Fig 2: FGFR1 associates with RNA polymerase II and promotes gene expression. A, FLAG-immunoprecipitation followed by MS was performed on sonicated and nuclease-treated mixed nuclear and chromatin fractions of CAMA1FGFR13XFLAG cells. FGFR1 and RNA Polymerase II subunits are highlighted (red). The Western blot on the right shows FLAG-FGFR1 overexpression in CAMA1FGFR13XFLAG cells. B, List of RNA polymerase II subunits that coprecipitate with FLAG-FGFR1. Coverage (%) indicates the percentage of the protein sequence that was covered by the identified peptides. #PSMs indicates the number of Peptide Spectrum Matches or the number of spectra assigned to peptides that contributed to inference of the protein. C, Coprecipitation of FGFR1 and RNA Polymerase phosphorylated on Serine residue 5 (Pol II S5P), with or without sonication and nuclease treatment. D, Heatmaps of ChIP-seq read densities around the FGFR1 (red) and Pol II S5P bound regions (green) in CAMA1 cells. E, Venn diagram and (F) density plots related to FGFR1- and Pol II S5P-ChIP-seq shown in D. G, Distribution of FGFR1 and Pol II S5P binding peaks at the VEGFA and CCND1 promoters (UCSC genome browser). H, ChIP-reChIP assay performed by sequential ChIP-qPCR with an FGFR1 antibody followed by an antibody against Pol II S5P (Rpb1) or normal rabbit IgG (control), at the VEGFA and CCND1 promoters. Enrichment values expressed as percent (%) of input. I, Venn diagram of FGFR1-bound genes by ChIP-seq in MCF7FGFR1(SP-)(NLS) cells (red circle), and genes upregulated (green), and downregulated (blue) in MCF7FGFR1(SP-)(NLS) versus MCF7EV cells. J, Prediction of activating or repressing transcription function of FGFR1 by Binding and Expression Target Analysis (BETA). ChIP-Seq and RNA-seq data from I were integrated. Genomic regions bound by FGFR1 are predicted to regulate the expression of upregulated genes, but not of downregulated genes. P values indicate the significance of the associations compared with background nonregulated genes.

Fig 3: WSTF-RPB1 is strongly associated with the anti-AML activity. Triptolide downregulates the WSTF and RPB1 signaling pathway without affecting TAB1/P38 signaling in a dose dependent manner in MV-4-11 (A) and THP-1 (B). Time-dependent experiments show that triptolide inhibits the WSTF-RPB1 signaling pathway with no effect on TAB1/P38 signaling in MV-4-11 (C) and THP-1 (D). (E) The different expression levels of WSTF, XPB, and RPB1 is shown.

Fig 4: hDT806 induces transcription inhibition and DNA damage. (A) Total protein extracts were prepared from the cells treated with vehicle or hDT806 (20 nM). Western blot analysis was performed for RNAPII CTD p-Ser2/5 (a), RNAPII CTD p-Ser7 (b), RNAPII large subunit Rpb1 (c), and ß-actin (d) in the cells treated with vehicle or hDT806 for 48 h. (B) Western blot analysis was performed for ?H2A.X (e), H2A.X (f), and ß-actin (g). (C) Band intensities of p-Ser2/5 (a), p-Ser7 (b), RNAPII large subunit Rpb1 (c), ?H2A.X (e), and H2AX (f) were quantified relative to the corresponding ß-actin bands for comparisons. Data of three independent experiments are presented as mean ± SD (n = 4).

Fig 5: CBX4 occupancy and H2AK119ub modification of the GnRH promoter in GT1-7 cells treated with maleate(A) Venn diagram view of genes with significantly different enrichments of CBX4 and H2AK119ub. Red and green fonts indicate up- and downregulation of CBX4 and H2AK119ub enrichment in maleate versus control. Gene browser views of (B) H2AK119ub and (C) CBX4 enrichment on AGTPBP1, CCNG2, FOXK1, GNRH1, MEF2C, and RBP4 in maleate-treated GT1-7 cells. (D) KEGG pathway analysis of intersected genes in Venn diagram. (E) Enrichments of CBX4, H2AK119ub, and POLR2A in the GnRH promoter from ARC of PNW-3, -4, and -8 by ChIP-qPCR. The qPCR data are presented as the mean ± SEM of three separate experiments. * represents the significant difference with a p value less than 0.05 compared to PNW-3 by one-way ANOVA.