Role for PAD2 in Gene Regulation localization by cellular fractionation. Truncated FLAG-PAD2 constructs were overexpressed in HEK 293 cells after which cellular proteins were separated by fractionation methods and examined by western blot. Cleanliness of fractionation was determined by stripping membranes and re- probing with antibodies for Histone H3 (nuclear) and SOD4 (cytoplasmic) proteins. Truncated FLAG-PAD2 constructs were also transiently transfected into HEK 293 cells and subcellular localization examined by IF (bottom panel of B) using an anti-FLAG antibody (green fluorescent signal) to corroborate cellular fractionation studies. (C) Truncation of PAD2 reveals regions of the protein necessary for subcellular localization by IF in mammary epithelial cells. Truncated FLAG-PAD2 constructs were over expressed in MCF-7 cells and examined by IF using an anti-PAD2 (green) and anti-cytokeratin (red) antibodies while DAPI nuclear stain is in blue. doi:10.1371/journal.pone.0041242.g004 chromatin fraction, suggesting that a motif between 665 and 437 amino acids is important for retention of PAD2 in the cytoplasm, and thus prevents PAD2 from being exclusively targeted to chromatin (Figure 4B, Figure S4). Further, a region located in the first 437 amino acids is sufficient for nuclear localization; however truncation to 140 amino acids results in a reacquisition of PAD2 in the cytoplasm. Chromatin fractionation studies were further validated by transiently transfecting HEK 293 cells with the FLAG-tagged PAD2 truncation mutants and examining them by IF which corroborated fractionation studies (Figure 4B bottom panel). Since differences in subcellular localization and staining intensity could be seen between kidney (Figure 1B) and normal breast tissue (Figure 1D) in IHC, we also wanted to examine PAD2’s subcellular localization in mammary epithelial cells and, thus, examined transiently transfected MCF-7 cells by IF. Results show that as PAD2 is truncated from full length to 1–437 and then to 1–278 amino acids, there is a progressive concentration of PAD2 signal within the nuclear compartment (Figure 4C). Although the PAD2 truncation of 140 amino acids retains nuclear localization, an increasing amount of PAD2 is again detected in the cytoplasm of cells. These results further support the hypothesis that PAD2 is associated with chromatin within the cell nucleus. PAD2 Regulates Gene Expression and Likely Promotes Cell Proliferation Since PAD2 associates with chromatin, we next asked if PAD2 plays a role in mediating gene expression. To address this question, we created stable shRNA control and PAD2 depleted MFC-7 cell lines. Validation of the cell lines revealed that PAD2 mRNA and protein levels were significantly reduced in the knock down cell line compared to the shRNA scrambled control (Figure 5A, Figure S5). Due to the high degree of homology between PAD2 and PAD4, we also examined PAD4 mRNA and proteins levels and found that PAD4 levels were not significantly reduced (Figure S2). Next, we examined genome-wide differences in the gene expression patterns between the control and PAD2 knockdown MCF-7 cell lines. Microarray data identified a number of candidate genes that were either up- or down-regulated in the control compared to PAD2 knockdown MCF-7 cells with P,0.001and a false discovery rate of ,0.01 (Figure 5B). We also examined our microarray data to examine the levels of TFF1 and p21, known targets of PAD4 mediated gene regulation. Neither PAD4 target gene was differentially regulated in the PAD2- depleted versus control MCF-7 cell line raising the possibility that PAD2 may target different gene promoters compared to PAD4. Of the candidate genes identified in our microarray screen, we picked a range of significantly up and down regulated genes to validate by qPCR (Table S1). QPCR validation studies corrob- orated microarray results showing that expression of PTN, GJA1, and SLCO1A2 are significantly upregulated while MAGEA12 is significantly down regulated (P,0.05) (Figure 5B). These studies are the first to suggest that PAD2 may play a role in gene regulation in MCF-7 cells. We next used chromatin immunoprecipitation (ChIP) analysis to determine if PAD2 can associate with gene promoters in cells. For initial ChIP assays we tried multiple anti-PAD2 antibodies using the shRNA control and PAD2 knock down MFC-7 cell lines, however none of the antibodies showed sufficient ability to immunoprecipitate chromatin. Thus, we created a stable MCF- 7 cell line that overexpresses FLAG-tagged PAD2 and used this cell line for ChIP analysis. We chose to examine if PAD2 can associate with the PTN and MAGEA12 gene promoters as these two genes showed the largest differential regulation in control versus PAD2 depleted cells in our microarray and qPCR validation studies (Figure 5B). Our results show an increase in PAD2 enrichment on both the PTN and MAGEA12 gene promoters in the FLAG-PAD2 MCF-7 cells compared to the control cell line (Figure 5C, Table S1). Given our previous observation that histone citrullination correlated with PAD2 expression across the estrous cycle in the canine mammary epithelium (20), we next tested whether PAD2 might regulate gene activity via histone tail citrullination. To do this we compared citrullination levels on histone H3 and H4 tails at the PTN and MAGEA12 gene promoters in both wild type and PAD2-depleted MCF-7 cells. Results show that depletion of PAD2 results in a decrease in citrullination on the histone H3 tail (at arginine residues 2–8–17) on both the MAGEA12 and PTN gene promoters while citrullination at arginine 3 on histone H4 was not affected (Figure 5D). Interestingly, we also found that the growth rate of the PAD2-depleted MCF-7 cell line was signif- icantly reduced (Figure 6), thus raising the possibility that the observed PAD2-mediated gene regulatory activity may facilitate cell proliferation. Discussion Our previous work identified a novel role for PAD2 in the canine mammary gland by showing that PAD2 likely targets epithelial cell histones for citrullination during the diestrus phase of the estrous cycle. Based on this novel finding, we expanded our studies here to test whether PAD2 might associate with chromatin and regulate gene activity in human breast epithelial cells. Strikingly, we found that PAD2 is robustly expressed in the luminal epithelium of terminal ductal-lobular units in human mammary tissue and localizes, in part, to the nucleus of these cells. Using tissue culture, we then confirmed PAD2’s nuclear localiza- tion and also demonstrated that a portion of endogenous PAD2 partitions in the chromatin fraction of MCF-7 cells. Given this observation, we next attempted to refine the localization of PAD2 to distinct sub-populations of chromatin. We found that PAD2 signal was detected in DAPI poor regions of the MCF-7 cell nucleus, suggesting that PAD2 primarily localizes to euchromatin within the nucleus. This prediction is supported by our finding that PAD2 does not co-localize with markers for heterochromatin (HP1), or facultative heterochromatin (H3K9 me2 and H3K27 me3) but does partially co-localize with the euchromatic marker, H3K9acetyl. Investigations are currently underway to further refine the localization of PAD2 within the nucleus. To expand upon these findings, we examined a full length 665 amino acid FLAG-tagged PAD2 protein expression vector and truncated FLAG-tagged PAD2 isoforms to identify regions of the PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e41242