Role for PAD2 in Gene Regulation Figure 3. PAD2 associates with chromatin in the nucleus of MCF-7 cells. (A) PAD2 is endogenously expressed in MCF-7 cells and is detected in multiple cellular compartments. Wild type and PAD2 over-expressing MCF-7 whole cell lysates were subject to SDS-PAGE and probed with an anti- PAD2 antibody. Anti-Rabbit IgG was used as a negative control. (left panel). Endogenous MCF-7 cellular proteins were also separated into cytoplasmic, chromatin, and soluble nuclear pools by fractionation methods and examined by western blot (right panel). Cleanliness of fractionation was determined by stripping membranes and re-probing with antibodies for TBP (nuclear) and SOD4 (cytoplasmic) proteins. (B) Punctate PAD2 expression is detected in the nucleus of MCF-7 cells. MCF-7 cells were subject to IF using anti-PAD2 (green), anti-cytokeratin (luminal-red), anti-H3K9 acetyl (euchromatin-red), anti-HP1 (heterochromatin-red), anti-H3K9 dimethyl (facultative heterochromatin-red), and anti-H3K27 trimethyl (facultative heterochromatin-red) antibodies while DAPI nuclear stain is in blue. White arrows denote punctuate PAD2 staining in DAPI poor nuclear regions. doi:10.1371/journal.pone.0041242.g003 1% paraformaldehyde for 10 min at 37uC, and quenched in 125 mM glycine for 5 min at 4uC. The cells were lysed (1% SDS, 10 mM EDTA, 50 mM Tris?HCl, pH 7.9, 1x protease inhibitor cocktail) and sonicated under conditions yielding fragments ranging from 200bp to 800bp. The material was clarified by centrifugation, diluted 10-fold in dilution buffer (0.5% Triton X- 100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris?HCl, pH 7.9, 1x protease inhibitor cocktail), and pre-cleared with protein A- agarose beads. The pre-cleared, chromatin-containing superna- tant was used in immunoprecipitation reactions with FLAG-M2 beads (Sigma Aldrich) or protein A-agarose beads loaded with anti-H4 cit 3 (Milipore, 07–596) or anti-H3 cit 2–8–17 (Abcam, ab77164). Ten percent of the supernatant was saved as reference control. The immunoprecipitated genomic DNA was cleared of protein and residual RNA by digestion with proteinase K and RNase (Roche), respectively. The DNA was then extracted with phenol:chloroform:isoamyl alcohol and precipitated with ethanol. For gene-specific ChIP analyses, quantitative real-time PCR (qPCR) was used to determine the enrichment of immunoprecip- itated DNA relative to the input DNA using gene-specific primer sets (Table S1). Each ChIP experiment was conducted a minimum of three times with independent chromatin isolates to ensure reproducibility. Statistical Analysis All experiments were independently repeated at least three times. Values were expressed as the mean 6 the SEM. Means were separated using Two-tailed Student’s T-Test with * indicating means significantly different (P,0.05) from control and ** indicating P,0.01. Results Human PAD2 is Expressed in the Luminal Epithelium of Mammary Lobulo-alveolar Units Currently, relatively little is known regarding PAD2 expression patterns in normal human tissues. Therefore, we first probed a human tissue array by immunohistochemistry (IHC) using a previously validated anti-PAD2 antibody (20) or an equal mass of rabbit IgG as a control (Figure S1) and determined that PAD2 is primarily expressed in the cytoplasm of epithelial cell populations in the human endometrium, kidney, mammary duct, and mammary lobulo-alveolar units (Figure 1A, B, C, D). Of these tissues, we found that the strongest PAD2 signal was observed in the mammary gland and was largely confined to the epithelium of the terminal ductal-lobular unit (Figure 1D). Interestingly, higher magnification revealed that a significant fraction of PAD2 also appears to localize to the nucleus in the mammary epithelium (Figure 1D arrows). Our immunofluorescence (IF) studies in the dog found that PAD2 was predominately expressed in luminal (cytokeratin), but not myoepithelial (p63) mammary epithelial cells in this species. We conducted similar experiments on human breast tissue and found that PAD2 retained the luminal localization pattern in humans (Figure 2A). Further, at higher magnification the localization of PAD2 to the nucleus of human mammary epithelial cells was evident. We also found that PAD2 mainly appears to localize to regions of the nucleus that stain poorly for DAPI suggesting that PAD2 may be primarily associated with euchro- matin (Figure 2B). Endogenous PAD2 Fractionates, in Part, with Chromatin in the MCF-7 Breast Cancer Cell Line To further test the hypothesis that PAD2 localizes to the nucleus and possibly plays a role in gene regulation, we next continued our studies using the luminal subtype MCF-7 breast cancer cell line. We first demonstrated by western blotting that PAD2 is expressed in this cell line using an anti-PAD2 antibody that is not cross- reactive with other PAD family members (Figure 3A, left panel, Figure S3A) (20). Next, we resolved endogenous PAD2’s subcellular localization within MCF-7 cells by small-scale chro- matin fractionation, which separates cellular proteins into three distinct pools: cytoplasmic, chromatin, and soluble nuclear proteins. We found that PAD2 is detected in all three subcellular compartments in MCF-7 cells, with a portion of PAD2 being associated with the chromatin fraction (Figure 3A, right panel, Figure S3). Accuracy of cell fractionation was confirmed by stripping the membranes and re-probing for Superoxide Dis- mutase 4 (SOD4), a cytoplasmic protein, and TATA Binding Protein (TBP), a nuclear protein. To further refine PAD2’s nuclear localization in MCF-7 cells, we used antibodies to histone modifiers and modifications, which have been previously shown to localize to specific sub-nuclear domains. As with primary mammary epithelial tissue, we found that PAD2 localizes to DAPI-poor regions of the nucleus (Figure 3B 1). While little co- localization was observed between PAD2 and the repressive histone markers (H3K9Me2, HP1, and H3K27Me3), partial co- localization was found between PAD2 and the euchromatic marker, H3K9 acetyl, thus supporting the hypothesis that PAD2 associates with euchromatin in the nucleus (Figure 3B 2–5). Truncated Mutants Reveal the N-terminus of PAD2 is Important for Targeting to the Nucleus Next, we examined which domains within PAD2 are important for targeting PAD2 to the nucleus using the highly transfectable Human Embyonic Kidney (HEK) 293 cell line. In addition to examining a full length FLAG-tagged PAD2 expression vector (665 aa), premature stop codons were inserted into a FLAG-tagged 437 amino acid PAD2 isoform to create truncated PAD2 proteins of 278 and 140 amino acids and these constructs were then transiently transfected into HEK 293 cells. Following sequencing and confirmation that the mass of these PAD2 proteins corresponded to their predicted molecular weight (Figure 4A, Figure S4), the constructs were then transfected into HEK 293 cells and subjected to chromatin fractionation. Supporting our endogenous PAD2 fractionation studies in MCF-7 cells, full length FLAG-tagged PAD2 is detected in the cytoplasmic and the chromatin fraction of 293 cells. Interestingly, truncation of PAD2 to 437 or 278 amino acids results in localization only in the PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e41242