Fig. 3: IGS-associated PAF1 mediates Pol II transcription elongation.

a, b Validation of PAF1 knockdown via immunoblotting (a) and RT-qPCR (b). c, d Heatmaps showing the effect of PAF1 knockdown on IGS ncRNA levels in cells treated with vehicle (VEH), iPol I, or iPol II. e Heatmap presenting the strand-specific RT-qPCR (ssRT-qPCR) analysis of IGS ncRNAs following PAF1 knockdown. f–h ChIP analysis assessing the impact of PAF1 knockdown on the localization of total Pol II (f), Pol II pSer2 (g), and H3K36me3 (h) across the IGS and the 28S rRNA-coding control. i–k Impact of PAF1 knockdown on R-loops detected using DRIP (i), Pol I in ChIP (j), and nascent ncRNA levels in RT-qPCR (k) at the IGS. The 28S rRNA-coding site served as control i, j. l, m Use of the LasR system employing the RED fusion protein together with sg20 represses R-loops at the IGS20 site in DRIP (l) and increases IGS ncRNA levels across the IGS in RT-qPCR (m). a–k Experiments were performed using HEK293T cells except for (k, m) where HEK293 TRex^TM were used; blots (a) are representative of three biologically independent experiments; quantitative data are shown as means (heat maps) or mean±s.d. (graphs); n = 3 except for (e, k) where n = 4 biologically independent experiments; data were analyzed using unpaired two-tailed t-tests (b), two-way ANOVA with Dunnett’s multiple comparisons test (e) or with uncorrected Fisher’s LSD test (f–m), and for (c, d) shown is two-way ANOVA with Tukey’s multiple comparisons tests (below heatmaps), multiple unpaired two-tailed t-tests comparing VEH-treated samples (orange asterisks), and two-way ANOVA for iPol I- or iPol II-treated samples (black asterisks). Source data are provided as a Source Data file.