Role for PAD2 in Gene Regulation Figure 5. PAD2 plays a role in gene expression in MCF-7 cells. (A) PAD2 levels are significantly reduced in PAD2 knock down MCF-7 cells compared to shRNA controls. Whole cell lysates from shRNA control and PAD2 knock down MCF-7 cells were analyzed by western blot using an anti- PAD2 antibody and an anti-b-actin antibody for loading control. RNA was purified from PAD2 knock down MCF-7 and shRNA control cells, reverse transcribed, and resulting cDNA used in qPCR reactions containing TaqMan assays to PAD2 and GAPDH as the reference gene control. Data represent the means 6 SEM of four independent experiments performed in triplicate. All values are normalized to shRNA control samples and bars represent the means 6 SEM. Means were separated by Student’s T-Test and ** represent significant differences (P,0.01). (B) Microarray and qPCR validation studies indicate that PAD2 is involved in gene regulation in MCF-7 cells. The table shows a sampling of genes whose expression profiles were significantly altered between shRNA control and PAD2 knockdown MCF-7 cells. Numbers represent log2 ratio of fold change of expression between control and PAD2 knockdown cell lines with P,0.001 and FDR,0.01. To validate microarray studies, total RNA was harvested from shRNA control and PAD2 knockdown MCF-7 cells and reverse transcribed. Resulting cDNA was used in qPCR reactions with specific primers listed in Table S1. The graph shows fold change of log2 ratio of expression between control and PAD2 knockdown cell lines with P,0.05. (C) PAD2 associates with the PTN and MAGEA12 gene promoters in MCF-7 cells stably overexpressing FLAG-tagged PAD2. ChIP-qPCR studies were done using an anti-FLAG antibody and data is presented as fold change of signal from MCF-7 cells overexpressing FLAG-tagged PAD2 over control cells with b-actin and GAPDH serving as controls. Data represent the means 6 SEM of three independent experiments. (D) The PTN and MAGEA12 gene promoters show citrullination of histone H3, but not histone H4 tail arginine residues using shRNA control and PAD2 knockdown MCF-7 cells. ChIP-qPCR studies were done using an anti-H3 cit 2–8–17 and H4 cit 3 antibodies and data is presented as % input with the GAPDH promoter serving as the control. Data represent the means 6 SEM of three independent experiments and means separated by one-tailed T-tests. doi:10.1371/journal.pone.0041242.g005 PLoS ONE | www.plosone.org 9 July 2012 | Volume 7 | Issue 7 | e41242