Extended Data Fig. 8: An LLPS-competent IDR within the leukaemia-related transcription factor chimera is essential for potentiating transcriptional activation of the downstream oncogenic gene-expression program.
From: Phase separation drives aberrant chromatin looping and cancer development
a, Fixed cell immunostaining for the 3×HA-3×Flag-tagged N-IDRWT/A9 (left; anti-Flag) and the indicated histone modification (middle) in the 293FT stable expression cells. Top panels show the enlarged images of an example region within the white dotted box shown in the bottom panels, in which the transcription factor chimera is co-localized with H3K27ac (top) and not H3K9me3 (bottom). Scale bars, 10 μm. b, Pearson’s correlation coefficient values between N-IDRWT/A9 and the indicated histone modification. The red dotted line indicates the calculated average value of each plot. The calculated means (red dotted lines) were compared with an independent two-tailed Student’s t-test. n, the number of cells analysed. c, RT–qPCR to assess the effect of phase separation in target gene expression in 293FT cells. All of the tested HOX and MEIS2 genes are direct targets of both N-IDRWT/A9 and N-IDRFS/A9 based on ChIP–seq, whereas MYC is not and serves as a negative control. Note that LLPS-competent N-IDRWT/A9 induces significantly more upregulation of target genes, relative to LLPS-incompetent N-IDRFS/A9. PCR signals were normalized first to those of an internal control (18S RNA) and then to vector-expressing cells and presented as mean ± s.d. of three replicated experiments. ***P < 0.001; ****P < 0.0001; two-sided t-test. n.s., not significant. d, Heat map illustrating relative expression of the 374 genes that show significant upregulation post-transduction of F-IDRWT/A9, compared to empty vector and its IDR-mutant form (F-IDRYS/A9), in 293FT stable expression cells. e, Venn diagrams showing the overlap of the significantly downregulated genes identified 7 days after transduction of the indicated construct into mouse HPSCs. f, Gene set enrichment analysis (GSEA) shows that, compared with that of N-IDRFS/A9, the expression N-IDRWT/A9 in mouse HPSCs is positively correlated with the indicated leukaemia- or HSPC-related gene sets (top) and negatively correlated with the indicated differentiation-related gene sets (bottom). The P value was calculated by an empirical phenotype-based permutation test; the false discovery rate (q) is adjusted for gene set size and several hypotheses testing whereas the P value is not. g, Venn diagrams showing the overlap of the significantly upregulated (left) or downregulated (right) genes identified after transduction of the indicated construct into mouse HPSCs.
