RESUMEN
Delivering macromolecules across biological barriers such as the blood-brain barrier limits their application in vivo. Previous work has demonstrated that Toxoplasma gondii, a parasite that naturally travels from the human gut to the central nervous system (CNS), can deliver proteins to host cells. Here we engineered T. gondii's endogenous secretion systems, the rhoptries and dense granules, to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16. We demonstrate delivery in cultured cells, brain organoids and in vivo, and probe protein activity using imaging, pull-down assays, scRNA-seq and fluorescent reporters. We demonstrate robust delivery after intraperitoneal administration in mice and characterize 3D distribution throughout the brain. As proof of concept, we demonstrate GRA16-mediated brain delivery of the MeCP2 protein, a putative therapeutic target for Rett syndrome. By characterizing the potential and current limitations of the system, we aim to guide future improvements that will be required for broader application.
Asunto(s)
Encéfalo , Neuronas , Proteínas Protozoarias , Toxoplasma , Toxoplasma/genética , Toxoplasma/metabolismo , Animales , Neuronas/metabolismo , Neuronas/parasitología , Ratones , Humanos , Encéfalo/metabolismo , Encéfalo/parasitología , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/genética , Proteína 2 de Unión a Metil-CpG/genética , Proteína 2 de Unión a Metil-CpG/metabolismo , Sistemas de Liberación de MedicamentosRESUMEN
The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.
Asunto(s)
Blastocisto/metabolismo , Linaje de la Célula , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Animales , Blastocisto/citología , Cromatina/genética , Técnicas de Cultivo de Embriones , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Histonas/genética , Humanos , Ratones , Fenotipo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Ubiquitinas/genética , Ubiquitinas/metabolismoRESUMEN
Isolating human MEK/ERK signaling-independent pluripotent stem cells (PSCs) with naive pluripotency characteristics while maintaining differentiation competence and (epi)genetic integrity remains challenging. Here, we engineer reporter systems that allow the screening for defined conditions that induce molecular and functional features of human naive pluripotency. Synergistic inhibition of WNT/ß-CATENIN, protein kinase C (PKC), and SRC signaling consolidates the induction of teratoma-competent naive human PSCs, with the capacity to differentiate into trophoblast stem cells (TSCs) and extraembryonic naive endodermal (nEND) cells in vitro. Divergent signaling and transcriptional requirements for boosting naive pluripotency were found between mouse and human. P53 depletion in naive hPSCs increased their contribution to mouse-human cross-species chimeric embryos upon priming and differentiation. Finally, MEK/ERK inhibition can be substituted with the inhibition of NOTCH/RBPj, which induces alternative naive-like hPSCs with a diminished risk for deleterious global DNA hypomethylation. Our findings set a framework for defining the signaling foundations of human naive pluripotency.
Asunto(s)
Células Madre Pluripotentes , Animales , Diferenciación Celular , Embrión de Mamíferos , Humanos , Ratones , Transducción de Señal , TrofoblastosRESUMEN
The N6-methyladenosine (m6A) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous m6A-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, Mettl3-KO severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers' expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers' role during early development that is Ythdf1/2/3 gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.
Asunto(s)
Compensación de Dosificación (Genética) , Gametogénesis/genética , Metiltransferasas/genética , Metiltransferasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular , Células Madre Embrionarias , Fertilidad/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Ratones , Ratones NoqueadosRESUMEN
Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that ß-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by ß-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.
RESUMEN
The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.
Asunto(s)
Reprogramación Celular/genética , Epigénesis Genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética , Animales , Linaje de la Célula/genética , Cromatina/metabolismo , Desmetilación , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Ratones , Unión Proteica , ARN de Transferencia/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The role of mitochondria dynamics and its molecular regulators remains largely unknown during naïve-to-primed pluripotent cell interconversion. Here we report that mitochondrial MTCH2 is a regulator of mitochondrial fusion, essential for the naïve-to-primed interconversion of murine embryonic stem cells (ESCs). During this interconversion, wild-type ESCs elongate their mitochondria and slightly alter their glutamine utilization. In contrast, MTCH2-/- ESCs fail to elongate their mitochondria and to alter their metabolism, maintaining high levels of histone acetylation and expression of naïve pluripotency markers. Importantly, enforced mitochondria elongation by the pro-fusion protein Mitofusin (MFN) 2 or by a dominant negative form of the pro-fission protein dynamin-related protein (DRP) 1 is sufficient to drive the exit from naïve pluripotency of both MTCH2-/- and wild-type ESCs. Taken together, our data indicate that mitochondria elongation, governed by MTCH2, plays a critical role and constitutes an early driving force in the naïve-to-primed pluripotency interconversion of murine ESCs.
Asunto(s)
Dinámicas Mitocondriales/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Pluripotentes/metabolismo , Animales , Células Cultivadas , Dinaminas/genética , Dinaminas/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Expresión Génica , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Confocal , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Células Madre Embrionarias de Ratones/citología , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Células Madre Pluripotentes/citologíaRESUMEN
Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.
Asunto(s)
ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción GATA/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Femenino , Células Madre Pluripotentes Inducidas/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Ratones TransgénicosRESUMEN
Ectopic expression of lineage master regulators induces transdifferentiation. Whether cell fate transitions can be induced during various developmental stages has not been systemically examined. Here we discover that amongst different developmental stages, mouse embryonic stem cells (mESCs) are resistant to cell fate conversion induced by the melanocyte lineage master regulator MITF. By generating a transgenic system we exhibit that in mESCs, the pluripotency master regulator Oct4, counteracts pro-differentiation induced by Mitf by physical interference with MITF transcriptional activity. We further demonstrate that mESCs must be released from Oct4-maintained pluripotency prior to ectopically induced differentiation. Moreover, Oct4 induction in various differentiated cells represses their lineage identity in vivo. Alongside, chromatin architecture combined with ChIP-seq analysis suggest that Oct4 competes with various lineage master regulators for binding promoters and enhancers. Our analysis reveals pluripotency and transdifferentiation regulatory principles and could open new opportunities in the field of regenerative medicine.
Asunto(s)
Diferenciación Celular/genética , Factor de Transcripción Asociado a Microftalmía/genética , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Animales , Línea Celular Tumoral , Transdiferenciación Celular/genética , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , Factor de Transcripción Asociado a Microftalmía/metabolismo , Células Madre Embrionarias de Ratones/citología , Factor 3 de Transcripción de Unión a Octámeros/metabolismoRESUMEN
Measurement of Alkaline Phosphatase (ALP) level is a widely used procedure in clinical and basic research. We present a simple and inexpensive luminescence-based method that allows multiplexed measurement and normalization of intracellular ALP levels in one sample well. The method comprises two commercially available reagents enabling quantification of ALP levels and cell number by two sequential luminescence readouts. Using this method we were able to detect and analyze somatic reprogramming into pluripotent stem cells. The method is highly applicable for High Throughput Screening (HTS) campaigns and analysis.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Mediciones Luminiscentes/métodos , Adenosina Trifosfato/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Línea Celular , Técnicas de Cocultivo , Luminiscencia , RatonesRESUMEN
Histone modifications play an important role in chromatin organization and transcriptional regulation, but despite the large amount of genome-wide histone modification data collected in different cells and tissues, little is known about co-occurrence of modifications on the same nucleosome. Here we present a genome-wide quantitative method for combinatorial indexed chromatin immunoprecipitation (co-ChIP) to characterize co-occurrence of histone modifications on nucleosomes. Using co-ChIP, we study the genome-wide co-occurrence of 14 chromatin marks (70 pairwise combinations), and find previously undescribed co-occurrence patterns, including the co-occurrence of H3K9me1 and H3K27ac in super-enhancers. Finally, we apply co-ChIP to measure the distribution of the bivalent H3K4me3-H3K27me3 domains in two distinct mouse embryonic stem cell (mESC) states and in four adult tissues. We observe dynamic changes in 5,786 regions and discover both loss and de novo gain of bivalency in key tissue-specific regulatory genes, suggesting a functional role for bivalent domains during different stages of development. These results show that co-ChIP can reveal the complex interactions between histone modifications.
Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Mapeo Cromosómico/métodos , Genoma/genética , Código de Histonas/genética , Histonas/genética , Microquímica/métodos , Animales , Sitios de Unión , Técnicas Químicas Combinatorias/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Unión Proteica , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadAsunto(s)
Ácidos/farmacología , Diferenciación Celular , Reprogramación Celular/efectos de los fármacos , Células Madre Embrionarias/citología , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Placenta/citología , Trofoblastos/citología , Animales , Femenino , Masculino , EmbarazoRESUMEN
Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency. We show that the vast majority of reprogrammed cardiomyocytes or neural stem cells obtained from mouse fibroblasts by OSKM-induced 'transdifferentiation' pass through a transient pluripotent state, and that their derivation is molecularly coupled to iPSC formation mechanisms. Our findings underscore the importance of defining trajectories during cell reprogramming by various methods.
Asunto(s)
Transdiferenciación Celular/genética , Reprogramación Celular/genética , Células Madre Pluripotentes Inducidas/fisiología , Factores de Transcripción/metabolismo , Animales , Células Cultivadas , Femenino , Factor 4 Similar a Kruppel , Masculino , Ratones , Ratones TransgénicosRESUMEN
Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner.
Asunto(s)
Adenosina/análogos & derivados , Diferenciación Celular/fisiología , Metiltransferasas/fisiología , Células Madre Pluripotentes/citología , ARN Mensajero/metabolismo , Adenosina/metabolismo , Animales , Blastocisto/enzimología , Diferenciación Celular/genética , Línea Celular , Pérdida del Embrión/genética , Epigénesis Genética , Femenino , Técnicas de Inactivación de Genes , Masculino , Metilación , Metiltransferasas/genética , Ratones , Ratones Noqueados , Células Madre Pluripotentes/enzimologíaRESUMEN
Mouse embryonic stem (ES) cells are isolated from the inner cell mass of blastocysts, and can be preserved in vitro in a naive inner-cell-mass-like configuration by providing exogenous stimulation with leukaemia inhibitory factor (LIF) and small molecule inhibition of ERK1/ERK2 and GSK3ß signalling (termed 2i/LIF conditions). Hallmarks of naive pluripotency include driving Oct4 (also known as Pou5f1) transcription by its distal enhancer, retaining a pre-inactivation X chromosome state, and global reduction in DNA methylation and in H3K27me3 repressive chromatin mark deposition on developmental regulatory gene promoters. Upon withdrawal of 2i/LIF, naive mouse ES cells can drift towards a primed pluripotent state resembling that of the post-implantation epiblast. Although human ES cells share several molecular features with naive mouse ES cells, they also share a variety of epigenetic properties with primed murine epiblast stem cells (EpiSCs). These include predominant use of the proximal enhancer element to maintain OCT4 expression, pronounced tendency for X chromosome inactivation in most female human ES cells, increase in DNA methylation and prominent deposition of H3K27me3 and bivalent domain acquisition on lineage regulatory genes. The feasibility of establishing human ground state naive pluripotency in vitro with equivalent molecular and functional features to those characterized in mouse ES cells remains to be defined. Here we establish defined conditions that facilitate the derivation of genetically unmodified human naive pluripotent stem cells from already established primed human ES cells, from somatic cells through induced pluripotent stem (iPS) cell reprogramming or directly from blastocysts. The novel naive pluripotent cells validated herein retain molecular characteristics and functional properties that are highly similar to mouse naive ES cells, and distinct from conventional primed human pluripotent cells. This includes competence in the generation of cross-species chimaeric mouse embryos that underwent organogenesis following microinjection of human naive iPS cells into mouse morulas. Collectively, our findings establish new avenues for regenerative medicine, patient-specific iPS cell disease modelling and the study of early human development in vitro and in vivo.
Asunto(s)
Células Madre Pluripotentes Inducidas/citología , Animales , Blastocisto/citología , Reprogramación Celular , Quimera/embriología , Cromatina/metabolismo , Metilación de ADN , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Epigénesis Genética , Femenino , Estratos Germinativos/citología , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Masculino , Ratones , Mórula/citología , Organogénesis , Regiones Promotoras Genéticas/genética , Medicina Regenerativa , Reproducibilidad de los Resultados , Transducción de Señal , Inactivación del Cromosoma XRESUMEN
Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells). Our findings uncover a dichotomous molecular function for the reprogramming factors, serving to reactivate endogenous pluripotency networks while simultaneously directly recruiting the Mbd3/NuRD repressor complex that potently restrains the reactivation of OSKM downstream target genes. Subsequently, the latter interactions, which are largely depleted during early pre-implantation development in vivo, lead to a stochastic and protracted reprogramming trajectory towards pluripotency in vitro. The deterministic reprogramming approach devised here offers a novel platform for the dissection of molecular dynamics leading to establishing pluripotency at unprecedented flexibility and resolution.