Xuesen Zhang

Peptidylarginine deiminase 2-catalyzed histone H3 arginine 26 citrullination facilitates estrogen receptor α target gene activation

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.

Genome-wide analysis reveals PADI4 cooperates with Elk-1 to activate c-Fos expression in breast cancer cells.

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.

Synthesis and Screening of a Haloacetamidine Containing Library To Identify PAD4 Selective Inhibitors.

Protein arginine deiminase activity (PAD) is increased in cancer, rheumatoid arthritis, and ulcerative colitis. Although the link between abnormal PAD activity and disease is clear, the relative contribution of the individual PADs to human disease is not known; there are 5 PAD isozymes in humans. Building on our previous development of F- and Cl-amidine as potent pan-PAD irreversible inhibitors, we describe herein a library approach that was used to identify PAD-selective inhibitors. Specifically, we describe the identification of Thr-Asp-F-amidine (TDFA) as a highly potent PAD4 inactivator that displays ≥15-fold selectivity for PAD4 versus PAD1 and ≥50-fold versus PADs 2 and 3. This compound is active in cells and can be used to inhibit PAD4 activity in cellulo. The structure of the PAD4·TDFA complex has also been solved, and the structure and mutagenesis data indicate that the enhanced potency is due to interactions between the side chains of Q346, R374, and R639. Finally, we converted TDFA into a PAD4-selective ABPP and demonstrated that this compound, biotin-TDFA, can be used to selectively isolate purified PAD4 in vitro. In total, TDFA and biotin-TDFA represent PAD4-selective chemical probes that can be used to study the physiological roles of this enzyme.

Potential Role for PAD2 in Gene Regulation in Breast Cancer Cells

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.

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

Peptidylarginine deiminase 4 (PAD4) is a Ca2+-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.

Transient estrogen receptor binding and p300 redistribution support a squelching mechanism for estradiol-repressed genes.

Proper gene regulation is essential for proper organismal development and appropriate responses to external stimuli. Specialized factors, termed master regulators, are often responsible for orchestrating the molecular events that result from signaling cascades. Master regulators coordinate the activation and repression of specific gene classes. Estrogen receptor α (ER) precipitates the signaling cascade that results from endogenous or exogenous estrogen hormones. ER is a classic transcriptional activator and the mechanisms by which ER coordinates gene activation are well characterized. However, it remains unclear how ER coordinates the immediate repression of genes. We integrated genomic transcription, chromosome looping, transcription factor binding, and chromatin structure data to analyze the molecular cascade that results from estradiol (E2)-induced signaling in human MCF-7 breast cancer cells and addressed the context-specific nature of gene regulation. We defined a class of genes that are immediately repressed upon estrogen stimulation, and we compared and contrasted the molecular characteristics of these repressed genes vs activated and unregulated genes. The most striking and unique feature of the repressed gene class is transient binding of ER at early time points after estrogen stimulation. We also found that p300, a coactivator and acetyltransferase, quantitatively redistributes from non-ER enhancers to ER enhancers after E2 treatment. These data support an extension of the classic physiological squelching model, whereby ER hijacks coactivators from repressed genes and redistributes the coactivators to ER enhancers that activate transcription.

The role of MATER in endoplasmic reticulum distribution and calcium homeostasis in mouse oocytes.

Ca(2+) oscillations are a hallmark of mammalian fertilization and play a central role in the activation of development. The calcium required for these oscillations is primarily derived from the endoplasmic reticulum (ER), which accumulates in clusters at the microvillar subcortex during oocyte maturation. The migration of the ER to the cortex during maturation is thought to play an important role in rendering the ER competent to generate the calcium transients, and the redistribution of ER is believed to be primarily mediated by microtubules and microfilaments. We have previously shown that the oocyte- and early embryo-restricted maternal effect gene Mater (Nlrp5) localizes to, and is required for, formation of the oocyte cytoplasmic lattices, a tubulin-containing structure that appears to play an important role in organelle positioning and distribution during oocyte maturation. Given these observations, we hypothesized that Mater may also be required for ER redistribution and Ca(2+) homeostasis in oocytes. To test this hypothesis, we first investigated ER localization in metaphase-II Mater(tm/tm) (hypomorph) oocytes and found ER clusters to be less abundant at the microvillar cortex when compared to wild type oocytes. To examine the potential mechanisms by which MATER mediates ER redistribution, we tested whether tubulin expression levels and localization were affected in the mutant oocytes and found that the Triton-insoluble fraction of tubulin was significantly decreased in Mater(tm/tm) oocytes. To identify potential functional defects associated with these ER abnormalities, we next set out to investigate if the pattern of Ca(2+) oscillations was altered in Mater(tm/tm) oocytes after fertilization in vitro. Intriguingly, Ca(2+) oscillations in Mater(tm/tm) oocytes exhibited a significantly lower first peak amplitude and a higher frequency when compared to wild type oocytes. We then found that the Ca(2+) oscillation defect in Mater(tm/tm) oocytes was likely caused by a reduced amount of Ca(2+) in the ER stores. Taken together, these observations support the hypothesis that MATER is required for ER distribution and Ca(2+) homeostasis in oocytes, likely due to defects in lattice-mediated ER positioning and/or redistribution.

Targeted H3R26 Deimination Specifically Facilitates Estrogen Receptor Binding by Modifying Nucleosome Structure

Transcription factor binding to DNA in vivo causes the recruitment of chromatin modifiers that can cause changes in chromatin structure, including the modification of histone tails. We previously showed that estrogen receptor (ER) target gene activation is facilitated by peptidylarginine deiminase 2 (PAD2)-catalyzed histone H3R26 deimination (H3R26Cit). Here we report that the genomic distributions of ER and H3R26Cit in breast cancer cells are strikingly coincident, linearly correlated, and observed as early as 2 minutes following estradiol treatment. The H3R26Cit profile is unlike that of previously described histone modifications and is characterized by sharp, narrow peaks. Paired-end MNase ChIP-seq indicates that the charge-neutral H3R26Cit modification facilitates ER binding to DNA by altering the fine structure of the nucleosome. Clinically, we find that PAD2 and H3R26Cit levels correlate with ER expression in breast tumors and that high PAD2 expression is associated with increased survival in ER+ breast cancer patients. These findings provide insight into how transcription factors gain access to nucleosomal DNA and implicate PAD2 as a novel therapeutic target for ER+ breast cancer.

Peptidylarginine deiminase 1-catalyzed histone citrullination is essential for early embryo development

Peptidylarginine deiminase (PADI) enzymes are increasingly being associated with the regulation of chromatin structure and gene activity via histone citrullination. As one of the PADI family members, PADI1 has been mainly reported to be expressed in the epidermis and uterus, where the protein in keratinocytes is thought to promote differentiation by citrullinating filament proteins. However, the roles of PADI1 in preimplantation development have not been addressed. Using a PADI1-specific inhibitor and Padi1-morpholino knockdown, we found that citrullination of histone tails at H4R3 and H3R2/8/17 were markedly reduced in the 2- and 4-cell embryos. Consistent with this observation, early embryo development was also arrested at the 4-cell stage upon depletion of PADI1 or inhibition of PADI1 enzyme activity. Additionally, by employing 5-ethynyl uridine (EU) incorporation analysis, ablation of PADI1 function led to a dramatic decrease in overall transcriptional activity, correlating well with the reduced levels of phosphorylation of RNA Pol II at Ser2 observed at 2- or 4-cell stage of embryos under Padi1 knockdown or inhibiting PADI1. Thus, our data reveal a novel function of PADI1 during early embryo development transitions by catalyzing histone tail citrullination, which facilitates early embryo genome transactivation.

Matrix metalloproteinase 9 is a distal-less 3 target-gene in placental trophoblast cells

Matrix metalloproteinases (MMPs) are enzymes that regulate extracellular matrix composition and contribute to cell migration. Microarray studies in mouse placenta suggested that MMP-9 transcript abundance was dependent on distal-less 3 (Dlx3), a placental-specific transcriptional regulator; however, it was not clear if this was a direct or indirect effect. Here we investigate mechanism(s) for Dlx3-dependent MMP-9 gene transcription and gelatinase activity in placental trophoblasts. Initial studies confirmed that MMP-9 activity was reduced in placental explants from Dlx3(-/-) mice and that murine MMP-9 promoter activity was induced by Dlx3 overexpression. Two binding sites within a murine MMP-9 promoter fragment bound Dlx3, and mutations in both elements reduced basal MMP-9-luciferase reporter activity and abolished regulation by Dlx3. Chromatin immunoprecipitation studies in JEG3 cells confirmed Dlx3 binding to the endogenous human MMP-9 promoter at three distinct sites and knockdown of human Dlx3 resulted in reduced endogenous MMP-9 transcripts and secreted activity. These studies provide novel evidence that Dlx3 is involved directly in the transcriptional regulation of mouse and human MMP-9 gene expression in placental trophoblasts.