Cofactors for estrogen receptor α (ERα) can modulate gene activity by posttranslationally modifying histone tails at target promoters. Here, we found that stimulation of ERα-positive cells with 17β-estradiol (E2) promotes global citrullination of histone H3 arginine 26 (H3R26) on chromatin. Additionally, we found that the H3 citrulline 26 (H3Cit26) modification colocalizes with ERα at decondensed chromatin loci surrounding the estrogen-response elements of target promoters. Surprisingly, we also found that citrullination of H3R26 is catalyzed by peptidylarginine deiminase (PAD) 2 and not by PAD4 (which citrullinates H4R3). Further, we showed that PAD2 interacts with ERα after E2 stimulation and that inhibition of either PAD2 or ERα strongly suppresses E2-induced H3R26 citrullination and ERα recruitment at target gene promoters. Collectively, our data suggest that E2 stimulation induces the recruitment of PAD2 to target promoters by ERα, whereby PAD2 then citrullinates H3R26, which leads to local chromatin decondensation and transcriptional activation. C ancers of the female reproductive system are serious human health problems, and estrogen plays a critical role in the initiation and progression of these diseases (1). Despite decades of research into mechanisms of 17β-estradiol (E2)-responsive gene transcription, our understanding of this process is far from complete (2). It is generally believed that, upon E2 binding, the nuclear hormone receptor estrogen receptor α (hereafter called ER) undergoes major structural reorganization, associates with estrogen-response elements (ERE) within target gene promoters, and recruits a range of coactivators including histone modification enzymes (3-6). After deposition, the resulting histone modifications can then modulate target gene activity by affecting local chromatin structure and regulating the accessibility of chromatin to transcription factors (2, 5, 7-9).Peptidylarginine deiminase (PAD) enzymes convert arginine and methylarginine residues to citrulline via a hydrolytic process termed citrullination or deimination (10, 11). We and others have shown that one such PAD, PAD4, appears to play a repressive role in regulating the expression of the canonical ER target gene, TFF1, via citrullination of histone H4 methylarginine 3, thus suggesting that PADs potentially function as ER cofactors (12, 13). Given that these previous studies were limited to a single ER target promoter, we chose to take a more comprehesive approach to test whether PAD-mediated histone tail citrullination may be more fundamental to ER target gene regulation than previously realized. In this study, we show that citrullination of histone H3R26 at ER targets is closely associated with gene transcription and that citrullination at this residue is catalyzed by PAD2, as opposed to PAD4. Additionally, we show that PAD2 interacts with ER and that PAD2-mediated citrullination of H3R26 likely facilitates transcriptional activation by creating an open, permissive, chromatin architecture around the EREs of E2-i...

BackgroundWe have recently reported that the expression of peptidylarginine deiminase 2 (PADI2) is regulated by EGF in mammary cancer cells and appears to play a role in the proliferation of normal mammary epithelium; however, the role of PADI2 in the pathogenesis of human breast cancer has yet to be investigated. Thus, the goals of this study were to examine whether PADI2 plays a role in mammary tumor progression, and whether the inhibition of PADI activity has anti-tumor effects.MethodsRNA-seq data from a collection of 57 breast cancer cell lines was queried for PADI2 levels, and correlations with known subtype and HER2/ERBB2 status were evaluated. To examine PADI2 expression levels during breast cancer progression, the cell lines from the MCF10AT model were used. The efficacy of the PADI inhibitor, Cl-amidine, was tested in vitro using MCF10DCIS cells grown in 2D-monolayers and 3D-spheroids, and in vivo using MCF10DCIS tumor xenografts. Treated MCF10DCIS cells were examined by flow-cytometry to determine the extent of apoptosis and by RT2 Profiler PCR Cell Cycle Array to detect alterations in cell cycle associated genes.ResultsWe show by RNA-seq that PADI2 mRNA expression is highly correlated with HER2/ERBB2 (p = 2.2 × 106) in luminal breast cancer cell lines. Using the MCF10AT model of breast cancer progression, we then demonstrate that PADI2 expression increases during the transition of normal mammary epithelium to fully malignant breast carcinomas, with a strong peak of PADI2 expression and activity being observed in the MCF10DCIS cell line, which models human comedo-DCIS lesions. Next, we show that a PADI inhibitor, Cl-amidine, strongly suppresses the growth of MCF10DCIS monolayers and tumor spheroids in culture. We then carried out preclinical studies in nude (nu/nu) mice and found that Cl-amidine also suppressed the growth of xenografted MCF10DCIS tumors by more than 3-fold. Lastly, we performed cell cycle array analysis of Cl-amidine treated and control MCF10DCIS cells, and found that the PADI inhibitor strongly affects the expression of several cell cycle genes implicated in tumor progression, including p21, GADD45α, and Ki67.ConclusionTogether, these results suggest that PADI2 may function as an important new biomarker for HER2/ERBB2+ tumors and that Cl-amidine represents a new candidate for breast cancer therapy.

Reproduction and fertility are regulated via hormones of the hypothalamic-pituitary-gonadal (HPG) axis. Control of this reproductive axis occurs at all levels, including the brain and pituitary, and allows for the promotion or inhibition of gonadal sex steroid secretion and function. In addition to guiding proper gonadal development and function, gonadal sex steroids also act in negative- and positive-feedback loops to regulate reproductive circuitry in the brain, including kisspeptin neurones, thereby modulating overall HPG axis status. Additional regulation is also provided by sex steroids made within the brain, including neuroprogestins. Furthermore, because reproduction and survival need to be coordinated and balanced, the HPG axis is able to modulate (and be modulated by) stress hormone signalling, including cortiscosterone, from the hypothalamic-pituitary-adrenal (HPA) axis. This review covers recent data related to the neural, hormonal and stress regulation of the HPG axis and emerging interactions between the HPG and HPA axes, focusing on actions at the level of the brain and pituitary.

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 (PAD4) is a Ca 2+ -dependent enzyme that converts arginine and methylarginine residues to citrulline, with histone proteins being among its best-described substrates to date. However, the biological function of this posttranslational modification, either in histones or in nonhistone proteins, is poorly understood. Here, we show that PAD4 recognizes, binds, and citrullinates glycogen synthase kinase-3β (GSK3β), both in vitro and in vivo. Among other functions, GSK3β is a key regulator of transcription factors involved in tumor progression, and its dysregulation has been associated with progression of human cancers. We demonstrate that silencing of PAD4 in breast cancer cells leads to a striking reduction of nuclear GSK3β protein levels, increased TGF-β signaling, induction of epithelial-to-mesenchymal transition, and production of more invasive tumors in xenograft assays. Moreover, in breast cancer patients, reduction of PAD4 and nuclear GSK3β is associated with increased tumor invasiveness. We propose that PAD4-mediated citrullination of GSK3β is a unique posttranslational modification that regulates its nuclear localization and thereby plays a critical role in maintaining an epithelial phenotype. We demonstrate a dynamic and previously unappreciated interplay between histone-modifying enzymes, citrullination of nonhistone proteins, and epithelial-to-mesenchymal transition.