RESUMO
The BAP1 tumor suppressor gene encodes a deubiquitinase enzyme involved in several cellular activities, including DNA repair and apoptosis. Germline pathogenic variants in BAP1 have been associated with heritable conditions including BAP1 tumor predisposition syndrome 1 (BAP1-TPDS1) and a neurodevelopmental disorder known as Kury-Isidor syndrome (KURIS). Both these conditions are caused by monoallelic, dominant alterations of BAP1 but have never been reported in the same subject or family, suggesting a mutually exclusive genotype-phenotype correlation. This distinction is extremely important considering the early onset and aggressive nature of the types of cancer reported in individuals with TPDS1. Genetic counseling in subjects with germline BAP1 variants is fundamental to predicting the effect of the variant and the expected phenotype, assessing the potential risk of developing cancer for the tested subject and the family members who may carry the same variant and providing the multidisciplinary clinical team with the proper information to establish precise surveillance and management protocols.
Assuntos
Estudos de Associação Genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Proteínas Supressoras de Tumor , Ubiquitina Tiolesterase , Humanos , Mutação em Linhagem Germinativa/genética , Ubiquitina Tiolesterase/genética , Proteínas Supressoras de Tumor/genética , Fenótipo , Aconselhamento Genético , Síndromes Neoplásicas Hereditárias/genética , Transtornos do Neurodesenvolvimento/genética , Proteína BRCA1/genética , FemininoRESUMO
Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation.
Assuntos
Alelos , Metilação de DNA/genética , Elementos Facilitadores Genéticos/genética , Receptores de Superfície Celular/genética , Antígenos CD34/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Imunoprecipitação da Cromatina , Ilhas de CpG/genética , Epigênese Genética , Células HEK293 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Humanos , Íntrons/genética , Megacariócitos/citologia , Megacariócitos/metabolismo , Modelos Genéticos , Polimorfismo de Nucleotídeo Único/genética , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/metabolismo , Transcrição GênicaRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is a common muscle disease whose molecular pathogenesis remains largely unknown. Over-expression of FSHD region gene 1 (FRG1) in mice, frogs, and worms perturbs muscle development and causes FSHD-like phenotypes. FRG1 has been implicated in splicing, and we asked how splicing might be involved in FSHD by conducting a genome-wide analysis in FRG1 mice. We find that splicing perturbations parallel the responses of different muscles to FRG1 over-expression and disease progression. Interestingly, binding sites for the Rbfox family of splicing factors are over-represented in a subset of FRG1-affected splicing events. Rbfox1 knockdown, over-expression, and RNA-IP confirm that these are direct Rbfox1 targets. We find that FRG1 is associated to the Rbfox1 RNA and decreases its stability. Consistent with this, Rbfox1 expression is down-regulated in mice and cells over-expressing FRG1 as well as in FSHD patients. Among the genes affected is Calpain 3, which is mutated in limb girdle muscular dystrophy, a disease phenotypically similar to FSHD. In FRG1 mice and FSHD patients, the Calpain 3 isoform lacking exon 6 (Capn3 E6-) is increased. Finally, Rbfox1 knockdown and over-expression of Capn3 E6- inhibit muscle differentiation. Collectively, our results suggest that a component of FSHD pathogenesis may arise by over-expression of FRG1, reducing Rbfox1 levels and leading to aberrant expression of an altered Calpain 3 protein through dysregulated splicing.
Assuntos
Calpaína , Proteínas Musculares , Distrofia Muscular Facioescapuloumeral , Proteínas , Proteínas de Ligação a RNA/genética , Processamento Alternativo/genética , Animais , Calpaína/genética , Calpaína/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Camundongos , Proteínas dos Microfilamentos , Desenvolvimento Muscular/genética , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Distrofia Muscular Facioescapuloumeral/genética , Distrofia Muscular Facioescapuloumeral/metabolismo , Distrofia Muscular Facioescapuloumeral/patologia , Mioblastos/citologia , Mioblastos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Fatores de Processamento de RNA , Proteínas de Ligação a RNA/metabolismoRESUMO
We recently demonstrated that ex vivo activation of SMAD-independent bone morphogenetic protein 4 (BMP4) signaling in hematopoietic stem/progenitor cells (HSPCs) influences their homing into the bone marrow (BM). Here, we assessed whether alterations in BMP signaling in vivo affects adult hematopoiesis by affecting the BM niche. We demonstrate that systemic inhibition of SMAD-dependent BMP signaling by infusion of the BMP antagonist noggin (NGN) significantly increased CXCL12 levels in BM plasma leading to enhanced homing and engraftment of transplanted HSPCs. Conversely, the infusion of BMP7 but not BMP4, resulted in decreased HSPC homing. Using ST2 cells as an in vitro model of BM niche, we found that incubation with neutralizing anti-BMP4 antibodies, NGN, or dorsomorphin (DM) as well as knockdown of Smad1/5 and Bmp4, all enhanced CXCL12 production. Chromatin immunoprecipitation identified the SMAD-binding element in the CXCL12 promoter to which SMAD4 binds. When deleted, increased CXCL12 promoter activity was observed, and NGN or DM no longer affected Cxcl12 expression. Interestingly, BMP7 infusion resulted in mobilization of only short-term HSCs, likely because BMP7 affected CXCL12 expression only in osteoblasts but not in other niche components. Hence, we describe SMAD-dependent BMP signaling as a novel regulator of CXCL12 production in the BM niche, influencing HSPC homing, engraftment, and mobilization.
Assuntos
Células da Medula Óssea/metabolismo , Medula Óssea/metabolismo , Quimiocina CXCL12/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Transdução de Sinais , Proteínas Smad/metabolismo , Nicho de Células-Tronco , Animais , Proteína Morfogenética Óssea 4/metabolismo , Linhagem da Célula , Movimento Celular/fisiologia , Células Cultivadas , Regulação da Expressão Gênica/fisiologia , Transplante de Células-Tronco Hematopoéticas/métodos , Camundongos , Receptores CXCR4/metabolismoRESUMO
RasL11a and RasL11b are Ras super-family proteins of unknown function. Here, we show that RasL11a is a chromatin-associated modulator of pre-ribosomal RNA (pre-rRNA) synthesis. RasL11a was found in the nucleolus of interphase mouse fibroblasts, where it co-localized with the RNA polymerase I-specific transcription factor UBF. Similar to UBF, RasL11a also marked the active subset of rDNA repeats (also called nucleolar organizers, or NORs) on mitotic chromosomes. In cells, RasL11a existed in stable complexes with UBF and, as shown by chromatin immunoprecipitation, distributed along the rDNA transcription unit. Upon treatment of cells with actinomycin D, RasL11a and UBF persisted on the transcription unit beyond the release of RNA polymerase I, and remained co-localized in peri-nucleolar cap structures. Ectopic expression of RasL11a enhanced pre-rRNA levels in cells, whereas RasL11a knockdown had the opposite effect. In transient transfection experiments, RasL11a enhanced the transcriptional activity of an RNA polymerase I-specific reporter controlled by the rDNA enhancer/promoter region. We speculate that RasL11a acts in concert with UBF to facilitate initiation and/or elongation by RNA polymerase I in response to specific upstream stimuli.
Assuntos
Cromatina/metabolismo , DNA Ribossômico/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transcrição Gênica , Sequência de Aminoácidos , Animais , Núcleo Celular/química , DNA Ribossômico/genética , Regulação da Expressão Gênica , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/análise , Proteínas Monoméricas de Ligação ao GTP/genética , Células NIH 3T3 , Proteínas Pol1 do Complexo de Iniciação de Transcrição/análise , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , RNA Polimerase I/metabolismoRESUMO
BACKGROUND: Triple-Negative Breast Cancer (TNBC) is a subtype of breast cancer that differs from other types of breast cancers in the faster spread and worse outcome. TNBC presented limited treatment options. BET (Bromodomain and extra-terminal domain) proteins are epigenetic readers that control the expression of different oncogenic proteins, and their inhibition (BETi) is considered a promising anti-cancer strategy. Recent evidence demonstrated the involvement of BET proteins in regulation of metabolic processes. METHODS: MDA-MB231 cells treated with JQ1 followed by RNA-sequencing analysis showed altered expression of lipid metabolic genes; among these, we focused on ATGL, a lipase required for efficient mobilization of triglyceride. Different in vitro approaches were performed to validate the RNA-sequencing data (qRT-PCR, immunofluorescence and flow cytometry). NMR (Nuclear Magnetic Resonance) was used to analyze the lipid reprogramming upon treatment. ATGL expression was determined by immunoblot and qRT-PCR, and the impact of ATGL function or protein knockdown, alone and in combination with BETi, was assessed by analyzing cell proliferation, mitochondrial function, and metabolic activity in TNBC and non-TNBC cells culture models. RESULTS: TNBC cells treated with two BETi markedly increased ATGL expression and lipolytic function and decreased intracellular lipid content in a dose and time-dependent manner. The intracellular composition of fatty acids (FAs) after BETi treatment reflected a significant reduction in neutral lipids. The short-chain FA propionate entered directly into the mitochondria mimicking ATGL activity. ATGL KD (knockdown) modulated the levels of SOD1 and CPT1a decreasing ROS and helped to downregulate the expression of mitochondrial ß-oxidation genes in favor of the upregulation of glycolytic markers. The enhanced glycolysis is reflected by the increased of the mitochondrial activity (MTT assay). Finally, we found that after BETi treatment, the FoxO1 protein is upregulated and binds to the PNPLA2 promoter leading to the induction of ATGL. However, FoxO1 only partially prompted the induction of ATGL expression by BETi. CONCLUSIONS: The anti-proliferative effect achieved by BETi is helped by ATGL mediating lipolysis. This study showed that BETi altered the mitochondrial dynamics taking advantage of ATGL function to induce cell cycle arrest and cell death. Schematic representation of BETi mechanism of action on ATGL in TNBC cells. BETi induce the expression of FoxO1 and ATGL, lowering the expression of G0G2, leading to a switch in metabolic status. The induced expression of ATGL leads to increased lipolysis and a decrease in lipid droplet content and bioavailability of neutral lipid. At the same time, the mitochondria are enriched with fatty acids. This cellular status inhibits cell proliferation and increases ROS production and mitochondrial stress. Interfering for ATGL expression, the oxidative phenotypic status mildly reverted to a glycolytic status where neutral lipids are stored into lipid droplets with a consequent reduction of oxidative stress in the mitochondrial.
Assuntos
Aciltransferases , Lipase , Neoplasias de Mama Triplo Negativas , Humanos , Linhagem Celular Tumoral , Ácidos Graxos , Lipase/genética , Lipase/metabolismo , Lipídeos , Proteínas , Espécies Reativas de Oxigênio , Neoplasias de Mama Triplo Negativas/patologia , Aciltransferases/genética , Aciltransferases/metabolismoRESUMO
G protein-coupled estrogen receptor 1 (GPER1) activation is emerging as a promising therapeutic strategy against several cancer types. While GPER targeting has been widely studied in the context of solid tumors, its effect on hematological malignancies remains to be fully understood. Here, we show that GPER1 mRNA is down-regulated in plasma cells from overt multiple myeloma (MM) and plasma cell leukemia patients as compared to normal donors or pre-malignant conditions (monoclonal gammopathy of undetermined significance and smoldering MM); moreover, lower GPER1 expression associates with worse overall survival of MM patients. Using the clinically applicable GPER1-selective agonist G-1, we demonstrate that the pharmacological activation of GPER1 triggered in vitro anti-MM activity through apoptosis induction, also overcoming the protective effects exerted by bone marrow stromal cells. Noteworthy, G-1 treatment reduced in vivo MM growth in two distinct xenograft models, even bearing bortezomib-resistant MM cells. Mechanistically, G-1 upregulated the miR-29b oncosuppressive network, blunting an established miR-29b-Sp1 feedback loop operative in MM cells. Overall, this study highlights the druggability of GPER1 in MM, providing the first preclinical framework for further development of GPER1 agonists to treat this malignancy.
Assuntos
Neoplasias Hematológicas , MicroRNAs , Mieloma Múltiplo , Mieloma Múltiplo Latente , Humanos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , PlasmócitosRESUMO
Promoter methylation represents one of the major epigenetic mechanisms responsible for the regulation of gene expression. Hypomethylating drugs are currently approved for the treatment of myelodysplastic syndromes and acute myeloid leukemia, and some studies have recently been carried out on diffuse large B cell lymphoma (DLBCL). DLBCL is a type of NonHodgkin lymphoma. The aim of the present study was to assess the role of DNA methyltransferase (DNMT)1 in mediating the epigenetic regulation of some key targets previously emerged as hypermethylated in NonHodgkin lymphoma. Reverse transcriptionquantitative PCR, genomewide arrays and methylationspecific PCR were used to determine the level of methylation of specific targets. Gene silencing, gene expression and immunoblotting were used to investigate the role of DNMT1 and DNMT3a in lymphoma cells. The present study showed that lymphoma cell lines displayed a completely different methylation profile on selected targets compared with primary B lymphocytes and peripheral blood mononuclear cells. 5'azacytidine (5AZA) and 5'aza2deoxycitidine (decitabine) exerted their activity through, at least in part, mechanisms independent of DNMT1 downregulation. Despite a global hypomethylating effect of 5AZA and decitabine, DNMT1 was not found to be necessary to maintain the hypermethylation of Krüppellike factor 4 (KLF4), death associated protein 1 (DAPK1) and spastic paraplegia 20 (SPG20). SPG20 was found to be a completely methylated target in all the tested cell lines, but not in peripheral blood mononuclear cells, suggesting its association with malignancy. The highest methylation was clustered upstream of the transcription starting site in a panel of 28 DLBCL cell lines and the results were unaffected by the silencing of DNMT1 expression. These data demonstrated the epigenetic regulation of SPG20 in lymphoid cells and identified a number of novel markers associated with lymphomas that deserve further investigation.
Assuntos
Proteínas de Ciclo Celular/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Proteínas Quinases Associadas com Morte Celular/genética , Decitabina/farmacologia , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/genética , Antimetabólitos Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/efeitos dos fármacos , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Proteínas Quinases Associadas com Morte Celular/efeitos dos fármacos , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Fator 4 Semelhante a Kruppel/efeitos dos fármacos , Fator 4 Semelhante a Kruppel/genéticaRESUMO
BACKGROUND: Anaplastic Thyroid Cancer (ATC) is an undifferentiated and aggressive tumor that often originates from well-Differentiated Thyroid Carcinoma (DTC) through a trans-differentiation process. Epithelial-to-Mesenchymal Transition (EMT) is recognized as one of the major players of this process. OVOL2 is a transcription factor (TF) that promotes epithelial differentiation and restrains EMT during embryonic development. OVOL2 loss in some types of cancers is linked to aggressiveness and poor prognosis. Here, we aim to clarify the unexplored role of OVOL2 in ATC. METHODS: Gene expression analysis in thyroid cancer patients and cell lines showed that OVOL2 is mainly associated with epithelial features and its expression is deeply impaired in ATC. To assess OVOL2 function, we established an OVOL2-overexpression model in ATC cell lines and evaluated its effects by analyzing gene expression, proliferation, invasion and migration abilities, cell cycle, specific protein localization through immunofluorescence staining. RNA-seq profiling showed that OVOL2 controls a complex network of genes converging on cell cycle and mitosis regulation and Chromatin Immunoprecipitation identified new OVOL2 target genes. RESULTS: Coherently with its reported function, OVOL2 re-expression restrained EMT and aggressiveness in ATC cells. Unexpectedly, we observed that it caused G2/M block, a consequent reduction in cell proliferation and an increase in cell death. This phenotype was associated to generalized abnormalities in the mitotic spindle structure and cytoskeletal organization. By RNA-seq experiments, we showed that many pathways related to cytoskeleton and migration, cell cycle and mitosis are profoundly affected by OVOL2 expression, in particular the RHO-GTPase pathway resulted as the most interesting. We demonstrated that RHO GTPase pathway is the central hub of OVOL2-mediated program in ATC and that OVOL2 transcriptionally inhibits RhoU and RhoJ. Silencing of RhoU recapitulated the OVOL2-driven phenotype pointing to this protein as a crucial target of OVOL2 in ATC. CONCLUSIONS: Collectively, these data describe the role of OVOL2 in ATC and uncover a novel function of this TF in inhibiting the RHO GTPase pathway interlacing its effects on EMT, cytoskeleton dynamics and mitosis.
Assuntos
Carcinoma Anaplásico da Tireoide , Neoplasias da Glândula Tireoide , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Mitose , Gravidez , Carcinoma Anaplásico da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Fatores de Transcrição/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
BRD4 is an epigenome reader known to exert key roles at the interface between chromatin remodeling and transcriptional regulation, and is primarily known for its role in promoting gene expression. In selective contexts, however, BRD4 may work as negative regulator of transcription. Here, we reported that BRD4 binds several long noncoding RNAs (lncRNA). Among these, the lncRNA NEAT1 was found to interfere with BRD4 transcriptional activity. Mechanistically, lncNEAT1 forms a complex with BRD4 and WDR5 and maintains them in a low-activity state. Treatment with Bromodomains and Extraterminal (BET) inhibitor caused the lncRNA NEAT1 to dissociate from the BRD4/WDR5 complex, restored the acetyl-transferase capacity of BRD4, and restored the availability of WDR5 to promote histone trimethylation, thereby promoting BRD4/WDR5 transcriptional activity and activation of target gene expression. In addition, the lncRNA NEAT1 then became available to bind and to inhibit EZH2, cooperatively increasing transcriptional activation. IMPLICATIONS: Our results revealed an epigenetic program that involves the interaction between the lncRNA NEAT1 and BRD4, functioning as a molecular switch between BRD4's activator and repressor chromatin complexes.
Assuntos
Proteínas de Ciclo Celular/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Melanoma/genética , RNA Longo não Codificante/genética , Fatores de Transcrição/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma/metabolismo , Melanoma/patologia , RNA Longo não Codificante/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Alternative splicing of pre-mRNAs is a major contributor to proteomic diversity and to the control of gene expression in higher eukaryotic cells. For this reasons, alternative splicing is tightly regulated in different tissues and developmental stages and its disruption can lead to a wide range of human disorders. The aim of this review is to focus on the relevance of alternative splicing for muscle function and muscle disease. We begin by giving a brief overview of alternative splicing, muscle-specific gene expression and muscular dystrophy. Next, to illustrate these concepts we focus on two muscular dystrophy, myotonic muscular dystrophy and facioscapulohumeral muscular dystrophy, both associated to disruption of splicing regulation in muscle.
Assuntos
Processamento Alternativo , Distrofias Musculares/genética , Animais , Humanos , Músculo Esquelético/metabolismo , Distrofias Musculares/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Enhancer (ENH)-associated long noncoding RNAs (lncRNA) are a peculiar class of RNAs produced by transcriptionally active ENHs, owning potential gene-regulatory function. Here, we characterized RAIN, a novel ENH-associated lncRNA. Analysis of RAIN expression in a retrospective cohort of human thyroid cancers showed that the expression of this lncRNA is restricted to cancer cells and strongly correlates with the expression of the cancer-promoting transcription factor RUNX2. We showed that RAIN, serving as a cis-regulatory element, promotes RUNX2 expression by two mechanisms. Binding WDR5 and facilitating its localization on the RUNX2 promoter, RAIN modifies the transcriptional status of the RUNX2 locus facilitating transcription initiation. In parallel, RAIN acts as decoy for negative elongation factor complex, restraining its inhibitory function on transcription elongation. In both thyroid and breast cancer cells, RAIN promotes oncogenic features. Using RNA-sequencing profiling, we showed that RAIN orchestrates the expression of a network of cancer-promoting transcription regulators, suggesting that RAIN affects cancer cell phenotype by coordinating the expression of a complex transcriptional network. IMPLICATIONS: Our data contribute to understand lncRNA function in gene regulation and to consolidate their role in cancer.
Assuntos
Neoplasias da Mama/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/efeitos adversos , RNA Longo não Codificante/genética , Neoplasias da Glândula Tireoide/genética , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Feminino , Humanos , MasculinoRESUMO
Therapeutic failures in cancer therapy are often associated with metabolic plasticity. The use of metabolic modulators as anti-cancer agents has been effective in correcting metabolic alterations; however, molecular events behind metabolic switch are still largely unexplored. Herein, we characterize the molecular and functional events that follow prolonged oxidative phosphorylation inhibition by phenformin in order to study how melanoma cells adapt to this specific metabolic pressure. We show that melanoma cells cultured up to 3 months with high doses of phenformin (R-cells) are less viable and migrate and invade less than parental (S-) cells. Microarray analysis of R-melanoma cells reveals a switch in the energy production strategy accompanied by the modulation of several immunological-associated genes. R-cells display low oxygen consumption rate and high basal extracellular acidification rate. When treated with vemurafenib, R-cell viability, growth and extracellular signal-regulated kinase activation decrease. Finally, phenformin withdrawal reverts R-cells phenotype. In summary, our study provides an in vitro model of on-off metabolic switch in melanoma and reveals interesting molecular signatures controlling metabolic reprogramming in this tumour.
Assuntos
Reprogramação Celular/imunologia , Hipoglicemiantes/uso terapêutico , Melanoma/genética , Fenformin/uso terapêutico , Humanos , Hipoglicemiantes/farmacologia , Fenformin/farmacologiaRESUMO
Currently, drug metabolization and toxicity studies rely on the use of primary human hepatocytes and hepatoma cell lines, which both have conceivable limitations. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) are an alternative and valuable source of hepatocytes that can overcome these limitations. EZH2 (enhancer of zeste homolog 2), a transcriptional repressor of the polycomb repressive complex 2 (PRC2), may play an important role in hepatocyte development, but its role during in vitro hPSC-HLC differentiation has not yet been assessed. We here demonstrate dynamic regulation of EZH2 during hepatic differentiation of hPSC. To enhance EZH2 expression, we inducibly overexpressed EZH2 between d0 and d8, demonstrating a significant improvement in definitive endoderm formation, and improved generation of HLCs. Despite induction of EZH2 overexpression until d8, EZH2 transcript and protein levels decreased from d4 onwards, which might be caused by expression of microRNAs predicted to inhibit EZH2 expression. In conclusion, our studies demonstrate that EZH2 plays a role in endoderm formation and hepatocyte differentiation, but its expression is tightly post-transcriptionally regulated during this process.
Assuntos
Linhagem da Célula , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Hepatócitos/metabolismo , Células-Tronco Pluripotentes/citologia , Diferenciação Celular , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Células HEK293 , Hepatócitos/citologia , Humanos , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Diffuse large-B cell lymphomas (DLBCL) and follicular lymphomas (FL) are the most represented subtypes among mature B-cell neoplasms and originate from malignant B lymphocytes. Methylation represents one of the major epigenetic mechanisms of gene regulation. Silent information regulator 1 (SIRT1) is a class III lysine-deacetylase playing several functions and considered to be a context-dependent tumor promoter. We present the quantitative methylation, gene expression and tissue distribution of SIRT1 and some key mediators related to lymphoma pathogenesis in B lymphocytes purified from biopsies of follicular hyperplasias, FL and DLBCL. SIRT1 mRNA levels are higher in FL than follicular hyperplasias and DLBCL. B cell lymphoma 6 (BCL6) positively correlates with SIRT1. SIRT1 promoter shows a methylation decrease in the order: follicular hyperplasia - FL - DLBCL. Kruppel-like factor 4 (KLF4), Death-associated protein kinase 1 (DAPK1) and Spastic Paraplegia 20 (SPG20) methylation increase significantly in FL and DLBCL compared to follicular hyperplasias. Gene expression of DAPK1 and SPG20 inversely correlates with their degree of methylation. Our findings evidence a positive correlation between SIRT1 and BCL6 expression increase in FL. SIRT1 methylation decreases in FL and DLBCL accordingly and this parallels the increase of KLF4, DAPK1 and SPG20 methylation.
Assuntos
Linfócitos B/metabolismo , Metilação de DNA , Linfoma Folicular/genética , Linfoma Difuso de Grandes Células B/genética , Proteínas de Ciclo Celular , Proteínas Quinases Associadas com Morte Celular/genética , Epigênese Genética , Humanos , Imuno-Histoquímica , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Linfonodos/patologia , Linfoma Folicular/patologia , Linfoma Difuso de Grandes Células B/patologia , Proteínas/genética , Sirtuína 1/genéticaRESUMO
Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells (hPSCs) is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.
Assuntos
Células-Tronco Pluripotentes , Recombinação Genética , Linhagem Celular , Marcação de Genes , Humanos , Recombinases , TransgenesRESUMO
Although pluripotent stem cells can be differentiated into the hepatocyte lineages, such cells retain an immature phenotype. As the chromatin state of regulatory regions controls spatiotemporal gene expression during development, we evaluated changes in epigenetic histone marks in lineage-specific genes throughout in vitro hepatocyte differentiation from human embryonic stem cells (hESCs). Active acetylation and methylation marks at promoters and enhancers correlated with progressive changes in gene expression. However, repression-associated H3K27me3 marks at these control regions showed an inverse correlation with gene repression during transition from hepatic endoderm to a hepatocyte-like state. Inhibitor of Enhancer of Zeste Homolog 2 (EZH2) reduced H3K27me3 decoration but did not improve hepatocyte maturation. Thus, H3K27me3 at regulatory regions does not regulate transcription and appears dispensable for hepatocyte lineage differentiation of hESCs in vitro.
Assuntos
Biomarcadores/metabolismo , Linhagem da Célula , Hepatócitos/citologia , Hepatócitos/metabolismo , Histonas/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Lisina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Dimetil Sulfóxido/farmacologia , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hepatócitos/efeitos dos fármacos , Humanos , Metilação , Sequências Reguladoras de Ácido Nucleico/genética , Transcrição Gênica/efeitos dos fármacosRESUMO
Tools for rapid and efficient transgenesis in "safe harbor" loci in an isogenic context remain important to exploit the possibilities of human pluripotent stem cells (hPSCs). We created hPSC master cell lines suitable for FLPe recombinase-mediated cassette exchange (RMCE) in the AAVS1 locus that allow generation of transgenic lines within 15 days with 100% efficiency and without random integrations. Using RMCE, we successfully incorporated several transgenes useful for lineage identification, cell toxicity studies, and gene overexpression to study the hepatocyte lineage. However, we observed unexpected and variable transgene expression inhibition in vitro, due to DNA methylation and other unknown mechanisms, both in undifferentiated hESC and differentiating hepatocytes. Therefore, the AAVS1 locus cannot be considered a universally safe harbor locus for reliable transgene expression in vitro, and using it for transgenesis in hPSC will require careful assessment of the function of individual transgenes.
Assuntos
Células-Tronco Embrionárias/metabolismo , Marcação de Genes/métodos , Hepatócitos/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Recombinases/metabolismo , Transgenes , Células Cultivadas , Metilação de DNA , Dependovirus/genética , Células-Tronco Embrionárias/citologia , Inativação Gênica , Loci Gênicos , Hepatócitos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Recombinases/genéticaRESUMO
Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant myopathy with a strong epigenetic component. It is associated with deletion of a macrosatellite repeat leading to over-expression of the nearby genes. Among them, we focused on FSHD region gene 1 (FRG1) since its over-expression in mice, Xenopus laevis and Caenorhabditis elegans, leads to muscular dystrophy-like defects, suggesting that FRG1 plays a relevant role in muscle biology. Here we show that, when over-expressed, FRG1 binds and interferes with the activity of the histone methyltransferase Suv4-20h1 both in mammals and Drosophila. Accordingly, FRG1 over-expression or Suv4-20h1 knockdown inhibits myogenesis. Moreover, Suv4-20h KO mice develop muscular dystrophy signs. Finally, we identify the FRG1/Suv4-20h1 target Eid3 as a novel myogenic inhibitor that contributes to the muscle differentiation defects. Our study suggests a novel role of FRG1 as epigenetic regulator of muscle differentiation and indicates that Suv4-20h1 has a gene-specific function in myogenesis.