RESUMO
Intronic splicing silencer N1 (ISS-N1) located within Survival Motor Neuron 2 (SMN2) intron 7 is the target of a therapeutic antisense oligonucleotide (ASO), nusinersen (Spinraza), which is currently being used for the treatment of spinal muscular atrophy (SMA), a leading genetic disease associated with infant mortality. The discovery of ISS-N1 as a promising therapeutic target was enabled in part by Anti-N1, a 20-mer ASO that restored SMN2 exon 7 inclusion by annealing to ISS-N1. Here, we analyzed the transcriptome of SMA patient cells treated with 100 nM of Anti-N1 for 30 h. Such concentrations are routinely used to demonstrate the efficacy of an ASO. While 100 nM of Anti-N1 substantially stimulated SMN2 exon 7 inclusion, it also caused massive perturbations in the transcriptome and triggered widespread aberrant splicing, affecting expression of essential genes associated with multiple cellular processes such as transcription, splicing, translation, cell signaling, cell cycle, macromolecular trafficking, cytoskeletal dynamics, and innate immunity. We validated our findings with quantitative and semiquantitative PCR of 39 candidate genes associated with diverse pathways. We also showed a substantial reduction in off-target effects with shorter ISS-N1-targeting ASOs. Our findings are significant for implementing better ASO design and dosing regimens of ASO-based drugs.
Assuntos
Atrofia Muscular Espinal/terapia , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos/farmacologia , Transcriptoma , Células Cultivadas , Terapia Genética , Humanos , Íntrons/efeitos dos fármacos , Atrofia Muscular Espinal/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Transcriptoma/efeitos dos fármacosRESUMO
Cereblon is the direct binding target of the immunomodulatory drugs (IMiDs) that are commonly used to treat multiple myeloma (MM), the second most frequent hematologic malignancy. Patients respond well to initial treatment with IMiDs, but virtually all patients develop drug resistance over time, and the underlying mechanisms are poorly understood. We identified an as yet undescribed DNA hypermethylation in an active intronic CRBN enhancer. Differential hypermethylation in this region was found to be increased in healthy plasma cells, but was more pronounced in IMiD-refractory MM. Methylation significantly correlated with decreased CRBN expression levels. DNA methyltransferase inhibitor (DNTMi) in vitro experiments induced CRBN enhancer demethylation, and sensitizing effects on lenalidomide treatment were observed in 2 MM cell lines. Thus, we provide first evidence that aberrant CRBN DNA methylation is a novel mechanism of IMiD resistance in MM and may predict IMiD response prior to treatment.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Antineoplásicos Imunológicos/uso terapêutico , Agentes de Imunomodulação/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Ubiquitina-Proteína Ligases/genética , Metilação de DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Elementos Facilitadores Genéticos/efeitos dos fármacos , Humanos , Íntrons/efeitos dos fármacos , Mieloma Múltiplo/genéticaRESUMO
Response to anti-TNF therapy is of pivotal importance in patients with Crohn's disease (CD). Here we integrated our and previously reported PBMC derived transcriptomic and genomic data for identification of biomarkers for discrimination between responders and non-responders to anti-TNF therapy. CD patients, who were naïve with respect to the treatment with biologicals, were enrolled in the study. DNA and RNA were extracted from peripheral blood mononuclear cells. RNA-seq was performed using BGISEQ-500. Genotyping was performed using Infinium Global Screening Array. Association regressions were carried out with 12 week response to adalimumab as an outcome variable. RNA-seq analysis confirmed 7 out of 65 previously suggested genes involved in anti-TNF response. Subsequently, analysis of single nucleotide variants in regions of confirmed genes identified 5 variants near MMD and two in ELOVL7 intronic regions associated with treatment response to anti-TNF. Functional analysis has shown that rs1465352, rs4422035 and rs78620886 are listed at H3K9ac_Pro histone modification epigenetic mark. The present study confirmed MMD and ELOVL7 involvement in anti-TNF response and revealed that the regulation of MMD and ELOVL7 gene regions in ADA response may be a part of a complex interplay extending from genetic to epigenetic and to transcriptomic level.
Assuntos
Adalimumab/uso terapêutico , Doença de Crohn/tratamento farmacológico , Elongases de Ácidos Graxos/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Inibidores do Fator de Necrose Tumoral/uso terapêutico , Adulto , Doença de Crohn/genética , Feminino , Loci Gênicos/efeitos dos fármacos , Genômica , Humanos , Íntrons/efeitos dos fármacos , Masculino , Polimorfismo de Nucleotídeo Único/efeitos dos fármacos , Transcriptoma/efeitos dos fármacos , Adulto JovemRESUMO
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.
Assuntos
Adenosina Desaminase/metabolismo , Elementos Alu/efeitos dos fármacos , Elementos Alu/genética , Decitabina/farmacologia , Decitabina/uso terapêutico , Epigênese Genética/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Transcrição Gênica/efeitos dos fármacos , Imunidade Adaptativa/efeitos dos fármacos , Adenosina Desaminase/deficiência , Elementos Alu/imunologia , Animais , Linhagem Celular Tumoral , Ilhas de CpG/efeitos dos fármacos , Ilhas de CpG/genética , DNA Intergênico/efeitos dos fármacos , DNA Intergênico/genética , DNA Intergênico/imunologia , DNA-Citosina Metilases/antagonistas & inibidores , Retroalimentação Fisiológica , Humanos , Imunidade Inata/efeitos dos fármacos , Helicase IFIH1 Induzida por Interferon/metabolismo , Íntrons/efeitos dos fármacos , Íntrons/genética , Íntrons/imunologia , Sequências Repetidas Invertidas/efeitos dos fármacos , Sequências Repetidas Invertidas/genética , Sequências Repetidas Invertidas/imunologia , Masculino , Camundongos , Mimetismo Molecular/efeitos dos fármacos , Mimetismo Molecular/imunologia , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , RNA de Cadeia Dupla/efeitos dos fármacos , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , Proteínas de Ligação a RNA/antagonistas & inibidores , Vírus/efeitos dos fármacos , Vírus/imunologiaRESUMO
Epigenetic regulation enables tumors to respond to changing environments during tumor progression and metastases and facilitates treatment resistance. Targeting chromatin modifiers or catalytic effectors of transcription is an emerging anti-cancer strategy. The cyclin-dependent kinases (CDKs) 12 and 13 phosphorylate the C-terminal domain of RNA polymerase II, regulating transcription and co-transcriptional processes. Here we report the development of SR-4835, a highly selective dual inhibitor of CDK12 and CDK13, which disables triple-negative breast cancer (TNBC) cells. Mechanistically, inhibition or loss of CDK12/CDK13 triggers intronic polyadenylation site cleavage that suppresses the expression of core DNA damage response proteins. This provokes a "BRCAness" phenotype that results in deficiencies in DNA damage repair, promoting synergy with DNA-damaging chemotherapy and PARP inhibitors.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Quinases Ciclina-Dependentes/metabolismo , Dano ao DNA/efeitos dos fármacos , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Humanos , Íntrons/efeitos dos fármacos , Íntrons/genética , Camundongos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Poliadenilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/uso terapêutico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
FOXP3+ regulatory T (Treg) cells are essential for immunological tolerance and immune homeostasis. Despite a great deal of interest in modulating their number and function for the treatment of autoimmune disease or cancer, the precise mechanisms that control the homeostasis of Treg cells remain unclear. We report a new ENU-induced mutant mouse, lack of costimulation (loco), with atopic dermatitis and Treg cell deficiency typical of Card11 loss-of-function mutants. Three distinct single nucleotide variants were found in the Card11 introns 2, 10 and 20 that cause the loss of CARD11 expression in these mutant mice. These mutations caused the loss of thymic-derived, Neuropilin-1+ (NRP1+ ) Treg cells in neonatal and adult loco mice; however, residual peripherally induced NRP1- Treg cells remained. These peripherally generated Treg cells could be expanded in vivo by the administration of IL-2:anti-IL-2 complexes, indicating that this key homeostatic signaling axis remained intact in CARD11-deficient Treg cells. Furthermore, these expanded Treg cells could mediate near-normal suppression of activated, conventional CD4+ T cells, suggesting that CARD11 is dispensable for Treg cell function. In addition to shedding light on the requirements for CARD11 in Treg cell homeostasis and function, these data reveal novel noncoding Card11 loss-of-function mutations that impair the expression of this critical immune-regulatory protein.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/deficiência , Dermatite Atópica/imunologia , Homeostase/imunologia , Transdução de Sinais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Proteínas Adaptadoras de Sinalização CARD/imunologia , Dermatite Atópica/genética , Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Homeostase/genética , Humanos , Íntrons/efeitos dos fármacos , Íntrons/genética , Íntrons/imunologia , Mutação com Perda de Função/efeitos dos fármacos , Mutação com Perda de Função/imunologia , Camundongos , Camundongos Transgênicos , Mutagênese/imunologia , Mutagênicos/toxicidade , Neuropilina-1/imunologia , Neuropilina-1/metabolismo , Polimorfismo de Nucleotídeo Único/efeitos dos fármacos , Polimorfismo de Nucleotídeo Único/imunologia , Transdução de Sinais/genética , Linfócitos T Reguladores/metabolismoRESUMO
OBJECTIVE: Dysferlin is a large transmembrane protein that functions in critical processes of membrane repair and vesicle fusion. Dysferlin-deficiency due to mutations in the dysferlin gene leads to muscular dystrophy (Miyoshi myopathy (MM), limb girdle muscular dystrophy type 2B (LGMD2B), distal myopathy with anterior tibial onset (DMAT)), typically with early adult onset. At least 416 pathogenic dysferlin mutations are known, but for approximately 17% of patients, one or both of their pathogenic variants remain undefined following standard exon sequencing methods that interrogate exons and nearby flanking intronic regions but not the majority of intronic regions. METHODS: We sequenced RNA from myogenic cells to identify a novel dysferlin pathogenic variant in two affected siblings that previously had only one disease-causing variant identified. We designed antisense oligonucleotides (AONs) to bypass the effects of this mutation on RNA splicing. RESULTS: We identified a new pathogenic point mutation deep within dysferlin intron 50i. This intronic variant causes aberrant mRNA splicing and inclusion of an additional pseudoexon (PE, we term PE50.1) within the mature dysferlin mRNA. PE50.1 inclusion alters the protein sequence, causing premature translation termination. We identified this mutation in 23 dysferlinopathy patients (seventeen families), revealing it to be one of the more prevalent dysferlin mutations. We used AON-mediated exon skipping to correct the aberrant PE50.1 splicing events in vitro, which increased normal mRNA production and significantly restored dysferlin protein expression. INTERPRETATION: Deep intronic mutations can be a common underlying cause of dysferlinopathy, and importantly, could be treatable with AON-based exon-skipping strategies.
Assuntos
Disferlina/genética , Íntrons/genética , Distrofia Muscular do Cíngulo dos Membros/etiologia , Mutação/genética , Miopatias Distais/genética , Humanos , Íntrons/efeitos dos fármacos , Proteínas de Membrana/deficiência , Atrofia Muscular/genética , Distrofia Muscular do Cíngulo dos Membros/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Splicing de RNA/efeitos dos fármacosRESUMO
Fuchs' endothelial corneal dystrophy (FECD) leads to vision loss and is one of the most common inherited eye diseases. Corneal transplants are the only curative treatment available, and there is a major unmet need for treatments that are less invasive and independent of donor tissue. Most cases of FECD are associated with an expanded CUG repeat within the intronic region of TCF4 and the mutant RNA has been implicated as the cause of the disease. We previously presented preliminary data suggesting that single-stranded antisense oligonucleotides (ASOs) can inhibit CUG RNA foci in patient-derived cells and tissue. We now show that duplex RNAs and single-stranded silencing RNAs (ss-siRNAs) reduce the number of cells with foci and the number of foci per cells. Potencies are similar to those that are achieved with chemically modified ASOs designed to block foci. These data widen the potential for synthetic nucleic acids to be used to treat a widely prevalent and debilitating disease.
Assuntos
Distrofia Endotelial de Fuchs/genética , Oligonucleotídeos Antissenso/farmacologia , RNA Interferente Pequeno/farmacologia , Linhagem Celular , Distrofia Endotelial de Fuchs/patologia , Distrofia Endotelial de Fuchs/terapia , Inativação Gênica/efeitos dos fármacos , Predisposição Genética para Doença , Humanos , Íntrons/efeitos dos fármacos , Oligonucleotídeos Antissenso/genética , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/farmacologia , RNA Interferente Pequeno/genética , Fator de Transcrição 4/antagonistas & inibidores , Fator de Transcrição 4/genética , Expansão das Repetições de Trinucleotídeos/efeitos dos fármacos , Expansão das Repetições de Trinucleotídeos/genéticaRESUMO
Specific RNA structures control numerous metabolic processes that impact human health, and yet efforts to target RNA structures de novo have been limited. In eukaryotes, the self-splicing group II intron is a mitochondrial RNA tertiary structure that is absent in vertebrates but essential for respiration in plants, fungi and yeast. Here we show that this RNA can be targeted through a process of high-throughput in vitro screening, SAR and lead optimization, resulting in high-affinity compounds that specifically inhibit group IIB intron splicing in vitro and in vivo and lack toxicity in human cells. The compounds are potent growth inhibitors of the pathogen Candida parapsilosis, displaying antifungal activity comparable to that of amphotericin B. These studies demonstrate that RNA tertiary structures can be successfully targeted de novo, resulting in pharmacologically valuable compounds.
Assuntos
Antifúngicos/química , Antifúngicos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Íntrons/efeitos dos fármacos , Splicing de RNA/efeitos dos fármacos , Candida parapsilosis/efeitos dos fármacos , Candida parapsilosis/genética , Candida parapsilosis/crescimento & desenvolvimento , Complexo IV da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Íntrons/genética , Testes de Sensibilidade Microbiana , RNA Catalítico/genética , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-AtividadeRESUMO
A disintegrin and metalloproteinase domain 33 (ADAM33) gene is a transmembrane glycoprotein that mediates changes in cell adhesion and plays an important role in cancer progression. Since bisphenol A (BPA) and phthalates are epigenetically toxic, the purpose of this study was to examine whether BPA and phthalate metabolites, including monoethyl phthalate (MEP), mononbutyl phthalate (MBP), monoisobutyl phthalate (MIBP), mono(2ethylhexyl) phthalate (MEHP), mono(2ethyl5hydroxyhexyl) phthalate (MEHHP), mono(2ethyl5carboxypentyl) phthalate (MECPP), and mono(2ethyl5oxohexyl) phthalate (MEOHP), have an epigenetic impact on ADAM33 and the incidence of breast cancer. CpG islands of breast cancer microarray datasets obtained from the Gene Expression Omnibus (GEO) were used to assess the ADAM33 methylation profile. We designed a casecontrol study including 44 cases and 22 agematched controls to detect the methylation status of intron 1 in ADAM33 from peripheral blood mononuclear cells (PBMCs) in blood, using BSP, nested PCR, and bisulfite sequencing, and measured the in vivo gene expression of ADAM33 and the urinary concentrations of endocrinedisrupting chemicals (EDCs), using realtime PCR, highperformance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LCMS). Only one dataset, GSE32393, reached significance (P=0.016). ADAM33 expression and methylation frequencies at CpG site 3 in intron 1 were higher in the control group. We found a positive association between intron 1 methylation level and ADAM33 expression as well as urinary concentrations of MEHHP, MECPP, MEOHP and Σ4MEHP (the sum of MEHP, MECPP, MEHHP, and MEOHP) in the cases. This study suggests that metabolites of phthalate such as MEHHP, MECPP, MEOHP and Σ4MEHP may increase the intron 1 methylation level to elevate ADAM33 gene expression and have a protective effect on reducing the risk of breast cancer.
Assuntos
Proteínas ADAM/genética , Neoplasias da Mama/genética , Disruptores Endócrinos/toxicidade , Ácidos Ftálicos/toxicidade , Adulto , Idoso , Compostos Benzidrílicos/toxicidade , Neoplasias da Mama/induzido quimicamente , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Cromatografia Líquida de Alta Pressão , Ilhas de CpG/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Íntrons/efeitos dos fármacos , Pessoa de Meia-Idade , Fenóis/toxicidade , Ácidos Ftálicos/urina , Análise Serial de TecidosRESUMO
Treatment with tyrosine kinase inhibitors (TKI) may sequentially induce TKI-resistant BCR-ABL mutants in chronic myeloid leukemia (CML). Conventional PCR monitoring of BCR-ABL is an important indicator to determine therapeutic intervention for preventing disease progression. However, PCR cannot separately quantify amounts of BCR-ABL and its mutants, including alternatively spliced BCR-ABL with an insertion of 35 intronic nucleotides (BCR-ABLIns35bp ) between ABL exons 8 and 9, which introduces the premature termination and loss of kinase activity. To assess the clinical impact of BCR-ABL mutants, we performed deep sequencing analysis of BCR-ABL transcripts of 409 samples from 37 patients with suboptimal response to frontline imatinib who were switched to nilotinib. At baseline, TKI-resistant mutations were documented in 3 patients, whereas BCR-ABLIns35bp was detected in all patients. After switching to nilotinib, both BCR-ABL and BCR-ABLIns35bp became undetectable in 3 patients who attained complete molecular response (CMR), whereas in the remaining all 34 patients, BCR-ABLIns35bp was persistently detected, and minimal residual disease (MRD) fluctuated at low but detectable levels. PCR monitoring underestimated molecular response in 5 patients whose BCR-ABLIns35bp was persisted, although BCR-ABLIns35bp does not definitively mark TKI resistance. Therefore, quantification of BCR-ABLIns35bp is useful for evaluating "functional" MRD and determining the effectiveness of TKI with accuracy.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/administração & dosagem , Processamento Alternativo/efeitos dos fármacos , Processamento Alternativo/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Éxons/efeitos dos fármacos , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Masculino , Pirimidinas/administração & dosagemRESUMO
OBJECTIVES: We examined mechanisms that contribute to the rapid antidepressant effect of ketamine in mice that is dependent on glycogen synthase kinase-3 (GSK3) inhibition. METHODS: We measured serotonergic (5HT)-2C-receptor (5HTR2C) cluster microRNA (miRNA) levels in mouse hippocampus after administering an antidepressant dose of ketamine (10 mg/kg) in wild-type and GSK3 knockin mice, after GSK3 inhibition with L803-mts, and in learned helpless mice. RESULTS: Ketamine up-regulated cluster miRNAs 448-3p, 764-5p, 1264-3p, 1298-5p and 1912-3p (2- to 11-fold). This up-regulation was abolished in GSK3 knockin mice that express mutant constitutively active GSK3. The GSK3 specific inhibitor L803-mts was antidepressant in the learned helplessness and novelty suppressed feeding depression-like behaviours and up-regulated the 5HTR2C miRNA cluster in mouse hippocampus. After administration of the learned helplessness paradigm mice were divided into cohorts that were resilient (non-depressed) or were susceptible (depressed) to learned helplessness. The resilient, but not depressed, mice displayed increased hippocampal levels of miRNAs 448-3p and 1264-3p. Administration of an antagonist to miRNA 448-3p diminished the antidepressant effect of ketamine in the learned helplessness paradigm, indicating that up-regulation of miRNA 448-3p provides an antidepressant action. CONCLUSIONS: These findings identify a new outcome of GSK3 inhibition by ketamine that may contribute to antidepressant effects.
Assuntos
Antidepressivos/farmacologia , Comportamento Animal/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Íntrons/efeitos dos fármacos , Ketamina/farmacologia , MicroRNAs/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Receptor 5-HT2C de Serotonina/efeitos dos fármacos , Animais , Antidepressivos/administração & dosagem , Depressão/tratamento farmacológico , Modelos Animais de Doenças , Desamparo Aprendido , Ketamina/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Regulação para CimaRESUMO
CD4+ T cells in female MRL+/+ mice exposed to solvent and water pollutant trichloroethylene (TCE) skew toward effector/memory CD4+ T cells, and demonstrate seemingly non-monotonic alterations in IFN-γ production. In the current study we examined the mechanism for this immunotoxicity using effector/memory and naïve CD4+ T cells isolated every 6 weeks during a 40 week exposure to TCE (0.5mg/ml in drinking water). A time-dependent effect of TCE exposure on both Ifng gene expression and IFN-γ protein production was observed in effector/memory CD4+ T cells, with an increase after 22 weeks of exposure and a decrease after 40 weeks of exposure. No such effect of TCE was observed in naïve CD4+ T cells. A cumulative increase in DNA methylation in the CpG sites of the promoter of the Ifng gene was observed in effector/memory, but not naïve, CD4+ T cells over time. Also unique to the Ifng promoter was an increase in methylation variance in effector/memory compared to naïve CD4+ T cells. Taken together, the CpG sites of the Ifng promoter in effector/memory CD4+ T cells were especially sensitive to the effects of TCE exposure, which may help explain the regulatory effect of the chemical on this gene.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Interferon gama/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacos , Solventes/toxicidade , Tricloroetileno/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Ilhas de CpG/efeitos dos fármacos , Epigênese Genética/efeitos dos fármacos , Éxons/efeitos dos fármacos , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Hormese , Memória Imunológica/efeitos dos fármacos , Interferon gama/agonistas , Interferon gama/antagonistas & inibidores , Interferon gama/genética , Íntrons/efeitos dos fármacos , Camundongos Endogâmicos MRL lpr , Reprodutibilidade dos Testes , Solventes/administração & dosagem , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Fatores de Tempo , Tricloroetileno/administração & dosagem , Poluentes Químicos da Água/administração & dosagemRESUMO
The M-transcript of human choline acetyltransferase (ChAT) produces an 82-kDa protein (82-kDa ChAT) that concentrates in nuclei of cholinergic neurons. We assessed the effects of acute exposure to oligomeric amyloid-ß1-42 (Aß1-42) on 82-kDa ChAT disposition in SH-SY5Y neural cells, finding that acute exposure to Aß1-42 results in increased association of 82-kDa ChAT with chromatin and formation of 82-kDa ChAT aggregates in nuclei. When measured by chromatin immunoprecipitation with next-generation sequencing (ChIP-seq), we identified that Aß1-42-exposure increases 82-kDa ChAT association with gene promoters and introns. The Aß1-42-induced 82-kDa ChAT aggregates co-localize with special AT-rich binding protein 1 (SATB1), which anchors DNA to scaffolding/matrix attachment regions (S/MARs). SATB1 had a similar genomic association as 82-kDa ChAT, with both proteins associating with synapse and cell stress genes. After Aß1-42 -exposure, both SATB1 and 82-kDa ChAT are enriched at the same S/MAR on the APP gene, with 82-kDa ChAT expression attenuating an increase in an isoform-specific APP mRNA transcript. Finally, 82-kDa ChAT and SATB1 have patterned genomic association at regions enriched with S/MAR binding motifs. These results demonstrate that 82-kDa ChAT and SATB1 play critical roles in the response of neural cells to acute Aß-exposure.
Assuntos
Peptídeos beta-Amiloides/farmacologia , Colina O-Acetiltransferase/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Regiões de Interação com a Matriz/efeitos dos fármacos , Neurônios/citologia , Precursor de Proteína beta-Amiloide/genética , Linhagem Celular , Núcleo Celular/metabolismo , Imunoprecipitação da Cromatina , Epigênese Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Íntrons/efeitos dos fármacos , Peso Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
BACKGROUND: The majority of beta-defensin family members are exclusively expressed in the epididymis, and some members have been shown to play essential roles in sperm maturation and fertility in rats, mice and humans. Therefore, beta-defensins are hypothesized to be potential targets for contraception and infertility diagnosis and treatment. Clarifying the regulatory mechanisms for the expression of these genes is necessary. Androgen/androgen receptor (AR) signaling plays an important regulatory role in epididymal structure and function. However, very little is known about the androgenic regulation on the production and secretion of the epididymal beta-defensins. METHODS: The expression of beta-defensins was detected by quantitative RT-PCR. The androgen dependence of beta-defensins was determined by bilateral orchiectomy and androgen supplementation. The androgen response elements (AREs) in the promoters of beta-defensins were identified using the MatInspector software. The binding of AR to AREs was assayed by ChIP-PCR/qPCR. RESULTS: We demonstrated that 23 mouse caput epididymal beta-defensins were differentially regulated by androgen/androgen receptor. Six genes, Defb18, 19, 20, 39, 41, and 42, showed full regulation by androgens. Ten genes, Defb15, 30, 34, 37, 40, 45, 51, 52, 22 and Spag11a, were partially regulated by androgens. Defb15, 18, 19, 20, 30, 34, 37, 39, 41, 42, 22 and Spag11a were associated with androgen receptor binding sites in their promoter or intronic regions, indicating direct regulation of AR. Six genes, Defb1, 12, 13, 29, 35, and spag11b/c, exhibited an androgen-independent expression pattern. One gene, Defb25, was highly dependent on testicular factors rather on androgens. CONCLUSIONS: The present study provides novel insights into the mechanisms of androgen regulation on epididymal beta-defensins, enabling a better understanding of the function of beta-defensins in sperm maturation and fertility.
Assuntos
Androgênios/farmacologia , Epididimo/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Elementos de Resposta/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , beta-Defensinas/metabolismo , Androgênios/administração & dosagem , Androgênios/química , Androgênios/metabolismo , Animais , Sítios de Ligação , Castração , Imunoprecipitação da Cromatina , Biologia Computacional , Epididimo/metabolismo , Injeções Intraperitoneais , Íntrons/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/efeitos dos fármacos , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Androgênicos/química , Espermatogênese/efeitos dos fármacos , Propionato de Testosterona/administração & dosagem , Propionato de Testosterona/química , Propionato de Testosterona/metabolismo , Propionato de Testosterona/farmacologia , beta-Defensinas/agonistas , beta-Defensinas/antagonistas & inibidores , beta-Defensinas/genéticaRESUMO
In tsetse flies, nutrients for intrauterine larval development are synthesized by the modified accessory gland (milk gland) and provided in mother's milk during lactation. Interference with at least two milk proteins has been shown to extend larval development and reduce fecundity. The goal of this study was to perform a comprehensive characterization of tsetse milk proteins using lactation-specific transcriptome/milk proteome analyses and to define functional role(s) for the milk proteins during lactation. Differential analysis of RNA-seq data from lactating and dry (non-lactating) females revealed enrichment of transcripts coding for protein synthesis machinery, lipid metabolism and secretory proteins during lactation. Among the genes induced during lactation were those encoding the previously identified milk proteins (milk gland proteins 1-3, transferrin and acid sphingomyelinase 1) and seven new genes (mgp4-10). The genes encoding mgp2-10 are organized on a 40 kb syntenic block in the tsetse genome, have similar exon-intron arrangements, and share regions of amino acid sequence similarity. Expression of mgp2-10 is female-specific and high during milk secretion. While knockdown of a single mgp failed to reduce fecundity, simultaneous knockdown of multiple variants reduced milk protein levels and lowered fecundity. The genomic localization, gene structure similarities, and functional redundancy of MGP2-10 suggest that they constitute a novel highly divergent protein family. Our data indicates that MGP2-10 function both as the primary amino acid resource for the developing larva and in the maintenance of milk homeostasis, similar to the function of the mammalian casein family of milk proteins. This study underscores the dynamic nature of the lactation cycle and identifies a novel family of lactation-specific proteins, unique to Glossina sp., that are essential to larval development. The specificity of MGP2-10 to tsetse and their critical role during lactation suggests that these proteins may be an excellent target for tsetse-specific population control approaches.
Assuntos
Abortivos/farmacologia , Genes de Insetos/genética , Proteínas de Insetos/genética , Reprodução/efeitos dos fármacos , Reprodução/genética , Moscas Tsé-Tsé/efeitos dos fármacos , Moscas Tsé-Tsé/genética , Sequência de Aminoácidos , Animais , Éxons/efeitos dos fármacos , Éxons/genética , Feminino , Fertilidade/efeitos dos fármacos , Fertilidade/genética , Perfilação da Expressão Gênica/métodos , Técnicas de Silenciamento de Genes/métodos , Íntrons/efeitos dos fármacos , Íntrons/genética , Lactação/efeitos dos fármacos , Lactação/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Metabolismo dos Lipídeos/genética , Masculino , Proteínas do Leite/genética , Filogenia , Proteoma/genética , RNA/genética , Análise de Sequência de RNA/métodos , Transcriptoma/genéticaRESUMO
BACKGROUND: Spinal muscular atrophy (SMA) is a fatal motor neuron disease of childhood that is caused by mutations in the SMN1 gene. Currently, no effective treatment is available. One possible therapeutic approach is the use of antisense oligos (ASOs) to redirect the splicing of the paralogous gene SMN2, thus increasing functional SMN protein production. Various ASOs with different chemical properties are suitable for these applications, including a morpholino oligomer (MO) variant with a particularly excellent safety and efficacy profile. OBJECTIVE: We investigated a 25-nt MO sequence targeting the negative intronic splicing silencer (ISS-N1) 10 to 34 region. METHODS: We administered a 25-nt MO sequence against the ISS-N1 region of SMN2 (HSMN2Ex7D[-10-34]) in the SMAΔ7 mouse model and evaluated the effect and neuropathologic phenotype. We tested different concentrations (from 2 to 24 nM) and delivery protocols (intracerebroventricular injection, systemic injection, or both). We evaluated the treatment efficacy regarding SMN levels, survival, neuromuscular phenotype, and neuropathologic features. RESULTS: We found that a 25-nt MO sequence against the ISS-N1 region of SMN2 (HSMN2Ex7D[-10-34]) exhibited superior efficacy in transgenic SMAΔ7 mice compared with previously described sequences. In our experiments, the combination of local and systemic administration of MO (bare or conjugated to octaguanidine) was the most effective approach for increasing full-length SMN expression, leading to robust improvement in neuropathologic features and survival. Moreover, we found that several small nuclear RNAs were deregulated in SMA mice and that their levels were restored by MO treatment. CONCLUSION: These results indicate that MO-mediated SMA therapy is efficacious and can result in phenotypic rescue, providing important insights for further development of ASO-based therapeutic strategies in SMA patients.
Assuntos
Íntrons/efeitos dos fármacos , Morfolinos/administração & dosagem , Morfolinos/uso terapêutico , Atrofia Muscular Espinal/tratamento farmacológico , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/uso terapêutico , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Animais , Comportamento/efeitos dos fármacos , Modelos Animais de Doenças , Injeções , Camundongos , Camundongos Transgênicos , Neurônios Motores , Atrofia Muscular Espinal/genética , Fenótipo , Splicing de RNA , RNA Nuclear Pequeno/efeitos dos fármacos , Teste de Desempenho do Rota-Rod , Medula EspinalRESUMO
The gene encoding the receptor for macrophage colony-stimulating factor (CSF-1R) is expressed exclusively in cells of the myeloid lineages as well as trophoblasts. A conserved element in the second intron, Fms-Intronic Regulatory Element (FIRE), is essential for macrophage-specific transcription of the gene. However, the molecular details of how FIRE activity is regulated and how it impacts the Csf1r promoter have not been characterised. Here we show that agents that down-modulate Csf1r mRNA transcription regulated promoter activity altered the occupancy of key FIRE cis-acting elements including RUNX1, AP1, and Sp1 binding sites. We demonstrate that FIRE acts as an anti-sense promoter in macrophages and reversal of FIRE orientation within its native context greatly reduced enhancer activity in macrophages. Mutation of transcription initiation sites within FIRE also reduced transcription. These results demonstrate that FIRE is an orientation-specific transcribed enhancer element.
Assuntos
Sequência Conservada/genética , Loci Gênicos/genética , Íntrons/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Sequência de Bases , Sítios de Ligação , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/genética , Loci Gênicos/efeitos dos fármacos , Humanos , Íntrons/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/metabolismo , Camundongos , Dados de Sequência Molecular , RNA Polimerase II/genética , RNA Antissenso/genética , RNA Mensageiro/genética , Fator de Transcrição Sp1/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/metabolismoRESUMO
P2X2 plays an important role in ATP signaling in guinea pig myenteric plexus. Here, we cloned and characterized three P2X2 isoforms expressed in myenteric neurons. RT/PCR was used to amplify the cDNA of P2X2 variants. These were expressed in Xenopus oocytes, and nucleotide-induced membrane currents were recorded with the two-electrode voltage clamp technique. Three P2X2 cDNAs were identified in myenteric single neurons, named P2X2-1, P2X2-2 and P2X2-4. Based on the analysis of the structural organization of these variants we predicted that P2X2-2 is the fully processed variant, which lead us to propose a new exon-intron arrangement of P2X2 receptor gene with 12 exons and 11 introns. In agreement with this new model, the intron 11 is retained in P2X2-1 and P2X2-4 variants by alternative splicing. Expression of P2X2-1, P2X2-2 and P2X2-4 were found in 92, 42 and 37%, respectively, out of 40 analyzed single neurons. P2X2-4 does not form functional channels, and homomeric channels formed by P2X2-1 and P2X2-2 have different pharmacological profile. Thus, the former receptor is more sensitive to ATP, BzATP, and PPADS, whereas, suramin inhibited both receptors in a biphasic- and monophasic-manner, respectively. α,ß-meATP has very low efficacy on either channel. Furthermore, ionic currents mediated by P2X2-1 have slower desensitization than P2X2-2. These results indicate that P2X2-1 was the most common P2X2 transcript in myenteric neurons and displays significant phenotypical changes implicating that retention of the intron 11 plays a major role in ATP signaling in the intestinal myenteric plexus.
Assuntos
Íntrons/efeitos dos fármacos , Íntrons/genética , Plexo Mientérico/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P2X2/efeitos dos fármacos , Receptores Purinérgicos P2X2/genética , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Fenômenos Eletrofisiológicos , Éxons/genética , Éxons/fisiologia , Feminino , Cobaias , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Cinética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Plexo Mientérico/citologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Isoformas de Proteínas , Reação em Cadeia da Polimerase em Tempo Real , Xenopus laevisRESUMO
The androgen receptor (AR) signaling axis plays a key role in the pathogenesis of prostate cancer. In this study, we found that the protein tyrosine phosphatase PTP1B, a well-established regulator of metabolic signaling, was induced after androgen stimulation of AR-expressing prostate cancer cells. PTP1B induction by androgen occurred at the mRNA and protein levels to increase PTP1B activity. High-resolution chromosome mapping revealed AR recruitment to two response elements within the first intron of the PTP1B encoding gene PTPN1, correlating with an AR-mediated increase in RNA polymerase II recruitment to the PTPN1 transcriptional start site. We found that PTPN1 and AR genes were coamplified in metastatic tumors and that PTPN1 amplification was associated with a subset of high-risk primary tumors. Functionally, PTP1B depletion delayed the growth of androgen-dependent human prostate tumors and impaired androgen-induced cell migration and invasion in vitro. However, PTP1B was also required for optimal cell migration of androgen-independent cells. Collectively, our results established the AR as a transcriptional regulator of PTPN1 transcription and implicated PTP1B in a tumor-promoting role in prostate cancer. Our findings support the preclinical testing of PTP1B inhibitors for prostate cancer treatment.