epithelium suggesting that PAD2-mediated histone citrullination may play a role in gene regulation in the mammary gland [20]. To expand upon our findings in the dog, here we illustrate for the first time that PAD2 is also expressed in human breast luminal epithelial cells. Further, we present data indicating that a portion of PAD2 localizes to the nucleus of MCF-7 cells and binds to chromatin. Using a genome-wide microarray approach, we identified a subset of genes whose expression is affected by PAD2-depletion, indicating that these genes may be targeted for regulation by PAD2. Our ChIP analysis of the most highly up- and down-regulated genes in the PAD2 knockdown line (PTN and MAGEA12) shows that PAD2 appears to bind directly to their promoters and citrullinate histone H3 arginine residues at these sites, thus further supporting the hypothesis that PAD2 regulates gene expression. Interestingly, given that both PTN and MAGEA12 are strongly associated with mammary tumor progression, our findings also suggest that PAD2 may play a role in breast cancer development. This prediction is supported by our finding that depletion of PAD2 from MCF-7 cells significantly suppresses cell proliferation. Experimental Procedures Materials Human tissue microarray slides were obtained from the Cooper- ative Breast Cancer Tissue Resource (National Cancer Institute) and from the Cooperative Human Tissue Network (CHTN) (University of Virginia). MCF-7 and 293 HEK cells were grown in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), and 1% antibiotic antimycotic (Sigma- Aldrich, St. Louis, MO) and maintained in a humidified atmosphere of 5% CO2 at 37uC as previously described [19]. Stable shRNA control and PAD2 knockdown MCF-7 cell lines were created by transfecting cells with Sigma MISSION shRNA vectors using FuGENE6 (Roche, Pleasanton, CA). Clones were selected with 2 mg/ml of puromycin. The 437 amino acid truncated PAD2 cDNA was purchased from Open Biosystems (Hunstville, AL). The cDNA was removed from pCMV-SPORT6 by digestion with EcoRI and NotI. The resulting fragment was then ligated into pCDNA3.1+ previously digested with EcoRI and NotI and clones sequence verified. The PAD2 278 and 140 amino acid truncated isoforms were constructed following a QuikChange XL site-directed mutagenesis protocol (Agilent, Santa Clara, CA) and verified by sequencing. Primers to create 1 base pair mutations to insert stop codons were as follows: 278 59-CTGCTGGAGTACATGGCCTAGGA- CATTCCCCTG-39, 140 59-CCAAAGAAGGCATCCTA- GACCTGGGGCCCC-39. Immunohistochemistry and Immunofluorescence IHC and IF experiments were carried out using a standard protocol previously described [20]. Briefly, slides were rehydrated in 3X 5 minute washes in xylene followed by single sequential 5 minute washes in 100, 95, and 75% EtOH. Slides for IHC were then incubated for 10 minutes in 0.5% hydrogen peroxide in methanol to quench endogenous peroxidases, however this step was omitted for IF slides. Next, slides were submerged in 0.01 M sodium citrate and boiled 2X for 10 minutes to retrieve antigens. After cooling, slides were washed in 1X PBS and then blocked in 10% normal goat serum and 2X casein (Vector Labs, Burlingame, CA) for 20 minutes at room temperature in a humidified microprobe chamber. Next, slides were blotted to remove excess blocking solution and then primary antibody diluted in 1X PBS was added for 2 hours at room temperature. Antibody dilutions are as follows: anti-PAD2 1:100 (ProteinTech #122100-1-AP, Role for PAD2 in Gene Regulation Chicago, IL), anti-Cytokeratin 1:100 (Dako MS3515, Carpinteria, CA), anti-H3K9 acetyl 1:200 (Abcam, ab12179, Cambridge, MA), anti-HP1a 1:200 (Millipore, #05-689, Billerica, MA), anti-H3K9 dimethyl 1:200 (Abcam, ab1220), anti-H3K27 trimethyl 1:100 (Abcam, ab6002), anti-PAD4 1:100 (Sigma, P4749), anti-FLAG M2 1:200 (Sigma, F1804). After washing three times, slides were incubated with a secondary antibody (biotinylated secondary for PAD2) diluted 1:200 in 1X PBS for 1 hour at room temperature then washed 3X in PBS. IHC slides were incubated in DAB chromagen (Vector Labs) solutions according to the manufactur- er’s protocol, washed and then counterstained with hematoxylin and coverslip mounted. IF slides were incubated in streptavidin conjugated-488 (Invitrogen), washed and then mounted using Vectashield containing DAPI (Vector Labs). During each exper- iment, duplicate slides were treated with control rabbit or mouse IgG antibody at the appropriate concentrations as a negative control. Western Blotting, Chromatin Fractionation, and Cell Proliferation Assays Cells were lysed in RIPA buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, and 0.2 mM PMSF and 1X general protease inhibitor. Protein concentration in lysates was determined by Bradford Assay prior to gel loading to ensure equal protein loading. 6X sample buffer (300 mM Tris-HCl, pH 6.8, 60% glycerol, 30 mM DTT, 6% SDS) was added to yield a final concentration of 1X and lysates were boiled at 95uC for 5 min. Samples were subjected to SDS polyacrylamide gel electrophoresis on a 10% gel (acrylamide:bis-acrylamide ratio of 29:1) and electro- blotted to Immobilin PVDF membranes (Millipore). Membranes were blocked in 1X casein diluted in Tris buffered saline (TBS). Anti-PAD2 1:1000 (ProteinTech) and anti-FLAG M2 1:4000 (Sigma), were incubated overnight at 4uC. Blots were washed and then incubated with a 1:10,000 dilution of anti-rabbit or mouse conjugated HRP (Jackson ImmunoResearch Labs, West Grove, PA) for 2 hr at room temperature. All blots were washed for 60 min (6610 min) with TBS-Tween after secondary antibody and then visualized by chemiluminescence using Millipore Immobilon Western. To confirm equal protein loading, mem- branes were stripped and re-probed with anti-b-actin (Abcam, ab8227). Chromatin fractionation was carried out as previously described [21]. Protein concentrations for resulting fractions were determined by Bradford Assay and subject to western blot analysis as described above. Cleanliness of fractionation was determined by stripping membranes and re-probing with anti-TBP 1:1000 (Abcam, ab818) or anti-Histone H3 1:1000 (Millipore, 06–755) and SOD4 1:1000 (Abcam, ab16962). Cell proliferation studies on control versus PAD2 depleted MCF-7 cells were carried out using the standardized non-radioactive cell proliferation assay protocol (MTT) from Promega (Madison, WI). Briefly, ,5000 cells/well were plated in triplicate and allowed to grow for 24, 48 or 72 hours. Following incubation with the dye solution, formazan product was measured at 570 nm. qPCR RNA was purified from MCF-7 cells according to the Qiagen RNeasy protocol including on-column DNAse treatment to remove genomic DNA. The resulting RNA was quantified and 0.5 mgs reverse transcribed using ABI High Capacity RNA to cDNA kit according to manufacturer’s protocol (ABI, 4387406, Foster City, CA). Complementary DNA was subject to qPCR analysis with TaqMan assays from ABI for PAD2 (Hs00247108_m1), PAD4 (Hs00202612_m1), and GAPDH PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41242