ABSTRACT
Tumor suppressor genes are frequently silenced in cancer cells by enzymes catalyzing epigenetic histone modifications. The peptidylarginine deiminase family member PAD4 (also called PADI4) is markedly overexpressed in a majority of human cancers, suggesting that PAD4 is a putative target for cancer treatment. Here, we have generated novel PAD inhibitors with low micromolar IC(50) in PAD activity and cancer cell growth inhibition. The lead compound YW3-56 alters the expression of genes controlling the cell cycle and cell death, including SESN2 that encodes an upstream inhibitor of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. Guided by the gene expression profile analyses with YW3-56, we found that PAD4 functions as a corepressor of p53 to regulate SESN2 expression by histone citrullination in cancer cells. Consistent with the mTORC1 inhibition by SESN2, the phosphorylation of its substrates including p70S6 kinase (p70S6K) and 4E-BP1 was decreased. Furthermore, macroautophagy is perturbed after YW3-56 treatment in cancer cells. In a mouse xenograft model, YW3-56 demonstrates cancer growth inhibition activity with little if any detectable adverse effect to vital organs, whereas a combination of PAD4 and histone deacetylase inhibitors further decreases tumor growth. Taken together, our work found that PAD4 regulates the mTORC1 signaling pathway and that PAD inhibitors are potential anticancer reagents that activate tumor suppressor gene expression alone or in combination with histone deacetylase inhibitors.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Hydrolases/antagonists & inhibitors , Proteins/metabolism , Animals , Antineoplastic Agents/metabolism , Antineoplastic Agents/therapeutic use , Cell Line, Tumor , Cell Survival/drug effects , Drug Synergism , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/drug effects , Histone Deacetylase Inhibitors/pharmacology , Humans , Hydrolases/metabolism , Inhibitory Concentration 50 , Mechanistic Target of Rapamycin Complex 1 , Mice , Multiprotein Complexes , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , Sarcoma/drug therapy , Sarcoma/pathology , Signal Transduction , TOR Serine-Threonine Kinases , Transcriptional Activation/drug effects , Tumor Burden/drug effects , Tumor Suppressor Protein p53/metabolism , Xenograft Model Antitumor AssaysABSTRACT
An activity coefficient-based model was proposed to predict pertinent saturated concentrations in organic solid-liquid equilibrium, and the binary parameters of xylene mixtures were experimentally obtained. Also, a novel monocular 3D reconstruction technique was developed to measure crystal size and applied to derive the kinetics of nucleation and growth of para-xylene crystals. Subsequently, a multi-dimensional population balance equation was used to predict the particle size distribution in the crystallizer and an algorithm was designed to simulate and optimize the economic benefit of the crystallization separation process. Consequently, it became possible to predict the optimal coolant flowrate and inlet temperature, as well as the feed flowrate for a crystallization process with given operating conditions and device parameters.
ABSTRACT
Tumor suppressor p53 regulates transcription of stress-response genes. Many p53 targets remain undiscovered because of uncertainty as to where p53 binds in the genome and the fact that few genes reside near p53-bound recognition elements (REs). Using chromatin immunoprecipitation followed by exonuclease treatment (ChIP-exo), we associated p53 with 2,183 unsplit REs. REs were positionally constrained with other REs and other regulatory elements, which may reflect structurally organized p53 interactions. Surprisingly, stress resulted in increased occupancy of transcription factor IIB (TFIIB) and RNA polymerase (Pol) II near REs, which was reduced when p53 was present. A subset associated with antisense RNA near stress-response genes. The combination of high-confidence locations for p53/REs, TFIIB/Pol II, and their changes in response to stress allowed us to identify 151 high-confidence p53-regulated genes, substantially increasing the number of p53 targets. These genes composed a large portion of a predefined DNA-damage stress-response network. Thus, p53 plays a comprehensive role in regulating the stress-response network, including regulating noncoding transcription.
Subject(s)
Genome, Human , Response Elements , Stress, Physiological , Tumor Suppressor Protein p53/genetics , HCT116 Cells , Humans , Protein Binding , RNA Polymerase II/genetics , RNA Polymerase II/metabolism , RNA, Untranslated/genetics , RNA, Untranslated/metabolism , Transcription Factor TFIIB/genetics , Transcription Factor TFIIB/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Cytokine receptors are randomly distributed on the cell surface membrane and are activated upon binding of their extracellular ligands to mediate downstream cellular activities. We hypothesized that pharmaceutical clustering of ligand-bound, activated receptors may lead to heretofore unrealized gain-of-function with therapeutically desirable properties. We here describe an engineered bifunctional cytokine borne of the fusion of Granulocyte Macrophage Colony Stimulating Factor (GMCSF) and Interleukin-9 (IL9) (hereafter GIFT9 fusokine) and demonstrate that it chaperones co-clustering of the functionally unrelated GMCSF receptor (GMCSFR) and IL9 receptor (IL9R) on cell surface of target cells. We demonstrate that GIFT9 treatment of MC/9 cells leads to transhyperphosphorylation of IL9R-associated STAT1 by GMCSFR-associated JAK2. We also show that IL9R-associated JAK1 and JAK3 augment phosphorylation of GMCSFR-linked STAT5. The functional relevance of these synergistic JAK/STAT transphosphorylation events translates to an increased mitogenic response by GMCSFR/IL9R-expressing primary marrow mast cells. The notion of inducing heterologous receptor clustering by engineered fusokines such as GIFT9 opens the door to a novel type of biopharmaceutical platform where designer fusokines modulate cell physiology through clustering of targeted receptor complexes.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Interleukin-9/pharmacology , Janus Kinase 2/metabolism , Recombinant Fusion Proteins/pharmacology , STAT1 Transcription Factor/metabolism , Animals , Cells, Cultured , Cloning, Molecular , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Interleukin-9/genetics , Mice , Mice, Inbred C57BL , Phosphorylation/drug effects , Receptors, Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Receptors, Interleukin-9/metabolism , Recombinant Fusion Proteins/geneticsABSTRACT
Histone posttranslational modifications play significant roles in regulating chromatin structure and gene expression. One of the histone modifications, histone citrullination, is catalyzed by an enzyme called peptidylarginine deiminase 4 (PAD4, also called PADI4), which converts both histone arginine (Arg) and mono-methyl arginine residues to citrulline. Recent studies have found that histone citrullination counteracts the effect of histone arginine methylation and functions as a repressive marker to turn off gene expression. Here, we describe assays to study histone citrullination by PAD4 in vitro and in vivo. We also describe approaches to measure histone citrullination levels at gene promoters using chromatin immunoprecipitation assay and analyze the effects of PAD4 inhibitor on cell cycle and apoptosis by flow cytometry. These methods would be useful techniques to study this unique histone modification.
Subject(s)
Histones/metabolism , Cell Cycle , Chromatin Immunoprecipitation/methods , Flow Cytometry , HL-60 Cells , Humans , Hydrolases/metabolism , Membrane Potential, Mitochondrial , Nucleosomes/metabolism , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , Reactive Oxygen Species/metabolismABSTRACT
Neutrophils trap and kill bacteria by forming highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs). We previously reported that histone hypercitrullination catalyzed by peptidylarginine deiminase 4 (PAD4) correlates with chromatin decondensation during NET formation. However, the role of PAD4 in NET-mediated bacterial trapping and killing has not been tested. Here, we use PAD4 knockout mice to show that PAD4 is essential for NET-mediated antibacterial function. Unlike PAD4(+/+) neutrophils, PAD4(-/-) neutrophils cannot form NETs after stimulation with chemokines or incubation with bacteria, and are deficient in bacterial killing by NETs. In a mouse infectious disease model of necrotizing fasciitis, PAD4(-/-) mice are more susceptible to bacterial infection than PAD4(+/+) mice due to a lack of NET formation. Moreover, we found that citrullination decreased the bacterial killing activity of histones and nucleosomes, which suggests that PAD4 mainly plays a role in chromatin decondensation to form NETs instead of increasing histone-mediated bacterial killing. Our results define a role for histone hypercitrullination in innate immunity during bacterial infection.
Subject(s)
Dysentery, Bacillary/immunology , Hydrolases/immunology , Immunity, Innate , Neutrophils/immunology , Shigella flexneri/immunology , Animals , Cell Differentiation , Extracellular Space/immunology , Extracellular Space/metabolism , Histones/metabolism , Hydrolases/deficiency , Hydrolases/metabolism , Mice , Mice, Knockout , Neutrophils/cytology , Neutrophils/metabolism , Protein-Arginine Deiminase Type 4ABSTRACT
Peripheral blood neutrophils form highly decondensed chromatin structures, termed neutrophil extracellular traps (NETs), that have been implicated in innate immune response to bacterial infection. Neutrophils express high levels of peptidylarginine deiminase 4 (PAD4), which catalyzes histone citrullination. However, whether PAD4 or histone citrullination plays a role in chromatin structure in neutrophils is unclear. In this study, we show that the hypercitrullination of histones by PAD4 mediates chromatin decondensation. Histone hypercitrullination is detected on highly decondensed chromatin in HL-60 granulocytes and blood neutrophils. The inhibition of PAD4 decreases histone hypercitrullination and the formation of NET-like structures, whereas PAD4 treatment of HL-60 cells facilitates these processes. The loss of heterochromatin and multilobular nuclear structures is detected in HL-60 granulocytes after PAD4 activation. Importantly, citrullination of biochemically defined avian nucleosome arrays inhibits their compaction by the linker histone H5 to form higher order chromatin structures. Together, these results suggest that histone hypercitrullination has important functions in chromatin decondensation in granulocytes/neutrophils.
Subject(s)
Chromatin/metabolism , Histones/metabolism , Neutrophils/ultrastructure , Citrulline/metabolism , Granulocytes/metabolism , HL-60 Cells , Humans , Hydrolases/metabolism , Neutrophils/metabolism , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , Tumor Necrosis Factor-alpha/metabolismABSTRACT
Histone Arg methylation has been correlated with transcriptional activation of p53 target genes. However, whether this modification is reversed to repress the expression of p53 target genes is unclear. Here, we report that peptidylarginine deiminase 4, a histone citrullination enzyme, is involved in the repression of p53 target genes. Inhibition or depletion of PAD4 elevated the expression of a subset of p53 target genes, including p21/CIP1/WAF1, leading to cell cycle arrest and apoptosis. Moreover, the induction of p21, cell cycle arrest, and apoptosis by PAD4 depletion is p53 dependent. Protein-protein interaction studies showed an interaction between p53 and PAD4. Chromatin immunoprecipitation assays showed that PAD4 is recruited to the p21 promoter in a p53-dependent manner. RNA polymerase II (Pol II) activities and the association of PAD4 are dynamically regulated at the p21 promoter during UV irradiation. Paused RNA Pol II and high levels of PAD4 were detected before UV treatment. At early time points after UV treatment, an increase of histone Arg methylation and a decrease of citrullination were correlated with a transient activation of p21. At later times after UV irradiation, a loss of RNA Pol II and an increase of PAD4 were detected at the p21 promoter. The dynamics of RNA Pol II activities after UV treatment were further corroborated by permanganate footprinting. Together, these results suggest a role of PAD4 in the regulation of p53 target gene expression.
Subject(s)
Gene Expression Regulation, Neoplastic , Hydrolases/metabolism , Tumor Suppressor Protein p53/metabolism , Apoptosis/drug effects , Apoptosis/radiation effects , Arginine/metabolism , Cell Cycle/drug effects , Cell Cycle/radiation effects , Cell Line, Tumor , Chromatin Immunoprecipitation , Cyclin-Dependent Kinase Inhibitor p21/genetics , DNA Footprinting , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/radiation effects , Histones/metabolism , Humans , Hydrolases/antagonists & inhibitors , Hydrolases/deficiency , Hydrolases/genetics , Manganese Compounds/pharmacology , Oxides/pharmacology , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Protein Binding/radiation effects , Protein-Arginine Deiminase Type 4 , Protein-Arginine Deiminases , RNA Polymerase II/metabolism , RNA, Small Interfering/metabolism , Ultraviolet RaysABSTRACT
Protein Arg methyltransferases function as coactivators of the tumor suppressor p53 to regulate gene expression. Peptidylarginine deiminase 4 (PAD4/PADI4) counteracts the functions of protein Arg methyltransferases in gene regulation by deimination and demethylimination. Here we show that the expression of a tumor suppressor gene, OKL38, is activated by the inhibition of PAD4 or the activation of p53 following DNA damage. Chromatin immunoprecipitation assays showed a dynamic change of p53 and PAD4 occupancy and histone Arg modifications at the OKL38 promoter during DNA damage, suggesting a direct role of PAD4 and p53 in the expression of OKL38. Furthermore, we found that OKL38 induces apoptosis through localization to mitochondria and induction of cytochrome c release. Together, our studies identify OKL38 as a novel p53 target gene that is regulated by PAD4 and plays a role in apoptosis.