RESUMO
BACKGROUND: Potato (Solanum tuberosum L.), the third most important non-cereal crop, is sensitive to high temperature. Histone modifications have been known to regulate various abiotic stress responses. However, the role of histone methyltransferases and demethylases remain unexplored in potato under heat stress. METHODS: Potato genome database was used for genome-wide analysis of StPRMT and StHDMA gene families, which were further characterized by analyzing gene structure, conserved motif, domain organization, sub-cellular localization, promoter region and phylogenetic relationships. Additionally, expression profiling under high-temperature stress in leaf and stolon tissue of heat contrasting potato genotypes was done to study their role in response to high temperature stress. RESULTS: The genome-wide analysis led to identification of nine StPRMT and eleven StHDMA genes. Structural analysis, including conserved motifs, exon/intron structure and phylogenetic relationships classified StPRMT and StHDMA gene families into two classes viz. Class I and Class II. A variety of cis-regulatory elements were explored in the promoter region associated with light, developmental, hormonal and stress responses. Prediction of sub-cellular localization of StPRMT proteins revealed their occurrence in nucleus and cytoplasm, whereas StHDMA proteins were observed in different sub-cellular compartments. Furthermore, expression profiling of StPRMT and StHDMA gene family members revealed genes responding to heat stress. Heat-inducible expression of StPRMT1, StPRMT3, StPRMT4 and StPRMT5 in leaf and stolon tissues of HS and HT cultivar indicated them as probable candidates for enhancing thermotolerance in potato. However, StHDMAs responded dynamically in leaf and stolon tissue of heat contrasting genotypes under high temperature. CONCLUSION: The current study presents a detailed analysis of histone modifiers in potato and indicates their role as an important epigenetic regulators modulating heat tolerance. GENERAL SIGNIFICANCE: Understanding epigenetic mechanisms underlying heat tolerance in potato will contribute towards breeding of thermotolerant potato varieties.
Assuntos
Solanum tuberosum , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Filogenia , Histona Metiltransferases/metabolismo , Resposta ao Choque Térmico/genética , Estresse Fisiológico/genéticaRESUMO
RATIONALE: Sotos syndrome is an congenital overgrowth syndrome characterized by the primary features including overgrowth, distinctive facial features, learning disability, and accompanied with various second features. NSD1 deletion or mutation is a major pathogenic cause. Although there are some reports on treatment of this disease worldwide, less cases under treatment have been published in China. PATIENT CONCERNS: A 1-year-old boy had macrocephaly, gigantism, excessive high body height, a particular face and delayed development, with a pathogenic gene of NSD1 (NM_022455.5:c.3536delA in exon 5). DIAGNOSIS AND INTERVENTIONS: The child was definitely diagnosed as Sotos syndrome and have 3 months' combination treatment of traditional Chinese medicine and rehabilitation. OUTCOMES: The child made a great progress in global development. LESSONS: This case firstly describes the traditional Chinese medicine and rehabilitation to treat Sotos syndrome in China. There is no radical cure, but our therapy could improve the prognosis and the life quality of the patient. Therefore, this case provides a reference to the clinical treatment of Sotos syndrome.
Assuntos
Síndrome de Sotos , Criança , Masculino , Humanos , Lactente , Síndrome de Sotos/genética , Histona Metiltransferases/genética , Histona-Lisina N-Metiltransferase/genética , Medicina Tradicional Chinesa , MutaçãoRESUMO
Previous studies have shown that inhibition of TNF family member FN14 (gene: TNFRSF12A) in colon tumors decreases inflammatory cytokine expression and mitigates cancer-induced cachexia. However, the molecular mechanisms underlying the regulation of FN14 expression remain unclear. Tumor microenvironments are often devoid of nutrients and oxygen, yet how the cachexic response relates to the tumor microenvironment and, importantly, nutrient stress is unknown. Here, we looked at the connections between metabolic stress and FN14 expression. We found that TNFRSF12A expression was transcriptionally induced during glutamine deprivation in cancer cell lines. We also show that the downstream glutaminolysis metabolite, alpha-ketoglutarate (aKG), is sufficient to rescue glutamine-deprivation-promoted TNFRSF12A induction. As aKG is a co-factor for histone de-methylase, we looked at histone methylation and found that histone H3K4me3 at the Tnfrsf12a promoter is increased under glutamine-deprived conditions and rescued via DM-aKG supplementation. Finally, expression of Tnfrsf12a and cachexia-induced weight loss can be inhibited in vivo by DM-aKG in a mouse cancer cachexia model. These findings highlight a connection between metabolic stress and cancer cachexia development.
Assuntos
Caquexia , Neoplasias do Colo , Receptor de TWEAK , Animais , Camundongos , Caquexia/genética , Caquexia/prevenção & controle , Modelos Animais de Doenças , Glutamina/farmacologia , Código das Histonas , Histona Metiltransferases , Histonas/genética , Ácidos Cetoglutáricos/farmacologia , Microambiente Tumoral , Humanos , Linhagem Celular Tumoral/metabolismo , Receptor de TWEAK/genética , Receptor de TWEAK/metabolismoRESUMO
The present study aimed to identify the function and expression of trimethylated protein histone H3 lysine 36 (H3K36)me3 and the upstream specific enzyme histone methyltransferase SET domain containing 2 (SETD2), during the differentiation of hepatic oval cells (HOCs) into cholangiocytes in mice following partial liver resection and fed with 2acetamidofluorene. HOCs were isolated from Kunming male mice fed with 2acetamidofluorene for 10 days. Their liver tissues were then isolated following partial liver resection and another week of 2acetamidofluorene treatment. HOCs were collected following a twostep enzyme digestion procedure involving protease E and collagenase 4. The target cells were cultured in DMEM/F12 supplemented with 10 µg/ml EGF, 5 µg/ml stem cell growth factor and 5 µg/ml leukemia inhibitory factor. Target cells using the markers OV6, CK19, SETD2, H3K36me3, were detected with flow cytometry and immunofluorescence microscopy; reverse transcriptionquantitative PCR and western blotting were used to quantify the protein levels of SETD2 and H3K36me3. The retrieved primary hepatocytes developed into cholangiocytes with increasing CK19 and decreasing OV6 expression in each subsequent passage, whereas the SETD2 and H3K36me3 levels gradually increased, suggesting the possible involvement of both of these factors in differentiation.
Assuntos
Histonas , Lisina , Camundongos , Masculino , Animais , Histonas/metabolismo , Histona Metiltransferases/metabolismo , Lisina/metabolismo , Domínios PR-SET , Diferenciação Celular , Células Epiteliais/metabolismo , Ductos Biliares/metabolismoRESUMO
As discussed in previous chapters, the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases, the Protein Arginine Methyltransferase (PRMT) family for arginine, and the SET domain family for lysine. The methylation of H3K79 by Dot1 is a notable outlier. In all cases, X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism, kinetics, regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a therapeutic setting. Notably, in combination with the structures of the complementary recognition domains that recognize their products, these structures have provided an important insight into how integral the number of methyl groups added to the acceptor amine is to making histone methylation a key process in epigenetic regulation of gene transcription. Here the concepts applied to determine their structure by X-ray crystallography are outlined, with particular emphasis on lysine methylation by the SET domain.
Assuntos
Histona-Lisina N-Metiltransferase , Lisina , Arginina/metabolismo , Cristalografia por Raios X , Epigênese Genética , Histona Metiltransferases/metabolismo , Histona-Lisina N-Metiltransferase/química , Histonas/metabolismo , Lisina/metabolismoRESUMO
Histone 3 lysine 4 trimethylation (H3K4me3) is an epigenetic mark found at gene promoters and CpG islands. H3K4me3 is essential for mammalian development, yet mechanisms underlying its genomic targeting are poorly understood. H3K4me3 methyltransferases SETD1B and MLL2 (KMT2B) are essential for oogenesis. We investigated changes in H3K4me3 in Setd1b conditional knockout (cKO) oocytes using ultra-low input ChIP-seq, with comparisons to DNA methylation and gene expression analyses. H3K4me3 was redistributed in Setd1b cKO oocytes showing losses at active gene promoters associated with downregulated gene expression. Remarkably, many regions also gained H3K4me3, in particular those that were DNA hypomethylated, transcriptionally inactive and CpG-rich, which are hallmarks of MLL2 targets. Consequently, loss of SETD1B disrupts the balance between MLL2 and de novo DNA methyltransferases in determining the epigenetic landscape during oogenesis. Our work reveals two distinct, complementary mechanisms of genomic targeting of H3K4me3 in oogenesis, with SETD1B linked to gene expression and MLL2 to CpG content.
Assuntos
Histonas , Lisina , Animais , Ilhas de CpG/genética , Metilação de DNA , Histona Metiltransferases/genética , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Mamíferos/genética , Oogênese/genéticaRESUMO
Aberrant levels of preadipocyte differentiation, triggered by adipocyte hyperplasia and hypertrophy, results in the obesogenic phenotype. Obesity is a risk factor for several metabolic disorders. In this paper, dehydroleucodine inhibited the accumulation of lipid droplets and decreased the elevations of triglycerides, and this inhibitory effect occurred during the early stage of adipogenesis. Thus, not only did dehydroleucodine downregulate the expression of C/EBPα and PPARγ, it also strongly blocked the expression of C/EBPß, an early stage biomarker of early adipogenesis, in a concentration-dependent manner. The proliferation of preadipocytes was dramatically suppressed when dehydroleucodine was added to the medium as early as 24 hr. These results indicate that dehydroleucodine may specifically affect mitotic clonal expansion to inhibit preadipocyte differentiation. Dehydroleucodine arrested the cell cycle at the G0 /G1 phase, increased p27 and decreased both cyclins A and D and their partners (e.g., CDK2 and CDK4). Additionally, dehydroleucodine decreased phosphorylation of Erk1/2 and Akt. Furthermore, dehydroleucodine downregulated expression of histone demethylase JMJD2B as well as repressed the expression of histone methyltransferase MLL4, which in turn diminished the expression of C/EBPß and PPARγ, respectively. Collectively, our results indicate that dehydroleucodine inhibits preadipocyte differentiation by blocking mitotic clonal expansion via cell cycle arrest, which may be mediated by regulation of selective histone methylation/demethylation in transcription activation during the early step of adipogenesis.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular , Lactonas/farmacologia , Mitose/efeitos dos fármacos , Sesquiterpenos/farmacologia , Células 3T3-L1 , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Camundongos , PPAR gama/metabolismo , Ativação TranscricionalRESUMO
Hepatitis B virus (HBV) infection remains a major health problem worldwide. Maintenance of the covalently closed circular DNA (cccDNA), which serves as a template for HBV RNA transcription, is responsible for the failure of eradicating chronic HBV during current antiviral therapy. cccDNA is assembled with cellular histone proteins into chromatin, but little is known about the regulation of HBV chromatin by histone posttranslational modifications. In this study, we identified silent mating type information regulation 2 homolog 3 (SIRT3) as a host factor restricting HBV transcription and replication by screening seven members of the sirtuin family, which is the class III histone deacetylase. Ectopic SIRT3 expression significantly reduced total HBV RNAs, 3.5-kb RNA, as well as replicative intermediate DNA in HBV-infected HepG2-Na+ /taurocholate cotransporting polypeptide cells and primary human hepatocytes. In contrast, gene silencing of SIRT3 promoted HBV transcription and replication. A mechanistic study found that nuclear SIRT3 was recruited to the HBV cccDNA, where it deacetylated histone 3 lysine 9. Importantly, occupancy of SIRT3 on cccDNA could increase the recruitment of histone methyltransferase suppressor of variegation 3-9 homolog 1 to cccDNA and decrease recruitment of SET domain containing 1A, leading to a marked increase of trimethyl-histone H3 (Lys9) and a decrease of trimethyl-histone H3 (Lys4) on cccDNA. Moreover, SIRT3-mediated HBV cccDNA transcriptional repression involved decreased binding of host RNA polymerase II and transcription factor Yin Yang 1 to cccDNA. Finally, hepatitis B viral X protein could relieve SIRT3-mediated cccDNA transcriptional repression by inhibiting both SIRT3 expression and its recruitment to cccDNA. CONCLUSION: SIRT3 is a host factor epigenetically restricting HBV cccDNA transcription by acting cooperatively with histone methyltransferase; these data provide a rationale for the use of SIRT3 activators in the prevention or treatment of HBV infection. (Hepatology 2018).
Assuntos
DNA Viral/genética , Epigênese Genética/genética , Hepatite B/genética , Domínios PR-SET/genética , Sirtuína 3/genética , Replicação Viral/genética , DNA Complementar/genética , Hepatite B/fisiopatologia , Vírus da Hepatite B/genética , Histona Metiltransferases/metabolismo , Humanos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e EspecificidadeRESUMO
Histone methylation is an epigenetic modification of chromatin undergoing dynamic changes and balancing tissue-specific demands of proliferation and differentiation. In cancer, aberrant histone methylation can facilitate oncogenic and tumor suppression programs by modulating gene expression. Histone remodelers such as lysine methyltransferases and lysine demethylases are seemingly opposite or contrary forces but may be part of an interconnected network complementing each other. We identify several layers of molecular communication where epigenetic master regulators engage in crosstalk between tumor metabolism and histone remodeling. Epigenetic master regulators have the ability to cooperate with members of the transcriptional machinery, DNA methyltransferases, as well as other histone modifiers. High-throughput sequencing and omics data in combination with cancer systems biology analysis have the power to prioritize regulatory events epigenome-wide.
Assuntos
Epigênese Genética , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Neoplasias/enzimologia , Sequenciamento de Nucleotídeos em Larga Escala , Histona Desmetilases/genética , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Humanos , Neoplasias/genéticaRESUMO
While the current epigenetic drug development is still largely restricted to target DNA methylome, emerging evidence indicates that histone methylome is indeed another major epigenetic determinant for gene expression and frequently deregulated in acute myeloid leukaemia (AML). The recent advances in dissecting the molecular regulation and targeting histone methylome in AML together with the success in developing lead compounds specific to key histone methylation-modifying enzymes have revealed new opportunities for effective leukaemia treatment. In this article, we will review the emerging functions of histone methyltransferases and histone demethylases in AML, especially MLL-rearranged leukaemia. We will also examine recent preclinical and clinical studies that show significant promises of targeting these histone methylation-modifying enzymes for AML treatment.
Assuntos
Antineoplásicos/farmacologia , Epigênese Genética/efeitos dos fármacos , Histona Desmetilases/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histonas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Acetilação , Animais , Antineoplásicos/uso terapêutico , Estudos Clínicos como Assunto , Metilação de DNA/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Histona Desmetilases/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Terapia de Alvo Molecular , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , Resultado do TratamentoRESUMO
Twenty-three histone methyltransferase genes were obtained from transcriptome dataset of Lonicera japonica. The nucleotide and proteins characteristics, subcellular localization, senior structural domains and conservative forecasting were analyzed. The result of phylogenetic tree showed that 23 histone methyltransferases were mainly divided into two groups: lysine methyltransferase and arginine methyltransferases. The result of gene expression showed that 23 histone methyltransferases showed preference in terms of interspecies and organs. They were more expressed in buds of L. japonica than in L. japonica var. chinensis and lower in leaves of L. japonica than in L. japonica var. chinensis. Eight genes were specific expressed in flower. These results provided basis for further understanding the function of histone methyltransferase and epigenetic regulation of active ingredients of L. japonica.
Assuntos
Biologia Computacional , Histona-Lisina N-Metiltransferase/genética , Lonicera/genética , Expressão Gênica , Histona Metiltransferases , Lonicera/enzimologia , FilogeniaRESUMO
Histone methylation is a prevalent and dynamic chromatin modification, executed by the action of histone methyltransferases (HMTs) and demethylases (HDMs). Aberrant activity of many of these enzymes is associated with human disease, hence, there is a growing interest in identifying corresponding small molecule inhibitors with therapeutic potential. To date, most of the technologies supporting the identification of these inhibitors constitute in vitro biochemical assays which, although robust and sensitive, do not study HMTs and HDMs in their native cellular state nor provide information of inhibitor's cell permeability and toxicity. The evident need for complementary cellular approaches has recently propelled the development of cell-based assays that enable screening of HMT and HDM enzymes in a more relevant environment. Here, we highlight current cellular methodologies for HMT and HDM drug discovery support. We anticipate that implementation of these cell-based assays will positively impact the discovery of pharmacologically potent HMT and HDM inhibitors.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Histona Desmetilases/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histonas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Bioensaio , Células Cultivadas , Perfilação da Expressão Gênica , Histona Desmetilases/genética , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Humanos , Metilação , Ligação ProteicaRESUMO
We evaluated the potential of an investigational histone methylation reversal agent, 3-deazaneplanocin A (DZNep), in improving the chemosensitivity of pancreatic cancer to nucleoside analogs (i.e., gemcitabine). DZNep brought delayed but selective cytotoxicity to pancreatic cancer cells without affecting normal human pancreatic ductal epithelial (HPDE) cells. Co-exposure of DZNep and gemcitabine induced cytotoxic additivity or synergism in both well- and poorly-differentiated pancreatic cell lines by increased apoptosis. In contrast, DZNep exerted antagonism with gemcitabine against HPDE cells with significant reduction in cytotoxicity compared with the gemcitabine-alone regimen. DZNep marginally depended on purine nucleoside transporters for its cytotoxicity, but the transport dependence was circumvented by acyl derivatization. Drug exposure studies revealed that a short priming with DZNep followed by gemcitabine treatment rather than co-treatment of both agents to produce a maximal chemosensitization response in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. DZNep rapidly and reversibly decreased trimethylation of histone H3 lysine 27 but increased trimethylation of lysine 9 in an EZH2- and JMJD1A/2C-dependent manner, respectively. However, DZNep potentiation of nucleoside analog chemosensitization was found to be temporally coupled to trimethylation changes in lysine 27 and not lysine 9. Polymeric nanoparticles engineered to chronologically release DZNep followed by gemcitabine produced pronounced chemosensitization and dose-lowering effects. Together, our results identify that an optimized DZNep exposure can presensitize pancreatic cancer cells to anticancer nucleoside analogs through the reversal of histone methylation, emphasizing the promising clinical utilities of epigenetic reversal agents in future pancreatic cancer combination therapies.
Assuntos
Adenosina/análogos & derivados , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Sistemas de Liberação de Medicamentos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Nucleosídeos/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Adenosina/farmacologia , Animais , Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Células Cultivadas , Metilação de DNA/efeitos dos fármacos , Preparações de Ação Retardada , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Humanos , Nanopartículas , Neoplasias Pancreáticas/patologia , Xenopus , GencitabinaRESUMO
Unfavorable nutritional conditions during early developmental periods may cause neuronal network remodeling in the hypothalamus, which influences subsequent adaptability to those same stressful conditions. Alterations in hypothalamic plasticity as a result of neuronal remodeling are achieved by variations in the repertoire of proteins expressed via gene transcriptional activation or repression, both of which are modulated by histone methylation status. This study demonstrates that fasting had a stimulatory effect on dimethylation and trimethylation of histone 3 at lysine 27 (H3K27) in preoptic/anterior hypothalamus (PO/AH) of 3-day-old chicks. The expression of enhancer of zeste 2 (EZH2), a H3K27-specific histone methyltransferase (HMT), was significantly increased by fasting in the paraventricular nucleus (PVN) and PO/AH, which is consistent with the upregulation of H3K27 dimethylation and trimethylation. Furthermore, in the PVN, corticotrophin-releasing hormone (CRH) mRNA expression was significantly inhibited, while mRNA expressions of thyrotropin-releasing hormone (TRH) and type 2 deiodinase (D2) were significantly stimulated by fasting. These findings highlight the potential role of H3K27 methylation status in early feed stress responses in chicks and may be indicative of an epigenetic mechanism for later adaptation to feed intake stress.
Assuntos
Jejum/fisiologia , Histonas/metabolismo , Hipotálamo/metabolismo , Animais , Animais Recém-Nascidos , Galinhas , Hormônio Liberador da Corticotropina/genética , Hormônio Liberador da Corticotropina/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Lisina/metabolismo , Masculino , Metilação , RNA Mensageiro , Hormônio Liberador de Tireotropina/genética , Hormônio Liberador de Tireotropina/metabolismo , Iodotironina Desiodinase Tipo IIRESUMO
For many novel epigenetics targets the chemical ligand space and structural information were limited until recently and are still largely unknown for some targets. Hit-finding campaigns are therefore dependent on large and chemically diverse libraries. In the specific case of the histone methyltransferase G9a, the authors have been able to apply an efficient process of intelligent selection of compounds for primary screening, rather than screening the full diverse deck of 900 000 compounds to identify hit compounds. A number of different virtual screening methods have been applied for the compound selection, and the results have been analyzed in the context of their individual success rates. For the primary screening of 2112 compounds, a FlashPlate assay format and full-length histone H3.1 substrate were employed. Validation of hit compounds was performed using the orthogonal fluorescence lifetime technology. Rated by purity and IC(50) value, 18 compounds (0.9% of compound screening deck) were finally considered validated primary G9a hits. The hit-finding approach has led to novel chemotypes being identified, which can facilitate hit-to-lead projects. This study demonstrates the power of virtual screening technologies for novel, therapeutically relevant epigenetics protein targets.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Epigênese Genética/efeitos dos fármacos , Fluorescência , Antígenos de Histocompatibilidade , Histona Metiltransferases , Histonas/metabolismo , Concentração Inibidora 50 , Ligantes , Radiometria/métodos , Reprodutibilidade dos Testes , Bibliotecas de Moléculas PequenasRESUMO
The histone methyltransferase (HMT) family of proteins consists of enzymes that methylate lysine or arginine residues on histone tails as well as other proteins. Such modifications affect chromatin structure and play a significant regulatory role in gene expression. Many HMTs have been implicated in tumorigenesis and progression of multiple malignancies and play essential roles in embryonic development and stem cell renewal. Overexpression of some HMTs has been observed and is correlated positively with various types of cancer. Here the authors report development of a continuous fluorescence-based methyltransferase assay in a 384-well format and its application in determining kinetic parameters for EHMT1, G9a, PRMT3, SETD7, and SUV39H2 as well as for screening against libraries of small molecules to identify enzyme inhibitors. They also report the development of a peptide displacement assay using fluorescence polarization in a 384-well format to assay and screen protein peptide interactions such as those of WDR5 and EED, components of MLL and EZH2 methyltransferase complexes. Using these high-throughput screening methods, the authors have identified potent inhibitors and ligands for some of these proteins.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Adenosil-Homocisteinase/metabolismo , Sequência de Aminoácidos , Fluorescência , Antígenos de Histocompatibilidade/análise , Antígenos de Histocompatibilidade/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/análise , Peptídeos e Proteínas de Sinalização Intracelular , Cinética , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Complexo Repressor Polycomb 2 , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Bibliotecas de Moléculas PequenasRESUMO
Gene silencing may have a direct or indirect impact on many biological processes in eukaryotic cells, and is a useful tool for the determination of the roles of specific genes. In this article, we report silencing in Phytophthora infestans, an oomycete pathogen of potato and tomato. Gene silencing is known to occur in P. infestans, but its genetic basis has yet to be determined. Genes encoding the major components of the RNA interference (RNAi) pathway, Dicer-like (Pidcl1), Argonaute (Piago1-5) and RNA-directed RNA polymerase (Pirdr1), were identified in the P. infestans genome by comparative genomics, together with families of other genes potentially involved in gene silencing, such as histone deacetylases, histone methyltransferases, DEAD helicases, chromodomain proteins and a class 1 RNaseIII. Real-time reverse transcription-polymerase chain reaction demonstrated transcript accumulation for all candidate genes throughout the asexual lifecycle and plant infection, but at different levels of mRNA abundance. A functional assay was developed in which silencing of the sporulation-associated Picdc14 gene was released by the treatment of protoplasts with in vitro-synthesized double-stranded RNAs homologous to Pidcl1, Piago1/2 and histone deacetylase Pihda1. These results suggest that the components of gene silencing, namely Dicer-like, Argonaute and histone deacetylase, are functional in P. infestans. Our data demonstrate that this oomycete possesses canonical gene silencing pathways similar to those of other eukaryotes.
Assuntos
Phytophthora infestans/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Inativação Gênica , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Solanum lycopersicum/microbiologia , Filogenia , Phytophthora infestans/genética , Doenças das Plantas/microbiologia , Interferência de RNA , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Solanum tuberosum/microbiologiaRESUMO
Epigenetic effects are considered heritable but may also be modified by environmental factors. Arecoline (ARC), a major component of areca nut alkaloids, is an important environmental risk factor for oral cancer and hepatocellular carcinomain Taiwan. The aim of this study was to determine the influence of ARC on the epigenome. The mRNA expression of histone methyltransferases, acetyltransferases, and demethylases were assessed in K-562 cells following exposure to ARC. Results demonstrated that ARC produced changes in the expressions of several genes catalyzing histone methylation (Mll, Setdb1, and Suv39h2), acetylation (Atf2), and demethylation (JMJD6). Since H3K9 methylation is involved in maintaining the stability of heterochromatin structures and inactivating euchromatic gene expressions, data suggest that the ARC-induced epigenetic changes play a role in the mechanisms underlying chemical-mediated cytotoxicity and genotoxicity.
Assuntos
Arecolina/toxicidade , Citotoxinas/toxicidade , Extratos Vegetais/toxicidade , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Epigênese Genética , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: SET-domain proteins are histone lysine (K) methyltransferases (HMTase) implicated in defining transcriptionally permissive or repressive chromatin. The Arabidopsis ASH1 HOMOLOG 2 (ASHH2) protein (also called SDG8, EFS and CCR1) has been suggested to methylate H3K4 and/or H3K36 and is similar to Drosophila ASH1, a positive maintainer of gene expression, and yeast Set2, a H3K36 HMTase. Mutation of the ASHH2 gene has pleiotropic developmental effects. Here we focus on the role of ASHH2 in plant reproduction. METHODOLOGY/PRINCIPAL FINDINGS: A slightly reduced transmission of the ashh2 allele in reciprocal crosses implied involvement in gametogenesis or gamete function. However, the main requirement of ASHH2 is sporophytic. On the female side, close to 80% of mature ovules lack embryo sac. On the male side, anthers frequently develop without pollen sacs or with specific defects in the tapetum layer, resulting in reduction in the number of functional pollen per anther by up to approximately 90%. In consistence with the phenotypic findings, an ASHH2 promoter-reporter gene was expressed at the site of megaspore mother cell formation as well as tapetum layers and pollen. ashh2 mutations also result in homeotic changes in floral organ identity. Transcriptional profiling identified more than 300 up-regulated and 600 down-regulated genes in ashh2 mutant inflorescences, whereof the latter included genes involved in determination of floral organ identity, embryo sac and anther/pollen development. This was confirmed by real-time PCR. In the chromatin of such genes (AP1, AtDMC1 and MYB99) we observed a reduction of H3K36 trimethylation (me3), but not H3K4me3 or H3K36me2. CONCLUSIONS/SIGNIFICANCE: The severe distortion of reproductive organ development in ashh2 mutants, argues that ASHH2 is required for the correct expression of genes essential to reproductive development. The reduction in the ashh2 mutant of H3K36me3 on down-regulated genes relevant to the observed defects, implicates ASHH2 in regulation of gene expression via H3K36 trimethylation in chromatin of Arabidopsis inflorescences.
Assuntos
Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Histona-Lisina N-Metiltransferase/genética , Proteínas de Plantas/metabolismo , Alelos , Cromatina/química , Cruzamentos Genéticos , Regulação para Baixo , Perfilação da Expressão Gênica , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/fisiologia , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Óvulo Vegetal/genética , Fenótipo , Pólen , Transcrição GênicaRESUMO
The polycomb repressive complex (PRC) 2 contains 3 core proteins, EZH2, SUZ12, and EED, in which the SET (suppressor of variegation-enhancer of zeste-trithorax) domain of EZH2 mediates the histone methyltransferase activity. This induces trimethylation of lysine 27 on histone H3, regulates the expression of HOX genes, and promotes proliferation and aggressiveness of neoplastic cells. In this study, we demonstrate that treatment with the S-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin A (DZNep) depletes EZH2 levels, and inhibits trimethylation of lysine 27 on histone H3 in the cultured human acute myeloid leukemia (AML) HL-60 and OCI-AML3 cells and in primary AML cells. DZNep treatment induced p16, p21, p27, and FBXO32 while depleting cyclin E and HOXA9 levels. Similar findings were observed after treatment with small interfering RNA to EZH2. In addition, DZNep treatment induced apoptosis in cultured and primary AML cells. Furthermore, compared with treatment with each agent alone, cotreatment with DZNep and the pan-histone deacetylase inhibitor panobinostat caused more depletion of EZH2, induced more apoptosis of AML, but not normal CD34(+) bone marrow progenitor cells, and significantly improved survival of nonobese diabetic/severe combined immunodeficiency mice with HL-60 leukemia. These findings indicate that the combination of DZNep and panobinostat is effective and relatively selective epigenetic therapy against AML cells.