RESUMO
Stem cell transplantation presents a potentially curative strategy for genetic disorders of skeletal muscle, but this approach is limited by the deleterious effects of cell expansion in vitro and consequent poor engraftment efficiency. In an effort to overcome this limitation, we sought to identify molecular signals that enhance the myogenic activity of cultured muscle progenitors. Here, we report the development and application of a cross-species small-molecule screening platform employing zebrafish and mice, which enables rapid, direct evaluation of the effects of chemical compounds on the engraftment of transplanted muscle precursor cells. Using this system, we screened a library of bioactive lipids to discriminate those that could increase myogenic engraftment in vivo in zebrafish and mice. This effort identified two lipids, lysophosphatidic acid and niflumic acid, both linked to the activation of intracellular calcium-ion flux, which showed conserved, dose-dependent, and synergistic effects in promoting muscle engraftment across these vertebrate species.
Assuntos
Células Satélites de Músculo Esquelético , Peixe-Zebra , Camundongos , Animais , Músculo Esquelético/fisiologia , Transplante de Células-Tronco , Lipídeos/farmacologia , Diferenciação Celular , Desenvolvimento MuscularRESUMO
Dyskeratosis congenita (DC) is a rare inherited bone marrow failure and cancer predisposition syndrome caused by mutations in telomerase or telomeric proteins. Here, we report that zebrafish telomerase RNA (terc) binds to specific DNA sequences of master myeloid genes and controls their expression by recruiting RNA Polymerase II (Pol II). Zebrafish terc harboring the CR4-CR5 domain mutation found in DC patients hardly interacted with Pol II and failed to regulate myeloid gene expression in vivo and to increase their transcription rates in vitro. Similarly, TERC regulated myeloid gene expression and Pol II promoter occupancy in human myeloid progenitor cells. Strikingly, induced pluripotent stem cells derived from DC patients with a TERC mutation in the CR4-CR5 domain showed impaired myelopoiesis, while those with mutated telomerase catalytic subunit differentiated normally. Our findings show that TERC acts as a transcription factor, revealing a target for therapeutic intervention in DC patients.
Assuntos
Disceratose Congênita/genética , Mielopoese/fisiologia , RNA Polimerase II/genética , RNA/metabolismo , Telomerase/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Células Cultivadas , Disceratose Congênita/patologia , Regulação da Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Larva/genética , Mutação , Mielopoese/genética , Regiões Promotoras Genéticas , Domínios Proteicos , RNA/genética , RNA Polimerase II/metabolismo , Telomerase/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The availability of nucleotides has a direct impact on transcription. The inhibition of dihydroorotate dehydrogenase (DHODH) with leflunomide impacts nucleotide pools by reducing pyrimidine levels. Leflunomide abrogates the effective transcription elongation of genes required for neural crest development and melanoma growth in vivo1. To define the mechanism of action, we undertook an in vivo chemical suppressor screen for restoration of neural crest after leflunomide treatment. Surprisingly, we found that alterations in progesterone and progesterone receptor (Pgr) signalling strongly suppressed leflunomide-mediated neural crest effects in zebrafish. In addition, progesterone bypasses the transcriptional elongation block resulting from Paf complex deficiency, rescuing neural crest defects in ctr9 morphant and paf1(alnz24) mutant embryos. Using proteomics, we found that Pgr binds the RNA helicase protein Ddx21. ddx21-deficient zebrafish show resistance to leflunomide-induced stress. At a molecular level, nucleotide depletion reduced the chromatin occupancy of DDX21 in human A375 melanoma cells. Nucleotide supplementation reversed the gene expression signature and DDX21 occupancy changes prompted by leflunomide. Together, our results show that DDX21 acts as a sensor and mediator of transcription during nucleotide stress.
Assuntos
RNA Helicases DEAD-box/genética , Melanócitos/metabolismo , Crista Neural/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/genética , Receptores de Progesterona/genética , Proteínas de Peixe-Zebra/genética , Animais , Linhagem Celular Tumoral , RNA Helicases DEAD-box/metabolismo , Di-Hidro-Orotato Desidrogenase , Embrião não Mamífero , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Leflunomida/farmacologia , Melanócitos/efeitos dos fármacos , Melanócitos/patologia , Crista Neural/efeitos dos fármacos , Crista Neural/crescimento & desenvolvimento , Nucleotídeos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Progesterona/metabolismo , Ligação Proteica , Receptores de Progesterona/metabolismo , Transdução de Sinais , Estresse Fisiológico/genética , Elongação da Transcrição Genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.