Fig. 1

Putative enhancer identified upstream of Mmp9 drives luciferase reporter activity in mouse and human trophoblast cell lines. (a) H3K27ac ChIP-seq data (in red) was used to identify a putative enhancer (light blue bar) 7025 bp upstream of Mmp9 in e7.5 mouse ectoplacental cones. The distance was measured from the middle of the enhancer to the gene transcription start site (determined using the eukaryotic promoter database⁵¹). Black bars below the putative enhancer show the E1-E7 (~ 1 kb length each) segments that were cloned into the pGL4.23 reporter vector. Data was visualized on the mm9 genome using the UCSC genome browser^40,41. (b,c) E1 has significantly higher luciferase reporter activity than all other constructs in mTGCs (p-value < 0.001) in (b) and the HTR-8/SVneo cell line (p-value < 0.01) in (c). (d,e) When E1 is divided into E1.1 and E1.2, E1.2 luciferase reporter activity is significantly higher than E1.1 in mTGCs (d) and the HTR-8/SVneo cell line (e). Primers and genomic coordinates used for the design of mE1-mE7, mE1.1 and mE1.2 are in Supplementary Table S1, S2 and Supplementary Fig. S1. For Fig. b-e: the y-axis shows luciferase activity relative to the empty pGL4.23 vector. (**) indicates p-value < 0.01 and (***) indicates p-value < 0.001. The black dots represent individual biological replicate values for the experiment. All experiments were performed with three biological replicates. Results are displayed as the mean +/- standard error (SE), and p-values were calculated using the two-tailed student’s t-test.