The miR-290-295 cluster as multi-faceted players in mouse embryonic stem cells.
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ABSTRACT:
SUBMITTER: Yuan K
PROVIDER: S-EPMC5547456 | biostudies-other | 2017
REPOSITORIES: biostudies-other
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Project description:MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.
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Project description:The DiGeorge syndrome critical region gene 8 (Dgcr8) knockout strategy has been widely used to study the function of canonical microRNAs (miRNAs) in vitro and in vivo. However, primary miRNA (pri-miRNA) transcripts are accumulated in Dgcr8 knockout cells due to interrupted processing. Whether abnormally accumulated pri-miRNAs have any function is unknown. Here, using clustered regularly interspaced short palindromic repeats system/CRISPR-associated protein 9 (CRISPR/Cas9), we successfully knocked out the primary microRNA-290~295 (pri-miR-290~295) cluster, the most highly expressed miRNA cluster in mouse embryonic stem cells (ESCs), in Dgcr8 knockout background. We found that the major defects associated with Dgcr8 knockout in mouse ESCs, including higher expression of epithelial-to-mesenchymal transition (EMT) markers, slower proliferation, G1 accumulation, and defects in silencing self-renewal, were not affected by the deletion of pri-miR-290~290 cluster. Interestingly, the transcription of neighboring gene nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 12(Nlrp12) was upregulated upon the deletion of the pri-miR-290~295 cluster. Together, our results suggested that the major defects in Dgcr8 knockout ESCs were not due to the accumulation of pri-miR-290~295, and the deletion of miRNA genes could affect the transcription of neighboring DNA elements.
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Project description:Numerous developmentally regulated genes in mouse embryonic stem cells (ESCs) are marked by both active (H3K4me3)- and polycomb group (PcG)-mediated repressive (H3K27me3) histone modifications. This bivalent state is thought to be important for transcriptional poising, but the mechanisms that regulate bivalent genes and the bivalent state remain incompletely understood. Examining the contribution of microRNAs (miRNAs) to the regulation of bivalent genes, we found that the miRNA biogenesis enzyme DICER was required for the binding of the PRC2 core components EZH2 and SUZ12, and for the presence of the PRC2-mediated histone modification H3K27me3 at many bivalent genes. Genes that lost bivalency were preferentially upregulated at the mRNA and protein levels. Finally, reconstituting Dicer-deficient ESCs with ESC miRNAs restored bivalent gene repression and PRC2 binding at formerly bivalent genes. Therefore, miRNAs regulate bivalent genes and the bivalent state itself.
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Project description:MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target.
2011-06-18 | GSE30012 | GEO
Project description:MicroRNAs (miRNAs) post-transcriptionally regulate the expression of thousands of distinct mRNAs. While some regulatory interactions help to maintain basal cellular functions, others are likely relevant in more specific settings, such as response to stress. Here we describe such a role for the mir-290-295 cluster, the dominant miRNA cluster in mouse embryonic stem cells (mESCs). Examination of a target list generated from bioinformatic prediction, as well as expression data following miRNA loss, revealed strong enrichment for apoptotic regulators, two of which we validated directly: Caspase 2, the most highly conserved mammalian caspase, and Ei24, a p53 transcriptional target. Consistent with these predictions, mESCs lacking miRNAs were more likely to initiate apoptosis following genotoxic exposure to gamma irradiation or doxorubicin. Knockdown of either candidate partially rescued this pro-apoptotic phenotype, as did transfection of members of the mir-290-295 cluster. These findings were recapitulated in a specific mir-290-295 deletion line, confirming that they reflect miRNA functions at physiological levels. In contrast to the basal regulatory roles previously identified, the pro-survival phenotype shown here may be most relevant to stressful gestations, where pro-oxidant metabolic states induce DNA damage. Similarly, this cluster may mediate chemotherapeutic resistance in a neoplastic context, making it a useful clinical target. Three replicates of mouse embryonic stem cells with Dicer (CTRL) compared to three replicates of mouse embryonic stem cells lacking Dicer (CRE)
2011-06-18 | E-GEOD-30012 | biostudies-arrayexpress
Project description:We compared the expression levels of 307 miRNAs in six different B16F1 melanoma cell lines of differing malignant properties and found that the miR-290-295 cluster showed a strong upregulation in the more malignant B16F1 daughter cell lines. Its overexpression in B16F1 cells had no major effects on cell proliferation, migration or anchorage-independent growth, but conferred resistance to glucose starvation. This was mediated by miR-290-295-induced downregulation of several essential autophagy genes, including Atg7 and ULK1, which resulted in inhibition of autophagic cell death induced by glucose starvation. Similar effects were observed after knockdown of Atg7 or ULK1 in B16F1 melanoma cells, and after treatment with two chemical inhibitors of autophagy. Together, these results indicate that autophagy mediates cell death of melanoma cells under chronic nutrient deprivation, and they reveal an unanticipated role of the miR-290-295 cluster in conferring a survival advantage to melanoma cells by inhibiting autophagic cell death.
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Project description:Expression of the mouse miR-290-295 cluster and its miR-371-373 homolog in human is restricted to early embryos, primordial germ cells, the germ line stem cell compartment of the adult testis and to stem cell lines derived from the early embryonic lineages. Sequencing data suggest considerable seed diversification between the seven homologous pre-miRNAs of miR-290-295 but it is not clear if all of the implied miR-290-295 seeds are also conserved in the human miR-371-373 cluster, which consists of only three homologous pre-miRNAs. By employing miRNA target reporters we show that most, if not all, seeds in miR-290-295 are represented in miR-371-373. In the mouse, pre-miR-290, pre-miR-292 and pre-miR-293 express subsets of the miRNA isoforms processed from the single human pre-miR-371. Comparison of the possible miR-290-295/miR-371-373 seed repertoires in placental mammals suggests a model for the evolution of this miRNA cluster family, which would be otherwise difficult to deduce based solely on pre-miRNA sequence comparisons. The conservation of co-expressed seeds that is characteristic of miR-290-295/miR-371-373 should be taken into account in models of the corresponding miRNA-target interaction networks.
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