Protein subunits of RNase P occupy the promoter and coding region of rDNA.

<p>A. Structure of a rRNA gene, showing 18S, 5.8S and 28S rRNA flanked by 5′ETS and 3′ETS and intervened by ITS1 and ITS2. RNase MRP cleaves at the A3 site in the ITS1. Small arrows indicate locations of primers used for PCR. B. ChIP was performed with rapidly dividing HeLa cells using affinity-purified polyclonal antibodies against Rpp20 (lane 2), Rpp21 (lane 3), Rpp29 (lane 4), Rpp38 (lane 5), RPB8 (lane 6), preimmune serum (lane 7) or p53 (lane 8). DNA of the precipitated chromatin was analyzed by PCR for the presence of 5.8S rDNA, tRNA^Tyr, U1 snRNA and ARPP P0 genes. Positive PCR products for each gene using 0.01, 0.1 and 1% of input DNA are shown (lanes 9–11). This panel is a composite of two mini-agarose gels with the same exposure time. C. HeLa cells were transfected with siRNA25 (lanes 1–6), which caused marked knockdown of Rpp25 (data not shown), or with control luciferase siRNA (lanes 7–12) for 48 h. ChIP analysis was then performed for 5.8S rDNA and U1 snRNA gene using antibodies against Rpp20, Rpp25, Rpp29, Rpp38, RPB8 and preimmune serum. Positive PCR reactions using 0.01, 0.1 and 1% of input DNA are shown (lanes 13–18). D. ChIP analysis was performed with cells as in B using antibodies against Rpp20 (lane 1), Rpp21 (lane 2), Rpp29 (lane 3), Rpp30 (lane 4), RPA194 (lane 5), preimmune serum (lane 6) or p53 (lane 7). DNA of the precipitated chromatin was analyzed by PCR for the presence of the promoter, 18S, 5.8S, 28S or IGS DNA using the corresponding primers shown in A. Positive PCR reactions for each rDNA region using 0.01, 0.1 and 1% of input DNA are shown (lanes 8–10).