature development. (**c**) Examples of e9.5-specific enhancers and their corresponding H3k27ac activity at e9.5 and e7.5, shown using the UCSC genome browser. Boxes correspond to regions identified as e9.5-specific. (**d**) E9.5-specific enhancers are associated with genes involved in proliferation, response to hormones, and placenta vasculature development. **Figure 1.** Genes and enhancers enriched in the placenta at e9.5 are associated with vasculature development. (**a**) Examples of genes significantly upregulated at e9.5 (FPKM ≥ 10, fold ≥ 2, and FDR ≤ 0.05) that are also associated with placental functions. (**b**) Top 10 GO biological process terms, according to GREAT, using the hypergeometric fold (≥2), FDR (≤0.05), and gene association (≥5 associated genes) cutoffs. Genes significantly upregulated at e9.5 are related to lipid metabolism, and vasculature development. (**c**) Examples of e9.5-specific enhancers and their corresponding H3k27ac activity at e9.5 and e7.5, shown using the UCSC genome browser. Boxes correspond to regions identified as e9.5-specific. (**d**) E9.5-specific enhancers are associated with genes involved in proliferation, response to hormones, and placenta vasculature development.

Next, we analyzed e9.5-specific enhancers. We observed that e9.5-specific enhancers were located near genes with known roles in the midgestation placenta (Figure 1c). For example, *Notch1* plays a role in promoting trophoblast differentiation, and regulates angiogenesis and placental branching [17,26]. *Dlc1*, a tumor suppressing gene, also contributes to the development of placental vasculature [27]. Ontology analysis of the e9.5-specific enhancers showed that many terms, such as 'response to insulin stimulus' and 'abnormal placental vasculature', are more significantly enriched in e9.5-specific enhancers compared to e7.5-specific enhancers (Figure 1d; Supplemental Table S1). Next, we analyzed e9.5-specific enhancers. We observed that e9.5-specific enhancers were located near genes with known roles in the midgestation placenta (Figure 1c). For example, *Notch1* plays a role in promoting trophoblast differentiation, and regulates angiogenesis and placental branching [17,26]. *Dlc1*, a tumor suppressing gene, also contributes to the development of placental vasculature [27]. Ontology analysis of the e9.5-specific enhancers showed that many terms, such as 'response to insulin stimulus' and 'abnormal placental vasculature', are more significantly enriched in e9.5-specific enhancers compared to e7.5-specific enhancers (Figure 1d; Supplemental Table S1).

Since both upregulated genes and enhancers specific to e9.5 were associated with placental development terms, such as vasculature and labyrinth morphology, we next investigated which TFs could be regulating these processes. Since both upregulated genes and enhancers specific to e9.5 were associated with placental development terms, such as vasculature and labyrinth morphology, we next investigated which TFs could be regulating these processes.

#### *2.2. Plagl1 Is Highly Expressed in the e9.5 Placenta and the PLAGL1 Binding Motif Is Enriched in e9.5- Specific Enhancer Regions 2.2. Plagl1 Is Highly Expressed in the e9.5 Placenta and the PLAGL1 Binding Motif Is Enriched in e9.5-Specific Enhancer Regions*

To identify TFs that could be regulating e9.5-specific enhancers, we first determined which ones were upregulated at e9.5. Based on expression thresholds (FPKM ≥ 10), fold (≥2), and q-value (≤0.05), we identified 37 TFs upregulated at e9.5 (Figure 2a; Supplemental Figure S1a; Supplemental Table S2]. We then used a phylofootprinting approach [28] to determine which of these TFs had motifs To identify TFs that could be regulating e9.5-specific enhancers, we first determined which ones were upregulated at e9.5. Based on expression thresholds (FPKM ≥ 10), fold (≥2), and q-value (≤0.05), we identified 37 TFs upregulated at e9.5 (Figure 2a; Supplemental Figure S1a; Supplemental Table S2). We then used a phylofootprinting approach [28] to determine which of these TFs had motifs

enriched in e9.5-specific enhancers. We ensured that the binding site predictions were conserved

enriched in e9.5-specific enhancers. We ensured that the binding site predictions were conserved between the human and mouse genome since conserved binding sites are more likely to be functionally important [29]. Four TFs passed our motif fold (≥1.5) and *p*-value (≤0.05; Bonferonni correction) cutoffs: PLAGL1, GCM1, PPARγ, and BHLHB2 (Figure 2b). Interestingly, each of these TFs has a known role in the placenta, though some TFs are better studied. GCM1 is a well-known, important transcription factor expressed within labyrinthine trophoblast [30] that contributes to syncytiotrophoblast differentiation, chorionic branching [31], and hormone production [20]. PPARγ is also well-studied, known to play a variety of roles in the placenta including fatty acid uptake, differentiation, and vascularization [32–34]. BHLHB2 is expressed in cytotrophoblast and has been found to be upregulated in preeclampsia [35]. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 4 of 21 correction) cutoffs: PLAGL1, GCM1, PPARγ, and BHLHB2 (Figure 2b). Interestingly, each of these TFs has a known role in the placenta, though some TFs are better studied. GCM1 is a well-known, important transcription factor expressed within labyrinthine trophoblast [30] that contributes to syncytiotrophoblast differentiation, chorionic branching [31], and hormone production [20]. PPARγ is also well-studied, known to play a variety of roles in the placenta including fatty acid uptake, differentiation, and vascularization [32–34]. BHLHB2 is expressed in cytotrophoblast and has been found to be upregulated in preeclampsia [35].

**Figure 2.** The PLAGL1 motif is enriched in e9.5-specific enhancers and is associated with blood vessel development genes. (**a**) Scatterplot showing the expression of all TFs at e7.5 and e9.5. Upregulated TFs at e9.5 are indicated by maroon triangles (fold ≥ 2, and *p*-value ≤ 0.05). (**b**) Of the 37 transcription factors upregulated at e9.5, four TFs (maroon triangles) pass the fold and Bonferroni corrected *p*-value thresholds (black lines). **Figure 2.** The PLAGL1 motif is enriched in e9.5-specific enhancers and is associated with blood vessel development genes. (**a**) Scatterplot showing the expression of all TFs at e7.5 and e9.5. Upregulated TFs at e9.5 are indicated by maroon triangles (fold ≥ 2, and *p*-value ≤ 0.05). (**b**) Of the 37 transcription factors upregulated at e9.5, four TFs (maroon triangles) pass the fold and Bonferroni corrected *p*-value thresholds (black lines).

PLAGL1 had a high expression fold change (fold change: 46.93; e7.5 FPKM: 0.99; e9.5 FPKM 45.26) as well as the highest, most significant fold change among binding sites of the four TFs (Figure 2b). To confirm expression differences, we performed qPCR using e7.5 and e9.5 mouse placenta and found *Plagl1* was indeed significantly more highly expressed at e9.5 (*p*-value = 0.015) (Supplemental Figure S1b). PLAGL1 is an imprinted zinc-finger transcription factor known to play roles in regulating glucose uptake [36], apoptosis [37], and proliferation [38] and peaks in expression in the midgestation human placenta [39]. Although several functions of PLAGL1 have been identified, its role in placenta has not been thoroughly studied. Therefore, we performed additional experiments in both the mouse placenta as well as human trophoblast cells. PLAGL1 had a high expression fold change (fold change: 46.93; e7.5 FPKM: 0.99; e9.5 FPKM 45.26) as well as the highest, most significant fold change among binding sites of the four TFs (Figure 2b). To confirm expression differences, we performed qPCR using e7.5 and e9.5 mouse placenta and found *Plagl1* was indeed significantly more highly expressed at e9.5 (*p*-value = 0.015) (Supplemental Figure S1b). PLAGL1 is an imprinted zinc-finger transcription factor known to play roles in regulating glucose uptake [36], apoptosis [37], and proliferation [38] and peaks in expression in the midgestation human placenta [39]. Although several functions of PLAGL1 have been identified, its role in placenta has not been thoroughly studied. Therefore, we performed additional experiments in both the mouse placenta as well as human trophoblast cells.
