RESUMO
Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.
Assuntos
Neoplasias Cerebelares , Células-Tronco Pluripotentes Induzidas , Meduloblastoma , Humanos , Camundongos , Animais , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Neoplasias Cerebelares/genética , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Proteínas Hedgehog/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Transformação Celular Neoplásica , Carcinogênese/genética , Organoides/metabolismo , Receptores PatchedRESUMO
AIMS: Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell-cell signalling and are thus a potential mechanism for communicating with remote tissues. METHODS AND RESULTS: Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. CONCLUSIONS: Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.
Assuntos
Vesículas Extracelulares , MicroRNAs , Infarto do Miocárdio , Camundongos , Animais , Neutrófilos/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Vesículas Extracelulares/metabolismo , Infarto do Miocárdio/metabolismo , Células Endoteliais/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismoRESUMO
Promiscuous activity of the Streptococcus pyogenes DNA nuclease CRISPR-Cas9 can result in destruction of a successfully modified sequence obtained by templated repair of a Cas9-induced DNA double-strand break. To avoid re-cutting, additional target-site-disruptions (TSDs) are often introduced on top of the desired base-pair alteration in order to suppress target recognition. These TSDs may lower the efficiency of introducing the intended mutation and can cause unexpected phenotypes. Alternatively, successfully edited sites can be protected against Cas9 re-cutting activity. This method exploits the finding that Cas9 complexed to trimmed guideRNAs can still tightly bind specific genomic sequences but lacks nuclease activity. We show here that the presence of a guideRNA plus a trimmed guideRNA that matches the successfully mutated sequence, which we call hideRNA, can enhance the recovery of precise single base-pair substitution events tenfold. The benefit of hideRNAs in generating a single point mutation was demonstrated in cell lines using plasmid-based delivery of CRISPR-Cas9 components and in mouse zygotes injected with Cas9/guideRNA plus Cas9/hideRNA ribonucleoprotein complexes. However, hRNA protection sometimes failed, which likely reflects an unfavorable affinity of hRNA/Cas9 versus gRNA/Cas9 for the DNA target site. HideRNAs can easily be implemented into current gene editing protocols and facilitate the recovery of single base-pair substitution. As such, hideRNAs are of great value in gene editing experiments demanding high accuracy.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Quebras de DNA de Cadeia Dupla , Endonucleases/genética , Edição de Genes/métodos , Camundongos , RNA Guia de Cinetoplastídeos/genéticaRESUMO
Directing the fates of human pluripotent stem cells (hPSC) to generate a multitude of differentiated cell types allows the study of the genetic regulation of human development and disease. The translational potential of hPSC is maximized by exploiting CRISPR to silence or activate genes with spatial and temporal precision permanently or reversibly. Here, we summarize the increasingly refined and diverse CRISPR toolkit for the latter forms of gene perturbation in hPSC and their downstream applications. We discuss newer methods to install edits efficiently with single nucleotide resolution and describe pooled CRISPR screens as a powerful means of unbiased discovery of genes associated with a phenotype of interest. Last, we discuss the potential of these combined technologies in the treatment of hitherto intractable human diseases and the challenges to their implementation in the clinic.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/metabolismo , Reparo do DNA , Humanos , Células-Tronco Pluripotentes Induzidas/citologiaRESUMO
Precise gene editing of mitochondrial DNA (mtDNA) is essential for the generation of model systems to study rare mitochondrial diseases but was long deemed impossible - until now. A recent publication by Mok et al. describes a gene editing tool capable of installing point mutations in mtDNA, and it does not involve CRISPR.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Genoma Mitocondrial , Sistemas CRISPR-Cas/genética , Citidina Desaminase , Edição de Genes , Genoma Mitocondrial/genéticaRESUMO
The pervasive nature of RNA polymerase II (Pol II) transcription requires efficient termination. A key player in this process is the cleavage and polyadenylation (CPA) factor PCF11, which directly binds to the Pol II C-terminal domain and dismantles elongating Pol II from DNA in vitro. We demonstrate that PCF11-mediated termination is essential for vertebrate development. A range of genomic analyses, including mNET-seq, 3' mRNA-seq, chromatin RNA-seq, and ChIP-seq, reveals that PCF11 enhances transcription termination and stimulates early polyadenylation genome-wide. PCF11 binds preferentially between closely spaced genes, where it prevents transcriptional interference and consequent gene downregulation. Notably, PCF11 is sub-stoichiometric to the CPA complex. Low levels of PCF11 are maintained by an auto-regulatory mechanism involving premature termination of its own transcript and are important for normal development. Both in human cell culture and during zebrafish development, PCF11 selectively attenuates the expression of other transcriptional regulators by premature CPA and termination.
Assuntos
RNA Mensageiro/biossíntese , Terminação da Transcrição Genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Fatores de Poliadenilação e Clivagem de mRNA/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Humanos , Mutação , Poliadenilação , Ligação Proteica , Clivagem do RNA , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Fatores de Poliadenilação e Clivagem de mRNA/genéticaRESUMO
Fanconi anemia (FA) is a cancer predisposition syndrome characterized by congenital abnormalities, bone marrow failure, and hypersensitivity to aldehydes and crosslinking agents. For FA patients, gene editing holds promise for therapeutic applications aimed at functionally restoring mutated genes in hematopoietic stem cells. However, intrinsic FA DNA repair defects may obstruct gene editing feasibility. Here, we report on the CRISPR/Cas9-mediated correction of a disruptive mutation in Fancf. Our experiments revealed that gene editing could effectively restore Fancf function via error-prone end joining resulting in a 27% increased survival in the presence of mitomycin C. In addition, templated gene correction could be achieved after double strand or single strand break formation. Although templated gene editing efficiencies were low (≤6%), FA corrected embryonic stem cells acquired a strong proliferative advantage over non-corrected cells, even without imposing genotoxic stress. Notably, Cas9 nickase activity resulted in mono-allelic gene editing and avoidance of undesired mutagenesis. In conclusion: DNA repair defects associated with FANCF deficiency do not prohibit CRISPR/Cas9 gene correction. Our data provide a solid basis for the application of pre-clinical models to further explore the potential of gene editing against FA, with the eventual aim to obtain therapeutic strategies against bone marrow failure.
Assuntos
Sistemas CRISPR-Cas/genética , Proteína do Grupo de Complementação F da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Edição de Genes/métodos , Terapia Genética/métodos , Animais , Células Cultivadas , Reparo do DNA , Orelha , Fibroblastos , Camundongos , Células-Tronco Embrionárias MurinasRESUMO
Although Astyanax mexicanus surface fish regenerate their hearts after injury, their Pachón cave-dwelling counterparts cannot and, instead, form a permanent fibrotic scar, similar to the human heart. Myocardial proliferation peaks at similar levels in both surface fish and Pachón 1 week after injury. However, in Pachón, this peak coincides with a strong scarring and immune response, and ultimately, cavefish cardiomyocytes fail to replace the scar. We identified lrrc10 to be upregulated in surface fish compared with Pachón after injury. Similar to cavefish, knockout of lrrc10 in zebrafish impairs heart regeneration without affecting wound cardiomyocyte proliferation. Furthermore, using quantitative trait locus (QTL) analysis, we have linked the degree of heart regeneration to three loci in the genome, identifying candidate genes fundamental to the difference between scarring and regeneration. Our study provides evidence that successful heart regeneration entails a delicate interplay between cardiomyocyte proliferation and scarring.
Assuntos
Characidae/fisiologia , Coração/fisiologia , Regeneração/fisiologia , Animais , Proliferação de Células , Characidae/genética , Cinética , Mutação/genética , Miocárdio/citologia , Miócitos Cardíacos/citologia , Locos de Características Quantitativas/genética , Regulação para Cima , Cicatrização , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
HERV-K HML-2 (HK2) has been proliferating in the germ line of humans at least as recently as 250,000 years ago, with some integrations that remain polymorphic in the modern human population. One of the solitary HK2 LTR polymorphic integrations lies between exons 17 and 18 of RASGRF2, a gene that affects dopaminergic activity and is thus related to addiction. Here we show that this antisense HK2 integration (namely RASGRF2-int) is found more frequently in persons who inject drugs compared with the general population. In a Greek HIV-1-positive population (n = 202), we found RASGRF2-int 2.5 times (14 versus 6%) more frequently in patients infected through i.v. drug use compared with other transmission route controls (P = 0.03). Independently, in a United Kingdom-based hepatitis C virus-positive population (n = 184), we found RASGRF2-int 3.6 times (34 versus 9.5%) more frequently in patients infected during chronic drug abuse compared with controls (P < 0.001). We then tested whether RASGRF2-int could be mechanistically responsible for this association by modulating transcription of RASGRF2 We show that the CRISPR/Cas9-mediated insertion of HK2 in HEK293 cells in the exact RASGRF2 intronic position found in the population resulted in significant transcriptional and phenotypic changes. We also explored mechanistic features of other intronic HK2 integrations and show that HK2 LTRs can be responsible for generation of cis-natural antisense transcripts, which could interfere with the transcription of nearby genes. Our findings suggest that RASGRF2-int is a strong candidate for dopaminergic manipulation, and emphasize the importance of accurate mapping of neglected HERV polymorphisms in human genomic studies.
Assuntos
Células-Tronco de Carcinoma Embrionário/metabolismo , Retrovirus Endógenos/genética , Abuso de Substâncias por Via Intravenosa/genética , Transcrição Gênica , Integração Viral/genética , Fatores ras de Troca de Nucleotídeo Guanina/genética , Estudos de Casos e Controles , Criança , Estudos de Coortes , Células-Tronco de Carcinoma Embrionário/patologia , Feminino , Genoma Humano , Humanos , Masculino , Células Tumorais CultivadasRESUMO
Transcription factor (TF) networks determine cell-type identity by establishing and maintaining lineage-specific expression profiles, yet reconstruction of mammalian regulatory network models has been hampered by a lack of comprehensive functional validation of regulatory interactions. Here, we report comprehensive ChIP-Seq, transgenic and reporter gene experimental data that have allowed us to construct an experimentally validated regulatory network model for haematopoietic stem/progenitor cells (HSPCs). Model simulation coupled with subsequent experimental validation using single cell expression profiling revealed potential mechanisms for cell state stabilisation, and also how a leukaemogenic TF fusion protein perturbs key HSPC regulators. The approach presented here should help to improve our understanding of both normal physiological and disease processes.
Assuntos
Redes Reguladoras de Genes , Hematopoese , Células-Tronco Hematopoéticas/fisiologia , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Simulação por Computador , Perfilação da Expressão Gênica , Camundongos , Modelos Teóricos , Análise de Sequência de DNARESUMO
The neuromuscular junction (NMJ) consists of a tripartite synapse with a presynaptic nerve terminal, Schwann cells that ensheathe the terminal bouton, and a highly specialized postsynaptic membrane. Synaptic structural integrity is crucial for efficient signal transmission. Congenital myasthenic syndromes (CMSs) are a heterogeneous group of inherited disorders that result from impaired neuromuscular transmission, caused by mutations in genes encoding proteins that are involved in synaptic transmission and in forming and maintaining the structural integrity of NMJs. To identify further causes of CMSs, we performed whole-exome sequencing (WES) in families without an identified mutation in known CMS-associated genes. In two families affected by a previously undefined CMS, we identified homozygous loss-of-function mutations in COL13A1, which encodes the alpha chain of an atypical non-fibrillar collagen with a single transmembrane domain. COL13A1 localized to the human muscle motor endplate. Using CRISPR-Cas9 genome editing, modeling of the COL13A1 c.1171delG (p.Leu392Sfs(∗)71) frameshift mutation in the C2C12 cell line reduced acetylcholine receptor (AChR) clustering during myotube differentiation. This highlights the crucial role of collagen XIII in the formation and maintenance of the NMJ. Our results therefore delineate a myasthenic disorder that is caused by loss-of-function mutations in COL13A1, encoding a protein involved in organization of the NMJ, and emphasize the importance of appropriate symptomatic treatment for these individuals.
Assuntos
Colágeno Tipo XIII/genética , Mutação , Síndromes Miastênicas Congênitas/genética , Mioblastos/metabolismo , Junção Neuromuscular/metabolismo , Adulto , Animais , Linhagem Celular , Pré-Escolar , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Colágeno Tipo XIII/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Exoma , Feminino , Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Humanos , Masculino , Camundongos , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/patologia , Mioblastos/patologia , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/patologia , Linhagem , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Sinapses/genética , Sinapses/metabolismo , Sinapses/patologia , Transmissão SinápticaRESUMO
Dendritic cell (DC) maturation is a tightly regulated process that requires coordinated and timed developmental cues. Here we investigate whether microRNAs are involved in this process. We identify microRNAs in mouse GM-CSF-generated, monocyte-related DC (GM-DC) that are differentially expressed during both spontaneous and LPS-induced maturation and characterize M-CSF receptor (M-CSFR), encoded by the Csf1r gene, as a key target for microRNA-mediated regulation in the final step toward mature DC. MicroRNA-22, -34a, and -155 are up-regulated in mature MHCII(hi) CD86(hi) DC and mediate Csf1r mRNA and protein down-regulation. Experimental inhibition of Csf1r-targeting microRNAs in vitro results not only in sustained high level M-CSFR protein expression but also in impaired DC maturation upon stimulation by LPS. Accordingly, over-expression of Csf1r in GM-DC inhibits terminal differentiation. Taken together, these results show that developmentally regulated microRNAs control Csf1r expression, supplementing previously identified mechanisms that regulate its transcription and protein surface expression. Furthermore, our data indicate a novel function for Csf1r in mouse monocyte-derived DC, showing that down-regulation of M-CSFR expression is essential for final DC maturation.
RESUMO
Glutathione S-transferase pi (GST, E.C.2.5.1.18) overexpression contributes to resistance of cancer cells towards cytostatic drugs. Furthermore, GSTpi is involved in the cellular stress response through inhibition of Jun N-terminal-kinase (JNK), a process that can be modulated by GST inhibitors. GSH conjugates are potent GST inhibitors, but are sensitive towards gamma-glutamyltranspeptidase (gammaGT)-mediated breakdown. In search for new peptidase stable GST inhibitors we employed the following strategy: (1) selection of a suitable (GST inhibiting) peptide-bond isostere from a series of previously synthesized gammaGT stabilized GSH-analogs. (2) The use of this peptidomimetic strategy to prepare a GSTpi selective inhibitor. Two gammaGT stable GSH conjugate analogs inhibited human GSTs, although non-selectively. One of these, a urethane-type peptide-bond is well accepted by GSTs and we selected this modification for the development of a gammaGT stable, GSTpi selective inhibitor, UrPhg-Et(2). This compound displayed selectivity for GSTpi compared to alpha and mu class enzymes. Furthermore, the inhibitor reversed GSTpi-mediated drug resistance (MDR) in breast tumor cells. In addition, short-term exposure of cells to UrPhg-Et(2) led to GSTpi oligomerization and JNK activation, suggesting that it activates the JNK-cJun signaling module through GSTpi dissociation. Altogether, we show the successful use of peptidomimetic glutathione conjugate analogs as GST inhibitors and MDR-modifiers. As many MDR related enzymes, such as MRP1, glyoxalase 1 and DNA-pk are also inhibited by GSH conjugates, these peptidomimetic compounds can be used as scaffolds for the development of multi-target MDR drugs.