Reassociation of protein subunits of RNase P with chromatin of target genes.

<p>A. HeLa cells were synchronized by nocodazole for 16 h before mitotic cells were separated from adhered G2 cells. Mitotic cells were released from the inhibitor for 2, 6 and 14 h. Cells were at early G1, mid G1 and S phase as determined by flow cytometry analysis (data not shown). ChIP was then performed with synchronized cells using antibodies against Rpp20 (lanes 1, 6 and 10), Rpp25 (lanes 2, 7 and 11), Rpp29 (lanes 3, 8 and 12), RPB6 (lanes 4, 9 and 13) or control IgG antibody (lane 5). DNA extracted from the precipitated chromatin was analyzed by PCR for the presence of 5.8S rDNA, 5S rDNA and tRNA^Leu genes. Linear PCR reactions using 0.01, 0.1 and 1% of input DNA are shown (lanes 14–22). B. HeLa cells were synchronized as described in A and at the indicated time points after release from nocodazole they were subjected to FACS analysis. C. Whole extracts obtained from HeLa cells described in B were examined for transcription of a human tRNA^Tyr gene as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004072#pone.0004072-Reiner1" target="_blank">[10]</a>. The resulted primary transcript is 112 nt in length and has an intron that is promptly excised to release the exons with 5′ leader and 3′ trailer sequences (42 and 50 nt in length, respectively). The exons are ligated to produce a 92-nt intermediate precursor tRNA^Tyr, which is processed by RNase P to remove the 5′ leader sequence (5 nt) and by a 3′ endonuclease that eliminates the 3′ trailer sequence (11 nt) to generate a mature 76-nt tRNA^Tyr. D. Whole HeLa extracts described in B were assayed for 5S rRNA gene transcription. E. HeLa cells were synchronized to G1/S phase by treatment with hydroxyurea for 16 h. Cells were then released from inhibitor and analyzed by FACS for their DNA content at the indicated time points. Cells were at mitosis at 12 h. F. Western blot analysis of whole extracts obtained from cells described in E using antibodies against Rpp29, Rpp25, Rpp20 and Rpp40 and cyclin B1. Cell cycle progression shown on the top was deduced from the FACS data shown in E and from the levels of cyclin B1. Similar results were obtained in three independent synchronization experiments.