Bay et al. 2022 Figure 2F, 3 and 6

We wanted to investigate changes in RNAPII levels on chromatin after UV irradiation. To address whether cell cycle phase differences in RNAPII chromatin binding after UV might be caused by changes in RNAPII degradation on chromatin, we added the proteasome inhibitor MG132. In addition, we added the transcription inhibitor THZ1, to inhibit transcription prior to promoter proximal stalling and further investigate effects on RNAPII chromatin levels.
Conclusion:
Using this flow cytometry method we observed that elongating RNAPII (pRNAPII S2) becomes more stable in S phase after UV. Furthermore, we observed that pRNAPII S5 is specifically targeted for degradation both in the presence and absence of UV. In addition, proteasome mediated degradation of pRNAPII S5 was higher in S phase vs G2 phase after UV. Moreover, pRNAPII S5 appeared to be slightly more stable after UV in the presence of THZ1, especially in S phase.
Notes:
Cells were treated with 1 uM EdU for 1h or longer to visualize cell cycle phases. Cells were extracted prior to fixation to release unbound factors. Non-treated HeLa cells were used as barcoding controls in this experiment. In brief, non-treated HeLa cells were incubated with Alexa Fluor 647 Succinimidyl Ester. Barcoded cells were then distributed equally among all the samples prior to antibody staining. All samples were normalized to the barcoded control upon analysis to eliminate sample to sample variation during staining. In addition, we included samples with non-treated HeLa cells stained with only secondary antibodies. CST run prior to analysis