Extended Data Fig. 4: Interaction between PAX8 and HIF2A in ccRCC.

a. Network presentation of the highest confidence experimental and database-derived physical connections between the 89 shared nuclear proteins from HIF2A and PAX8 interactomes as determined by String 11.0. Shading reflects MCL clustering. Isolated nodes are removed. b. PAX8 locus. Median ATAC-seq signal from shRen control (N = 6) and shPAX8 (N = 6) cells. Median HIF2A and PAX8 ChIP-seq signal from 786-M1A and OS-LM1 xenografts, 3 tumours each. c. Overlap of HIF2A and PAX8 ChIP-seq peaks in 786-M1A (top) and OS-LM1 (bottom) cells. d. Enrichment of known DNA motifs in the 1,948 peaks with most significantly increased DNA accessibility in ccRCCs when compared to KIRP tumours in the TCGA data set (Fold change 2, two-sided padj < 0.001 as determined by DESeq2). e. The most significant de novo DNA motif enriched in the ccRCC-specific peaks as described in panel (d). f. Distribution of distances between the centres of PAX8 and HIF2A DNA motifs in ccRCC-specific ATAC-seq regions. Cartoons in each quadrant demonstrate the motif orientation. The 18bp distance seen in the common ERV1 endogenous retrovirus highlighted. g. Co-immunoprecipitation with antibodies targeting HIF2A and PAX8 in C-M1A^HIF2A-/- cells with HIF2A reintroduction (PAX8-HIF2A interaction, N = 3 independent IP reactions; HIF2A-HIF1B interaction N = 1). h. Global gene expression changes by RNA-seq in 786-M1A and OS-LM1 ccRCC cells upon PAX8 and HNF1B depletion when compared to non-targeting controls. Pooled analysis of both cell lines and targeting constructs. Adjusted two-sided p-value derived by DESeq2. i. Pseudotime analysis of the different stages of the proximal renal epithelium lineage in fetal human kidney for the cell types shown in Extended Data Fig. 2f. j. Expression of PAX8, HNF1B and the respective gene signatures from ccRCC cell lines as a function of the proximal renal epithelium lineage pseudotime in the fetal human kidney.

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