Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells.

Howard Hughes Medical Institute at Massachusetts General Hospital, Center for Regenerative Medicine
Nature (Impact Factor: 42.35). 04/2010; 465(7295):175-81. DOI: 10.1038/nature09017
Source: PubMed

ABSTRACT Induced pluripotent stem cells (iPSCs) have been generated by enforced expression of defined sets of transcription factors in somatic cells. It remains controversial whether iPSCs are molecularly and functionally equivalent to blastocyst-derived embryonic stem (ES) cells. By comparing genetically identical mouse ES cells and iPSCs, we show here that their overall messenger RNA and microRNA expression patterns are indistinguishable with the exception of a few transcripts encoded within the imprinted Dlk1-Dio3 gene cluster on chromosome 12qF1, which were aberrantly silenced in most of the iPSC clones. Consistent with a developmental role of the Dlk1-Dio3 gene cluster, these iPSC clones contributed poorly to chimaeras and failed to support the development of entirely iPSC-derived animals ('all-iPSC mice'). In contrast, iPSC clones with normal expression of the Dlk1-Dio3 cluster contributed to high-grade chimaeras and generated viable all-iPSC mice. Notably, treatment of an iPSC clone that had silenced Dlk1-Dio3 with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of all-iPSC mice. Thus, the expression state of a single imprinted gene cluster seems to distinguish most murine iPSCs from ES cells and allows for the prospective identification of iPSC clones that have the full development potential of ES cells.

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    ABSTRACT: Although viable mice can be generated from induced pluripotent stem cells (iPSCs), the impact of accumulated mutations on the developmental potential of the resulting iPSCs remains to be determined. Here, we demonstrate that all-iPSC mice generated through tetraploid blastocysts complementation can tolerate the accumulation of somatic mutations for up to six generations using a Tet-on inducible reprogramming system. But, the viability of the all-iPS mice decreased with increasing generations. A whole-genome sequencing survey revealed that thousands of single-nucleotide variations (SNVs), including 44 non-synonymous ones, accumulated throughout the sequential reprogramming process. Subsequent analysis provides evidence that these accumulated SNVs account for the gradual reduction in viability of the resultant all-iPSC mice. Unexpectedly, our present reprogramming system revealed that pluripotent stem cells are heterogeneous in terms of possessing a set of copy-number alterations (CNAs). These CNAs are unique for pluripotent cells and subsequently disappear in the differentiating progenies.
    Nature Communications 02/2015; 6:6318. DOI:10.1038/ncomms7318 · 10.74 Impact Factor
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    ABSTRACT: MicroRNAs (miRNAs) simultaneously target many transcripts through partial complementarity binding, and have emerged as a key type of post-transcriptional regulator for gene expression. How miRNA accomplishes its pleiotropic effects largely depends on its expression and its target repertoire. Previous studies discovered thousands of miRNAs and numerous miRNA target genes mainly through computation and prediction methods which produced high rates of false positive prediction. The development of Argonaute cross-linked immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) provides a system to effectively determine miRNA target genes. Likewise, the accuracy of dissecting the transcriptional regulation of miRNA genes has been greatly improved by chromatin immunoprecipitation of the transcription factors coupled with sequencing (ChIP-Seq). Elucidation of the miRNA target repertoire will provide an in-depth understanding of the functional roles of microRNA pathways. To reliably reconstruct a miRNA-mediated regulatory network, we established a computational framework using publicly available, sequence-based transcription factor-miRNA databases, including ChIPBase and TransmiR for the TF-miRNA interactions, along with miRNA-target databases, including miRTarBase, TarBase and starBase, for the miRNA-target interactions. We applied the computational framework to elucidate the miRNA-mediated regulatory network in the Mir122a-/- mouse model, which has an altered transcriptome and progressive liver disease. We applied our computational framework to the expression profiles of miRNA/mRNA of Mir122a-/- mutant mice and wild-type mice. The miRNA-mediated network involves 40 curated TFs contributing to the aberrant expression of 65 miRNAs and 723 curated miRNA target genes, of which 56% was found in the differentially-expressed genes of Mir122a--mice. Hence, the regulatory network disclosed previously-known and also many previously-unidentified miRNA-mediated regulations in mutant mice. Moreover, we demonstrate that loss of imprinting at the chromosome 12qF1 region is associated with miRNA overexpression in human hepatocellular carcinoma and stem cells, suggesting initiation of precancerous changes in young mice deficient in miR-122. A group of 9 miRNAs was found to share miR-122 target genes, indicating synergy between miRNAs and target genes by way of multiplicity and cooperativity. The study provides significant insight into miRNA-mediated regulatory networks. Based on experimentally verified data, this network is highly reliable and effective in revealing previously-undetermined disease-associated molecular mechanisms. This computational framework can be applied to explore the significant TF-miRNA-miRNA target interactions in any complex biological systems with high degrees of confidence.

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Jun 3, 2014