Potential Role for PAD2 in Gene Regulation in Breast Cancer Cells Brian D. Cherrington2., Xuesen Zhang1., John L. McElwee1, Eric Morency1, Lynne J. Anguish1, 1 Scott A. Coonrod * 1 James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York, United States of America, 2 Department of Zoology and Physiology, University of Wyoming, Laramie, Wyoming, United States of America Abstract The peptidylarginine deiminase (PAD) family of enzymes post-translationally convert positively charged arginine residues in substrate proteins to the neutral, non-standard residue citrulline. PAD family members 1, 2, 3, and 6 have previously been localized to the cell cytoplasm and, thus, their potential to regulate gene activity has not been described. We recently demonstrated that PAD2 is expressed in the canine mammary gland epithelium and that levels of histone citrullination in this tissue correlate with PAD2 expression. Given these observations, we decided to test whether PAD2 might localize to the nuclear compartment of the human mammary epithelium and regulate gene activity in these cells. Here we show, for the first time, that PAD2 is specifically expressed in human mammary gland epithelial cells and that a portion of PAD2 associates with chromatin in MCF-7 breast cancer cells. We investigated a potential nuclear function for PAD2 by microarray, qPCR, and chromatin immunoprecipitation analysis. Results show that the expression of a unique subset of genes is disregulated following depletion of PAD2 from MCF-7 cells. Further, ChIP analysis of two of the most highly up- and down-regulated genes (PTN and MAGEA12, respectively) found that PAD2 binds directly to these gene promoters and that the likely mechanism by which PAD2 regulates expression of these genes is via citrullination of arginine residues 2–8–17 on histone H3 tails. Thus, our findings define a novel role for PAD2 in gene expression in human mammary epithelial cells. Citation: Cherrington BD, Zhang X, McElwee JL, Morency E, Anguish LJ, et al. (2012) Potential Role for PAD2 in Gene Regulation in Breast Cancer Cells. PLoS ONE 7(7): e41242. doi:10.1371/journal.pone.0041242 Editor: Michelina Plateroti, University Claude Bernard Lyon 1, France Received January 18, 2012; Accepted June 24, 2012; Published July 24, 2012 Copyright: 2012 Cherrington et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a DOD Era of Hope Scholar Award (grant W871XWH-07-1-0372) to SAC. XZ was supported by a Post Doctoral Fellowship from Susan G. Komen for the Cure, KG101303. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Dr. Coonrod is a PLoS ONE Editorial Board member. This does not alter the authors‘ adherence to all the PLoS ONE policies on sharing data and materials. * E-mail: sac269@cornell.edu . These authors contributed equally to this work. Introduction The post-translational conversion of positively charged arginine to neutral citrulline residues in proteins is catalyzed exclusively by the peptidylarginine deiminase (PAD) enzyme family. PAD activity is alternatively termed citrullination or deimination, and mediates wide ranging effects on protein structure, function, and protein-protein interactions [1–5]. PAD2 appears to be the ancestral PAD homologue and is widely expressed in mammalian tissues [6]. Regarding targets and function, PAD2 can citrullinate vimentin in macrophages resulting in filament network breakdown and possibly apoptosis [7]. PAD2 also citrullinates IKKc in macrophages which suppresses NF-kB activity [8]. In the brain, PAD2 citrullinates myelin basic protein (MBP), a major component of the myelin sheath and this activity has been found to be elevated in multiple sclerosis [9,10]. PAD2 activity has also been described in reproductive tissues. PAD2 protein is expressed in the epithelium of the uterine endometrium and the pituitary gland in rodents [11,12]. Interestingly, PAD2 expression and citrullination activity in reproductive tissues is strongly tied to the estrous cycle, with both expression and enzymatic activity reaching their peak during the secretory phase [11–13]. To date, PAD4 is the only PAD family member with a documented role in gene expression. PAD4 was initially found to target histone tail arginine and methyl-arginine residues for citrullination [14]. At a promoter-specific level, PAD4 catalyzed histone citrullination has been correlated with repression of the estrogen-responsive gene TFF1 and apoptosis-associated p21 and OKL38 genes [14–18]. In addition to gene repression, we recently carried out a genome-wide ChIP-chip study and found that PAD4 also appears to play an important role in gene transactivation in MCF-7 breast cancer cells. Further, we found that PAD4 is enriched at the Elk-1 transcription factor binding site motif within the serum response element (SRE) of the c-fos gene promoter [19]. This work further characterized the role of PAD4 in gene expression and defined it as a novel transcriptional co-activator. We also recently found that PAD2 expression in the canine mammary gland initiates within a subset of epithelial alveolar cells during estrus and reaches peak expression levels during diestrus, with strong PAD2 staining detected in the nuclei at this stage. In this tissue, citrullination activity is primarily seen during diestrus and is confined to epithelial cell nuclei at this stage [20]. Interestingly, the N-termini of histone H3, but not H4, appear to be the primary target of PAD activity in canine mammary PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41242