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.

Dysregulation of PAD4-mediated citrullination of nuclear GSK3β activates TGF-β signaling and induces epithelial-to-mesenchymal transition in breast cancer cells

“…PAD4 was recently also shown to citrullinate the nonhistone proteins p300, nucleophosmin (NPM1), ING4, Elk-1 and RPS2, thereby partially influencing the activity or localization of these proteins (16)(17)(18)(19). Despite this progress, in-depth studies linking experiments that examine cell morphology, mouse xenograft, and human tissue array analyses to elucidate the role of PAD4 in breast cancer progression have largely been lacking.…”

“…Citrullination has been linked to either transcriptional repression or activation, depending on the context (12)(13)(14)(15)(16). Recently PAD4 was also shown to citrullinate nonhistone proteins, raising important issues as to what other substrates may be targets of citrullination in different biological contexts (16)(17)(18)(19). Because PAD4 is expressed in breast cancer cells, we aimed to investigate whether PAD4 activity might play a role in breast cancer initiation or progression.…”

Anticancer Peptidylarginine Deiminase (PAD) Inhibitors Regulate the Autophagy Flux and the Mammalian Target of Rapamycin Complex 1 Activity

“…PAD6 (also called ePAD) is a protein uniquely expressed in eggs and regulates the early stage of embryonic development, but it misses a conserved cysteine residue at the active center of all other PAD enzymes (12,13). PAD2 is widely expressed in many tissues, including muscle, brain, and mammary gland, and is found to citrullinate myelin basic protein and actin (10,11,14). PAD4 is the only PAD family member containing a nuclear localization signal and citrullinates many substrates including histones (e.g.…”

“…Moreover, PAD4 was recently reported to cooperate with Elk-1 to facilitate c-fos expression. As such, PAD4 can positively and negatively regulate transcription in a promoter context-dependent manner (14,24).…”

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

“…Likewise, dysregulated PAD4 activity has recently been observed in a number of human cancers including ovarian, breast, lung, and esophageal cancers [19][20][21][22] . The link between PAD4 and cancer has been shown to be mediated through the ELK1 oncogene or via the p53 tumor suppressor protein 22,23 and previous work has suggested that PAD4 could be a novel anti-cancer therapeutic target . Quite remarkably, PAD4 inhibition appeared to act as a targeted therapy that resulted in selective killing of cancerous cells while sparing untransformed cells.…”