Fig. 1

Rapid depletion of endogenous Pol I, Pol II, and Pol III proteins in the mESCs. a Schematic of auxin-inducible degron technology. Endogenous RNA polymerases are fused to the mAID-GFP tag in the C terminal with CRISPR genome editing. The fusion protein is recognized by OsTIR1 and subsequently degraded in the presence of auxin. Upon the removal of auxin, the Pol II protein was restored. b Western blot analyses of Pol I (RPA1), Pol II (RPB1), and Pol III (RPC1) protein levels after auxin treatment at different time points. Pol I (RPA1), Pol II (RPB1), Pol III (RPC1), Cohesin (SMC1), CTCF, and TIR1 (OsTIR1) protein levels were also examined. β-actin served as a loading control. c Lamin b1 immunofluorescence, DAPI, and GFP fluorescence signals for the RNA polymerases before and after a 6 h auxin treatment for Pol II and a 24 h auxin treatment for Pol I and Pol III. Images were obtained using a × 100 objective. d Left: Heatmap of the normalized ChIP-Seq signal centered at the Pol I peaks (n = 605) detected in the untreated cells, showing the marked reduction in Pol I binding in the degraded cells; the middle panel shows the Pol II peaks (n = 13,816); and the right panel shows the Pol III peaks (n = 1845). Heatmaps are ordered by descending ChIP-Seq signal intensity. RNA polymerases lost chromatin binding ability after auxin treatment (6 h for Pol II and 24 h for Pol I and Pol III)