CTCF activates RNA polymerase I-mediated rRNA gene transcription and increases cell size and proliferation.
A, Western blot analysis of FLAG-CTCF expression in NEs with CTCF stably expressed HeLa-S3 cells. β-actin was used as a loading control. B, RT-qPCR analysis of pre-rRNA and CTCF expression after CTCF overexpression. Vertical bars represent mean ± S.D. C, RT-qPCR analysis of 5.8 S, 18 S, and 28 S RNA levels after ectopic CTCF expression. Vertical bars represent mean ± S.D. D, Western blot analysis of the CTCF level in whole cell extracts from control, CTCF-depleted HeLa-S3 cells, and CTCF-depleted HeLa-S3 cells with ectopic expression of mouse CTCF. β-actin was used as a loading control. E, RT-qPCR analysis of pre-rRNA expression from control (Ctrl), CTCF-depleted HeLa-S3 cells, and CTCF-depleted HeLa-S3 cells with ectopic expression of mouse CTCF. GAPDH was used as an internal control. Mean values of three independent experiments ± S.D. are included. F, RT-qPCR analysis of pre-rRNA expression in cells with pOZN or pOZN-CTCF overexpression after either actinomycin D or α-amanitin treatment. Vertical bars represent mean ± S.D. DMSO, dimethyl sulfoxide. G, cell size was determined by FACS (forward scatter) in HeLa-S3 cells retrovirally infected with empty vector (black) or constructs encoding wild-type FLAG-CTCF (red), as indicated. FSC-H, forward scatter height. H, cell growth analysis in pOZN- and pOZN-CTCF-overexpressed HeLa-S3 cells.