Journal: Nature Communications
Article Title: Topoisomerase II? promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation
Figure Lengend Snippet: Top2α facilitates formation of PICs in Pol I transcription activation. ( a , b ) Top2 inhibition reduces activation of Pol I transcription in serum-refed cells. H1299 cells (p53 null) ( a ) or U2OS cells ( b ) were serum-starved for 20 h and (starting at –1 h) incubated with Top2 inhibitors etoposide (Et) or merbarone (Me), respectively, or without inhibitors, for 1 h. At 0 h, serum was added to the cells. RNA was extracted 15, 30, 60 and 90 min after serum addition. Pre-rRNA levels were analysed by S1 nuclease protection (complementary assay, 3 H-uridine labelling of RNA, Supplementary Fig. S6a,b ); s.d. is shown. ( c ) Top2α depletion reduces activation of Pol I transcription in serum-refed cells. HTETOP cells were incubated for 48 h +/− Tet, with serum starvation for the last 20 h. Serum was added back and Pol I transcription assayed as in a (complementary assay, Supplementary Fig. S6c ). ( d , e ) Inhibition of Top2 or depletion of Top2α reduces the accumulation of PIC components SL1, UBF and Pol I at the rDNA promoter in serum-refed cells. ChIP assays were performed on chromatin from cells treated as in a and c , respectively, using antibodies for Top2α, TAF I 63 (TAF1B; SL1), UBF and A135 (Pol I), and normalized to control IgG samples. s.d. is shown, n =3; *** P
Article Snippet: For Top2 inhibition, Top2 poison etoposide (100 μM final concentration; Merck) or catalytic inhibitors ICRF-193 (50 μM) and merbarone (100 μM; Merck) were added (except ).
Techniques: Activation Assay, Inhibition, Incubation, Chromatin Immunoprecipitation