ABSTRACT
Heterochromatin is critical for maintaining genome stability, especially in flowering plants, where it relies on a feedback loop involving the H3K9 methyltransferase, KRYPTONITE (KYP), and the DNA methyltransferase CHROMOMETHYLASE3 (CMT3). The H3K9 demethylase INCREASED IN BONSAI METHYLATION 1 (IBM1) counteracts the detrimental consequences of KYP-CMT3 activity in transcribed genes. IBM1 expression in Arabidopsis is uniquely regulated by methylation of the 7th intron, allowing it to monitor global H3K9me2 levels. We show the methylated intron is prevalent across flowering plants and its underlying sequence exhibits dynamic evolution. We also find extensive genetic and expression variations in KYP, CMT3, and IBM1 across flowering plants. We identify Arabidopsis accessions resembling weak ibm1 mutants and Brassicaceae species with reduced IBM1 expression or deletions. Evolution towards reduced IBM1 activity in some flowering plants could explain the frequent natural occurrence of diminished or lost CMT3 activity and loss of gene body DNA methylation, as cmt3 mutants in A. thaliana mitigate the deleterious effects of IBM1.
Subject(s)
Arabidopsis Proteins , Arabidopsis , DNA Methylation , Evolution, Molecular , Gene Expression Regulation, Plant , Heterochromatin , Heterochromatin/genetics , Heterochromatin/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , DNA Methylation/genetics , Jumonji Domain-Containing Histone Demethylases/genetics , Jumonji Domain-Containing Histone Demethylases/metabolism , Introns/genetics , Histones/metabolism , Histones/genetics , Mutation , DNA-Cytosine Methylases/metabolism , DNA-Cytosine Methylases/genetics , Genomic InstabilityABSTRACT
Much is known about the generation, removal, and roles of 5-methylcytosine (5mC) in eukaryote DNA, and there is a growing body of evidence regarding N6-methyladenine, but very little is known about N4-methylcytosine (4mC) in the DNA of eukaryotes. The gene for the first metazoan DNA methyltransferase generating 4mC (N4CMT) was reported and characterized recently by others, in tiny freshwater invertebrates called bdelloid rotifers. Bdelloid rotifers are ancient, apparently asexual animals, and lack canonical 5mC DNA methyltransferases. Here, we characterize the kinetic properties and structural features of the catalytic domain of the N4CMT protein from the bdelloid rotifer Adineta vaga. We find that N4CMT generates high-level methylation at preferred sites, (a/c)CG(t/c/a), and low-level methylation at disfavored sites, exemplified by ACGG. Like the mammalian de novo 5mC DNA methyltransferase 3A/3B (DNMT3A/3B), N4CMT methylates CpG dinucleotides on both DNA strands, generating hemimethylated intermediates and eventually fully methylated CpG sites, particularly in the context of favored symmetric sites. In addition, like DNMT3A/3B, N4CMT methylates non-CpG sites, mainly CpA/TpG, though at a lower rate. Both N4CMT and DNMT3A/3B even prefer similar CpG-flanking sequences. Structurally, the catalytic domain of N4CMT closely resembles the Caulobacter crescentus cell cycle-regulated DNA methyltransferase. The symmetric methylation of CpG, and similarity to a cell cycle-regulated DNA methyltransferase, together suggest that N4CMT might also carry out DNA synthesis-dependent methylation following DNA replication.
Subject(s)
DNA-Cytosine Methylases , Rotifera , Animals , DNA Methylation , DNA-Cytosine Methylases/chemistry , DNA-Cytosine Methylases/isolation & purification , Mammals/metabolism , Rotifera/classification , Rotifera/enzymologyABSTRACT
DNA methyltransferase inhibitors (DNMTis) reexpress hypermethylated genes in cancers and leukemias and also activate endogenous retroviruses (ERVs), leading to interferon (IFN) signaling, in a process known as viral mimicry. In the present study we show that in the subset of acute myeloid leukemias (AMLs) with mutations in TP53, associated with poor prognosis, DNMTis, important drugs for treatment of AML, enable expression of ERVs and IFN and inflammasome signaling in a STING-dependent manner. We previously reported that in solid tumors poly ADP ribose polymerase inhibitors (PARPis) combined with DNMTis to induce an IFN/inflammasome response that is dependent on STING1 and is mechanistically linked to generation of a homologous recombination defect (HRD). We now show that STING1 activity is actually increased in TP53 mutant compared with wild-type (WT) TP53 AML. Moreover, in TP53 mutant AML, STING1-dependent IFN/inflammatory signaling is increased by DNMTi treatment, whereas in AMLs with WT TP53, DNMTis alone have no effect. While combining DNMTis with PARPis increases IFN/inflammatory gene expression in WT TP53 AML cells, signaling induced in TP53 mutant AML is still several-fold higher. Notably, induction of HRD in both TP53 mutant and WT AMLs follows the pattern of STING1-dependent IFN and inflammatory signaling that we have observed with drug treatments. These findings increase our understanding of the mechanisms that underlie DNMTi + PARPi treatment, and also DNMTi combinations with immune therapies, suggesting a personalized approach that statifies by TP53 status, for use of such therapies, including potential immune activation of STING1 in AML and other cancers.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , DNA-Cytosine Methylases , Leukemia, Myeloid, Acute , Membrane Proteins , Poly(ADP-ribose) Polymerase Inhibitors , Tumor Suppressor Protein p53 , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , DNA-Cytosine Methylases/antagonists & inhibitors , Homologous Recombination/genetics , Humans , Inflammasomes/metabolism , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Membrane Proteins/immunology , Mutation , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Signal Transduction , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
Herein, a hand-drawing paper-based bipolar electrode (BPE) electrochemiluminescence (ECL) platform for M.SssI methyltransferase (M.SssI MTase) assay was proposed via employing high electrocatalytic Pt@CeO2 as an ECL co-reaction accelerator and pencil-drawing graphite electric circuits as wires and electrodes. Notably, the introduction of pencil-drawing trace not only simplified the manufacturing process but also reduced the cost and saved fabricating time. Meanwhile, Pt@CeO2 with good electrocatalytic activity and satisfactory chemical stability was used at the anode of the closed BPE-ECL device to accelerate the oxidation rate of uric acid. Due to the balanced charges of the bipolar electrode, the ECL response of the MnS: CdS@ZnS/S2O82- system emitted on the cathode was enhanced. In situ growth of gold nanoparticles in the two electrode areas was convenient for DNA immobilization. With the above points in mind, the specific DNA double strands functionalized via Pt@CeO2 were employed to identify M.SssI MTase. The unmethylated DNA double strands were cut by HpaII endonuclease, resulting in the quenching of the ECL signal. Under the optimal conditions, sensitive detection of M.SssI MTase in a wide linear range of 0.01-100 U·mL-1 with a satisfactory detection limit of 0.008 U·mL-1 was realized. The reliable and versatile BPE-ECL tool for the determination of M.SssI MTase with easy-to-operate pencil-drawing traces and independent solution systems provides a new opportunity to develop paper-based devices applied in early disease diagnosis and pathogenesis research.
Subject(s)
Biosensing Techniques , Metal Nanoparticles , Biosensing Techniques/methods , DNA , DNA-Cytosine Methylases , Electrochemical Techniques/methods , Electrodes , Gold , Luminescent Measurements/methods , MethyltransferasesABSTRACT
Little is known regarding the DNA methyltransferases (MTases) in hyperthermophilic archaea. In this study, we focus on an MTase from Aeropyrum pernix K1, a hyperthermophilic archaeon that is found in hydrothermal vents and whose optimum growth temperature is 90°C to 95°C. From genomic sequence analysis, A. pernix K1 has been predicted to have a restriction-modification system (R-M system). The restriction endonuclease from A. pernix K1 (known as ApeKI from New England BioLabs Inc. [catalog code R06435]) has been described previously, but the properties of the MTase from A. pernix K1 (M.ApeKI) have not yet been clarified. Thus, we demonstrated the properties of M.ApeKI. In this study, M.ApeKI was expressed in Escherichia coli strain JM109 and affinity purified using its His tag. The recognition sequence of M.ApeKI was determined by methylation activity and bisulfite sequencing (BS-seq). High-performance liquid chromatography (HPLC) was used to detect the position of the methyl group in methylated cytosine. As a result, it was clarified that M.ApeKI adds the methyl group at the C-5 position of the second cytosine in 5'-GCWGC-3'. Moreover, we also determined that the MTase optimum temperature was over 70°C and that it is strongly tolerant to high temperatures. M.ApeKI is the first highly thermostable DNA (cytosine-5)-methyltransferase to be evaluated by experimental evidence. IMPORTANCE In general, thermophilic bacteria with optimum growth temperatures over or equal to 60°C have been predicted to include only N4-methylcytosine or N6-methyladenine as methylated bases in their DNA, because 5-methylcytosine is susceptible to deamination by heat. However, from this study, A. pernix K1, with an optimum growth temperature at 95°C, was demonstrated to produce a DNA (cytosine-5)-methyltransferase. Thus, A. pernix K1 presumably has 5-methylcytosine in its DNA and may produce an original repair system for the expected C-to-T mutations. M.ApeKI was demonstrated to be tolerant to high temperatures; thus, we expect that M.ApeKI may be valuable for the development of a novel analysis system or epigenetic editing tool.
Subject(s)
Aeropyrum/enzymology , DNA Methylation/genetics , DNA-Cytosine Methylases/metabolism , Aeropyrum/genetics , Aeropyrum/metabolism , Amino Acid Sequence , DNA-Cytosine Methylases/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression/genetics , Hot Temperature , Hydrothermal Vents/microbiologyABSTRACT
Non-coding transcription is an important determinant of heterochromatin formation. In Arabidopsis thaliana a specialized RNA polymerase V (Pol V) transcribes pervasively and produces long non-coding RNAs. These transcripts work with small interfering RNA to facilitate locus-specific establishment of RNA-directed DNA methylation (RdDM). Subsequent maintenance of RdDM is associated with elevated levels of Pol V transcription. However, the impact of DNA methylation on Pol V transcription remained unresolved. We found that DNA methylation strongly enhances Pol V transcription. The level of Pol V transcription is reduced in mutants defective in RdDM components working downstream of Pol V, indicating that RdDM is maintained by a mutual reinforcement of DNA methylation and Pol V transcription. Pol V transcription is affected only on loci that lose DNA methylation in all sequence contexts in a particular mutant, including mutants lacking maintenance DNA methyltransferases, which suggests that RdDM works in a complex crosstalk with other silencing pathways.
Subject(s)
Arabidopsis/genetics , DNA Methylation , Gene Expression Regulation, Plant , Gene Silencing , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Transposable Elements , DNA-Cytosine Methylases/metabolism , DNA-Directed RNA Polymerases/metabolism , Feedback, Physiological , Methyltransferases/metabolism , RNA, Long Noncoding/biosynthesis , Transcription, GeneticABSTRACT
DNA methylation plays crucial roles in transposon silencing and genome integrity. CHROMOMETHYLASE3 (CMT3) is a plant-specific DNA methyltransferase responsible for catalyzing DNA methylation at the CHG (H = A, T, C) context. Here, we identified a positive role of CMT3 in heat-induced activation of retrotransposon ONSEN. We found that the full transcription of ONSEN under heat stress requires CMT3. Interestingly, loss-of-function CMT3 mutation led to increased CHH methylation at ONSEN. The CHH methylation is mediated by CMT2, as evidenced by greatly reduced CHH methylation in cmt2 and cmt2 cmt3 mutants coupled with increased ONSEN transcription. Furthermore, we found more CMT2 binding at ONSEN chromatin in cmt3 compared to wild-type accompanied with an ectopic accumulation of H3K9me2 under heat stress, suggesting a collaborative role of H3K9me2 and CHH methylation in preventing heat-induced ONSEN activation. In summary, this study identifies a non-canonical role of CMT3 in preventing transposon silencing and provides new insights into how DNA methyltransferases regulate transcription under stress conditions.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/physiology , DNA Transposable Elements/ethics , DNA-Cytosine Methylases/genetics , Arabidopsis/genetics , DNA Methylation , Gene Expression Regulation, Plant , Gene Silencing , Heat-Shock Response , Sequence Analysis, DNA , Transcription, GeneticABSTRACT
Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.
Subject(s)
DNA Methylation , DNA, Bacterial/metabolism , DNA-Cytosine Methylases/metabolism , Escherichia coli/genetics , Base Sequence , Binding Sites , Protein Binding , Zinc FingersABSTRACT
DNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Our results are also consistent with CMT3-induced ectopic methylation of promoters or bodies of genes undergoing transcriptional activation reducing their expression. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , DNA Methylation , DNA-Cytosine Methylases/genetics , MicroRNAs/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Chromatin/metabolism , DNA-Cytosine Methylases/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Plant , MicroRNAs/metabolismABSTRACT
CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.
Subject(s)
Arabidopsis , Bacterial Proteins , CRISPR-Cas Systems , DNA Methylation , DNA, Plant/metabolism , DNA-Cytosine Methylases , Plants, Genetically Modified , Tenericutes/genetics , Arabidopsis/enzymology , Arabidopsis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA, Plant/genetics , DNA-Cytosine Methylases/genetics , DNA-Cytosine Methylases/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Tenericutes/enzymologyABSTRACT
Patients with long-standing diabetes have a high risk for cardiac complications that is exacerbated by increased reactive oxygen species (ROS) production. We found that feeding cyanocobalamin (B12), a scavenger of superoxide, not only prevented but reversed signs of cardiomyopathy in type 1 diabetic Elmo1H/H Ins2Akita/+ mice. ROS reductions in plasma and hearts were comparable to those in mice treated with other antioxidants, N-acetyl-L-cysteine or tempol, but B12 produced better cardioprotective effects. Diabetes markedly decreased plasma insulin-like growth factor (IGF)-1 levels, while B12, but not N-acetyl-L-cysteine nor tempol, restored them. B12 activated hepatic IGF-1 production via normalization of S-adenosylmethionine levels, DNA methyltransferase (DNMT)-1/3a/3b mRNA, and DNA methylation of promoters for suppressor of cytokine signaling (SOCS)-1/3. Reductions of cardiac IGF-1 mRNA and phosphorylated IGF-1 receptors were also restored. Thus, B12 is a promising option for preventing diabetic cardiomyopathy via ROS reduction and IGF-1 retrieval through DNMT-SOCS1/3 signaling.
Subject(s)
Cardiomyopathies/prevention & control , DNA-Cytosine Methylases/physiology , Diabetes Mellitus, Type 1/complications , Insulin-Like Growth Factor I/physiology , Oxidative Stress/drug effects , Suppressor of Cytokine Signaling 1 Protein/physiology , Suppressor of Cytokine Signaling 3 Protein/physiology , Vitamin B 12/pharmacology , Adaptor Proteins, Signal Transducing/physiology , Animals , Diabetes Mellitus, Type 1/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Signal Transduction/drug effectsABSTRACT
DNA methylation plays an important role in modulating plant growth plasticity in response to stress, but mechanisms involved in such control need further investigation. We used drm1 drm2 cmt3 mutant of Arabidopsis thaliana, defective in DNA methylation, to explore metabolic pathways downstream epigenetic modulation under cadmium (Cd) stress. To this aim, a transcriptomic analysis was performed on ddc and WT plants exposed to a long-lasting (21 d) Cd treatment (25/50 µM), focusing on hormone genetic pathways. Growth parameters and hormones amount were also estimated. Transcriptomic data and hormone quantification showed that, under prolonged Cd treatment, level and signalling of growth-sustaining hormones (auxins, CKs, GAs) were enhanced and/or maintained, while a decrease was detected for stress-related hormones (JA, ABA, SA), likely as a strategy to avoid the side effects of their long-lasting activation. Such picture was more effective in ddc than WT, already at 25 µM Cd, in line with its better growth performance. A tight relationship between methylation status and the modulation of hormone genetic pathways under Cd stress was assessed. We propose that the higher genome plasticity conferred to ddc by DNA hypomethylated status underlies its prompt response to modulate hormones genetic pathways and activity and assure a flexible growth.
Subject(s)
Arabidopsis Proteins/physiology , Arabidopsis/genetics , Cadmium/pharmacology , DNA-Cytosine Methylases/physiology , Gene Expression Regulation, Plant/drug effects , Methyltransferases/physiology , Plant Growth Regulators/genetics , Arabidopsis/drug effects , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Base Sequence , Cytokinins/biosynthesis , Cytokinins/genetics , DNA Methylation , DNA, Plant/genetics , DNA-Cytosine Methylases/deficiency , DNA-Cytosine Methylases/genetics , Genes, Plant , Methyltransferases/deficiency , Methyltransferases/genetics , Mutation , Plant Roots/growth & development , RNA, Messenger/genetics , RNA, Plant/genetics , Soil Pollutants/pharmacology , Stress, Physiological/genetics , Transcriptome/drug effectsABSTRACT
The ability to target epigenetic marks like DNA methylation to specific loci is important in both basic research and in crop plant engineering. However, heritability of targeted DNA methylation, how it impacts gene expression, and which epigenetic features are required for proper establishment are mostly unknown. Here, we show that targeting the CG-specific methyltransferase M.SssI with an artificial zinc finger protein can establish heritable CG methylation and silencing of a targeted locus in Arabidopsis. In addition, we observe highly heritable widespread ectopic CG methylation mainly over euchromatic regions. This hypermethylation shows little effect on transcription while it triggers a mild but significant reduction in the accumulation of H2A.Z and H3K27me3. Moreover, ectopic methylation occurs preferentially at less open chromatin that lacks positive histone marks. These results outline general principles of the heritability and interaction of CG methylation with other epigenomic features that should help guide future efforts to engineer epigenomes.
Subject(s)
Arabidopsis/genetics , Bacterial Proteins/genetics , DNA Methylation , DNA-Cytosine Methylases/genetics , Gene Expression Regulation, Plant , Spiroplasma/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Bacterial Proteins/metabolism , Chromatin/genetics , Chromatin/metabolism , Chromatin Immunoprecipitation Sequencing/methods , DNA-Cytosine Methylases/metabolism , Histones/metabolism , Plants, Genetically Modified , RNA-Seq/methods , Spiroplasma/enzymologyABSTRACT
BACKGROUND: DNA methylation is a conserved and important epigenetic modification involved in the regulation of numerous biological processes, including plant development, secondary metabolism, and response to stresses. However, no information is available regarding the identification of cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase) genes in the orchid Dendrobium officinale. RESULTS: In this study, we performed a genome-wide analysis of DoC5-MTase and DodMTase gene families in D. officinale. Integrated analysis of conserved motifs, gene structures and phylogenetic analysis showed that eight DoC5-MTases were divided into four subfamilies (DoCMT, DoDNMT, DoDRM, DoMET) while three DodMTases were divided into two subfamilies (DoDML3, DoROS1). Multiple cis-acting elements, especially stress-responsive and hormone-responsive ones, were found in the promoter region of DoC5-MTase and DodMTase genes. Furthermore, we investigated the expression profiles of DoC5-MTase and DodMTase in 10 different tissues, as well as their transcript abundance under abiotic stresses (cold and drought) and at the seedling stage, in protocorm-like bodies, shoots, and plantlets. Interestingly, most DoC5-MTases were downregulated whereas DodMTases were upregulated by cold stress. At the seedling stage, DoC5-MTase expression decreased as growth proceeded, but DodMTase expression increased. CONCLUSIONS: These results provide a basis for elucidating the role of DoC5-MTase and DodMTase in secondary metabolite production and responses to abiotic stresses in D. officinale.
Subject(s)
DNA Methylation/genetics , DNA-Cytosine Methylases/genetics , Dendrobium/enzymology , Dendrobium/genetics , Oxidoreductases/genetics , Polysaccharides/genetics , Polysaccharides/metabolism , Arabidopsis/genetics , DNA-Cytosine Methylases/metabolism , Gene Expression Regulation, Plant , Genes, Plant , Genome-Wide Association Study , Oryza/genetics , Oxidoreductases/metabolismABSTRACT
BACKGROUND: Zinc-finger protein 471 (ZNF471) is a member of the Krüppel-associated box domain zinc finger protein (KRAB-ZFP) family. ZNF471 is methylated in squamous cell carcinomas of tongue, stomach and esophageal. However, its role in breast carcinogenesis remains elusive. Here, we studied its expression, functions, and molecular mechanisms in breast cancer. METHODS: We examined ZNF471 expression by RT-PCR and qPCR. Methylation-specific PCR determined its promoter methylation. Its biological functions and related molecular mechanisms were assessed by CCK-8, clonogenicity, wound healing, Transwell, nude mice tumorigenicity, flow cytometry, BrdU-ELISA, immunohistochemistry and Western blot assays. RESULTS: ZNF471 was significantly downregulated in breast cell lines and tissues due to its promoter CpG methylation, compared with normal mammary epithelial cells and paired surgical-margin tissues. Ectopic expression of ZNF471 substantially inhibited breast tumor cell growth in vitro and in vivo, arrested cell cycle at S phase, and promoted cell apoptosis, as well as suppressed metastasis. Further knockdown of ZNF471 verified its tumor-suppressive effects. We also found that ZNF471 exerted its tumor-suppressive functions through suppressing epithelial-mesenchymal transition, tumor cell stemness and AKT and Wnt/ß-catenin signaling. CONCLUSIONS: ZNF471 functions as a tumor suppressor that was epigenetically inactivated in breast cancer. Its inhibition of AKT and Wnt/ß-catenin signaling pathways is one of the mechanisms underlying its anti-cancer effects.
Subject(s)
Breast Neoplasms/genetics , Neoplasm Metastasis/genetics , Proto-Oncogene Proteins c-akt/genetics , Repressor Proteins/genetics , Wnt Signaling Pathway/genetics , Animals , Apoptosis/genetics , Breast Neoplasms/pathology , Cell Cycle Checkpoints/genetics , Cell Line, Tumor/metabolism , Cell Proliferation/genetics , DNA Methylation , DNA-Cytosine Methylases/metabolism , Down-Regulation , Epigenomics , Epithelial-Mesenchymal Transition/genetics , Female , Gene Expression Regulation, Neoplastic , Genes, Tumor Suppressor , Humans , Mice , Mice, Nude/genetics , Models, Animal , Neoplasm Metastasis/pathology , Promoter Regions, Genetic , Proto-Oncogene Proteins c-akt/metabolism , Repressor Proteins/pharmacology , Zinc Fingers/geneticsABSTRACT
Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor1-6. This state of viral mimicry leads to loss of cancer cell fitness and stimulates innate and adaptive immune responses7,8. However, the clinical efficacy of epigenetic therapies has been limited. To find targets that would synergize with the viral mimicry response, we sought to identify the immunogenic retroelements that are activated by epigenetic therapies. Here we show that intronic and intergenic SINE elements, specifically inverted-repeat Alus, are the major source of drug-induced immunogenic dsRNA. These inverted-repeat Alus are frequently located downstream of 'orphan' CpG islands9. In mammals, the ADAR1 enzyme targets and destabilizes inverted-repeat Alu dsRNA10, which prevents activation of the MDA5 receptor11. We found that ADAR1 establishes a negative-feedback loop, restricting the viral mimicry response to epigenetic therapy. Depletion of ADAR1 in patient-derived cancer cells potentiates the efficacy of epigenetic therapy, restraining tumour growth and reducing cancer initiation. Therefore, epigenetic therapies trigger viral mimicry by inducing a subset of inverted-repeats Alus, leading to an ADAR1 dependency. Our findings suggest that combining epigenetic therapies with ADAR1 inhibitors represents a promising strategy for cancer treatment.
Subject(s)
Adenosine Deaminase/metabolism , Alu Elements/drug effects , Alu Elements/genetics , Decitabine/pharmacology , Decitabine/therapeutic use , Epigenesis, Genetic/drug effects , RNA-Binding Proteins/metabolism , Transcription, Genetic/drug effects , Adaptive Immunity/drug effects , Adenosine Deaminase/deficiency , Alu Elements/immunology , Animals , Cell Line, Tumor , CpG Islands/drug effects , CpG Islands/genetics , DNA, Intergenic/drug effects , DNA, Intergenic/genetics , DNA, Intergenic/immunology , DNA-Cytosine Methylases/antagonists & inhibitors , Feedback, Physiological , Humans , Immunity, Innate/drug effects , Interferon-Induced Helicase, IFIH1/metabolism , Introns/drug effects , Introns/genetics , Introns/immunology , Inverted Repeat Sequences/drug effects , Inverted Repeat Sequences/genetics , Inverted Repeat Sequences/immunology , Male , Mice , Molecular Mimicry/drug effects , Molecular Mimicry/immunology , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , RNA, Double-Stranded/drug effects , RNA, Double-Stranded/genetics , RNA, Double-Stranded/immunology , RNA-Binding Proteins/antagonists & inhibitors , Viruses/drug effects , Viruses/immunologyABSTRACT
DNA methylation plays important roles in various biological processes, and the alteration of DNA methyltransferase activity can induce the aberrant DNA methylation patterns. Despite the progress in methyltransferase activity assays, few methods enable the detection of both bacteria and human methyltransferases. Herein, we construct a universal and label-free chemiluminescent sensor for accurate quantification of both bacteria methyltransferases (e.g., M. SssI methyltransferase (M.SssI MTase)) and human methyltransferases (e.g., DNA (cytosine-5)-methyltransferase 1, (Dnmt1)) by integrating a dumbbell probe with BssHII endonuclease-mediated rolling circle amplification (RCA). We ingeniously design a structure-switchable dumbbell probe which integrates target-recognition, BssHII endonuclease-cleavage, RCA amplification and signal transduction in one probe for the detection of both M.SssI MTase and Dnmt1. Moreover, the introduction of two BssHII endonuclease recognition sites in a dumbbell probe can greatly reduce the false positivity resulting from the incomplete cleavage of dumbbell probe by BssHII, because once one of two recognition sites is identified by BssHII, the dumbbell probe can be completely digested by Exonuclease III (Exo III) and Exonuclease I (Exo I) to prevent the nonspecific RCA. This chemiluminescent sensor can accurately quantify M.SssI MTase in both 10% serum and various cell lysis buffers, and even sensitively detect Dnmt1 activity in MCF-7 cells. Furthermore, this chemiluminescent sensor can be used to screen the inhibitors of Dnmt1 and M.SssI MTase, with promising applications in disease diagnosis and drug discovery.
Subject(s)
DNA (Cytosine-5-)-Methyltransferase 1/analysis , DNA-Cytosine Methylases/analysis , Luminescent Measurements , Spiroplasma/enzymology , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA-Cytosine Methylases/metabolism , HumansABSTRACT
Flowering plants and mammals contain imprinted genes that are primarily expressed in the endosperm and placenta in a parent-of-origin manner. In this study, we show that early activation of the geminivirus genes C2 and C3 in Arabidopsis (Arabidopsis thaliana) plants, encoding a viral suppressor of RNA interference and a replication enhancer protein, respectively, is correlated with the transient vegetative expression of VARIANT IN METHYLATION5 (VIM5), an endosperm imprinted gene that is conserved in diverse plant species. VIM5 is a ubiquitin E3 ligase that directly targets the DNA methyltransferases MET1 and CMT3 for degradation by the ubiquitin-26S proteasome proteolytic pathway. Infection with Beet severe curly top virus induced VIM5 expression in rosette leaf tissues, possibly via the expression of the viral replication initiator protein, leading to the early activation of C2 and C3 coupled with reduced symmetric methylation in the C2-3 promoter and the onset of disease symptoms. These findings demonstrate how this small DNA virus recruits a host imprinted gene for the epigenetic activation of viral gene transcription. Our findings reveal a distinct strategy used by plant pathogens to exploit the host machinery in order to inhibit methylation-mediated defense responses when establishing infection.
Subject(s)
Arabidopsis/genetics , Arabidopsis/virology , Geminiviridae/pathogenicity , Plant Diseases/virology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , DNA (Cytosine-5-)-Methyltransferases/genetics , DNA (Cytosine-5-)-Methyltransferases/metabolism , DNA Methylation , DNA-Cytosine Methylases/genetics , DNA-Cytosine Methylases/metabolism , Epigenesis, Genetic , Gene Expression Regulation, Plant , Genomic Imprinting , Host-Pathogen Interactions/genetics , Plant Diseases/genetics , Plant Leaves/genetics , Plant Leaves/virology , Plants, Genetically Modified , Promoter Regions, Genetic , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Viral Proteins/geneticsABSTRACT
Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFß1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFß1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFß1-induced and uninduced PLOD2 expression.
Subject(s)
Gene Silencing , Heterochromatin/metabolism , Kruppel-Like Transcription Factors/metabolism , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics , Adult , Cells, Cultured , DNA-Cytosine Methylases/genetics , DNA-Cytosine Methylases/metabolism , Epigenesis, Genetic , HEK293 Cells , Humans , Kruppel-Like Transcription Factors/genetics , MCF-7 Cells , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Promoter Regions, Genetic , Transcriptional Activation , Transforming Growth Factor beta/metabolismABSTRACT
This phase 2 study was designed to compare systemic decitabine exposure, demethylation activity, and safety in the first 2 cycles with cedazuridine 100 mg/decitabine 35 mg vs standard decitabine 20 mg/m2 IV. Adults with International Prognostic Scoring System intermediate-1/2- or high-risk myelodysplastic syndromes (MDS) or chronic myelomonocytic leukemia (CMML) were randomized 1:1 to receive oral cedazuridine/decitabine or IV decitabine in cycle 1, followed by crossover to the other treatment in cycle 2. All patients received oral cedazuridine/decitabine in subsequent cycles. Cedazuridine and decitabine were given initially as separate capsules in a dose-confirmation stage and then as a single fixed-dose combination (FDC) tablet. Primary end points: mean decitabine systemic exposure (geometric least-squares mean [LSM]) of oral/IV 5-day area under curve from time 0 to last measurable concentration (AUClast), percentage long interspersed nuclear element 1 (LINE-1) DNA demethylation for oral cedazuridine/decitabine vs IV decitabine, and clinical response. Eighty patients were randomized and treated. Oral/IV ratios of geometric LSM 5-day AUClast (80% confidence interval) were 93.5% (82.1-106.5) and 97.6% (80.5-118.3) for the dose-confirmation and FDC stages, respectively. Differences in mean %LINE-1 demethylation between oral and IV were ≤1%. Clinical responses were observed in 48 patients (60%), including 17 (21%) with complete response. The most common grade ≥3 adverse events regardless of causality were neutropenia (46%), thrombocytopenia (38%), and febrile neutropenia (29%). Oral cedazuridine/decitabine (100/35 mg) produced similar systemic decitabine exposure, DNA demethylation, and safety vs decitabine 20 mg/m2 IV in the first 2 cycles, with similar efficacy. This study is registered at www.clinicaltrials.gov as #NCT02103478.