Fig. 9: HIF1α activation upon Rnf20 haploinsufficiency induces RNA polymerase II promoter-proximal pause release at metabolic genes.
From: RNF20 links the DNA damage response and metabolic rewiring in lung cancer through HIF1α
a Schematic representation of the method for calculating Pol II pausing index (PI). b Genome tracks of merged Pol II RNA-Seq reads of control and Rnf20 + /- MLE12 cells at genes involved in glycolysis. c Log2 of the PI at genes upregulated, unchanged or downregulated upon Rnf20 LOF (n = 3). d, e Venn diagram showing an overlap of genes with decreased PI and upregulation in Rnf20 + /- compared to control MLE12 cells (d) and GO analysis of the overlapping genes (e). f Log2 of the PI at HIF-1 signaling pathway (left) or EMT genes (right) in control and Rnf20 + /- MLE12 cells (n = 3). g Ratio of the Pol II enrichment at the TSS and TTS of Scl2a1, Eno1 and Pdk1 in control (n = 3) and Rnf20 + /- lungs (n = 5) determined by Pol II Chip-qPCR. TSS, Transcription Start Site; TTS, Transcription Termination Site. h Venn diagram showing the overlap of genes with decreased PI in Rnf20 + /- compared to control MLE12 cells and HIF1α-bound genes in A549 lung AD cells44 (left) and GO analysis of the overlap (right). i, j Genome tracks of merged Pol II ChIP-Seq reads of control, Rnf20 + /- and Rnf20 + /- MLE12 cells stably expressing shRNA against Hif1a (i) and PI at HIF1α-bound genes in lung A549 AD cells (j, n = 2). k Chip-qPCR for RNA Pol II CTD-pSer2 (left) and CTD-pSer5 (right) at Scl2a1, Ldha and Eno1 gene body in control, Rnf20 + /- MLE12 cells, or Rnf20 + /- MLE12 cells after shRNA mediated Hif1a silencing (n = 3). Statistical analysis in (c, f, g) was performed using a two-tailed Student’s t-test. Multiple comparisons were performed using one-way ANOVA with Šídák’s (j, k) multiple comparisons test. Boxplots in (c, f, j) represent the minimum, 25th percentile (Q1), median, 75th percentile (Q3), and maximum of the Log2 PI in control, Rnf20 + /- MLE12 cells, or Rnf20 + /- MLE12 cells after shRNA-mediated Hif1a silencing. GO analysis in e and h was performed using Metascape (Version 3.5). Data are shown as mean ± SEM. ns, non-significant. ‘n’ indicates biological replicates.
