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. Author manuscript; available in PMC: 2010 Nov 3.
Published in final edited form as: J Mol Biol. 2007 Jul 3;372(2):298–316. doi: 10.1016/j.jmb.2007.06.079

Figure 7.

Figure 7

SUZ12 rapidly associates with RAREs whereas RNA polymerase II disassociates from RAREs upon removal of RA. (A) Schematic diagram of the RA washout ChIP experiment protocol. F9 cells were treated with RA then rinsed with PBS, and fresh media was added for various times prior to formaldehyde crosslinking and cell harvest. The time of RA addition is denoted by +RA, and the wide grey bar. The times of the PBS rinses, followed by addition of fresh media, are denoted by the change to the white bar. The positive control (pos) was treated with RA for 48 hours and was not rinsed prior to crosslinking (x-link). The negative control (neg) was not treated with RA, nor rinsed prior to x-linking. The “0” sample was treated with RA for 24 hours and then immediately x-linked and cells harvested. The “0 rinse” sample was rinsed 3× after the 24 hour RA treatment and then medium containing 1% formaldehyde was immediately added to the culture dish. The addition of RA, and the subsequent steps were staggered so that all samples were x-linked at the same time. (B) Chromatin samples were immunoprecipitated with an anti-SUZ12 antibody or a (C) monoclonal antibody that recognizes phosphorylated serine 5 of the CTD of polymerase II, and bound DNA was quantitated by real time PCR. The percent of the total input of each locus was quantitated and normalized to the negative control locus. The osteopontin VDRE was used as a negative control for SUZ12 ChIP assays, and the Hoxb1 −18kb region was used as a negative control for pol II ChIP assays. The fold induction was then calculated relative to the 0 rinse sample. Each experiment was repeated at least three times, starting with cell culture and the quantitative PCR analyses were performed in triplicate for each experiment. The bars represent fold induction (±SE). Statistical analyses were performed with Graphpad Prism 4.0. A * symbol denotes statistical significance (p < 0.05), τ indicates a p value of 0.0685. Unless denoted otherwise, statistical significance is calculated relative to the 0 (rinse) sample.