RESUMO
Intestinal microbiota have been proposed to induce commensal-specific memory T cells that cross-react with tumor-associated antigens. We identified major histocompatibility complex (MHC) class I-binding epitopes in the tail length tape measure protein (TMP) of a prophage found in the genome of the bacteriophage Enterococcus hirae Mice bearing E. hirae harboring this prophage mounted a TMP-specific H-2Kb-restricted CD8+ T lymphocyte response upon immunotherapy with cyclophosphamide or anti-PD-1 antibodies. Administration of bacterial strains engineered to express the TMP epitope improved immunotherapy in mice. In renal and lung cancer patients, the presence of the enterococcal prophage in stools and expression of a TMP-cross-reactive antigen by tumors correlated with long-term benefit of PD-1 blockade therapy. In melanoma patients, T cell clones recognizing naturally processed cancer antigens that are cross-reactive with microbial peptides were detected.
Assuntos
Antígenos de Neoplasias/imunologia , Bacteriófagos/imunologia , Streptococcus faecium ATCC 9790/virologia , Microbioma Gastrointestinal/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Proteínas da Cauda Viral/imunologia , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Alquilantes/uso terapêutico , Linfócitos T CD8-Positivos/imunologia , Reações Cruzadas , Ciclofosfamida/uso terapêutico , Epitopos/imunologia , Fezes/virologia , Antígenos H-2/imunologia , Humanos , Camundongos , Neoplasias/dietoterapia , Neoplasias/imunologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Proteínas da Cauda Viral/uso terapêuticoRESUMO
The integrated stress response is characterized by the phosphorylation of eukaryotic initiation factor-2α (eIF2α) on serine 51 by one out of four specific kinases (EIF2AK1 to 4). Here we provide three series of evidence suggesting that macroautophagy (to which we refer to as autophagy) induced by a variety of distinct pharmacological agents generally requires this phosphorylation event. First, the induction of autophagic puncta by various distinct compounds was accompanied by eIF2α phosphorylation on serine 51. Second, the modulation of autophagy by >30 chemically unrelated agents was partially inhibited in cells expressing a non-phosphorylable (S51A) mutant of eIF2α or lacking all four eIF2α kinases, although distinct kinases were involved in the response to different autophagy inducers. Third, inhibition of eIF2α phosphatases was sufficient to stimulate autophagy. In synthesis, it appears that eIF2α phosphorylation is a central event for the stimulation of autophagy.
Assuntos
Fator de Iniciação 2 em Eucariotos/metabolismo , Animais , Autofagia/fisiologia , Linhagem Celular Tumoral , Humanos , Camundongos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação/efeitos dos fármacos , eIF-2 Quinase/antagonistas & inibidores , eIF-2 Quinase/metabolismoRESUMO
Chemotherapy still constitutes the standard of care for the treatment of most neoplastic diseases. Certain chemotherapeutics from the oncological armamentarium are able to trigger pre-mortem stress signals that lead to immunogenic cell death (ICD), thus inducing an antitumor immune response and mediating long-term tumor growth reduction. Here, we used an established model, built on artificial intelligence to identify, among a library of 50,000 compounds, anticancer agents that, based on their molecular descriptors, were predicted to induce ICD. This algorithm led us to the identification of dactinomycin (DACT, best known as actinomycin D), a highly potent cytotoxicant and ICD inducer that mediates immune-dependent anticancer effects in vivo. Since DACT is commonly used as an inhibitor of DNA to RNA transcription, we investigated whether other experimentally established or algorithm-selected, clinically employed ICD inducers would share this characteristic. As a common leitmotif, a panel of pharmacological ICD stimulators inhibited transcription and secondarily translation. These results establish the inhibition of RNA synthesis as an initial event for ICD induction.
Assuntos
Antineoplásicos , Neoplasias , Antineoplásicos/uso terapêutico , Inteligência Artificial , Dactinomicina/farmacologia , Dactinomicina/uso terapêutico , Humanos , Morte Celular Imunogênica , Neoplasias/tratamento farmacológicoRESUMO
Mutations affecting exon 9 of the CALR gene lead to the generation of a C-terminally modified calreticulin (CALR) protein that lacks the KDEL endoplasmic reticulum (ER) retention signal and consequently mislocalizes outside of the ER where it activates the thrombopoietin receptor in a cell-autonomous fashion, thus driving myeloproliferative diseases. Here, we used the retention using selective hooks (RUSH) assay to monitor the trafficking of CALR. We found that exon-9-mutated CALR was released from cells in response to the biotin-mediated detachment from its ER-localized hook, in vitro and in vivo. Cellular CALR release was confirmed in suitable mouse models bearing exon-9-mutated hematopoietic systems or tumors. Extracellular CALR mediated immunomodulatory effects and inhibited the phagocytosis of dying cancer cells by dendritic cells (DC), thereby suppressing antineoplastic immune responses elicited by chemotherapeutic agents or by PD-1 blockade. Altogether, our results demonstrate paracrine immunosuppressive effects for exon-9-mutated CALR.
Assuntos
Calreticulina/genética , Tolerância Imunológica/genética , Mutação , Neoplasias/genética , Neoplasias/imunologia , Animais , Calreticulina/metabolismo , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos C57BL , FagocitoseRESUMO
cis-Regulatory communication is crucial in mammalian development and is thought to be restricted by the spatial partitioning of the genome in topologically associating domains (TADs). Here, we discovered that the Xist locus is regulated by sequences in the neighboring TAD. In particular, the promoter of the noncoding RNA Linx (LinxP) acts as a long-range silencer and influences the choice of X chromosome to be inactivated. This is independent of Linx transcription and independent of any effect on Tsix, the antisense regulator of Xist that shares the same TAD as Linx. Unlike Tsix, LinxP is well conserved across mammals, suggesting an ancestral mechanism for random monoallelic Xist regulation. When introduced in the same TAD as Xist, LinxP switches from a silencer to an enhancer. Our study uncovers an unsuspected regulatory axis for X chromosome inactivation and a class of cis-regulatory effects that may exploit TAD partitioning to modulate developmental decisions.
Assuntos
Sequência Conservada/genética , RNA Longo não Codificante/genética , Cromossomo X/genética , Animais , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Camundongos , Regiões Promotoras Genéticas/genética , RNA Antissenso/genética , Elementos Silenciadores Transcricionais/genética , Transcrição Gênica/genéticaRESUMO
The retention using selective hooks (RUSH) system allows to retain a target protein fused to green fluorescent protein (GFP) and a streptavidin-binding peptide (SBP) due to the interaction with a molar excess of streptavidin molecules ("hooks") targeted to selected subcellular compartments. Supplementation of biotin competitively disrupts the interaction between the SBP moiety and streptavidin, liberating the chimeric target protein from its hooks, while addition of avidin causes the removal of biotin from the system and reestablishes the interaction. Based on this principle, we engineered two chimeric proteins involved in autophagy, namely microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B, best known as LC3) and sequestosome-1 (SQSTM1, best known as p62) to move them as SBP-GFP-LC3 and p62-SBP-GFP at will between the cytosol and two different organelles, the endoplasmic reticulum (ER) and the Golgi apparatus. Although both proteins were functional in thus far that SBP-GFP-LC3 and p62-SBP-GFP could recruit their endogenous binding partners, p62 and LC3, respectively, their enforced relocation to the ER or Golgi failed to induce organelle-specific autophagy. Hence, artificial tethering of LC3 or p62 to the surface of the ER and the Golgi is not sufficient to trigger autophagy.
Assuntos
Autofagia/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia/efeitos dos fármacos , Biotina/metabolismo , Linhagem Celular Tumoral , Citosol/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Ligação Proteica/genética , Ligação Proteica/fisiologia , Transporte Proteico/genética , Transporte Proteico/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estreptavidina/metabolismoRESUMO
Autophagy facilitates the adaptation to nutritional stress. Here, we show that short-term starvation of cultured cells or mice caused the autophagy-dependent cellular release of acyl-CoA-binding protein (ACBP, also known as diazepam-binding inhibitor, DBI) and consequent ACBP-mediated feedback inhibition of autophagy. Importantly, ACBP levels were elevated in obese patients and reduced in anorexia nervosa. In mice, systemic injection of ACBP protein inhibited autophagy, induced lipogenesis, reduced glycemia, and stimulated appetite as well as weight gain. We designed three approaches to neutralize ACBP, namely, inducible whole-body knockout, systemic administration of neutralizing antibodies, and induction of antiACBP autoantibodies in mice. ACBP neutralization enhanced autophagy, stimulated fatty acid oxidation, inhibited appetite, reduced weight gain in the context of a high-fat diet or leptin deficiency, and accelerated weight loss in response to dietary changes. In conclusion, neutralization of ACBP might constitute a strategy for treating obesity and its co-morbidities.
Assuntos
Inibidor da Ligação a Diazepam/metabolismo , Ingestão de Alimentos , Lipogênese , Macroautofagia , Obesidade/metabolismo , Animais , Anorexia Nervosa/metabolismo , Linhagem Celular , Ácidos Graxos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Aumento de Peso , Redução de PesoRESUMO
The retention using selective hooks (RUSH) system allows to withhold a fluorescent biosensor such as green fluorescent protein (GFP) fused to a streptavidin-binding peptide (SBP) by an excess of streptavidin molecules that are addressed to different subcellular localizations. Addition of biotin competitively disrupts this interaction, liberating the biosensor from its hook. We constructed a human cell line co-expressing soluble secretory-SBP-GFP (ss-SBP-GFP) and streptavidin within the endoplasmic reticulum (ER) lumen and then used this system to screen a compound library for inhibitors of the biotin-induced release of ss-SBP-GFP via the conventional Golgi-dependent protein secretion pathway into the culture supernatant. We identified and validated a series of molecularly unrelated drugs including antianginal, antidepressant, anthelmintic, antipsychotic, antiprotozoal and immunosuppressive agents that inhibit protein secretion. These compounds vary in their capacity to suppress protein synthesis and to compromise ER morphology and Golgi integrity, as well as in the degree of reversibility of such effects. In sum, we demonstrate the feasibility and utility of a novel RUSH-based phenotypic screening assay.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas/metabolismo , Linhagem Celular Tumoral , Análise por Conglomerados , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Análise de Componente Principal , Biossíntese de Proteínas , Estrutura Secundária de Proteína , Proteínas/química , Reprodutibilidade dos TestesRESUMO
A systematic search for anticancer agents that may induce the release of high mobility group box 1 (HMGB1) protein from cells into the extracellular space has led to the identification of several drugs capable of elevating plasma HMGB1 levels in vivo, in mice. Such agents include bona-fide immunogenic cell death inducers such as oxaliplatin, as well as a series of epigenetic modifiers, namely azacitidine, decitabine, and suberoylanilide hydroxamic acid (SAHA).
RESUMO
Preclinical evidence depicts the capacity of redaporfin (Redp) to act as potent photosensitizer, causing direct antineoplastic effects as well as indirect immune-dependent destruction of malignant lesions. Here, we investigated the mechanisms through which photodynamic therapy (PDT) with redaporfin kills cancer cells. Subcellular localization and fractionation studies based on the physicochemical properties of redaporfin revealed its selective tropism for the endoplasmic reticulum (ER) and the Golgi apparatus (GA). When activated, redaporfin caused rapid reactive oxygen species-dependent perturbation of ER/GA compartments, coupled to ER stress and an inhibition of the GA-dependent secretory pathway. This led to a general inhibition of protein secretion by PDT-treated cancer cells. The ER/GA play a role upstream of mitochondria in the lethal signaling pathway triggered by redaporfin-based PDT Pharmacological perturbation of GA function or homeostasis reduces mitochondrial permeabilization. In contrast, removal of the pro-apoptotic multidomain proteins BAX and BAK or pretreatment with protease inhibitors reduced cell killing, yet left the GA perturbation unaffected. Altogether, these results point to the capacity of redaporfin to kill tumor cells via destroying ER/GA function.
Assuntos
Retículo Endoplasmático/efeitos dos fármacos , Complexo de Golgi/efeitos dos fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Sulfonamidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Retículo Endoplasmático/fisiologia , Feminino , Complexo de Golgi/fisiologia , Humanos , Luz , Camundongos Endogâmicos C57BL , Fármacos Fotossensibilizantes/efeitos da radiação , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/efeitos da radiação , Porfirinas/uso terapêutico , Sulfonamidas/efeitos da radiação , Sulfonamidas/uso terapêuticoRESUMO
The age-associated deterioration in cellular and organismal functions associates with dysregulation of nutrient-sensing pathways and disabled autophagy. The reactivation of autophagic flux may prevent or ameliorate age-related metabolic dysfunctions. Non-toxic compounds endowed with the capacity to reduce the overall levels of protein acetylation and to induce autophagy have been categorized as caloric restriction mimetics (CRMs). Here, we show that aspirin or its active metabolite salicylate induce autophagy by virtue of their capacity to inhibit the acetyltransferase activity of EP300. While salicylate readily stimulates autophagic flux in control cells, it fails to further increase autophagy levels in EP300-deficient cells, as well as in cells in which endogenous EP300 has been replaced by salicylate-resistant EP300 mutants. Accordingly, the pro-autophagic activity of aspirin and salicylate on the nematode Caenorhabditis elegans is lost when the expression of the EP300 ortholog cpb-1 is reduced. Altogether, these findings identify aspirin as an evolutionary conserved CRM.
Assuntos
Aspirina/farmacologia , Restrição Calórica , Acetilcoenzima A/metabolismo , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Proteína p300 Associada a E1A/metabolismo , Humanos , Metaboloma/efeitos dos fármacos , Metabolômica , Camundongos Endogâmicos C57BLRESUMO
The translocation of the protein high mobility group box 1 (HMGB1) from the nucleus to the cytoplasm and its secretion or passive release through the permeabilized plasma membrane, constitutes a major cellular danger signal. Extracellular HMGB1 can interact with pattern recognition receptors to stimulate pro-inflammatory and immunostimulatory pathways. Here, we developed a screening assay to identify pharmacological agents endowed with HMGB1 releasing properties. For this, we took advantage of the "retention using selective hooks" (RUSH) system in which a streptavidin-NLS3 fusion protein was used as a nuclear hook to sequestrate streptavidin-binding peptide (SBP) fused with HMGB1 and green fluorescent protein (GFP). When combined with biotin, which competitively disrupts the interaction between streptavidin-NLS3 and HMGB1-SBP-GFP, immunogenic cell death (ICD) inducers such as anthracyclines were able to cause the nucleo-cytoplasmic translocation of HMGB1-SBP-GFP. This system, was used in a high-content screening (HCS) campaign for the identification of HMGB1 releasing agents. Hits fell into three functional categories: known ICD inducers, microtubule inhibitors and epigenetic modifiers. These agents induced ICD through a panoply of distinct mechanisms. Their effective action was confirmed by multiple methods monitoring nuclear, cytoplasmic and extracellular HMGB1 pools, both in cultured human or murine cells, as well as in mouse plasma.
Assuntos
Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Proteína HMGB1/metabolismo , Transporte Proteico/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Feminino , Proteína HMGB1/análise , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Moduladores de Tubulina/farmacologiaRESUMO
Some anticancer agents induce immunogenic cell death that is accompanied by the emission of danger signals into the tumor microenvironment, thus attracting and activating innate immune effectors and finally inducing anticancer immunity. The release of extracellular nucleosides such as adenosine triphosphate (ATP) from the tumor in response to anticancer therapy plays a pivotal role in the attraction of antigen presenting cells and the activation of inflammasome-mediated proinflammatory cascades. In contrast, the ectonucleotidase-catalyzed phosphohydrolysis of nucleotides to nucleosides reduces the extracellular availability of nucleotides, hence limiting the recruitment and activation of antigen-presenting cells. In addition, the (over-)production of nucleosides including adenosine by ectonucleotidases located on cancer cells and regulatory T cells can induce immunosuppression, as adenosine directly inhibits the proliferation and activation of effector T cells. Here, we discuss the importance of death metabolites for immunomodulation in general, and the role of the purine nucleotide ATP and its derivative adenosine in particular. In addition, we provide an overview on therapeutic interventions that reinstate tumor immunogenicity in conditions where nucleotide-dependent immunostimulation is obstructed.
Assuntos
Morte Celular , Fatores Imunológicos/metabolismo , Imunoterapia/métodos , Neoplasias/imunologia , Nucleosídeos/metabolismo , Nucleotídeos/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Apresentação de Antígeno , Espaço Extracelular , Humanos , Terapia de Imunossupressão , Ativação Linfocitária , Microambiente TumoralRESUMO
Xist is indispensable for X chromosome inactivation. However, how Xist RNA directs chromosome-wide silencing and why some regions are more efficiently silenced than others remains unknown. Here, we explore the function of Xist by inducing ectopic Xist expression from multiple different X-linked and autosomal loci in mouse aneuploid and female diploid embryonic stem cells in which Xist-mediated silencing does not lead to lethal functional monosomy. We show that ectopic Xist expression faithfully recapitulates endogenous X chromosome inactivation from any location on the X chromosome, whereas long-range silencing of autosomal genes is less efficient. Long interspersed elements facilitate inactivation of genes located far away from the Xist transcription locus, and genes escaping X chromosome inactivation show enrichment of CTCF on X chromosomal but not autosomal loci. Our findings highlight important genomic and epigenetic features acquired during sex chromosome evolution to facilitate an efficient X chromosome inactivation process.Xist RNA is required for X chromosome inactivation but it is not well understood how Xist silences some regions more efficiently than others. Here, the authors induce ectopic Xist expression from multiple different X-linked and autosomal loci in cells to explore Xist function.
Assuntos
Evolução Molecular , RNA Longo não Codificante/fisiologia , Inativação do Cromossomo X/genética , Animais , Fator de Ligação a CCCTC/metabolismo , Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Inativação Gênica , Elementos Nucleotídeos Longos e Dispersos/fisiologia , Camundongos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
In spite of the emergence of genome editing tools, ES cell mediated transgenesis remains the most controllable way of creating genetically modified animals. Although tetraploid (4N) complementation of 4N host embryos and ES cells, is the only method guaranteeing that offspring are entirely ES cell derived, this technique is challenging, not always successful and difficult to implement in some laboratory settings. The current study shows that pretreatment of host blastocysts with FGF4 prior to ES cell injection can provide an alternative method for the generation of animals displaying high rates of chimaerism. Chimaerism assessment in E11 fetuses and born pups shows that a large percentage of resulting conceptuses show a high ES cell contribution from implantation onwards and that developing pups do not necessitate c-section for delivery.
Assuntos
Animais Geneticamente Modificados/genética , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias , Fator 4 de Crescimento de Fibroblastos/genética , Animais , Animais Geneticamente Modificados/crescimento & desenvolvimento , Blastocisto/metabolismo , Quimera/genética , Quimera/crescimento & desenvolvimento , Clonagem de Organismos , Fator 4 de Crescimento de Fibroblastos/metabolismo , Edição de Genes/métodos , Técnicas de Transferência de Genes , CamundongosRESUMO
In female mammals, X chromosome inactivation (XCI) is a key process in the control of gene dosage compensation between X-linked genes and autosomes. Xist and Tsix, two overlapping antisense-transcribed noncoding genes, are central elements of the X inactivation center (Xic) regulating XCI. Xist upregulation results in the coating of the entire X chromosome by Xist RNA in cis, whereas Tsix transcription acts as a negative regulator of Xist Here, we generated Xist and Tsix reporter mouse embryonic stem (ES) cell lines to study the genetic and dynamic regulation of these genes upon differentiation. Our results revealed mutually antagonistic roles for Tsix on Xist and vice versa and indicate the presence of semistable transcriptional states of the Xic locus predicting the outcome of XCI. These transcriptional states are instructed by the X-to-autosome ratio, directed by regulators of XCI, and can be modulated by tissue culture conditions.
Assuntos
Cromossomos de Mamíferos/genética , RNA Longo não Codificante/genética , Transcrição Gênica , Cromossomo X/genética , Alelos , Animais , Linhagem Celular , Feminino , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Genes Reporter , Loci Gênicos , Camundongos , Modelos Genéticos , RNA Longo não Codificante/metabolismo , Inativação do Cromossomo X/genéticaRESUMO
Genome-wide gene expression studies have indicated that the eukaryotic genome contains many gene pairs showing overlapping sense and antisense transcription. Regulation of these coding and/or noncoding gene pairs involves intricate regulatory mechanisms. In the present study, we utilized an enhanced green fluorescent protein (EGFP)-tagged reporter plasmid cis linked to a doxycycline-inducible antisense promoter, generating antisense transcription that fully overlaps EGFP, to study the mechanism and dynamics of gene silencing after induction of noncoding antisense transcription in undifferentiated and differentiating mouse embryonic stem cells (ESCs). We found that EGFP silencing is reversible in ESCs but is locked into a stable state upon ESC differentiation. Reversible silencing in ESCs is chromatin dependent and is associated with accumulation of trimethylated lysine 36 on histone H3 (H3K36me3) at the EGFP promoter region. In differentiating ESCs, antisense transcription-induced accumulation of H3K36me3 was associated with an increase in CpG methylation at the EGFP promoter. Repression of the sense promoter was affected by small-molecule inhibitors which interfere with DNA methylation and histone demethylation pathways. Our results indicate a general mechanism for silencing of fully overlapping sense-antisense gene pairs involving antisense transcription-induced accumulation of H3K36me3 at the sense promoter, resulting in reversible silencing of the sense partner, which is stabilized during ESC differentiation by CpG methylation.
Assuntos
Diferenciação Celular/genética , Cromatina/genética , Células-Tronco Embrionárias/metabolismo , Inativação Gênica , Animais , Células Cultivadas , Cromatina/metabolismo , Ilhas de CpG/genética , Metilação de DNA , DNA Antissenso/genética , Doxiciclina/farmacologia , Células-Tronco Embrionárias/citologia , Citometria de Fluxo , Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Metilação , Camundongos da Linhagem 129 , Camundongos Transgênicos , Modelos Genéticos , Regiões Promotoras Genéticas/genéticaRESUMO
Silencing of the FMR1 gene leads to fragile X syndrome, the most common cause of inherited intellectual disability. To study the epigenetic modifications of the FMR1 gene during silencing in time, we used fibroblasts and induced pluripotent stem cells (iPSCs) of an unmethylated full mutation (uFM) individual with normal intelligence. The uFM fibroblast line carried an unmethylated FMR1 promoter region and expressed normal to slightly increased FMR1 mRNA levels. The FMR1 expression in the uFM line corresponds with the increased H3 acetylation and H3K4 methylation in combination with a reduced H3K9 methylation. After reprogramming, the FMR1 promoter region was methylated in all uFM iPSC clones. Two clones were analyzed further and showed a lack of FMR1 expression, whereas the presence of specific histone modifications also indicated a repressed FMR1 promoter. In conclusion, these findings demonstrate that the standard reprogramming procedure leads to epigenetic silencing of the fully mutated FMR1 gene.
Assuntos
Metilação de DNA , Fibroblastos/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Inativação Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Adolescente , Animais , Estudos de Casos e Controles , Linhagem Celular , Reprogramação Celular , Criança , Pré-Escolar , Feminino , Fibroblastos/citologia , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Masculino , Camundongos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
During replication, bypass of DNA lesions is orchestrated by the Rad6 pathway. Monoubiquitination of proliferating cell nuclear antigen (PCNA) by Rad6/Rad18 leads to recruitment of translesion polymerases for direct and potentially mutagenic damage bypass. An error-free bypass pathway may be initiated via K63-linked PCNA polyubiquitination by Ubc13/Mms2 and the E3 ligase Rad5 in yeast, or HLTF/SHPRH in vertebrates. For the latter two enzymes, redundancy with a third E3 ligase and alternative functions have been reported. We have previously shown that the Rad6 pathway is involved in somatic hypermutation of immunoglobulin genes in B lymphocytes. Here, we have used knockout strategies targeting expression of the entire SHPRH protein or functionally significant domains in chicken DT40 cells that do not harbor a HLTF ortholog. We show that SHPRH is apparently redundant with another E3 ligase during DNA damage-induced PCNA modification. SHPRH plays no substantial role in cellular resistance to drugs initiating excision repair and the Rad6 pathway, but is important in survival of topoisomerase II inhibitor treatment. Removal of only the C-terminal RING domain does not interfere with this SHPRH function. SHPRH inactivation does not substantially impact on the overall efficacy of Ig diversification. Redundancy of E3 ligases in the Rad6 pathway may be linked to its different functions in genome maintenance and genetic plasticity.