Genome-Wide Analysis Reveals PADI4 Cooperates with Elk-1 to Activate c-Fos Expression in Breast Cancer Cells

Abstract

Peptidylarginine deiminase IV (PADI4) catalyzes the conversion of positively charged arginine and methylarginine residues to neutrally charged citrulline, and this activity has been linked to the repression of a limited number of target genes. To broaden our knowledge of the regulatory potential of PADI4, we utilized chromatin immunoprecipitation coupled with promoter tiling array (ChIP-chip) analysis to more comprehensively investigate the range of PADI4 target genes across the genome in MCF-7 breast cancer cells. Results showed that PADI4 is enriched in gene promoter regions near transcription start sites (TSSs); and, surprisingly, this pattern of binding is primarily associated with actively transcribed genes. Computational analysis found potential binding sites for Elk-1, a member of the ETS oncogene family, to be highly enriched around PADI4 binding sites; and coimmunoprecipitation analysis then confirmed that Elk-1 physically associates with PADI4. To better understand how PADI4 may facilitate gene transactivation, we then show that PADI4 interacts with Elk-1 at the c-Fos promoter and that, following Epidermal Growth Factor (EGF) stimulation, PADI4 catalytic activity facilitates Elk-1 phosphorylation, histone H4 acetylation, and c-Fos transcriptional activation. These results define a novel role for PADI4 as a transcription factor co-activator. Peptidylarginine deiminase 4 (PADI4) converts positively charged arginine and methylarginine residues on histones to the neutrally charged non-standard amino acid citrulline. We and others have previously shown that citrullination of a small subset of gene promoters, such as the estrogen receptor target, TFF1, appears to downregulate gene expression. In this study, we looked across the human genome using ChIP-chip to better define the full repertoire of genes that are regulated by PADI4 in breast cancer cells. Surprisingly, we found that PADI4 appears to primarily be involved in gene activation, as opposed to gene repression. Further, we found that PADI4 is likely recruited as a co-activator to these target genes by a range of well-defined transcription factors, such as Elk-1. With respect to how PADI4 activates gene transcription, we show that PADI4 directly interacts with Elk-1 at its well-defined target, the c-Fos oncogene. Additionally, we found that, following stimulation with epidermal growth factor, PADI4 appears to directly target Elk-1 for citrullination, which in turn leads to increased histone acetylation and gene transcription. These novel genome-wide and gene-specific findings suggest that PADI4 plays a much broader role in gene activation than previously thought.