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Fig. 1 | Genome Biology

Fig. 1

From: Cross-regulome profiling of RNA polymerases highlights the regulatory role of polymerase III on mRNA transcription by maintaining local chromatin architecture

Fig. 1

Rapid disruption of Pol I, Pol II, and Pol III transcription in mESCs. A Western blot analyses of Pol II (RPB1) protein levels at different time points after IAA treatment in C-terminal domain (CTD, left) and N-terminal domain (NTD, right) degron mESCs. GFP was fused to the degron tag and used to confirm degradation. HA-tagged TIR1 (TIR1-HA) level were also examined to indicate efficient induction of TIR1. b-Actin served as the loading control. B Genome browser ChIP-Seq track at the 22,193,658–22,321,486 region on chromosome 16 in Pol II_CTD_degron cells under untreated (upper) and after 1 h of IAA treatment conditions (middle). ChIP-Seq was performed with an antibody recognizing the Pol II (RPB1) N-terminal (anti-Pol II-NTD). The input is shown in the bottom panel, all tracks are flipped horizontally, and the y-axis shows the normalized read density in reads per genome coverage (RPGC). C Average metagene profiles of Pol II occupancy on gene bodies and the adjacent regions 3 kb upstream and downstream in Pol II_CTD_degron cells at active mRNA genes under untreated (left) and after 1 h of IAA treatment conditions (middle) with an antibody recognizing the Pol II (RPB1) N-terminal (anti-Pol II-NTD). The input is shown in the right panel. D Genome browser PRO-Seq track at the same region described in Fig. 1B in Pol II_CTD_degron and Pol II_NTD_degron cells under untreated and after 1 h of IAA treatment conditions. The y-axis shows the normalized read density in reads per kilobase per million mapped reads (RPKM). Only sense strand signals are presented, and all tracks are flipped horizontally. E Average metagene profiles of spike-in-normalized PRO-Seq signals at active mRNA genes in Pol II_CTD_degron (left) and Pol II_NTD_degron (right) cells under untreated and after 1 h of IAA treatment conditions. F Western blot analysis of Pol I (RPA1) and Pol III (RPC1) protein levels at different time points after IAA treatment. Immunoblotting was performed with antibodies recognizing the N-terminal domains, as shown in Fig. 1A. G Normalized PRO-Seq read counts were summed and compared at rRNA (left, N = 3), active mRNAs (middle, N = 8845), and tRNA loci (right, N = 435) in Pol I degron, Pol II CTD_degron, and Pol III degron cells under untreated and after 1 h of IAA treatment conditions. The rRNA density was calculated as the sum of the 5.8S, 18S, and 28S rRNA densities. The boxplots show the range of the values, with the median indicated by a line. The whiskers on the boxplots show the lowest data value within IQR=1.5 of the lower quartile and the highest data value within IQR=1.5 of the upper quartile. The p value was calculated using the Mann–Whitney test. H Genome browser PRO-Seq track at the 39,840,997–39,850,829 region on chromosome 17, 34,648,531–34,653,537 region on chromosome 3 and 21,240,180–21,243,656 region on chromosome 13 in Pol I_degron, Pol II_CTD_degron, and Pol III_degron cells under untreated and after 1 h of IAA treatment conditions. The y-axis shows the normalized read density in reads per kilobase per million mapped reads (RPKM). Only sense strand signals are presented

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