RESUMEN
The evaluation of substances for their potency to induce embryotoxicity is controlled by safety regulations. Test guidelines for reproductive and developmental toxicity rely mainly on animal studies, which make up the majority of animal usage in regulatory toxicology. Therefore, there is an urgent need for alternative in vitro methods to follow the 3R principles. To improve human safety, cell models based on human cells are of great interest to overcome species differences. Here, human induced pluripotent stem cells (hiPSCs) are an ideal cell source as they largely recapitulate embryonic stem cells without bearing ethical concerns and they are able to differentiate into most cell types of the human body. Here, we set up and characterized a fetal bovine serum (FBS)-free hiPSC-based in vitro test method, called the human induced pluripotent stem cell test (hiPS Test), to evaluate the embryotoxic potential of substances. After 10 days in culture, hiPSCs develop into beating cardiomyocytes. As terminal endpoint evaluations, cell viability, qPCR analyses as well as beating frequency and area of beating cardiomyocytes by video analyses are measured. The embryotoxic positive and non-embryotoxic negative controls, 5-Fluorouracil (5-FU) and Penicillin G (PenG), respectively, were correctly assessed in the hiPS Test. More compounds need to be screened in the future for defining the assay's applicability domain, which will inform us of the suitability of the hiPS Test for detecting adverse effects of substances on embryonic development.
Asunto(s)
Células Madre Pluripotentes Inducidas , Animales , Diferenciación Celular , Células Madre Embrionarias , Fluorouracilo/farmacología , Humanos , Miocitos Cardíacos , Teratógenos/toxicidad , Pruebas de Toxicidad/métodosRESUMEN
Sulfite oxidase (SO) is encoded by the nuclear SUOX gene and catalyzes the final step in cysteine catabolism thereby oxidizing sulfite to sulfate. Oxidation of sulfite is dependent on two cofactors within SO, a heme and the molybdenum cofactor (Moco), the latter forming the catalytic site of sulfite oxidation. SO localizes to the intermembrane space of mitochondria where both-pre-SO processing and cofactor insertion-are essential steps during SO maturation. Isolated SO deficiency (iSOD) is a rare inborn error of metabolism caused by mutations in the SUOX gene that lead to non-functional SO. ISOD is characterized by rapidly progressive neurodegeneration and death in early infancy. We diagnosed an iSOD patient with homozygous mutation of SUOX at c.1084G>A replacing Gly362 to serine. To understand the mechanism of disease, we expressed patient-derived G362S SO in Escherichia coli and surprisingly found full catalytic activity, while in patient fibroblasts no SO activity was detected, suggesting differences between bacterial and human expression. Moco reconstitution of apo-G362S SO was found to be approximately 90-fold reduced in comparison to apo-WT SO in vitro. In line, levels of SO-bound Moco in cells overexpressing G362S SO were significantly reduced compared to cells expressing WT SO providing evidence for compromised maturation of G362S SO in cellulo. Addition of molybdate to culture medium partially rescued impaired Moco binding of G362S SO and restored SO activity in patient fibroblasts. Thus, this study demonstrates the importance of the orchestrated maturation of SO and provides a first case of Moco-responsive iSOD.
Asunto(s)
Errores Innatos del Metabolismo de los Aminoácidos/metabolismo , Mitocondrias/metabolismo , Sulfito-Oxidasa/deficiencia , Sulfito-Oxidasa/metabolismo , Alelos , Errores Innatos del Metabolismo de los Aminoácidos/diagnóstico , Errores Innatos del Metabolismo de los Aminoácidos/genética , Secuencia de Aminoácidos , Biomarcadores , Catálisis , Activación Enzimática , Fibroblastos/metabolismo , Genotipo , Humanos , Lactante , Recién Nacido , Imagen por Resonancia Magnética , Masculino , Modelos Moleculares , Mutación , Oxidación-Reducción , Conformación Proteica , Proteínas Recombinantes , Índice de Severidad de la Enfermedad , Sulfito-Oxidasa/química , Sulfito-Oxidasa/genéticaRESUMEN
Cardiac dysfunctions dramatically increase with age. Revealing a currently unknown contributor to cardiac ageing, we report the age-dependent, cardiac-specific accumulation of the lysosphingolipid sphinganine (dihydrosphingosine, DHS) as an evolutionarily conserved hallmark of the aged vertebrate heart. Mechanistically, the DHS-derivative sphinganine-1-phosphate (DHS1P) directly inhibits HDAC1, causing an aberrant elevation in histone acetylation and transcription levels, leading to DNA damage. Accordingly, the pharmacological interventions, preventing (i) the accumulation of DHS1P using SPHK2 inhibitors, (ii) the aberrant increase in histone acetylation using histone acetyltransferase (HAT) inhibitors, (iii) the DHS1P-dependent increase in transcription using an RNA polymerase II inhibitor, block DHS-induced DNA damage in human cardiomyocytes. Importantly, an increase in DHS levels in the hearts of healthy young adult mice leads to an impairment in cardiac functionality indicated by a significant reduction in left ventricular fractional shortening and ejection fraction, mimicking the functional deterioration of aged hearts. These molecular and functional defects can be partially prevented in vivo using HAT inhibitors. Together, we report an evolutionarily conserved mechanism by which increased DHS levels drive the decline in cardiac health.
Asunto(s)
Envejecimiento/genética , Envejecimiento/metabolismo , Variación Genética , Inestabilidad Genómica , Miocardio/metabolismo , Esfingolípidos/metabolismo , Animales , Curcumina/química , Curcumina/farmacología , Daño del ADN/efectos de los fármacos , Metabolismo Energético , Epigénesis Genética , Evolución Molecular , Fundulidae , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genómica/métodos , Histona Acetiltransferasas/química , Histona Acetiltransferasas/metabolismo , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/farmacología , Histonas/metabolismo , Humanos , Modelos Moleculares , Miocitos Cardíacos/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Relación Estructura-Actividad , Vertebrados/genética , Vertebrados/metabolismoRESUMEN
The blueprints of developing organs are preset at the early stages of embryogenesis. Transcriptional and epigenetic mechanisms are proposed to preset developmental trajectories. However, we reveal that the competence for the future cardiac fate of human embryonic stem cells (hESCs) is preset in pluripotency by a specialized mRNA translation circuit controlled by RBPMS. RBPMS is recruited to active ribosomes in hESCs to control the translation of essential factors needed for cardiac commitment program, including Wingless/Integrated (WNT) signaling. Consequently, RBPMS loss specifically and severely impedes cardiac mesoderm specification, leading to patterning and morphogenetic defects in human cardiac organoids. Mechanistically, RBPMS specializes mRNA translation, selectively via 3'UTR binding and globally by promoting translation initiation. Accordingly, RBPMS loss causes translation initiation defects highlighted by aberrant retention of the EIF3 complex and depletion of EIF5A from mRNAs, thereby abrogating ribosome recruitment. We demonstrate how future fate trajectories are programmed during embryogenesis by specialized mRNA translation.
Asunto(s)
Células Madre Embrionarias Humanas , Humanos , Células Madre Embrionarias Humanas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , Corazón , Transducción de Señal , Proteínas de Unión al ARN/metabolismoRESUMEN
Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions.
Asunto(s)
Linaje de la Célula/genética , Proteínas Fetales/metabolismo , Mesodermo/metabolismo , Células Madre Pluripotentes/metabolismo , ARN Largo no Codificante/genética , Proteínas de Dominio T Box/metabolismo , Transcripción Genética , Animales , Diferenciación Celular , Línea Celular , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Sitios Genéticos , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Ratones , ARN Largo no Codificante/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ADN Metiltransferasa 3BRESUMEN
A substantial proportion of the currently annotated genes in eukaryotes are proposed to function as RNA molecules (>200 bp) with no significant protein coding potential, currently classified as long noncoding RNAs (lncRNA). A distinct subgroup of lncRNAs is circular RNAs (circRNAs), which can be easily identified by unique junction reads, resulting from their biogenesis. CircRNAs are largely cytosolic and thought to either code for micro-peptides or facilitate gene regulation by sequestering microRNAs (miRNAs) or RNA-binding proteins (RBPs) from their targets. Interrogation of the interaction of circRNAs with cellular macromolecular machineries could indicate their mode of action. Here, we detail a sucrose gradient-based method to pinpoint association of a given circRNA (or any transcript of interest) with distinct ribosomal fractions. This method can evaluate the coding potential of candidate circRNAs (or any transcript of interest) and its association with the translation machinery.
Asunto(s)
Regulación de la Expresión Génica , MicroARNs/genética , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN/metabolismo , Humanos , ARN/genética , ARN Circular , ARN Mensajero/genética , Proteínas de Unión al ARN/genéticaRESUMEN
Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/genética , ARN Largo no Codificante/genética , Factor de Transcripción SOX9/genética , Animales , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica/métodos , Humanos , Ratones , Neuroblastoma/genética , Neuroblastoma/patología , Ubiquitina Tiolesterasa/genéticaRESUMEN
While the transcriptional network of human embryonic stem cells (hESCs) has been extensively studied, relatively little is known about how post-transcriptional modulations determine hESC function. RNA-binding proteins play central roles in RNA regulation, including translation and turnover. Here we show that the RNA-binding protein CSDE1 (cold shock domain containing E1) is highly expressed in hESCs to maintain their undifferentiated state and prevent default neural fate. Notably, loss of CSDE1 accelerates neural differentiation and potentiates neurogenesis. Conversely, ectopic expression of CSDE1 impairs neural differentiation. We find that CSDE1 post-transcriptionally modulates core components of multiple regulatory nodes of hESC identity, neuroectoderm commitment and neurogenesis. Among these key pro-neural/neuronal factors, CSDE1 binds fatty acid binding protein 7 (FABP7) and vimentin (VIM) mRNAs, as well as transcripts involved in neuron projection development regulating their stability and translation. Thus, our results uncover CSDE1 as a central post-transcriptional regulator of hESC identity and neurogenesis.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteína de Unión a los Ácidos Grasos 7/metabolismo , Células Madre Embrionarias Humanas/citología , Placa Neural/embriología , Neurogénesis/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Vimentina/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Proteína de Unión a los Ácidos Grasos 7/genética , Regulación de la Expresión Génica/genética , Humanos , Ratones , Sistema Nervioso/embriología , Placa Neural/citología , Células-Madre Neurales/citología , Neurogénesis/fisiología , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Proteínas Supresoras de Tumor/genética , Vimentina/genéticaRESUMEN
Activation of brown adipose tissue (BAT) controls energy homeostasis in rodents and humans and has emerged as an innovative strategy for the treatment of obesity and type 2 diabetes mellitus. Here we show that ageing- and obesity-associated dysfunction of brown fat coincides with global microRNA downregulation due to reduced expression of the microRNA-processing node Dicer1. Consequently, heterozygosity of Dicer1 in BAT aggravated diet-induced-obesity (DIO)-evoked deterioration of glucose metabolism. Analyses of differential microRNA expression during preadipocyte commitment and mouse models of progeria, longevity and DIO identified miR-328 as a regulator of BAT differentiation. Reducing miR-328 blocked preadipocyte commitment, whereas miR-328 overexpression instigated BAT differentiation and impaired muscle progenitor commitment-partly through silencing of the ß-secretase Bace1. Loss of Bace1 enhanced brown preadipocyte specification in vitro and was overexpressed in BAT of obese and progeroid mice. In vivo Bace1 inhibition delayed DIO-induced weight gain and improved glucose tolerance and insulin sensitivity. These experiments reveal Dicer1-miR-328-Bace1 signalling as a determinant of BAT function, and highlight the potential of Bace1 inhibition as a therapeutic approach to improve not only neurodegenerative diseases but also ageing- and obesity-associated impairments of BAT function.