Figure 6. Knockdown of endogenous PAD2 in MCF-7 cells alters cellular proliferation rates versus control cells. Equal numbers of shRNA control and PAD2 knockdown MCF-7 cells were plated in 96-well plates and allowed to grow for 24, 48, or 72 hours. At time of harvest, dye solution from MTT assay kit was allowed to incubate with cells for one hour at which point solubilization reagent was added and resulting formazan product measured at 570 nm. Data represent the means 6 SEM of three independent experiments performed in triplicate. Values represent the means 6 SEM. doi:10.1371/journal.pone.0041242.g006 protein that are important for targeting to chromatin. Given that the PAD4 crystal structure has been resolved and the high sequence homology between PAD4 and PAD2, we placed stop codons into the PAD2 sequence based on defined regions in PAD4 [23]. Truncation of PAD2 from 665 to 437 and to 278 amino acids resulted in increasing proportions of PAD2 being targeted to the cell nucleus. This finding suggests that a region of PAD2 from 438–665 may be important for cytoplasmic retention, potentially via protein-protein interaction, while a motif in the first 437 amino acids is sufficient for nuclear localization. Interestingly, truncation from 437 to 278 amino acids increases nuclear localization, while truncation to 140 amino acids retains nuclear localization but reacquires cytoplasmic expression, indicating that more than one motif may be necessary for subcellular localization. Our studies here utilized IIF, IHC, and most importantly, small scale chromatin fractionation to document that both endogenous and FLAG-Tagged PAD2 appear to localize to the nucleus and bind to chromatin. These well-accepted techniques, however, are not without limitation and future studies will likely need to be carried out to unequivocally confirm the presence of PAD2 in the nucleus. While much of the previous work on PAD2 has been biochemical in nature, one previous study by Nakashima et al. overexpressed GFP-tagged forms of PADs 1, 2, 3, and 4 in HeLa cells and concluded that PAD4 was the only PAD member that localized to the cell nucleus, likely via its canonical nuclear localization signal (NSL) [24]. It is not clear why our results differ from those of Nakashima, although the GFP tag, which is considerably larger than that of the FLAG tag, might have hindered the ability of the PAD2 fusion protein to be targeted to the nucleus. In further support of our conclusion that a portion of PAD2 localizes to the nucleus, a recent report by Jang et al. showed by cell fractionation and immunogold electron microscopy Role for PAD2 in Gene Regulation that PAD2 is found in both the cytoplasmic and nuclear compartment of mouse astrocytes and neurons [25]. Given that PAD2 does not possess a canonical NLS, it is currently unclear how PAD2 enters and is retained in the nucleus. While speculative, we hypothesize that either a distinct nuclear directing motif in the PAD2 coding sequence or an interaction between PAD2 and an NLS-containing protein may function to target and/or retain PAD2 in the nucleus. In support of this hypothesis, signaling proteins such as ERKs, MEKS, and SMADS translocate to the nucleus upon stimulation via an NLS-independent mechanism [26,27] while protein-protein interactions like the one between tissue transglutaminase (tTG) and vascular endothe- lial growth factor receptor 2 (VEGFR-2) can also facilitate nuclear import upon stimulation [28]. A recent study has shown the functional relevance of PAD4 dimerization [29]. Given the high degree of homology between PAD2 and PAD4 isoforms, it is intriguing to speculate that heterodimerization may occur between PAD2 and PAD4; thus, PAD2 may enter the nucleus by dimerizing with PAD4. Studies are currently underway to test this hypothesis. Given our finding that PAD2 associates with chromatin in MCF-7 cells, we next focused our attention on investigating the role of PAD2 in this subcellular compartment. To do this, we first used a non-biased, genome-wide approach to compare the gene expression profiles of wild type and PAD2-depleted MCF-7 cell lines and then validated candidate genes by qPCR. We found the most highly up-regulated gene in PAD2-depleted cells to be pleotropin (PTN), a secreted heparin-binding growth factor believed to be important in tumor angiogenesis and also described as a proto-oncogene [30,31]. We found the most highly down- regulated gene in the PAD2-depleted cells to be melanoma associated antigen A12 (MAGEA12), a member of the large MAGE family of proteins that have been observed in brain, liver, lung, prostate, ovarian, skin and thyroid cancers [32,33]. Interestingly, these proteins are normally only expressed in testis germline cells and aberrant expression of these factors is believed to promote survival, proliferation, and metastasis of cancer cells [34,35]. To address if PAD2 directly regulates the expression of the PTN and MAGEA12 genes, we performed ChIP assays using MCF-7 cells stably overexpressing a FLAG-tagged version of full- length PAD2. This approach was necessary as ChIP-quality antibodies to PAD2 are currently not available. Results from these ChIP experiments found that PAD2 binds both the PTN and MAGEA12 promoters, thus supporting a role for PAD2 in gene regulation in vivo. Given that PAD4 has been found to regulate gene activity by citrullinating specific arginine residues on histone H3 and H4 tails, we next tested whether PAD2 might also regulate the expression of MAGEA12 and PTN via similar mechanisms. Our ChIP studies using previously validated site-specific anti- citrullinated histone antibodies found that citrullination was suppressed at histone H3 arginine residues (2–8–17) on the MAGEA12 and PTN gene promoters in PAD2-depleted cells while citrullination at histone H4 arginine 3 was not observed in either cell line. This finding supports our previous observations in canine mammary tissue (20) and defines a new role for PAD2 as an epigenetic modifier of gene activity via histone tail citrullination. Regarding a potential function of PAD2-mediated gene regulation in mammary epithelial cells, we found that PAD2 depletion suppresses MCF-7 cell proliferation rates when com- pared to the shRNA control cells (Figure 6). Interestingly, the two genes found in this study to be most strongly affected by PAD2 depletion, MAGEA12 and PTN, are closely associated with tumor progression in a range of cancers (including breast cancer) (30–35). Therefore, our findings raise the possibility that PAD2-mediated PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e41242