Extended Data Fig. 9: Epistatic pathway of Tu-Stroma mediated exon 3 methylation and Flower Lose generation in stromal cells.
A) A functional genetic assay is performed on HGSC and Stromal cells cultured individually (row 1) or in co- culture using a series of loss-of-function experiments using Tu-Stroma (row 2) and DDX3X (row 3) shRNA (HGSC), double KO of Rab27a and Rab27b (row 4), Exosome (row 5) and DNMT3A (row 6) inhibitors (co- culture) or shRNA against Tu-Stroma (row 7), DDX3X (row 8), DNMT3A (row 9), G9a (row 10), GLP (row 11), HP1 (row 12) and SRSF3 (row 13). We used five sets of molecular experiments to determine the steps in this molecular mechanism. These experiments were a) ChOP on exon 3 region of HGSC stromal cells to identify the association of Tu-Stroma at this gene locus, b) MethyLight Assay to evaluate level of DNA methylation on this region, c) a series of ChIP experiments (with input, no Ab, Actin Ab, IgG Ab, and Scramble primers as controls) to identify DNA methylation status, and association of DNMT3A, G9a, GLP, H3K9Me3, HP1, and SRSF3 on hFwe exon 3 gene locus in HGSC stromal cells, d) qPCR-based analysis of hFwe-Lose isoform, hFwe-Win isoform, and exon 3 expression in HGSC stromal cells, and e) RT-PCR based validation of Exon 3 cassette deletion. Using the results from this assay, a series of hypotheses are generated to construct the epistatic relationship between individual components in the pathway and depicted as a model. The p-values shown in d) compare the expression of hFwe-Lose in each group with the expression in the control. Microscopy images of each experiment are shown in the first column (scale 10 µm). B) Pseudotime algorithm model to determine the sequence of epigenetic events at the exon 3 hFwe locus. The current model represents the most plausible series of steps (significance of the model (p) is 0.043, which is calculated as a sum of the type I and type II errors across all steps). This pseudo time analysis combined with the known biological function of individual components reveals the mechanism of Tu-Stroma mediated exclusion of exon 3 in stromal cells.
