RESUMEN
MicroRNAs (miRNAs) have been shown to be important in early development and maintenance of human embryonic stem cells (hESCs). The miRNA miR-302-367 is specifically expressed in hESCs, and its expression decays on differentiation. We previously identified the structure of the gene coding for the human miR-302-367 cluster and characterized its promoter. The promoter activity was functionally validated in hESCs, opening up new avenues to further investigate how these miRNA molecules fit in the complex molecular network conferring "stemness" properties to hESCs. The physiological roles of specific miRNA-mRNA interactions remain largely unknown. Here, we investigated putative miR-302-367 mRNA targets in hESCs, potentially relevant for ESC biology. We found that the Nodal inhibitors Lefty1 and Lefty2 are post-transcriptionally targeted by miR-302s in hESCs. Functional analyses indicate that miR-302s negatively modulate the level of lefties, and become upstream regulators of the TGFß/Nodal pathway, functioning via Smad-2/3 signaling. Overexpression of the miR-302-367 cluster in hESCs causes a delay in early hESC differentiation, as measured by enhanced levels of ESC-specific transcription factors, coupled to a faster teratoma formation in mice transplanted with miR-302-367-expressing hESCs and a concomitant impairment of germ layer specification, displaying robust decreased levels of early mesodermal, endodermal, and ectodermal specific markers. These findings suggest that Lefty is negatively modulated by miR-302s in hESCs, which plays an important role in maintaining the balance between pluripotency and germ layer specification.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Factores de Determinación Derecha-Izquierda/metabolismo , MicroARNs/genética , Regiones no Traducidas 3'/genética , Northern Blotting , Western Blotting , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular Tumoral , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Humanos , Inmunohistoquímica , Factores de Determinación Derecha-Izquierda/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
High-quality human DNA samples and associated information of individuals are necessary for biomedical research. Biobanks act as a support infrastructure for the scientific community by providing a large number of high-quality biological samples for specific downstream applications. For this purpose, biobank methods for sample preparation must ensure the usefulness and long-term functionality of the products obtained. Quality indicators are the tool to measure these parameters, the purity and integrity determination being those specifically used for DNA. This study analyzes the quality indicators in DNA samples derived from 118 frozen human tissues in optimal cutting temperature (OCT) reactive, 68 formalin-fixed paraffin-embedded (FFPE) tissues, 119 frozen blood samples, and 26 saliva samples. The results obtained for DNA quality are discussed in association with the usefulness for downstream applications and availability of the DNA source in the target study. In brief, if any material is valid, blood is the most approachable option of prospective collection of samples providing high-quality DNA. However, if diseased tissue is a requisite or samples are available, the recommended source of DNA would be frozen tissue. These conclusions will determine the best source of DNA, according to the planned downstream application. Furthermore our results support the conclusion that a complete procedure of DNA quantification and qualification is necessary to guarantee the appropriate management of the samples, avoiding low confidence results, high costs, and a waste of samples.
Asunto(s)
Criopreservación/normas , ADN/normas , Fijación del Tejido/normas , Bancos de Muestras Biológicas/normas , Criopreservación/métodos , ADN/análisis , Formaldehído , Perfilación de la Expresión Génica , Humanos , Adhesión en Parafina , Estudios Prospectivos , Fijación del Tejido/métodosRESUMEN
Long interspersed element 1s (LINE-1s or L1s) are a family of non-long-terminal-repeat retrotransposons that predominate in the human genome. Active LINE-1 elements encode proteins required for their mobilization. L1-encoded proteins also act in trans to mobilize short interspersed elements (SINEs), such as Alu elements. L1 and Alu insertions have been implicated in many human diseases, and their retrotransposition provides an ongoing source of human genetic diversity. L1/Alu elements are expected to ensure their transmission to subsequent generations by retrotransposing in germ cells or during early embryonic development. Here, we determined that several subfamilies of Alu elements are expressed in undifferentiated human embryonic stem cells (hESCs) and that most expressed Alu elements are active elements. We also exploited expression from the L1 antisense promoter to map expressed elements in hESCs. Remarkably, we found that expressed Alu elements are enriched in the youngest subfamily, Y, and that expressed L1s are mostly located within genes, suggesting an epigenetic control of retrotransposon expression in hESCs. Together, these data suggest that distinct subsets of active L1/Alu elements are expressed in hESCs and that the degree of somatic mosaicism attributable to L1 insertions during early development may be higher than previously anticipated.
Asunto(s)
Elementos Alu/genética , Células Madre Embrionarias/fisiología , Epigénesis Genética , Elementos de Nucleótido Esparcido Largo/genética , Retroelementos/genética , Animales , Células Cultivadas , Mapeo Cromosómico , Células Madre Embrionarias/citología , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Masculino , Ratones , Regiones Promotoras GenéticasRESUMEN
Increasing experimental evidence suggests an important role of miRNAs in embryonic stem cell (ESC) biology. The miR-302-367 cluster is exclusively expressed at high levels in ESCs but not in either somatic stem cells or adult/embryonic differentiated cells. The human miR-302-367 gene structure has been recently described and its promoter has been identified, characterized and functionally validated in human stem cells. The miR-302-367 promoter activity depends on the ontogeny and hierarchical cellular stage. The miR-302-367 promoter is transcriptionally regulated by the ESC-specific transcription factors Oct3/4, Sox2 and Nanog and, its activity restricted to the ESC compartment. Functionally, this cluster regulates cell cycle in ESCs promoting self-renewal and pluripotency, therefore representing a master regulator in the maintenance of hESC stemness. We envision this data may open up new avenues to investigate the transcriptional regulators upstream miR-302-367 cluster and to dissect the complex interplay by which this miR-302-367 cluster integrates in the molecular network conferring pluripotency to ESCs. In this perspective, we summarize recent progress in the genomic and functional characterization of the miR-302-367 cluster and discuss its potential as a stemness determinant.
Asunto(s)
Células Madre Embrionarias/fisiología , MicroARNs , Familia de Multigenes , Animales , Secuencia de Bases , Células Madre Embrionarias/citología , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Datos de Secuencia Molecular , Regiones Promotoras GenéticasRESUMEN
MicroRNAs (miRNAs) play a central role in the regulation of multiple biological processes including the maintenance of stem cell self-renewal and pluripotency. Recently, the miRNA cluster miR302-367 was shown to be differentially expressed in embryonic stem cells (ESCs). Unfortunately, very little is known about the genomic structure of miRNA-encoding genes and their transcriptional units. Here, we have characterized the structure of the gene coding for the human miR302-367 cluster. We identify the transcriptional start and functional core promoter region which specifically drives the expression of this miRNA cluster. The promoter activity depends on the ontogeny and hierarchical cellular stage. It is functional during embryonic development, but it is turned off later in development. From a hierarchical standpoint, its activity decays upon differentiation of ESCs, suggesting that its activity is restricted to the ESC compartment and that the ESC-specific expression of the miR302-367 cluster is fully conferred by its core promoter transcriptional activity. Furthermore, algorithmic prediction of transcription factor binding sites and knockdown studies suggest that ESC-associated transcription factors, including Nanog, Oct3/4, Sox2, and Rex1 may be upstream regulators of miR302-367 promoter. This study represents the first identification, characterization, and functional validation of a human miRNA promoter in stem cells. This study opens up new avenues to further investigate the upstream transcriptional regulation of the miR302-367 cluster and to dissect how these miRNAs integrate in the complex molecular network conferring stem cell properties to ESCs.