Fig. 1: Identification of DNA and RNA binding targets of NONO-TFE3. | Nature Communications

Fig. 1: Identification of DNA and RNA binding targets of NONO-TFE3.

From: RNA-mediated condensation of TFE3 oncofusions facilitates transcriptional hub formation to promote translocation renal cell carcinoma

Fig. 1

a Schematic depicting the CRISPR-homology directed degron (dTAG) knocking in (KI) strategy. GFP and Flag tags were also knocked in together with dTAG. LHA or RHA: left or right homology arm. b Representative immunoblotting showing time-dependent degradation of the endogenous NONO-TFE3 with dTAG13 (left) (500 nM) and reversible expression (right) after dTAG13 washing out at the indicated time points in UOK109 KI cells. n = 3 independent biological replicates. c Representative images (top) and quantification (bottom) of clone formation assay using UOK109 parental or KI cells with indicated conditions. n = 3 independent biological replicates, one-way ANOVA with Tukey’s post-hoc test. Data were shown as mean ± SD. d, e CUT&Tag heatmap signals of NONO-TFE3 DNA binding sites around the transcriptional start site (TSS, ± 5 kb) (d) and genome browser views of representative loci (e) in UOK109 KI cells treated with dTAG13 (500 nM) at indicated time points. n = 2 independent biological replicates. f Normalized RIP-seq intensities of NONO-TFE3 binding genes from −2 kb TSS to +2 kb. transcriptional end site (TES) with or without dTAG13 (500 nM) for 24 h. n = 2 independent biological replicates. g Genomic annotation of the NONO-TFE3 CUT&Tag peaks (left) and RIP-seq peaks (right) in UOK109 KI cells. h Venn diagram showing the overlapping bound genes of NONO-TFE3 identified from CUT&Tag and RIP-seq. i The binding profiles (left) and correlation analysis (right) between NONO-TFE3 CUT&Tag and RIP-seq signals. Heatmaps were sorted by NONO-TFE3 CUT&Tag and presented the signals of the bound genes from −2 kb TSS to +2 kb TES. The box plots indicated the median (center line), the third and first quartiles (box limits) and 1.5x interquartile range (IQR) above and below the box (whiskers). (n = 2 independent biological replicates; two-sided Wilcoxon test). j, k Venn diagram (j) and heatmap binding profiles of NONO-TFE3 or TFE3-S321A CUT&Tag signals (k) in dTAG-13 treated UOK109 KI cells transfected with Flag-tagged NLS-TFE3 S321A. n = 2 independent biological replicates. Source data are provided as a Source Data file.

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