Figure 3. PAD4 and HDAC2 dynamically associate with the p21 promoter during DNA damage response.
(a) Illustration of the p21 gene structure and its two p53-binding sites (PBS1 and PBS2). (b) The amount of p53, p21, PAD4, and HDAC2 was analyzed by western blot in U2OS cells (lanes 1 and 2) or U2OS/p53-shRNA cells (lanes 3 and 4) before and after doxorubicin treatment. β-actin was probed as a control to ensure equal loading. (c–d) Chromatin immunoprecipitation (ChIP) experiments were performed to analyze the level of p53, PAD4, HDAC2, histone H3 K9/14 acetylation (H3K9/14Ac), H3 citrullination (H3Cit), or H3 Arg 17 methylation (H3R17Me) in U2OS cells (c) or U2OS/p53-shRNA cells (d) before or after 0.4 μM doxorubicin treatment for 6 hr at the two p53 binding sites (PBS1 and PBS2) as well as the transcription start site (TSS). ChIP samples were analyzed using real-time PCR as previously described (Li et al., 2008). ChIP signals were calculated as percentages of the input (% IP). ChIP primers are available upon request. Protein A agarose beads were used to measure the background signals. Averages and standard deviations were shown. Throughout this manuscript, ChIP experiments were generally performed from two to three independent experiments of sample preparation and PCR reactions were performed at least 6 times. For those ChIP results with small fold of changes but key to the conclusion, p values were analyzed by student t-test. * denotes p<0.02. (e) Re-ChIP experiments detected simultaneous binding of PAD4 and HDAC2 to the p21 promoter. For re-ChIP analyses, the first step ChIP was performed using the M2 agarose beads to recover FLAG-PAD4 associated chromatin fragments. After elution with 200 ug/ml FLAG peptide, the second step ChIP was performed using the affinity purified PAD4 antibody or anti-HDAC2 antibody (Upstate, 05–814).