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ABSTRACT: SETBP1 mutations are found in various clonal myeloid disorders. However, it is unclear whether they can initiate leukemia, because SETBP1 mutations typically appear as later events during oncogenesis. To answer this question, we generated a mouse model expressing mutated SETBP1 in hematopoietic tissue: this model showed profound alterations in the differentiation program of hematopoietic progenitors and developed a myeloid neoplasm with megakaryocytic dysplasia, splenomegaly, and bone marrow fibrosis, prompting us to investigate SETBP1 mutations in a cohort of 36 triple-negative primary myelofibrosis (TN-PMF) cases. We identified 2 distinct subgroups, one carrying SETBP1 mutations and the other completely devoid of somatic variants. Clinically, a striking difference in disease aggressiveness was noted, with patients with SETBP1 mutation showing a much worse clinical course. In contrast to myelodysplastic/myeloproliferative neoplasms, in which SETBP1 mutations are mostly found as a late clonal event, single-cell clonal hierarchy reconstruction in 3 patients with TN-PMF from our cohort revealed SETBP1 to be a very early event, suggesting that the phenotype of the different SETBP1+ disorders may be shaped by the opposite hierarchy of the same clonal SETBP1 variants.
Assuntos
Sistema Hematopoético , Doenças Mieloproliferativas-Mielodisplásicas , Transtornos Mieloproliferativos , Mielofibrose Primária , Animais , Camundongos , Humanos , Mielofibrose Primária/genética , Transtornos Mieloproliferativos/genética , Mutação , Proteínas de Transporte/genética , Proteínas Nucleares/genéticaRESUMO
Within the chromatin, distal elements interact with promoters to regulate specific transcriptional programs. Histone acetylation, interfering with the net charges of the nucleosomes, is a key player in this regulation. Here, we report that the oncoprotein SET is a critical determinant for the levels of histone acetylation within enhancers. We disclose that a condition in which SET is accumulated, the severe Schinzel-Giedion Syndrome (SGS), is characterized by a failure in the usage of the distal regulatory regions typically employed during fate commitment. This is accompanied by the usage of alternative enhancers leading to a massive rewiring of the distal control of the gene transcription. This represents a (mal)adaptive mechanism that, on one side, allows to achieve a certain degree of differentiation, while on the other affects the fine and corrected maturation of the cells. Thus, we propose the differential in cis-regulation as a contributing factor to the pathological basis of SGS and possibly other the SET-related disorders in humans.
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Elementos Facilitadores Genéticos , Histonas , Humanos , Histonas/genética , Histonas/metabolismo , Elementos Facilitadores Genéticos/genética , Diferenciação Celular/genética , Cromatina/genética , Regiões Promotoras Genéticas/genéticaRESUMO
BACKGROUND: Neurodevelopmental disorders (NDDs) are heterogeneous conditions due to alterations of a variety of molecular mechanisms and cell dysfunctions. SETD5 haploinsufficiency leads to NDDs due to chromatin defects. Epigenetic basis of NDDs has been reported in an increasing number of cases while mitochondrial dysfunctions are more common within NDD patients than in the general population. METHODS: We investigated in vitro neural stem cells as well as the brain of the Setd5 haploinsufficiency mouse model interrogating its transcriptome, analyzing mitochondrial structure, biochemical composition, and dynamics, as well as mitochondrial functionality. RESULTS: Mitochondrial impairment is facilitated by transcriptional aberrations originated by the decrease of the SETD5 enzyme. Low levels of SETD5 resulted in fragmented mitochondria, reduced mitochondrial membrane potential, and ATP production both in neural precursors and neurons. Mitochondria were also mislocalized in mutant neurons, with reduced organelles within neurites and synapses. LIMITATIONS: We found several defects in the mitochondrial compartment; however, we can only speculate about their position in the hierarchy of the pathological mechanisms at the basis of the disease. CONCLUSIONS: Our study explores the interplay between chromatin regulation and mitochondria functions as a possible important aspect of SETD5-associated NDD pathophysiology. Our data, if confirmed in patient context, suggest that the mitochondrial activity and dynamics may represent new therapeutic targets for disorders associated with the loss of SETD5.
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Haploinsuficiência , Células-Tronco Neurais , Camundongos , Animais , Humanos , Neurônios/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neurais/metabolismo , Cromatina/metabolismo , Metiltransferases/genética , Metiltransferases/metabolismoRESUMO
Current therapies remain unsatisfactory in preventing the recurrence of glioblastoma multiforme (GBM), which leads to poor patient survival. By rational engineering of the transcription factor SOX2, a key promoter of GBM malignancy, together with the Kruppel-associated box and DNA methyltransferase3A/L catalytic domains, we generated a synthetic repressor named SOX2 epigenetic silencer (SES), which induces the transcriptional silencing of its original targets. By doing so, SES kills both glioma cell lines and patient-derived cancer stem cells in vitro and in vivo. SES expression, through local viral delivery in mouse xenografts, induces strong regression of human tumors and survival rescue. Conversely, SES is not harmful to neurons and glia, also thanks to a minimal promoter that restricts its expression in mitotically active cells, rarely present in the brain parenchyma. Collectively, SES produces a significant silencing of a large fraction of the SOX2 transcriptional network, achieving high levels of efficacy in repressing aggressive brain tumors.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Epigênese Genética , Glioblastoma/metabolismo , Glioma/patologia , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
The advent of next-generation sequencing (NGS) is heavily changing both the diagnosis of human conditions and basic biological research. It is now possible to dig deep inside the genome of hundreds of thousands or even millions of people and find both common and rare genomic variants and to perform detailed phenotypic characterizations of both physiological organs and experimental models. Recent years have seen the introduction of multiple techniques using NGS to profile transcription, DNA and chromatin modifications, protein binding, etc., that are now allowing us to profile cells in bulk or even at a single-cell level. Although rare and ultra-rare diseases only affect a few people, each of these diseases represent scholarly cases from which a great deal can be learned about the pathological and physiological function of genes, pathways, and mechanisms. Therefore, for rare diseases, state-of-the-art investigations using NGS have double valence: their genomic cause (new variants) and the characterize the underlining the mechanisms associated with them (discovery of gene function) can be found. In a non-exhaustive manner, this review will outline the main usage of NGS-based techniques for the diagnosis and characterization of neurodevelopmental disorders (NDDs), under whose umbrella many rare and ultra-rare diseases fall.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Doenças Raras , Humanos , Análise de Sequência de DNARESUMO
The investigation of genetic forms of juvenile neurodegeneration could shed light on the causative mechanisms of neuronal loss. Schinzel-Giedion syndrome (SGS) is a fatal developmental syndrome caused by mutations in the SETBP1 gene, inducing the accumulation of its protein product. SGS features multi-organ involvement with severe intellectual and physical deficits due, at least in part, to early neurodegeneration. Here we introduce a human SGS model that displays disease-relevant phenotypes. We show that SGS neural progenitors exhibit aberrant proliferation, deregulation of oncogenes and suppressors, unresolved DNA damage, and resistance to apoptosis. Mechanistically, we demonstrate that high SETBP1 levels inhibit P53 function through the stabilization of SET, which in turn hinders P53 acetylation. We find that the inheritance of unresolved DNA damage in SGS neurons triggers the neurodegenerative process that can be alleviated either by PARP-1 inhibition or by NAD + supplementation. These results implicate that neuronal death in SGS originates from developmental alterations mainly in safeguarding cell identity and homeostasis.
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Anormalidades Múltiplas/patologia , Proteínas de Transporte/metabolismo , Anormalidades Craniofaciais/patologia , Dano ao DNA , Deformidades Congênitas da Mão/patologia , Transtornos Heredodegenerativos do Sistema Nervoso/patologia , Deficiência Intelectual/patologia , Mutação , Unhas Malformadas/patologia , Células-Tronco Neurais/patologia , Proteínas Nucleares/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/metabolismo , Proteínas de Transporte/genética , Células Cultivadas , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Deformidades Congênitas da Mão/genética , Deformidades Congênitas da Mão/metabolismo , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Unhas Malformadas/genética , Unhas Malformadas/metabolismo , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/genética , OrganoidesRESUMO
TBL1XR1 gene is associated with multiple developmental disorders presenting several neurological aspects. The relative protein is involved in the modulation of important cellular pathways and master regulators of transcriptional output, including nuclear receptor repressors, Wnt signaling, and MECP2 protein. However, TBL1XR1 mutations (including complete loss of its functions) have not been experimentally studied in a neurological context, leaving a knowledge gap in the mechanisms at the basis of the diseases. Here, we show that Tbl1xr1 knock-out mice exhibit behavioral and neuronal abnormalities. Either the absence of TBL1XR1 or its point mutations interfering with stability/regulation of NCOR complex induced decreased proliferation and increased differentiation in neural progenitors. We suggest that this developmental unbalance is due to a failure in the regulation of the MAPK cascade. Taken together, our results broaden the molecular and functional aftermath of TBL1XR1 deficiency associated with human disorders.
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Human multipotent mesenchymal stromal cells (MSC) are isolated from a plethora of tissue sources for cell therapy purposes. In 2006, the International Society for Cellular Therapy (ISCT) published minimal guidelines to define MSC identity. Nevertheless, many independent studies demonstrated that cells meeting the ISCT criteria possessed heterogeneous phenotypes and functionalities, heavily influenced by culture conditions. In this study, human MSC derived from many adult (bone marrow and adipose tissue) or fetal (cord blood, Wharton's jelly, umbilical cord perivascular compartment and amniotic fluid) tissues were investigated. Their immunophenotype was analyzed to define consistent source-specific markers by extensive flow cytometry analysis and real-time qRT-PCR. CD271+ subpopulations were detected in adult MSC, whereas NG2 was significantly more expressed in fetal MSC but failed validation on independent samples coming from an external laboratory. The highest number of CD271+ adult MSC were detected soon after isolation in serum-based culture conditions. Furthermore, heterogeneous percentages of CD271 expression were found in platelet lysate-based or serum-free culture conditions. Finally, CD271+ adult MSC showed high clonogenic and osteogenic properties as compared to CD271- cells. To conclude, in this phenotype-function correlation study CD271+ subpopulation confers heterogeneity on adult MSC, confirming the need of more specific markers to address MSC properties.
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Adapaleno/metabolismo , Células-Tronco Adultas/metabolismo , Células-Tronco Fetais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Adulto , Biomarcadores/metabolismo , Forma Celular , Células Cultivadas , Células Clonais , Análise por Conglomerados , Humanos , Imunofenotipagem , Pessoa de Meia-Idade , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismoRESUMO
SETBP1 variants occur as somatic mutations in several hematological malignancies such as atypical chronic myeloid leukemia and as de novo germline mutations in the Schinzel-Giedion syndrome. Here we show that SETBP1 binds to gDNA in AT-rich promoter regions, causing activation of gene expression through recruitment of a HCF1/KMT2A/PHF8 epigenetic complex. Deletion of two AT-hooks abrogates the binding of SETBP1 to gDNA and impairs target gene upregulation. Genes controlled by SETBP1 such as MECOM are significantly upregulated in leukemias containing SETBP1 mutations. Gene ontology analysis of deregulated SETBP1 target genes indicates that they are also key controllers of visceral organ development and brain morphogenesis. In line with these findings, in utero brain electroporation of mutated SETBP1 causes impairment of mouse neurogenesis with a profound delay in neuronal migration. In summary, this work unveils a SETBP1 function that directly affects gene transcription and clarifies the mechanism operating in myeloid malignancies and in the Schinzel-Giedion syndrome caused by SETBP1 mutations.
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Proteínas de Transporte/genética , Epigênese Genética , Perfilação da Expressão Gênica , Mutação , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Anormalidades Múltiplas/genética , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Anormalidades Craniofaciais/genética , Ontologia Genética , Células HEK293 , Deformidades Congênitas da Mão/genética , Humanos , Deficiência Intelectual/genética , Leucemia/genética , Leucemia/patologia , Camundongos , Unhas Malformadas/genética , Neurogênese/genética , Proteínas Nucleares/metabolismo , Ligação ProteicaRESUMO
The molecular profile of human mesenchymal stem cells (MSCs) have emerged as a key factor in defining their identity. Nevertheless, the effect of fetal bovine serum (FBS) batches or origin on MSC molecular signature has been neglected. In this frame, chemical fingerprint of FBS batches from unrelated countries showed strong correlation between chemical composition and country of origin. Thus, the aim of this study was to evaluate in stem cells isolated from bone marrow (BMMSCs) and umbilical cord-blood (CBMSCs) the effects of independently collected FBS batches on both twelve commonly used reference genes (RGs) and a selected panel of thirty-eight genes crucial for MSC definition in both research and clinical settings. Gene expression stability was estimated comparing the outcomes of two applets: geNorm and NormFinder. The bioinformatics analysis emphasized that, in a panorama of general balance, few RG candidates (YWHAZ/UBC for BMMSCs, RPLP0/EF1A for CBMSCs and EF1A/TBP for both MSCs scored together) showed superior stability. In addition, a wider study on genes involved in differentiation/proliferation/stemness processes, often used to define MSC potency, showed that these genes exhibited no major transcriptional modulation after treatment with different FBS, and allowed the identification of genes strongly discriminating between BM- and CBMSC populations. Therefore, in conclusion, FBS origin does not dramatically impact the general molecular profile of MSCs, although we could identify validated candidates able to allow more reliable comparison of data regarding MSC identity and potency and obtained by research laboratories and clinical manufacturers using different sera.
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Antígenos de Diferenciação/biossíntese , Células da Medula Óssea , Técnicas de Cultura de Células/normas , Meios de Cultura/normas , Sangue Fetal , Células-Tronco Mesenquimais , Soro , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Bovinos , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Humanos , Masculino , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Pessoa de Meia-Idade , Padrões de ReferênciaRESUMO
Mesenchymal stem cells (MSC) are multipotent cells able to differentiate into several cell types, hence providing cell reservoirs for therapeutic applications. The absence of detectable MSC homing at injury sites suggests that paracrine functions could, at least in part, be mediated by extracellular vesicles (EVs); EVs are newly identified players that are studied mainly as predictive or diagnostic biomarkers. Together with their clinical interests, EVs have recently come to the fore for their role in cell-to-cell communication. In this context, we investigated gene-based communication mechanisms in EVs generated by bone marrow and umbilical cord blood MSC (BMMSC and CBMSC, respectively). Both MSC types released vesicles with similar physical properties, although CBMSC were able to secrete EVs with faster kinetics. A pattern of preferentially incorporated EV transcripts was detected with respect to random internalization from the cytosol, after a validated normalization procedure was established. In the paradigm where EVs act as bioeffectors educating target cells, we demonstrated that kidney tubular cells lacking IL-10 expression and exposed to BMMSC-EVs and CBMSC-EVs acquired the IL-10 mRNA, which was efficiently translated into the corresponding protein. These findings suggest that horizontal mRNA transfer through EVs is a new mechanism in the MSC restoring ability observed in vivo that is here further demonstrated in an in vitro rescue model after acute cisplatin injury of tubular cells. Stem Cells 2017;35:1093-1105.
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Comunicação Celular , Vesículas Extracelulares/metabolismo , Células-Tronco Mesenquimais/metabolismo , Regiões 3' não Traduzidas/genética , Vesículas Extracelulares/ultraestrutura , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Cinética , Células-Tronco Mesenquimais/ultraestrutura , Modelos Biológicos , Compostos Orgânicos/metabolismo , Biossíntese de Proteínas , Transporte de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes , Coloração e Rotulagem , Transcrição GênicaRESUMO
In a recent work, our group showed the existence of two distinct mesenchymal stem cell (MSC) subsets within human umbilical cord blood. One less proliferative and short-living (SL-CBMSC), the other with higher growth rate and long-living (LL-CBMSC), and therefore better suited for regenerative medicine applications. We examined whether LL-CBMSC possess peculiar paracrine properties able to affect angiogenesis or inflammatory processes. It was shown for the first time that pro-angiogenic, proliferation-stimulating and tissue repairing factors were released at high level not only as soluble cytokines, but also as mRNA precursors embedded in membrane vesicles. The combination of this primary (proteic factors interacting with surface receptors) and delayed (mRNA transferred and translated via vesicle fusion and cargo release) interaction in endothelial target cells resulted in strong blood vessel induction with the development of capillary-like structures. In addition, LL-CBMSC dynamically modulated their release of pro-angiogenic and anti-inflammatory factors in an in vitro model of damage. In conclusion, LL-CBMSC synthesize and secrete multiple factors that may be attuned in response to the status of the target cell, a crucial requisite when paracrine mechanisms are needed at onset of tissue regeneration.
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Sangue Fetal/citologia , Células-Tronco Mesenquimais/fisiologia , Regeneração/fisiologia , Proliferação de Células/fisiologia , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/fisiologia , Humanos , Recém-Nascido , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Neovascularização Fisiológica/fisiologia , Medicina RegenerativaRESUMO
Evaluation of the antibody responses induced by the 2004-2005 influenza vaccine strain against the homologous variant, the 2004-2005 field isolates, and a previous circulating strain showed that a correlation between neutralizing and hemagglutination-inhibiting activities exists only when the antigen is very close to the vaccine strain.
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Anticorpos Antivirais/imunologia , Vacinas contra Influenza/uso terapêutico , Orthomyxoviridae/classificação , Orthomyxoviridae/imunologia , Deriva Genética , Variação Genética , Testes de Inibição da Hemaglutinação , Humanos , Testes de Neutralização , Orthomyxoviridae/genética , FilogeniaRESUMO
Although the haemagglutination inhibition assay is considered the "gold standard" for antigenic characterisation of influenza viruses, some limitations of this technique are well known. A new microneutralisation assay, as a tool for antigenic characterisation of influenza B viruses, has been standardised and its performance evaluated in comparison with the haemagglutination inhibition test in the light of molecular characterisation of the haemagglutinin. Twelve B viruses belonging to the two lineages and the four sub-lineages discriminated by phylogenetic analysis of HA were tested. The microneutralisation assay clearly distinguishes viruses belonging to different lineages and, in addition, discriminates strains belonging to different sub-lineages that are poorly or not discriminated using the haemagglutination inhibition test. This new microneutralisation assay could provide a useful tool for antigenic characterisation of circulating influenza viruses and contribute, together with the haemagglutination inhibition test and sequence analysis of the haemagglutinin and neuraminidase, in the choice of the strain for use in vaccine composition.