Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE20576 | GEO | 2010/04/27
SECONDARY ACCESSION(S): PRJNA125269
REPOSITORIES: GEO
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Project description: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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Project description:This SuperSeries is composed of the following subset Series: GSE20572: mRNA profiling of genetically matched ESCs and iPSCs GSE20575: mRNA profiling of iPSCs and derivative NT-ESCs Refer to individual Series
2010-04-27 | E-GEOD-20576 | biostudies-arrayexpress
Project description:Patient-specific induced pluripotent stem cells (iPSCs) have the potential to be useful in the treatment of human diseases. While prior studies have reported multiple methods to generate iPSCs, DNA methylation continues to limit the totipotency and reprogramming efficiency of iPSCs. Here, we first show the competency of embryonic germ cells (EGCs) as a reprogramming catalyst capable of effectively promoting reprogramming induced by four defined factors, including Oct4, Sox2, Klf4 and c-Myc. Combining EGC extracts with these four factors resulted in formation of more embryonic stem cell-like colonies than did factors alone. Notably, expression of imprinted genes was higher with combined induction than with factors alone. Moreover, iPSCs derived from the combined inductors tended to have more global hypomethylation. Our research not only provides evidence that EGC extracts could activate DNA demethylation and reprogram imprinted genes, but also establishes a new way to enhance reprogramming of iPSCs, which remains a critical safety concern for potential use of iPSCs in regenerative medicine.
| S-EPMC6053380 | biostudies-literature
Project description:Induced pluripotent stem cells (iPSCs) offer immense potential for regenerative medicine and studies of disease and development. Somatic cell reprogramming involves epigenomic reconfiguration, conferring iPSCs with characteristics similar to embryonic stem (ES) cells. However, it remains unknown how complete the reestablishment of ES-cell-like DNA methylation patterns is throughout the genome. Here we report the first whole-genome profiles of DNA methylation at single-base resolution in five human iPSC lines, along with methylomes of ES cells, somatic cells, and differentiated iPSCs and ES cells. iPSCs show significant reprogramming variability, including somatic memory and aberrant reprogramming of DNA methylation. iPSCs share megabase-scale differentially methylated regions proximal to centromeres and telomeres that display incomplete reprogramming of non-CG methylation, and differences in CG methylation and histone modifications. Lastly, differentiation of iPSCs into trophoblast cells revealed that errors in reprogramming CG methylation are transmitted at a high frequency, providing an iPSC reprogramming signature that is maintained after differentiation.
| S-EPMC3100360 | biostudies-literature
Project description:BACKGROUND:Genomic imprinting, resulting in parent-of-origin specific gene expression, plays a critical role in mammalian development. Here, we apply allele-specific RNA-seq on isogenic B6D2F1 mice to assay imprinted genes in tissues from early embryonic tissues between E3.5 and E7.25 and in pluripotent cell lines to evaluate maintenance of imprinted gene expression. For the cell lines, we include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) derived from fertilized embryos and from embryos obtained after nuclear transfer (NT) or parthenogenetic activation (PGA). RESULTS:As homozygous genomic regions of PGA-derived cells are not compatible with allele-specific RNA-seq, we developed an RNA-seq-based genotyping strategy allowing identification of informative heterozygous regions. Global analysis shows that proper imprinted gene expression as observed in embryonic tissues is largely lost in the ESC lines included in this study, which mainly consisted of female ESCs. Differentiation of ESC lines to embryoid bodies or NPCs does not restore monoallelic expression of imprinted genes, neither did reprogramming of the serum-cultured ESCs to the pluripotent ground state by the use of 2 kinase inhibitors. Fertilized EpiSC and EpiSC-NT lines largely maintain imprinted gene expression, as did EpiSC-PGA lines that show known paternally expressed genes being silent and known maternally expressed genes consistently showing doubled expression. Notably, two EpiSC-NT lines show aberrant silencing of Rian and Meg3, two critically imprinted genes in mouse iPSCs. With respect to female EpiSC, most of the lines displayed completely skewed X inactivation suggesting a (near) clonal origin. CONCLUSIONS:Altogether, our analysis provides a comprehensive overview of imprinted gene expression in pluripotency and provides a benchmark to allow identification of cell lines that faithfully maintain imprinted gene expression and therefore retain full developmental potential.
| S-EPMC6376749 | biostudies-literature
Project description:Spermatogonial stem cells (SSCs), which are unipotent stem cells in the testes that give rise to sperm, can be converted into germline-derived pluripotent stem (gPS) by self-induction. The androgenetic imprinting pattern of SSCs is maintained even after their reprogramming into gPS cells. In this study, we used an in vitro neural differentiation model to investigate whether the imprinting patterns are maintained or altered during differentiation. The androgenetic patterns of H19, Snrpn, and Mest were maintained even after differentiation of gPS cells into NSCs (gPS-NSCs), whereas the fully unmethylated status of Ndn in SSCs was altered to somatic patterns in gPS cells and gPS-NSCs. Thus, our study demonstrates epigenetic alteration of genomic imprinting during the induction of pluripotency in SSCs and neural differentiation, suggesting that gPS-NSCs can be a useful model to study the roles of imprinted genes in brain development and human neurodevelopmental disorders.
| S-EPMC4854545 | biostudies-literature
Project description:BackgroundGenomic imprinting is an exception to Mendelian genetics in that imprinted genes are expressed monoallelically, dependent on parental origin. In mammals, imprinted genes are critical in numerous developmental and physiological processes. Aberrant imprinted gene expression is implicated in several diseases including Prader-Willi/Angelman syndromes and cancer.Methodology/principal findingsTo identify novel imprinted genes, transcription profiling was performed on two uniparentally derived cell lines, androgenetic and parthenogenetic primary mouse embryonic fibroblasts. A maternally expressed transcript termed Imprinted RNA near Meg3/Gtl2 (Irm) was identified and its expression studied by Northern blotting and whole mounts in situ hybridization. The imprinted region that contains Irm has a parent of origin effect in three mammalian species, including the sheep callipyge locus. In mice and humans, both maternal and paternal uniparental disomies (UPD) cause embryonic growth and musculoskeletal abnormalities, indicating that both alleles likely express essential genes. To catalog all imprinted genes in this chromosomal region, twenty-five mouse mRNAs in a 1.96Mb span were investigated for allele specific expression.Conclusions/significanceTen imprinted genes were elucidated. The imprinting of three paternally expressed protein coding genes (Dlk1, Peg11, and Dio3) was confirmed. Seven noncoding RNAs (Meg3/Gtl2, Anti-Peg11, Meg8, Irm/"Rian", AK050713, AK053394, and Meg9/Mirg) are characterized by exclusive maternal expression. Intriguingly, the majority of these noncoding RNA genes contain microRNAs and/or snoRNAs within their introns, as do their human orthologs. Of the 52 identified microRNAs that map to this region, six are predicted to regulate negatively Dlk1, suggesting an additional mechanism for interactions between allelic gene products. Since several previous studies relied heavily on in silico analysis and RT-PCR, our findings from Northerns and cDNA cloning clarify the genomic organization of this region. Our results expand the number of maternally expressed noncoding RNAs whose loss may be responsible for the phenotypes associated with mouse pUPD12 and human pUPD14 syndromes.
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Project description:Reprogramming of the somatic state to pluripotency can be induced by a defined set of transcription factors including Oct3/4, Sox2, Klf4, and c-Myc [Cell 2006;126:663-676]. These induced pluripotent stem cells (iPSCs) hold great promise in human therapy and disease modeling. However, tumor suppressive activities of p53, which are necessary to prevent persistence of DNA damage in mammalian cells, have proven a serious impediment to formation of iPSCs [Nat Methods 2011;8:409-412]. We examined the requirement for downstream p53 activities in suppressing efficiency of reprogramming as well as preventing persistence of DNA damage into the early iPSCs. We discovered that the majority of the p53 activation occurred through early reprogramming-induced DNA damage with the activated expression of the apoptotic inducer Puma and the cell cycle inhibitor p21. While Puma deficiency increases reprogramming efficiency only in the absence of c-Myc, double deficiency of Puma and p21 has achieved a level of efficiency that exceeded that of p53 deficiency alone. We further demonstrated that, in both the presence and absence of p21, Puma deficiency was able to prevent any increase in persistent DNA damage in early iPSCs. This may be due to a compensatory cellular senescent response to reprogramming-induced DNA damage in pre-iPSCs. Therefore, our findings provide a potentially safe approach to enhance iPSC derivation by transiently silencing Puma and p21 without compromising genomic integrity.
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Project description:Rett syndrome (RTT) is a neurodevelopmental disorder that affects girls due primarily to heterozygous mutations in the X-linked gene encoding methyl-CpG binding protein 2 (MECP2). Random X-chromosome inactivation (XCI) results in cellular mosaicism in which some cells express wild-type (WT) MECP2 while other cells express mutant MECP2. The generation of patient-specific human induced pluripotent stem cells (hiPSCs) facilitates the production of RTT-hiPSC-derived neurons in vitro to investigate disease mechanisms and identify novel drug treatments. The generation of RTT-hiPSCs has been reported by many laboratories, however, the XCI status of RTT-hiPSCs has been inconsistent. Some report RTT-hiPSCs retain the inactive X-chromosome (post-XCI) of the founder somatic cell allowing isogenic RTT-hiPSCs that express only the WT or mutant MECP2 allele to be isolated from the same patient. Post-XCI RTT-hiPSCs-derived neurons retain this allele-specific expression pattern of WT or mutant MECP2. Conversely, others report RTT-hiPSCs in which the inactive X-chromosome of the founder somatic cell reactivates (pre-XCI) upon reprogramming into RTT-hiPSCs. Pre-XCI RTT-hiPSC-derived neurons exhibit random XCI resulting in cellular mosaicism with respect to WT and mutant MECP2 expression. Here we review and attempt to interpret the inconsistencies in XCI status of RTT-hiPSCs generated to date by comparison to other pluripotent systems in vitro and in vivo and the methods used to analyze XCI. Finally, we discuss the relative strengths and weaknesses of post- and pre-XCI hiPSCs in the context of RTT, and other X-linked and autosomal disorders for translational medicine.
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Project description:In placental mammals, balanced expression of X-linked genes is accomplished by X chromosome inactivation (XCI) in female cells. In humans, random XCI is initiated early during embryonic development. To investigate whether reprogramming of female human fibroblasts into induced pluripotent stem cells (iPSCs) leads to reactivation of the inactive X chromosome (Xi), we have generated iPSC lines from fibroblasts heterozygous for large X-chromosomal deletions. These fibroblasts show completely skewed XCI of the mutated X chromosome, enabling monitoring of X chromosome reactivation (XCR) and XCI using allele-specific single-cell expression analysis. This approach revealed that XCR is robust under standard culture conditions, but does not prevent reinitiation of XCI, resulting in a mixed population of cells with either two active X chromosomes (Xas) or one Xa and one Xi. This mixed population of XaXa and XaXi cells is stabilized in naive human stem cell medium, allowing expansion of clones with two Xas.
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