Fig. 5: ESC domain of IWS1 stimulates Pol II elongation to support H3K36me3.
From: IWS1 positions downstream DNA to globally stimulate Pol II elongation
a Schematic 2D overview of IWS1 truncation mutants analyzed in RNA extension assays (T1-4). b Denaturing PAGE analysis of RNA extension assays. Fluorescently labeled RNA primer was extended by the activated elongation complex with ELOF1 ± IWS1 mutants. Labels as in Fig. 3b.c Quantification of the full-length transcripts from the RNA extension assay shown in a). Data represent mean ± SEM from four independent replicates. p-values were determined using one-way ANOVA followed by Tukey’s multiple comparisons test, representation as in Fig. 3c. Statistical significance was observed between multiple conditions including +/− IWS1 WT (p = 6.2 × 10−7) and +/− IWS1 T4 (p = 3.5 × 10−3). No significance was observed between +/− T1, T2 or T3 (p = 0.9, p = 0.9, p = 0.9, respectively). d Dotplot depicts fold changes in levels of Pol II and its associated factors in the chromatin fraction after 1 h depletion of IWS1. Each dot represents a factor/subunit, where colors denote p-value, non-significant changes are shown in gray. The horizontal line represents mean log2 fold-change of each set. p-values were assessed using two-sided Student’s t-test. e Boxplots show H3K36me3 ChIP-seq counts of expressed genes (n = 10,147) in 1 h control- or dTAG7-treated (dIWS1) cells. Representation as in Fig. 2e. p-value = 1.518 × 10−12. f Heatmap representation of changes in H3K36me3 ChIP-seq signal of expressed genes (n = 10,147) aligned at the gene centers after 1 h depletion of IWS1. Representation as in Fig. 2b. g As in e), but after 1 h depletion of RTF1 (n = 12,976). p < 2.2 × 10−16. h As in (f), but after 1 h depletion of RTF1 (n = 12,976). i As in (e), but after 4 h depletion of SPT6 (n = 14,002). p < 2.2 × 10−16. j As in (f), but after 4 h depletion of SPT6 (n = 14,002).
