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1.
Mol Cell ; 82(1): 106-122.e9, 2022 01 06.
Artículo en Inglés | MEDLINE | ID: mdl-34875212

RESUMEN

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/metabolismo
2.
Nature ; 622(7983): 562-573, 2023 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-37673118

RESUMEN

The ability to study human post-implantation development remains limited owing to ethical and technical challenges associated with intrauterine development after implantation1. Embryo-like models with spatially organized morphogenesis and structure of all defining embryonic and extra-embryonic tissues of the post-implantation human conceptus (that is, the embryonic disc, the bilaminar disc, the yolk sac, the chorionic sac and the surrounding trophoblast layer) remain lacking1,2. Mouse naive embryonic stem cells have recently been shown to give rise to embryonic and extra-embryonic stem cells capable of self-assembling into post-gastrulation structured stem-cell-based embryo models with spatially organized morphogenesis (called SEMs)3. Here we extend those findings to humans using only genetically unmodified human naive embryonic stem cells (cultured in human enhanced naive stem cell medium conditions)4. Such human fully integrated and complete SEMs recapitulate the organization of nearly all known lineages and compartments of post-implantation human embryos, including the epiblast, the hypoblast, the extra-embryonic mesoderm and the trophoblast layer surrounding the latter compartments. These human complete SEMs demonstrated developmental growth dynamics that resemble key hallmarks of post-implantation stage embryogenesis up to 13-14 days after fertilization (Carnegie stage 6a). These include embryonic disc and bilaminar disc formation, epiblast lumenogenesis, polarized amniogenesis, anterior-posterior symmetry breaking, primordial germ-cell specification, polarized yolk sac with visceral and parietal endoderm formation, extra-embryonic mesoderm expansion that defines a chorionic cavity and a connecting stalk, and a trophoblast-surrounding compartment demonstrating syncytium and lacunae formation. This SEM platform will probably enable the experimental investigation of previously inaccessible windows of human early post implantation up to peri-gastrulation development.


Asunto(s)
Implantación del Embrión , Embrión de Mamíferos , Desarrollo Embrionario , Células Madre Embrionarias Humanas , Humanos , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Fertilización , Gastrulación , Estratos Germinativos/citología , Estratos Germinativos/embriología , Células Madre Embrionarias Humanas/citología , Trofoblastos/citología , Saco Vitelino/citología , Saco Vitelino/embriología , Células Gigantes/citología
3.
Genes Dev ; 34(19-20): 1373-1391, 2020 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-32943573

RESUMEN

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 Noqueados
4.
Nature ; 593(7857): 119-124, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33731940

RESUMEN

The mammalian body plan is established shortly after the embryo implants into the maternal uterus, and our understanding of post-implantation developmental processes remains limited. Although pre- and peri-implantation mouse embryos are routinely cultured in vitro1,2, approaches for the robust culture of post-implantation embryos from egg cylinder stages until advanced organogenesis remain to be established. Here we present highly effective platforms for the ex utero culture of post-implantation mouse embryos, which enable the appropriate development of embryos from before gastrulation (embryonic day (E) 5.5) until the hindlimb formation stage (E11). Late gastrulating embryos (E7.5) are grown in three-dimensional rotating bottles, whereas extended culture from pre-gastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle culture platforms. Histological, molecular and single-cell RNA sequencing analyses confirm that the ex utero cultured embryos recapitulate in utero development precisely. This culture system is amenable to the introduction of a variety of embryonic perturbations and micro-manipulations, the results of which can be followed ex utero for up to six days. The establishment of a system for robustly growing normal mouse embryos ex utero from pre-gastrulation to advanced organogenesis represents a valuable tool for investigating embryogenesis, as it eliminates the uterine barrier and allows researchers to mechanistically interrogate post-implantation morphogenesis and artificial embryogenesis in mammals.


Asunto(s)
Técnicas de Cultivo de Embriones , Embrión de Mamíferos/embriología , Desarrollo Embrionario , Técnicas In Vitro , Organogénesis , Animales , Técnicas de Cultivo de Embriones/métodos , Embrión de Mamíferos/citología , Femenino , Gastrulación , Masculino , Ratones , Factores de Tiempo , Útero
5.
Stem Cell Reports ; 17(11): 2484-2500, 2022 11 08.
Artículo en Inglés | MEDLINE | ID: mdl-36270280

RESUMEN

The recent derivation of human trophoblast stem cells (TSCs) from placental cytotrophoblasts and blastocysts opened opportunities for studying the development and function of the human placenta. Recent reports have suggested that human naïve, but not primed, pluripotent stem cells (PSCs) retain an exclusive potential to generate TSCs. Here we report that, in the absence of WNT stimulation, transforming growth factor ß (TGF-ß) pathway inhibition leads to direct and robust conversion of primed human PSCs into TSCs. The resulting primed PSC-derived TSC lines exhibit self-renewal, can differentiate into the main trophoblast lineages, and present RNA and epigenetic profiles that are indistinguishable from recently established TSC lines derived from human placenta, blastocysts, or isogenic human naïve PSCs expanded under human enhanced naïve stem cell medium (HENSM) conditions. Activation of nuclear Yes-associated protein (YAP) signaling is sufficient for this conversion and necessary for human TSC maintenance. Our findings underscore a residual plasticity in primed human PSCs that allows their in vitro conversion into extra-embryonic trophoblast lineages.


Asunto(s)
Células Madre Pluripotentes , Trofoblastos , Femenino , Humanos , Embarazo , Blastocisto , Diferenciación Celular , Placenta , Células Madre Pluripotentes/metabolismo
6.
Cell Stem Cell ; 28(9): 1549-1565.e12, 2021 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-33915080

RESUMEN

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 , Trofoblastos
7.
Cell Death Dis ; 9(2): 107, 2018 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-29371591

RESUMEN

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease caused by the progressive loss of motor neurons in the brain and spinal cord. It has been suggested that toxicity of mutant SOD1 results from its misfolding, however, it is yet unclear why misfolded SOD1 accumulates specifically within motor neurons. We recently demonstrated that macrophage migration inhibitory factor (MIF)-a multifunctional protein with cytokine/chemokine activity and cytosolic chaperone-like properties-inhibits the accumulation of misfolded SOD1. Here, we show that MIF inhibits mutant SOD1 nuclear clearance when overexpressed in motor neuron-like NSC-34 cells. In addition, MIF alters the typical SOD1 amyloid aggregation pathway in vitro, and, instead, promotes the formation of disordered aggregates, as measured by Thioflavin T (ThT) assay and transmission electron microscopy (TEM) imaging. Moreover, we report that MIF reduces the toxicity of misfolded SOD1 by directly interacting with it, and that the chaperone function and protective effect of MIF in neuronal cultures do not require its intrinsic catalytic activities. Importantly, we report that the locked-trimeric MIFN110C mutant, which exhibits strongly impaired CD74-mediated cytokine functions, has strong chaperone activity, dissociating, for the first time, these two cellular functions. Altogether, our study implicates MIF as a potential therapeutic candidate in the treatment of ALS.


Asunto(s)
Amiloide/química , Esclerosis Amiotrófica Lateral/patología , Factores Inhibidores de la Migración de Macrófagos/farmacología , Agregado de Proteínas/efectos de los fármacos , Pliegue de Proteína , Superóxido Dismutasa-1/química , Superóxido Dismutasa-1/toxicidad , Transporte Activo de Núcleo Celular/efectos de los fármacos , Esclerosis Amiotrófica Lateral/metabolismo , Biocatálisis , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Humanos , Modelos Biológicos , Proteínas Mutantes/metabolismo , Proteínas Mutantes/toxicidad , Unión Proteica/efectos de los fármacos , Pliegue de Proteína/efectos de los fármacos , Multimerización de Proteína/efectos de los fármacos , Proteínas Recombinantes/farmacología
8.
Cell Stem Cell ; 23(3): 412-425.e10, 2018 09 06.
Artículo en Inglés | MEDLINE | ID: mdl-30122475

RESUMEN

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 Transgénicos
9.
J Vis Exp ; (123)2017 05 16.
Artículo en Inglés | MEDLINE | ID: mdl-28570551

RESUMEN

Nucleocytoplasmic transport refers to the import and export of large molecules from the cell nucleus. Recently, a number of studies have shown a connection between amyotrophic lateral sclerosis (ALS) and impairments in the nucleocytoplasmic pathway. ALS is a neurodegenerative disease affecting the motor neurons and resulting in paralysis and ultimately in death, within 2-5 years on average. Most cases of ALS are sporadic, lacking any apparent genetic linkage, but 10% are inherited in a dominant manner. Recently, hexanucleotide repeat expansions (HREs) in the chromosome 9 open reading frame 72 (C9orf72) gene were identified as a genetic cause of ALS and frontotemporal dementia (FTD). Importantly, different groups have recently proposed that these mutants affect nucleocytoplasmic transport. These studies have mostly shown the final outcome and manifestations caused by HREs on nucleocytoplasmic transport, but they do not demonstrate nuclear transport dysfunction in real time. As a result, only severe nucleocytoplasmic transport deficiency can be determined, mostly due to high overexpression or exogenous protein insertion. This protocol describes a new and very sensitive assay to evaluate and quantify nucleocytoplasmic transport dysfunction in real time. The rate of import of a NLS-NES-GFP protein (shuttle-GFP) can be quantified in real time using fluorescent microscopy. This is performed by using an exportin inhibitor, thus allowing the shuttle GFP only to enter the nucleus. To validate the assay, the C9orf72 HRE translated dipeptide repeats, poly(GR) and poly(PR), which have been previously shown to disrupt nucleocytoplasmic transport, were used. Using the described assay, a 50% decrease in the nuclear import rate was observed compared to the control. Using this system, minute changes in nucleocytoplasmic transport can be examined and the ability of different factors to rescue (even partially) a nucleocytoplasmic transport defect can be determined.


Asunto(s)
Proteína C9orf72/metabolismo , Neuronas Motoras/metabolismo , Transporte Activo de Núcleo Celular , Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proteína C9orf72/genética , Línea Celular Tumoral , Núcleo Celular/metabolismo , Dipéptidos/metabolismo , Demencia Frontotemporal/diagnóstico , Demencia Frontotemporal/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Ratones , Señales de Exportación Nuclear/fisiología , Señales de Localización Nuclear/metabolismo
10.
ACS Chem Neurosci ; 7(11): 1595-1606, 2016 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-27540759

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that leads to the death of the upper and lower motor neurons. Superoxide dismutase 1 (SOD1) is an ALS pathogenic protein, whose misfolding results in the formation of amyloid aggregates. The mechanism underlying SOD1 pathogenesis in ALS remains obscure, but one possible mechanism involves gain-of-interaction, in which the misfolded soluble SOD1 forms abnormal protein-protein interactions (PPIs) with various cellular proteins, including with other SOD1 molecules, thereby interfering with their function. The structural basis of this gain-of-interaction mechanism is unknown. Here, we characterized the backbone dynamics landscape of misfolded SOD1 to pinpoint surface areas predisposed to aberrant PPIs. This analysis enabled us to formulate a working hypothesis for the mechanism of the gain-of-function of misfolded SOD1, according to which an abnormal PPI potential results from the increased mobility of the SOD1 surface backbone. Guided by the backbone dynamics landscape, we have identified a SOD1-derived peptide that can bind SOD1 proteins and divert the typical amyloid aggregation of ALS-related SOD1 mutants toward a potentially less toxic amorphous aggregation pathway.


Asunto(s)
Superóxido Dismutasa-1/metabolismo , Secuencia de Aminoácidos , Esclerosis Amiotrófica Lateral/metabolismo , Escherichia coli , Humanos , Cinética , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , Péptidos/metabolismo , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Pliegue de Proteína , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Superóxido Dismutasa-1/genética , Propiedades de Superficie
11.
ChemMedChem ; 10(5): 850-61, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25772747

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective death of motor neurons and skeletal muscle atrophy. The majority of ALS cases are acquired spontaneously, with inherited disease accounting for only 10 % of all cases. Recent studies provide compelling evidence that aggregates of misfolded proteins underlie both types of ALS. Small molecules such as artificial chaperones can prevent or even reverse the aggregation of proteins associated with various human diseases. However, their very high active concentration (micromolar range) severely limits their utility as drugs. We synthesized several ester and amide derivatives of chemical chaperones. The lead compound 14, 3-((5-((4,6-dimethylpyridin-2-yl)methoxy)-5-oxopentanoyl)oxy)-N,N-dimethylpropan-1-amine oxide shows, in the micromolar concentration range, both neuronal and astrocyte protective effects in vitro; at daily doses of 10 mg kg(-1) 14 improved the neurological functions and delayed body weight loss in ALS mice. Members of this new chemical chaperone derivative class are strong candidates for the development of new drugs for ALS patients.


Asunto(s)
Amidas/uso terapéutico , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Amidas/síntesis química , Amidas/química , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Ratones Transgénicos , Estructura Molecular
12.
Neuron ; 86(1): 218-32, 2015 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-25801706

RESUMEN

Mutations in superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by loss of motor neurons and accompanied by accumulation of misfolded SOD1 onto the cytoplasmic faces of intracellular organelles, including mitochondria and the endoplasmic reticulum (ER). Using inhibition of misfolded SOD1 deposition onto mitochondria as an assay, a chaperone activity abundant in nonneuronal tissues is now purified and identified to be the multifunctional macrophage migration inhibitory factor (MIF), whose activities include an ATP-independent protein folding chaperone. Purified MIF is shown to directly inhibit mutant SOD1 misfolding. Elevating MIF in neuronal cells suppresses accumulation of misfolded SOD1 and its association with mitochondria and the ER and extends survival of mutant SOD1-expressing motor neurons. Accumulated MIF protein is identified to be low in motor neurons, implicating correspondingly low chaperone activity as a component of vulnerability to mutant SOD1 misfolding and supporting therapies to enhance intracellular MIF chaperone activity.


Asunto(s)
Factores Inhibidores de la Migración de Macrófagos/metabolismo , Pliegue de Proteína , Superóxido Dismutasa/metabolismo , Fosfatasa Ácida/genética , Animales , Diferenciación Celular/genética , Células Cultivadas , Colina O-Acetiltransferasa/genética , Colina O-Acetiltransferasa/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Células Madre Pluripotentes Inducidas , Isoenzimas/genética , Hígado/metabolismo , Hígado/ultraestructura , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , Neuronas Motoras/fisiología , Mutación/genética , Proteínas del Tejido Nervioso/metabolismo , Transporte de Proteínas/genética , Ratas , Ratas Transgénicas , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Médula Espinal/metabolismo , Médula Espinal/ultraestructura , Superóxido Dismutasa/genética , Superóxido Dismutasa-1 , Fosfatasa Ácida Tartratorresistente
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