RESUMEN
Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.
Asunto(s)
Diferenciación Celular , Factores de Transcripción , Humanos , Cromatina , Regulación de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , Factores de Transcripción/metabolismo , Atlas como AsuntoRESUMEN
Retrotransposons mediate gene regulation in important developmental and pathological processes. Here, we characterized the transient retrotransposon induction during preimplantation development of eight mammals. Induced retrotransposons exhibit similar preimplantation profiles across species, conferring gene regulatory activities, particularly through long terminal repeat (LTR) retrotransposon promoters. A mouse-specific MT2B2 retrotransposon promoter generates an N-terminally truncated Cdk2ap1ΔN that peaks in preimplantation embryos and promotes proliferation. In contrast, the canonical Cdk2ap1 peaks in mid-gestation and represses cell proliferation. This MT2B2 promoter, whose deletion abolishes Cdk2ap1ΔN production, reduces cell proliferation and impairs embryo implantation, is developmentally essential. Intriguingly, Cdk2ap1ΔN is evolutionarily conserved in sequence and function yet is driven by different promoters across mammals. The distinct preimplantation Cdk2ap1ΔN expression in each mammalian species correlates with the duration of its preimplantation development. Hence, species-specific transposon promoters can yield evolutionarily conserved, alternative protein isoforms, bestowing them with new functions and species-specific expression to govern essential biological divergence.
Asunto(s)
Secuencia Conservada , Desarrollo Embrionario/genética , Proteínas Quinasas/metabolismo , Retroelementos/genética , Proteínas Supresoras de Tumor/metabolismo , Animales , Secuencia de Bases , Blastocisto/metabolismo , Proliferación Celular , Evolución Molecular , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , Humanos , Mamíferos/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Biológicos , Regiones Promotoras Genéticas , Isoformas de Proteínas/metabolismoRESUMEN
RTN4-binding proteins were widely studied as "NoGo" receptors, but their physiological interactors and roles remain elusive. Similarly, BAI adhesion-GPCRs were associated with numerous activities, but their ligands and functions remain unclear. Using unbiased approaches, we observed an unexpected convergence: RTN4 receptors are high-affinity ligands for BAI adhesion-GPCRs. A single thrombospondin type 1-repeat (TSR) domain of BAIs binds to the leucine-rich repeat domain of all three RTN4-receptor isoforms with nanomolar affinity. In the 1.65 Å crystal structure of the BAI1/RTN4-receptor complex, C-mannosylation of tryptophan and O-fucosylation of threonine in the BAI TSR-domains creates a RTN4-receptor/BAI interface shaped by unusual glycoconjugates that enables high-affinity interactions. In human neurons, RTN4 receptors regulate dendritic arborization, axonal elongation, and synapse formation by differential binding to glial versus neuronal BAIs, thereby controlling neural network activity. Thus, BAI binding to RTN4/NoGo receptors represents a receptor-ligand axis that, enabled by rare post-translational modifications, controls development of synaptic circuits.
Asunto(s)
Inhibidores de la Angiogénesis/metabolismo , Encéfalo/metabolismo , Neurogénesis , Neuronas/metabolismo , Proteínas Nogo/metabolismo , Receptores Nogo/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adipoquinas/metabolismo , Secuencia de Aminoácidos , Animales , Axones/metabolismo , Adhesión Celular , Moléculas de Adhesión Celular Neuronal/metabolismo , Complemento C1q/metabolismo , Dendritas/metabolismo , Glicosilación , Células HEK293 , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ligandos , Ratones Endogámicos C57BL , Red Nerviosa/metabolismo , Polisacáridos/metabolismo , Unión Proteica , Dominios Proteicos , Eliminación de Secuencia , Sinapsis/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.
Asunto(s)
Alelos , Proteína 9 Asociada a CRISPR/metabolismo , Cromosomas Humanos/genética , Embrión de Mamíferos/metabolismo , Animales , Secuencia de Bases , Blastocisto/metabolismo , Ciclo Celular/genética , Línea Celular , Deleción Cromosómica , Roturas del ADN de Doble Cadena , Reparación del ADN por Unión de Extremidades/genética , Implantación del Embrión/genética , Proteínas del Ojo/genética , Fertilización , Edición Génica , Reordenamiento Génico/genética , Sitios Genéticos , Genoma Humano , Genotipo , Heterocigoto , Células Madre Embrionarias Humanas/metabolismo , Humanos , Mutación INDEL/genética , Ratones , Mitosis , Sistemas de Lectura Abierta/genética , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Long noncoding RNAs (lncRNAs) evolve more rapidly than mRNAs. Whether conserved lncRNAs undergo conserved processing, localization, and function remains unexplored. We report differing subcellular localization of lncRNAs in human and mouse embryonic stem cells (ESCs). A significantly higher fraction of lncRNAs is localized in the cytoplasm of hESCs than in mESCs. This turns out to be important for hESC pluripotency. FAST is a positionally conserved lncRNA but is not conserved in its processing and localization. In hESCs, cytoplasm-localized hFAST binds to the WD40 domain of the E3 ubiquitin ligase ß-TrCP and blocks its interaction with phosphorylated ß-catenin to prevent degradation, leading to activated WNT signaling, required for pluripotency. In contrast, mFast is nuclear retained in mESCs, and its processing is suppressed by the splicing factor PPIE, which is highly expressed in mESCs but not hESCs. These findings reveal that lncRNA processing and localization are previously under-appreciated contributors to the rapid evolution of function.
Asunto(s)
Espacio Intracelular/genética , ARN Largo no Codificante/metabolismo , Células Madre/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Células Cultivadas , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Empalme del ARN/genética , ARN Largo no Codificante/genética , ARN Mensajero/metabolismo , Transducción de Señal/genética , Células Madre/patologíaRESUMEN
Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.
Asunto(s)
Transferasas Intramoleculares/metabolismo , Biosíntesis de Proteínas , Seudouridina/metabolismo , ARN de Transferencia/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de Ciclo Celular , Diferenciación Celular , Factores Eucarióticos de Iniciación/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Transferasas Intramoleculares/antagonistas & inhibidores , Transferasas Intramoleculares/genética , Ratones , Ratones Endogámicos NOD , Ratones SCID , Síndromes Mielodisplásicos/patología , Conformación de Ácido Nucleico , Fosfoproteínas/metabolismo , Proteína I de Unión a Poli(A)/antagonistas & inhibidores , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Nicho de Células MadreRESUMEN
The pace of human brain development is highly protracted compared with most other species1-7. The maturation of cortical neurons is particularly slow, taking months to years to develop adult functions3-5. Remarkably, such protracted timing is retained in cortical neurons derived from human pluripotent stem cells (hPSCs) during in vitro differentiation or upon transplantation into the mouse brain4,8,9. Those findings suggest the presence of a cell-intrinsic clock setting the pace of neuronal maturation, although the molecular nature of this clock remains unknown. Here we identify an epigenetic developmental programme that sets the timing of human neuronal maturation. First, we developed a hPSC-based approach to synchronize the birth of cortical neurons in vitro which enabled us to define an atlas of morphological, functional and molecular maturation. We observed a slow unfolding of maturation programmes, limited by the retention of specific epigenetic factors. Loss of function of several of those factors in cortical neurons enables precocious maturation. Transient inhibition of EZH2, EHMT1 and EHMT2 or DOT1L, at progenitor stage primes newly born neurons to rapidly acquire mature properties upon differentiation. Thus our findings reveal that the rate at which human neurons mature is set well before neurogenesis through the establishment of an epigenetic barrier in progenitor cells. Mechanistically, this barrier holds transcriptional maturation programmes in a poised state that is gradually released to ensure the prolonged timeline of human cortical neuron maturation.
Asunto(s)
Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas , Células-Madre Neurales , Neurogénesis , Neuronas , Adulto , Animales , Humanos , Ratones , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Neuronas/citología , Neuronas/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
The nucleus is highly organized, such that factors involved in the transcription and processing of distinct classes of RNA are confined within specific nuclear bodies1,2. One example is the nuclear speckle, which is defined by high concentrations of protein and noncoding RNA regulators of pre-mRNA splicing3. What functional role, if any, speckles might play in the process of mRNA splicing is unclear4,5. Here we show that genes localized near nuclear speckles display higher spliceosome concentrations, increased spliceosome binding to their pre-mRNAs and higher co-transcriptional splicing levels than genes that are located farther from nuclear speckles. Gene organization around nuclear speckles is dynamic between cell types, and changes in speckle proximity lead to differences in splicing efficiency. Finally, directed recruitment of a pre-mRNA to nuclear speckles is sufficient to increase mRNA splicing levels. Together, our results integrate the long-standing observations of nuclear speckles with the biochemistry of mRNA splicing and demonstrate a crucial role for dynamic three-dimensional spatial organization of genomic DNA in driving spliceosome concentrations and controlling the efficiency of mRNA splicing.
Asunto(s)
Genoma , Motas Nucleares , Precursores del ARN , Empalme del ARN , ARN Mensajero , Empalmosomas , Animales , Humanos , Masculino , Ratones , Genes , Genoma/genética , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Motas Nucleares/genética , Motas Nucleares/metabolismo , Precursores del ARN/metabolismo , Precursores del ARN/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Empalmosomas/metabolismo , Transcripción GenéticaRESUMEN
Cells undergo a major epigenome reconfiguration when reprogrammed to human induced pluripotent stem cells (hiPS cells). However, the epigenomes of hiPS cells and human embryonic stem (hES) cells differ significantly, which affects hiPS cell function1-8. These differences include epigenetic memory and aberrations that emerge during reprogramming, for which the mechanisms remain unknown. Here we characterized the persistence and emergence of these epigenetic differences by performing genome-wide DNA methylation profiling throughout primed and naive reprogramming of human somatic cells to hiPS cells. We found that reprogramming-induced epigenetic aberrations emerge midway through primed reprogramming, whereas DNA demethylation begins early in naive reprogramming. Using this knowledge, we developed a transient-naive-treatment (TNT) reprogramming strategy that emulates the embryonic epigenetic reset. We show that the epigenetic memory in hiPS cells is concentrated in cell of origin-dependent repressive chromatin marked by H3K9me3, lamin-B1 and aberrant CpH methylation. TNT reprogramming reconfigures these domains to a hES cell-like state and does not disrupt genomic imprinting. Using an isogenic system, we demonstrate that TNT reprogramming can correct the transposable element overexpression and differential gene expression seen in conventional hiPS cells, and that TNT-reprogrammed hiPS and hES cells show similar differentiation efficiencies. Moreover, TNT reprogramming enhances the differentiation of hiPS cells derived from multiple cell types. Thus, TNT reprogramming corrects epigenetic memory and aberrations, producing hiPS cells that are molecularly and functionally more similar to hES cells than conventional hiPS cells. We foresee TNT reprogramming becoming a new standard for biomedical and therapeutic applications and providing a novel system for studying epigenetic memory.
Asunto(s)
Reprogramación Celular , Epigénesis Genética , Células Madre Pluripotentes Inducidas , Humanos , Cromatina/genética , Cromatina/metabolismo , Desmetilación del ADN , Metilación de ADN , Elementos Transponibles de ADN , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Lamina Tipo BRESUMEN
Telomere length control is critical for cellular lifespan and tumor suppression. Telomerase is transiently activated in the inner cell mass of the developing blastocyst to reset telomere reserves. Its silencing upon differentiation leads to gradual telomere shortening in somatic cells. Here, we report that transcriptional regulation through cis-regulatory elements only partially accounts for telomerase activation in pluripotent cells. Instead, developmental control of telomerase is primarily driven by an alternative splicing event, centered around hTERT exon 2. Skipping of exon 2 triggers hTERT mRNA decay in differentiated cells, and conversely, its retention promotes telomerase accumulation in pluripotent cells. We identify SON as a regulator of exon 2 alternative splicing and report a patient carrying a SON mutation and suffering from insufficient telomerase and short telomeres. In summary, our study highlights a critical role for hTERT alternative splicing in the developmental regulation of telomerase and implicates defective splicing in telomere biology disorders.
Asunto(s)
Empalme Alternativo , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Antígenos de Histocompatibilidad Menor/genética , Telomerasa/genética , Homeostasis del Telómero , Telómero/metabolismo , Blastocisto/metabolismo , Blastocisto/patología , Diferenciación Celular , Preescolar , Proteínas de Unión al ADN/deficiencia , Femenino , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Humanos , Linaje , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología , Cultivo Primario de Células , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Telomerasa/deficiencia , Telómero/patologíaRESUMEN
Cell state changes are associated with proteome remodeling to serve newly emergent cell functions. Here, we show that NGN2-driven conversion of human embryonic stem cells to induced neurons (iNeurons) is associated with increased PINK1-independent mitophagic flux that is temporally correlated with metabolic reprogramming to support oxidative phosphorylation. Global multiplex proteomics during neurogenesis revealed large-scale remodeling of functional modules linked with pluripotency, mitochondrial metabolism, and proteostasis. Differentiation-dependent mitophagic flux required BNIP3L and its LC3-interacting region (LIR) motif, and BNIP3L also promoted mitophagy in dopaminergic neurons. Proteomic analysis of ATG12-/- iNeurons revealed accumulation of endoplasmic reticulum, Golgi, and mitochondria during differentiation, indicative of widespread organelle remodeling during neurogenesis. This work reveals broad organelle remodeling of membrane-bound organelles during NGN2-driven neurogenesis via autophagy, identifies BNIP3L's central role in programmed mitophagic flux, and provides a proteomic resource for elucidating how organelle remodeling and autophagy alter the proteome during changes in cell state.
Asunto(s)
Células Madre Embrionarias Humanas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Mitofagia , Células-Madre Neurales/enzimología , Neurogénesis , Neuronas/enzimología , Proteoma , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína 12 Relacionada con la Autofagia/genética , Proteína 12 Relacionada con la Autofagia/metabolismo , Línea Celular , Humanos , Proteínas de la Membrana/genética , Mitocondrias/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteostasis , Proteínas Proto-Oncogénicas/genética , Factores de Tiempo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Experimental modelling of human disorders enables the definition of the cellular and molecular mechanisms underlying diseases and the development of therapies for treating them. The availability of human pluripotent stem cells (PSCs), which are capable of self-renewal and have the potential to differentiate into virtually any cell type, can now help to overcome the limitations of animal models for certain disorders. The ability to model human diseases using cultured PSCs has revolutionized the ways in which we study monogenic, complex and epigenetic disorders, as well as early- and late-onset diseases. Several strategies are used to generate such disease models using either embryonic stem cells (ES cells) or patient-specific induced PSCs (iPSCs), creating new possibilities for the establishment of models and their use in drug screening.
Asunto(s)
Enfermedades Genéticas Congénitas , Células Madre Embrionarias Humanas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Trasplante de Células Madre/métodos , Aloinjertos , Animales , Autoinjertos , Modelos Animales de Enfermedad , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Enfermedades Genéticas Congénitas/terapia , HumanosRESUMEN
Rett syndrome (RTT), mainly caused by mutations in methyl-CpG binding protein 2 (MeCP2), is one of the most prevalent intellectual disorders without effective therapies. Here, we used 2D and 3D human brain cultures to investigate MeCP2 function. We found that MeCP2 mutations cause severe abnormalities in human interneurons (INs). Surprisingly, treatment with a BET inhibitor, JQ1, rescued the molecular and functional phenotypes of MeCP2 mutant INs. We uncovered that abnormal increases in chromatin binding of BRD4 and enhancer-promoter interactions underlie the abnormal transcription in MeCP2 mutant INs, which were recovered to normal levels by JQ1. We revealed cell-type-specific transcriptome impairment in MeCP2 mutant region-specific human brain organoids that were rescued by JQ1. Finally, JQ1 ameliorated RTT-like phenotypes in mice. These data demonstrate that BRD4 dysregulation is a critical driver for RTT etiology and suggest that targeting BRD4 could be a potential therapeutic opportunity for RTT.
Asunto(s)
Azepinas/farmacología , Encéfalo/patología , Proteínas de Ciclo Celular/metabolismo , Interneuronas/patología , Proteína 2 de Unión a Metil-CpG/fisiología , Síndrome de Rett/patología , Factores de Transcripción/metabolismo , Transcriptoma/efectos de los fármacos , Triazoles/farmacología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Proteínas de Ciclo Celular/genética , Femenino , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Fenotipo , Síndrome de Rett/tratamiento farmacológico , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Factores de Transcripción/genéticaRESUMEN
Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio , Retina , Factores de Transcripción , Animales , Humanos , Ratones , Elementos de Facilitación Genéticos/genética , Evolución Molecular , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Microftalmía/genética , Microftalmía/patología , Neurogénesis/genética , Organoides/metabolismo , Retina/metabolismo , Factores de Transcripción/metabolismo , Factores de Transcripción/genéticaRESUMEN
Limited access to embryos has hampered the study of human embryogenesis and disorders that occur during early pregnancy. Human pluripotent stem cells provide an alternative means to study human development in a dish1-7. Recent advances in partial embryo models derived from human pluripotent stem cells have enabled human development to be examined at early post-implantation stages8-14. However, models of the pre-implantation human blastocyst are lacking. Starting from naive human pluripotent stem cells, here we developed an effective three-dimensional culture strategy with successive lineage differentiation and self-organization to generate blastocyst-like structures in vitro. These structures-which we term 'human blastoids'-resemble human blastocysts in terms of their morphology, size, cell number, and composition and allocation of different cell lineages. Single-cell RNA-sequencing analyses also reveal the transcriptomic similarity of blastoids to blastocysts. Human blastoids are amenable to embryonic and extra-embryonic stem cell derivation and can further develop into peri-implantation embryo-like structures in vitro. Using chemical perturbations, we show that specific isozymes of protein kinase C have a critical function in the formation of the blastoid cavity. Human blastoids provide a readily accessible, scalable, versatile and perturbable alternative to blastocysts for studying early human development, understanding early pregnancy loss and gaining insights into early developmental defects.
Asunto(s)
Blastocisto/citología , Blastocisto/metabolismo , Diferenciación Celular , Células Madre Pluripotentes/citología , Blastocisto/enzimología , Técnicas de Cultivo de Célula/métodos , Línea Celular , Linaje de la Célula , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/enzimología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Isoenzimas/metabolismo , Células Madre Pluripotentes/enzimología , Células Madre Pluripotentes/metabolismo , Proteína Quinasa C/metabolismo , Análisis de la Célula Individual , TranscriptomaRESUMEN
Transcription regulation underlies stem cell function and development. Here, we elucidate an unexpected role of an essential ribogenesis factor, WDR43, as a chromatin-associated RNA-binding protein (RBP) and release factor in modulating the polymerase (Pol) II activity for pluripotency regulation. WDR43 binds prominently to promoter-associated noncoding/nascent RNAs, occupies thousands of gene promoters and enhancers, and interacts with the Pol II machinery in embryonic stem cells (ESCs). Nascent transcripts and transcription recruit WDR43 to active promoters, where WDR43 facilitates releases of the elongation factor P-TEFb and paused Pol II. Knockdown of WDR43 causes genome-wide defects in Pol II release and pluripotency-associated gene expression. Importantly, auxin-mediated rapid degradation of WDR43 drastically reduces Pol II activity, precluding indirect consequences. These results reveal an RNA-mediated recruitment and feedforward regulation on transcription and demonstrate an unforeseen role of an RBP in promoting Pol II elongation and coordinating high-level transcription and translation in ESC pluripotency.
Asunto(s)
Proteínas de Transporte de Catión/genética , Cromatina/química , Regulación del Desarrollo de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo , ARN Polimerasa II/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Transcripción Genética , Proteínas de Pez Cebra/genética , Animales , Sitios de Unión , Proteínas de Transporte de Catión/metabolismo , Diferenciación Celular , Línea Celular , Cromatina/metabolismo , Embrión de Mamíferos , Elementos de Facilitación Genéticos , Eliminación de Gen , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Células Madre Embrionarias de Ratones/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Factor B de Elongación Transcripcional Positiva/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Biosíntesis de Proteínas , Proteolisis , ARN Polimerasa II/metabolismo , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Transducción de Señal , Proteínas de Pez Cebra/metabolismoRESUMEN
Mammalian SWI/SNF complexes are multi-subunit chromatin remodeling complexes associated with an ATPase (either SMARCA4 or SMARCA2). Heterozygous mutations in the SMARCA2 ATPase cause Nicolaides-Baraitser syndrome (NCBRS), an intellectual disability syndrome associated with delayed speech onset. We engineered human embryonic stem cells (hESCs) to carry NCBRS-associated heterozygous SMARCA2 K755R or R1159Q mutations. While SMARCA2 mutant hESCs were phenotypically normal, differentiation to neural progenitors cells (NPCs) was severely impaired. We find that SMARCA2 mutations cause enhancer reorganization with loss of SOX3-dependent neural enhancers and prominent emergence of astrocyte-specific de novo enhancers. Changes in chromatin accessibility at enhancers were associated with an increase in SMARCA2 binding and retargeting of SMARCA4. We show that the AP-1 family member FRA2 is aberrantly overexpressed in SMARCA2 mutant NPCs, where it functions as a pioneer factor at de novo enhancers. Together, our results demonstrate that SMARCA2 mutations cause impaired differentiation through enhancer reprogramming via inappropriate targeting of SMARCA4.
Asunto(s)
ADN Helicasas/metabolismo , Elementos de Facilitación Genéticos , Heterocigoto , Células Madre Embrionarias Humanas/metabolismo , Mutación Missense , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Sustitución de Aminoácidos , Diferenciación Celular/genética , Cromatina/genética , Cromatina/metabolismo , ADN Helicasas/genética , Facies , Deformidades Congénitas del Pie/genética , Deformidades Congénitas del Pie/metabolismo , Deformidades Congénitas del Pie/patología , Antígeno 2 Relacionado con Fos/biosíntesis , Antígeno 2 Relacionado con Fos/genética , Células HEK293 , Células Madre Embrionarias Humanas/patología , Humanos , Hipotricosis/genética , Hipotricosis/metabolismo , Hipotricosis/patología , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Discapacidad Intelectual/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Proteínas Nucleares/genética , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factores de Transcripción/genéticaRESUMEN
The complicated process of neuronal development is initiated early in life, with the genetic mechanisms governing this process yet to be fully elucidated. Single-cell RNA sequencing (scRNA-seq) is a potent instrument for pinpointing biomarkers that exhibit differential expression across various cell types and developmental stages. By employing scRNA-seq on human embryonic stem cells, we aim to identify differentially expressed genes (DEGs) crucial for early-stage neuronal development. Our focus extends beyond simply identifying DEGs. We strive to investigate the functional roles of these genes through enrichment analysis and construct gene regulatory networks to understand their interactions. Ultimately, this comprehensive approach aspires to illuminate the molecular mechanisms and transcriptional dynamics governing early human brain development. By uncovering potential links between these DEGs and intelligence, mental disorders, and neurodevelopmental disorders, we hope to shed light on human neurological health and disease. In this study, we have used scRNA-seq to identify DEGs involved in early-stage neuronal development in hESCs. The scRNA-seq data, collected on days 26 (D26) and 54 (D54), of the in vitro differentiation of hESCs to neurons were analyzed. Our analysis identified 539 DEGs between D26 and D54. Functional enrichment of those DEG biomarkers indicated that the up-regulated DEGs participated in neurogenesis, while the down-regulated DEGs were linked to synapse regulation. The Reactome pathway analysis revealed that down-regulated DEGs were involved in the interactions between proteins located in synapse pathways. We also discovered interactions between DEGs and miRNA, transcriptional factors (TFs) and DEGs, and between TF and miRNA. Our study identified 20 significant transcription factors, shedding light on early brain development genetics. The identified DEGs and gene regulatory networks are valuable resources for future research into human brain development and neurodevelopmental disorders.
Asunto(s)
Biomarcadores , Encéfalo , Redes Reguladoras de Genes , Células Madre Embrionarias Humanas , Análisis de la Célula Individual , Humanos , Análisis de la Célula Individual/métodos , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/citología , Encéfalo/metabolismo , Encéfalo/embriología , Encéfalo/citología , Biomarcadores/metabolismo , Neuronas/metabolismo , Neuronas/citología , Diferenciación Celular/genética , RNA-Seq , Neurogénesis/genética , Regulación del Desarrollo de la Expresión Génica , Perfilación de la Expresión Génica , Análisis de Secuencia de ARN/métodos , Análisis de Expresión Génica de una Sola CélulaRESUMEN
Metazoan development requires the robust proliferation of progenitor cells, the identities of which are established by tightly controlled transcriptional networks1. As gene expression is globally inhibited during mitosis, the transcriptional programs that define cell identity must be restarted in each cell cycle2-5 but how this is accomplished is poorly understood. Here we identify a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters6,7, recruit the anaphase-promoting complex (APC/C) to specific transcription start sites during mitosis. This allows APC/C to decorate histones with ubiquitin chains branched at Lys11 and Lys48 (K11/K48-branched ubiquitin chains) that recruit p97 (also known as VCP) and the proteasome, which ensures the rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and the re-initiation of transcription are thus controlled by a single regulator (APC/C), which provides a robust mechanism for maintaining cell identity throughout cell division.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Diferenciación Celular/genética , Regulación de la Expresión Génica , Complejos Multiproteicos/metabolismo , Anafase , División Celular , Células HEK293 , Células HeLa , Histonas/química , Histonas/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Interfase , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Mitosis , Organofosfatos/metabolismo , Poliubiquitina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Sitio de Iniciación de la Transcripción , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Cells respond to temperature stress via up- and downregulation of hundreds of genes. This process is thought to be regulated by the heat shock factor HSF1, which controls the release of RNAPII from promoter-proximal pausing. Here, we analyze the events taking place in hESCs upstream of RNAPII release. We find that temperature stress results in the activation or decommissioning of thousands of enhancers. This process involves alterations in the occupancy of transcription factors HSF1, AP-1, NANOG, KLF4, and OCT4 accompanied by nucleosome remodeling by BRG1 and changes in H3K27ac. Furthermore, redistribution of RAD21 and CTCF results in the formation and disassembly of interactions mediated by these two proteins. These alterations tether and untether enhancers to their cognate promoters or refashion insulated neighborhoods, thus transforming the landscape of enhancer-promoter interactions. Details of the 3D interactome remodeling process support loop extrusion initiating at random sites as a mechanism for the establishment of CTCF/cohesin loops.