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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.
Highlights
The post-translational conversion of positively charged arginine to neutral citrulline residues in proteins is catalyzed exclusively by the peptidylarginine deiminase (PAD) enzyme family
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)
We 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 euchromatin (Figure 2B)
Summary
The post-translational conversion of positively charged arginine to neutral citrulline residues in proteins is catalyzed exclusively by the peptidylarginine deiminase (PAD) enzyme family. We 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]. 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, further supporting the hypothesis that PAD2 regulates gene expression Given that both PTN and MAGEA12 are strongly associated with mammary tumor progression, our findings 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
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