CSB recruits PCAF to rDNA promoter.

<p>A. Inducible knockdown of CSB impairs Pol I and PCAF occupancy at rDNA promoter. Knockdown of CSB by CSB-specific shRNA (shCSB) was induced with tetracycline (doxycycline-treated, 1 µg/ml, 72 hr). qRT-PCR data show the associations of CSB, UBF, Pol I, PCAF, GCN5 and p300 with the rDNA promoter in CSB knockdown cells (shCSB) and control cells without treatment with tetracycline (shCtrl). The immunoprecipitated DNA with specific antibodies was normalized to input DNA. Values of the average %IP (±standard deviation) for specific antibodies are shown on the bar graph with the standard deviations from three independent experiments. *<i>P</i> value <0.05, **<i>P</i> value <0.01. B. Overexpression of CSB increases the associations of Pol I and PCAF with rDNA promoter. ChIP data were from NIH 3T3 cells infected with retroviruses encoding wildtype or mutant CSB. The immunoprecipitated DNA from CSB overexpressed cells and mock-transfected cells (Ctrl) was normalized to input DNA. The levels of indicated proteins are shown with the standard deviation from three independent experiments. **<i>P</i> value <0.01, ***<i>P</i> value <0.001. C. Knockdown of PCAF decreases the binding of Pol I, but not UBF and CSB to the rDNA promoter. ChIP assays show the associations of UBF, Pol I, PCAF and CSB with rDNA promoter after knockdown of PCAF in NIH 3T3 cells by PCAF-specific shRNA. The immunoprecipitated DNA with specific antibodies from PCAF knockdown cells (shPCAF) and control cells (shCtrl) was normalized to input DNA. Values of the average %IP (±standard deviation) are shown on the bar graph with the standard deviations from three independent experiments. **<i>P</i> value <0.01. D. Association of PCAF with Pol I depends on CSB. Nuclear extracts from NIH 3T3 cells transfected with either control duplex shRNA or CSB-specific shRNA were incubated with anti-PCAF antibodies or control IgGs. About 10% of input and 80% of precipitated proteins were analyzed on western blots.