Rapid degradation of RNAPII increased the chromatin dynamics. (A) A schematic illustration of the AID system (Natsume et al., 2016; Yesbolatova et al., 2019 Preprint). OsTIR1, which was expressed by addition of doxycycline, can form a functional SCFOsTIR1 E3 ligase complex with the endogenous components in human cells. In the presence of auxin, a protein of interest fused with mAID is rapidly degraded upon polyubiquitylation. (B) Experimental scheme used to introduce Tet-OsTIR1 at the safe-harbor AAVS1 locus in human colorectal carcinoma DLD-1 cells (top) and used to generate mAID-mClover-RPB1 (mAC-RPB1) cells (bottom) by a CRISPR/Cas9 genome editing method. Genomic PCR to test the genotype of clones after hygromycin selection was performed. Primer sets and expected PCR products are shown in B (bottom). After integration at the POLR2A gene encoding the largest subunit of RNAPII, RPB1, the PCR primers should give rise to ∼3.4-kb products in DLD-1 cells. (C) PCR confirmed that both alleles of POLR2A gene were tagged with mAID-mClover. (D) RNAPII degradation in DLD-1 cells (Clone 1 and Clone 5) after auxin treatment was verified by immunoblotting by using an antibody against the RPB1 CTD. α-Tubulin antibody was used as a control. Since the RPB1 in the AID cells was fused with mAID and mClover (totally ∼35 kD), the detected RPB1 in Clone 1 and Clone 5 has a slightly larger size than that of the parental cells. Note that the auxin treatment induced RNAPII degradation. (E) Fluorescent images of H2B-Halo-TMR (top) and mClover-RPB1 (middle) in living DLD-1 cells: From left to right, the cells before (untreated control) and after treatment with auxin for 1 h (+Auxin), the cells incubated for 6 h and 12 h after washing out auxin. Bottom: The median intensities of mClover-RPB1 in the indicated cells are the following: 931 (n = 10) in untreated control; 78.8 (n = 10) in +Auxin; 557 (n = 10) at 6 h after washing; 713 (n = 10) at 12 h after washing cells. ***, P < 0.0001 (P = 1.1 × 10−5), **, P < 0.01 (P = 7.2 × 10−4), and N.S. (P = 0.063) by the Wilcoxon rank sum test. (F) MSD plots (±SD among cells) of nucleosomes in DLD-1 cells with indicated conditions: The cells before (untreated control, black) and after treatment with auxin for 1 h (+Auxin, orange); after washing out auxin, the cells incubated for 6 h (dark green) and 12 h (light green). For each condition, n = 20 cells. The prompt degradation of RNAPII increased the chromatin dynamics. Note that DLD-1 cells have generally higher MSD values than RPE-1 cells due to unknown reasons. **, P < 0.01 for control versus +Auxin (P = 2.7 × 10−4) and N.S. (P = 0.83) by the Kolmogorov–Smirnov test.
