*2.6. Relationship between Mouse and Human Plagl1 Results*

The gene networks regulated by PLAGL1 in mouse placenta, where it is expressed in endothelial cells, and in HTR-8/SVneo human trophoblasts are at least partially conserved. For example, the GO terms enriched for genes downregulated upon *PLAGL1* knockdown in HTR-8/SVneo cells were similar to the terms associated with the genes we initially predicted to contain PLAGL1-binding motifs in their enhancer regions in the mouse genome (Figures 4a and 5g). Therefore, we tested whether the specific genes associated with terms enriched for predicted PLAGL1 enhancers decreased in expression after *PLAGL1* was knocked down in HTR-8/SVneo cells. To do this, we focused on the terms that were associated with *PLAGL1* enhancers—'abnormal placental labyrinth vasculature morphology', 'regulation of cellular response to insulin stimulus', and 'placental development'. Of the eight target genes associated with 'abnormal placental labyrinth vasculature morphology', four were downregulated upon *PLAGL1* knockdown in HTR-8/SVneo cells (*p*-value of overlap = 0.00188). Of the five predicted PLAGL1 target genes associated with 'regulation of cellular response to insulin stimulus', three were downregulated upon *PLAGL1* knockdown in HTR-8/SVneo cells (*p*-value of overlap = 0.00161). Of the 11 predicted PLAGL1 target genes associated with the more general term, 'placental development', five were downregulated upon *PLAGL1* knockdown (*p*-value of overlap = 0.00159) (Supplemental Figure S4a).

We further determined if the enhancers identified in mouse could drive gene activity in the HTR-8/SVneo cells. We performed a dual-glow luciferase assay using five enhancers predicted to be bound by PLAGL1 and target genes with varying functions, including blood vessel development (COL1A1 [18]), migration (DLC1 [61]), signal transduction (ARHGEF3 [62]), insulin regulation (IRS1 [63]) and fetal growth (IGF2BP1 [64]). We found that all of these regions were indeed acting as enhancers in the HTR-8/SVneo cells (relative luciferase activity ≥ 2) (Supplemental Figure S4b). To confirm PLAGL1 regulates the activity of these enhancers, we tested the activity with and without an siRNA-mediated knockdown. After *PLAGL1* knockdown, the enhancer activity significantly decreased in three out of five of these enhancers (Supplemental Figure S4c).
