Figure 5

UBF depletion does not affect DNA methylation and other heterochromatic markers.

(A) Knockdown of UBF expression by siRNA. Human liver cancer cell (HepG2) was transfected with control or UBF siRNA and protein samples were harvested and analyzed by Western blot (UBF and β-actin) 48 h after transfection. The gels have been run under the same experimental conditions. The full-length blot is presented in Supplementary Figure 2A. (B) Levels of UBF mRNA and pre-rRNA were determined by RT-QPCR. The data are derived from three different independent experiments. **p < 0.01, *0.01 < p < 0.05, Student’s t-test was done for cells transfected with or without UBF siRNA. (C) Depletion of UBF does not affect the methylation status of rDNA promoter. DNA was isolated from HepG2 cells transfected with control siRNA or UBF siRNA and digested with MspI or HpaII. The promoter region of human rDNA was then amplified with the primers flanking the CCGG site at −9. Only the methylated CpG dimer was resistant to cleavage with HpaII, it can yield a fragment of 100 nt that is amplified from −57 to +43 of rDNA, whereas unmethylated DNA would be cleaved and no PCR product would be amplified. Student’s t-test was performed between cells transfected with or without UBF siRNA. (D–F) Depletion of UBF does not alter the binding of SNF2H or histone modifications associated with the silencing of rDNA mediated by NoRC. ChIP-QPCR analysis of different regions of rDNA in HepG2 cells transfected with control or UBF siRNA using antibodies against SNF2H (D), H3K9m2 (E), H3K27me3 (F). The ratio of rDNA occupancy between UBF knockdown cells and control cells was calculated for each antibody. Student’s t-test was performed between cells transfected with or without UBF siRNA.