Fig. 6: RPB7 permits Pol II reinitiation by stabilizing Pol II promoter binding.

Meta-gene plots showing the mean Pol II ChIP-seq signal upon untreated (blue), auxin (red), dCDK9 (green), and auxin then dCDK9 (yellow) in Cul3^WT/WT (a), Cul3^−/− (g) RPB7 degron cells and CTDP1 degron cells (c) at −2 kb to 5 kb around TSS. b, d, h Left: Genome browser showing RPB1 ChIP-seq tracks in (a), (c), (g) at the same region shown in Fig. 1f. The y-axis shows the read counts normalized to the spike-in. Right: The Pol II ChIP enrichment relative to input (2.5%) at the Dhx9 gene promoter was shown as bar graphs (means, n = 2, biological replicates). e Western blot analyses of whole-cell lysates of Cul3^WT/WT or Cul3^−/− RPB7 degron cells. β-Actin served as a loading control. The fold changes in hypophosphorylated Pol II (IIA) and hyperphosphorylated Pol II (IIO) are shown as means in the right panel (n = 2, biological replicates). f Western blot analyses of two subcellular fractions in Cul3^WT/WT and Cul3^−/− RPB7 degron mESCs with or without auxin treatment. Right: Bar graph (means) showing the fold change in IIA and IIO (n = 2, biological replicates). i Western blot analyses of three fractions of dCDK9-pretreated RPB7/CTDP1 degron cells with or without auxin. Right: Bar graph (means) showing the fold change in RPB1 in the upper panel (n = 2, biological replicates). j Left: Boxplots same as Fig. 4d, showing Pol II ChIP-Seq signal at TSS region in three clusters during DRB release with or without CTDP1. Right top: A schematic diagram showing the reinitiation of Pol II upon DRB release. At 20 min most of the Pol II at TSS was released into gene body, at 40 min Pol II TSS signal recovered due to the reinitiation of Pol II in the pool. Right down: Boxplots showing the change of recovery rate of the three clusters. Statistics, two biological replicates, two-sided Wilcoxon test.