Figures and data
Contribution of RXRγ domains on repressing Nur77-mediated transcription.
(a) General scheme of the cellular transcriptional reporter assay. (b) 3xNBRE-luciferase assay performed in SK-N-BE(2)-C cells. Data are normalized to empty vector control (n=9 replicates), shown as a box and whiskers plot with boundaries of the box representing the 25th percentile and the 75th percentile, and representative of two or more independent experiments. Statistical testing was performed and p-values were calculated using the Brown-Forsythe and Welch multiple comparisons test of the FL Nur77 + RXRγ constructs conditions relative to FL Nur77 control condition. See Figure 1—source data 1 for data plotted.
Ligand profiling for Nur77-RXRγ heterodimer activation and pharmacological RXRγ agonism.
(a) General scheme and data from the Nur77-RXRγ/3xNBRE-luciferase cellular transcriptional reporter assay performed in SK-N-BE(2)-C cells treated with RXR ligand (1 µM) or DMSO (dotted line). Data are normalized to DMSO (n=6 replicates), represent the mean ± s.d., and representative of two or more independent experiments. See Figure 2— source data 1 for data plotted. (b) General scheme and data from RXRγ LBD time-resolved fluorescence resonance energy transfer (TR-FRET) coactivator peptide interaction assay. TR-FRET ratio measured in the presence of DMSO (dotted line) or compound (2–4 µM). Data are normalized to DMSO control (n=3 replicates), represent the mean ± s.d., representative of two or more independent experiments. See Figure 2—source data 2 for data plotted. (c) General scheme and data from the RXRγ/3xDR1-luciferase cellular transcriptional reporter assay performed in HEK293T cells treated with compound (1 µM) or DMSO control (dotted line). Data normalized to DMSO (n=6 replicates), represent the mean ± s.d., and representative of two or more independent experiments. See Figure 2—source data 3 for data plotted. (d,e,f) Correlation plots of (d) RXRγ transcriptional reporter data vs. RXRγ LBD TR-FRET data, (e) Nur77-RXRγ cellular transcription data vs. RXRγ LBD TR-FRET data, and (f) Nur77-RXRγ cellular transcription data vs. RXRγ transcriptional reporter data with calculated Pearson (rp) and Spearman (rs) correlation coefficients. For all comparisons, statistical testing was performed, and p-values were calculated, using the Brown-Forsythe and Welch (a,c) or ordinary one-way ANOVA (b) tests for multiple comparisons with Dunnett corrections relative to DMSO control treated condition. Data and RXR ligand label text in (a,b,c) are colored according to RXR ligand activity as grouped in Figure 2—figure supplement 1.
Ligand profiling for Nur77-RXRγ LBD heterodimer dissociation.
(a) 2D [1H,15N]-TROSY HSQC data of 15N-labeled Nur77 LBD heterodimerized with unlabeled RXRγ LBD in the presence of RXR ligands focused on the NMR peak of G544. The upper left shows an overlay of two spectra corresponding to 15N-labeled Nur77 LBD monomer (200 µM; purple) and 15N-labeled Nur77 LBD + unlabeled RXRγ LBD heterodimer (1:2 molar ratio; black) to demonstrate the shift of the G544 peak between Nur77 LBD monomer (m) and heterodimer (hd) forms; solid green and dotted blue arrows denote the complex chemical shift perturbation pattern. RXR ligand label text is colored according to RXR ligand activity as grouped in Figure 2—figure supplement 1. (b) Peak intensity estimated ligand-dependent Nur77 LBD monomer populations from G544 and G376 in the 2D [1H,15N]-TROSY HSQC data. Data and RXR ligand label text are colored according to RXR ligand activity as grouped in Figure 2—figure supplement 1. See Figure 3—source data 1 for data plotted. (c) Correlation plot of Nur77-RXRγ cellular transcription data vs. NMR estimated Nur77 LBD monomer populations for G544 and G376 with calculated Pearson (rp) and Spearman (rs) correlation coefficients; see Figure 3—figure supplement 3 for G376 2D [1H,15N]-TROSY HSQC data. (d) Analytical size exclusion chromatography (SEC) analysis of Nur77-RXRγ LBD in the presence of RXR ligands (solid colored lines) relative to Nur77 LBD monomer (dotted black line) and Nur77-RXRγ LBD heterodimer (solid black line).
Reanalysis of published Nurr1-RXRα correlation data excluding Nurr1-RXRα selective agonists
(a,b) Correlation plots of our previously reported (a) Nurr1-RXRα cellular transcription data vs. RXRα LBD TR-FRET data and (b) Nurr1-RXRα cellular transcription data vs. RXRα LBD cellular transcription data. Pearson (rp) and Spearman (rs) correlation coefficients calculated with or without the two Nurr1-RXRα specific agonists, BRF110 and HX600 (pink data points). (c-e) Principal component analysis (PCA) 2D biplots (left) and proportion of variance plots (right) for our previously published Nurr1-RXRα ligand profiling data (c) including or (d) excluding the Nurr1-RXRα selective agonists BRF110 and HX600; and the (e) Nur77-RXRγ ligand profiling data from this study. Biplots contain the loadings (data types; blue text and blue circles) and ligand-specific PC scores of the first two PCs. Data and RXR ligand label text is colored according to RXR ligand activity as grouped in Figure 2—figure supplement 1.
