Limits...
Potential role for PAD2 in gene regulation in breast cancer cells.

Cherrington BD, Zhang X, McElwee JL, Morency E, Anguish LJ, Coonrod SA - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3404060&req=5

pone-0041242-g003: 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.

Mentions: 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 chromatin 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 Dismutase 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).


Potential role for PAD2 in gene regulation in breast cancer cells.

Cherrington BD, Zhang X, McElwee JL, Morency E, Anguish LJ, Coonrod SA - PLoS ONE (2012)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3404060&req=5

pone-0041242-g003: 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.
Mentions: 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 chromatin 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 Dismutase 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).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 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.

Show MeSH
Related in: MedlinePlus