Fig 2: RXRA hot-spot mutations induce the PPAR signaling pathway by activating enhancer/promoters with a canonical PPAR response element.(A) JMSU-1 and 575A cells were transduced with pBABE retrovirus to express indicated RXRA alleles and expression confirmed by western blot (top) or RT-qPCR in triplicate ± SD (data expressed as a fraction of actin signal). (B) Protein coding transcripts up-regulated greater than or equal to twofold (FDR < 0.05) in cells expressing RXRAS427F compared to cells expressing RXRAwt were identified and then subjected to over representation analysis (ORA, GO-Elite) to discover enriched pathways relative to all other protein coding transcripts identified by RNA-seq. Experiment was done in two bladder cancer cell lines, JMSU-1 or 575A, using three RNA samples, each purified from an independent cell well, for each condition. (See also source data 1). (C) Transcriptome changes induced by RXRAS427F relative to RXRAwt were compared to expression changes of the same transcripts induced by 16 hr of pioglitazone (1 µM) treatment in the RXRAwt expressing cells. D) Relative expression of two PPAR targets with expression of indicated RXRA alleles. RT-qPCR performed in triplicate ±SD. Comparison by Student’s t-test. (E) RAEs were defined by the presence of overlapping ChIP-seq signal for RXRA and H3K27ac. RAEs identified by binding of RXRAwt and/or RXRAS427F are represented in grey. Hyperactive RAEs represented in red had elevated H3K27ac mean peak height in the mutant expressing cells compared to the wild-type cells (FDR < 0.05). All ChIP-seq peak callings were based on data from three independent immuno-precipitations, each utilizing input material from an independent cell plate. HOMER motif analysis was used to identify motifs enriched in hyperactive RAEs relative to the background of non-hyperactive RAEs. Source data 2 specifies number of peaks in each sector of the venn diagram. (F) Activity of a DR1 response element reporter (3X PPRE) transfected into JMSU-1 cells stably expressing either RXRAwt or RXRAS427F. RXRAwt cells were also treated with pioglitazone (1 µM) for 16 hr. For all reporter assays, Firefly luciferase expressing reporter was co-transfected with a constitutive Renilla luciferase expression vector to normalize for transfection efficiency. Data represents mean ± SEM of Firefly to Renilla luciferase signal from three independent experiments done on different days, each performed using triplicate cell wells. Statistical comparisons are by paired t-test.10.7554/eLife.30862.004Figure 1—source data 1.GO Elite Over Representation Analysis complete results.Pathways over-represented in genes up-regulated by RXRAS427Fcompared to RXRAwtin either JMSU-1 or 575A. Analysis was also done using genes up-regulated in both cell lines.10.7554/eLife.30862.005Figure 1—source data 2.ChIP-seq peak numbers used to generate venn diagram in Figure 1E.

Fig 3: PPARG or PPARD expression is necessary and sufficient for mutant RXRA activity.A) PPAR RNA expression in RXRA hot-spot mutant clinical samples from the TCGA dataset. Whisker plot shows 25th, median, and 75th percentile. (B) Data from panel A plotted per patient with hot-spot mutation. (C) Effects of siRNA-mediated knock-down of PPARD and PPARG in JMSU-1 and 575A cell lines on two target genes (PLIN2 and FABP3) up-regulated by mutant RXRA. Data by RT-qPCR in triplicate ±SD and indicated comparisons by Student’s t-test. (D) DR1 luciferase reporter activity in UM-UC-3 cells transfected with RXRA ±PPARD or PPARG. Cells were treated with 1 µM of the PPARG agonist pioglitazone or the PPARD agonist GW0742 for 16 hr. Data represents mean ±SEM of Firefly to Renilla luciferase signal from three independent experiments done on different days, each performed using triplicate cell wells. Statistical comparisons are by paired t-test.

Fig 4: Mutant RXRA induces allosteric activation of PPARs through their terminal tyrosine.A) RXRA S427 and other amino acids mutated for structure-function studies highlighted in green on a published full-length crystal structure of a RXRA/PPARG heterodimer. (B) DR1 reporter assay in UM-UC-3 co-transfecting indicated RXRA and PPARG alleles. Cells were treated with vehicle (DMSO) or pioglitazone 1 µM for 16 hr. Data represents mean normalized signal ±SEM of three independent experiments done on different days, each performed in triplicate, with data from each experiment normalized to the RXRAwt vehicle condition for each section. Statistical comparisons are by unpaired t-test. (C) Left, reporter assay performed with indicated RXRA alleles only and drug treatment with the RXRA agonist SR11237 (100 nM) for 16 hr. Right, reporter assay with wild-type PPARG co-transfected. Data represent mean ±SEM of Firefly to Renilla luciferase signal from three independent experiments done on different days, each performed using triplicate cell wells. Statistical comparisons are by paired t-test. (D) Published agonist structure of RXRA/PPARG heterodimer (PDB: 1FM6) in red and blue with key residues highlighted in bright green. Top three occupied microstate clusters from simulation experiments are superimposed. (E) Distance from starting agonist structure between alpha carbons of RXR 427 and PPARG 477 in the top 5% most-occupied microstates for wild-type and mutant RXRA. Mean ±SD, comparison is by Student’s t test. (F) Alignment of the AF2 region and C-terminus of all RXRA dimerization partners. Terminal tyrosine unique to PPARs is indicated. (G) Reporter assay similar to B, but using PPARD and the PPARD agonist GW0742.

Fig 5: PPARD agonist and mutant RXRA confer growth-factor-independent growth to urothelium in the context of tumor suppressor loss.(A) Organoids were derived from three independent wild-type mouse bladders and infected with Adeno-Cre. Growth was determined for each line in organoid media without EGF using CellTiter-Glo after treatment with vehicle, GW0742 (100 nM), or pioglitazone (100 nM). Data represent mean ±SEM from three independent experiments, each performed in triplicate cell wells. Statistical comparison is by unpaired t-test. (B) Similar to A but organoids were derived from Trp53flox/flox; Kdm6aflox mice. (C) CellTiter-Glo growth assay in media without EGF of a sub-clone from DKO431 (DKO 431.A) treated with GW0742 (10, 100 nM) or pioglitazone (100, 1000 nM). Mean values ± SEM from three independent experiments, each performed using triplicate organoid wells. Comparison is by paired t-test. (D) DKO 431.A organoids were plated in standard organoid media and then treated with, vehicle, GW0742 (10, 100 nM)) or pioglitazone (100, 1000 nM) for an additional 48 hr. Induction of PPAR target genes was determined by RT-qPCR in triplicate ± SD. (See also Figure 4—figure supplement 1C.) (E) DKO 431.A organoids were infected with a retroviral vector that was empty, expresses RXRAwt, or expresses RXRAS427F and expression of total RXRA (western blot) and human RXRA (RT-qPCR, triplicate +/SD) was determined. (F) Mean CellTiter-Glo signal ±SEM from three identical experiments, each performed using triplicate organoid wells. Comparison of D10 data is by paired t-test. (G) Identical number of indicated organoid cells were plated in media without EGF and then harvested with trypsin weekly and counted in duplicate using a BioRad TC20. Identical numbers of cells were then re-plated and this was repeated for 6 weeks. Total cell number doublings were calculated and plotted. Comparison of doublings is by paired t-test.