Summary 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 cells (ESCs). By comparing genetically identical mouse ESCs and iPSCs, we show here that the overall mRNA and miRNA expression patterns of these cell types are indistinguishable with the exception of a few transcripts and miRNAs encoded on chromosome 12qF1. Specifically, maternally expressed imprinted genes in the Dlk1-Dio3 cluster including Gtl2, Rian and Mirg as well as a larger number of miRNAs encoded within this region were aberrantly silenced in the majority of iPSC clones, irrespective of their cell type of origin. Consistent with a developmental role of the Dlk1-Dio3 gene cluster, iPSC clones with repressed Gtl2 contributed poorly to chimeras and failed to support the development of entirely iPSC-derived animals (“all-iPSC mice”). In contrast, iPSC clones with normal expression levels of these genes contributed to high-grade chimeras and generated viable all-iPSC mice. Importantly, treatment of an iPSC clone that had silenced Dlk1-Dio3 and failed to give rise to all-iPSC animals with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of exclusively iPSC-derived mice. Thus, the expression state of a single imprinted gene cluster distinguishes most murine iPSCs from ESCs and allows for the prospective identification of iPSC clones that have the full development potential of ESCs.

Genome-wide dynamic changes in DNA methylation are indispensable for germline development and genomic imprinting in mammals. Here, we report single-base resolution DNA methylome and transcriptome maps of mouse germ cells, generated using whole-genome shotgun bisulfite sequencing and cDNA sequencing (mRNA-seq). Oocyte genomes showed a significant positive correlation between mRNA transcript levels and methylation of the transcribed region. Sperm genomes had nearly complete coverage of methylation, except in the CpG-rich regions, and showed a significant negative correlation between gene expression and promoter methylation. Thus, these methylome maps revealed that oocytes and sperms are widely different in the extent and distribution of DNA methylation. Furthermore, a comparison of oocyte and sperm methylomes identified more than 1,600 CpG islands differentially methylated in oocytes and sperm (germline differentially methylated regions, gDMRs), in addition to the known imprinting control regions (ICRs). About half of these differentially methylated DNA sequences appear to be at least partially resistant to the global DNA demethylation that occurs during preimplantation development. In the absence of Dnmt3L, neither methylation of most oocyte-methylated gDMRs nor intragenic methylation was observed. There was also genome-wide hypomethylation, and partial methylation at particular retrotransposons, while maintaining global gene expression, in oocytes. Along with the identification of the many Dnmt3L-dependent gDMRs at intragenic regions, the present results suggest that oocyte methylation can be divided into 2 types: Dnmt3L-dependent methylation, which is required for maternal methylation imprinting, and Dnmt3L-independent methylation, which might be essential for endogenous retroviral DNA silencing. The present data provide entirely new perspectives on the evaluation of epigenetic markers in germline cells.

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Maintaining a single active X-chromosome by repressing Xist is crucial for embryonic development in mice. Although the Xist activator RNF12/RLIM is present as a maternal factor, maternal Xist (Xm-Xist) is repressed during preimplantation phases to establish imprinted X-chromosome inactivation (XCI). Here we show, using a highly reproducible chromatin immunoprecipitation method that facilitates chromatin analysis of preimplantation embryos, that H3K9me3 is enriched at the Xist promoter region, preventing Xm-Xist activation by RNF12. The high levels of H3K9me3 at the Xist promoter region are lost in embryonic stem (ES) cells, and ES-cloned embryos show RNF12-dependent Xist expression. Moreover, lack of Xm-XCI in the trophectoderm, rather than loss of paternally expressed imprinted genes, is the primary cause of embryonic lethality in 70–80% of parthenogenotes immediately after implantation. This study reveals that H3K9me3 is involved in the imprinting that silences Xm-Xist. Our findings highlight the role of maternal-specific H3K9me3 modification in embryo development.

In mammals, both the maternal and paternal genomes are necessary for normal embryogenesis due to parent-specific epigenetic modification of the genome during gametogenesis, which leads to non-equivalent expression of imprinted genes from the maternal and paternal alleles. In this study, we identified a paternally expressed imprinted gene, Zdbf2, by microarray-based screening using parthenogenetic and normal embryos. Expression analyses showed that Zdbf2 was paternally expressed in various embryonic and adult tissues, except for the placenta and adult testis, which showed biallelic expression of the gene. We also identified a differentially methylated region (DMR) at 10 kb upstream of exon 1 of the Zdbf2 gene and this differential methylation was derived from the germline. Furthermore, we also identified that the human homolog (ZDBF2) of the mouse Zdbf2 gene showed paternal allele-specific expression in human lymphocytes but not in the human placenta. Thus, our findings defined mouse chromosome 1 and human chromosome 2 as the loci for imprinted genes.

Patients with T1-2 benefited from surgical intervention without significant morbidity or mortality. Our findings also suggested that definitive CRT might be appropriate as the first-line treatment for T3-4, especially in cases with unresectable tumors.

Although cloned embryos generated by somatic/embryonic stem cell nuclear transfer (SECNT) certainly give rise to viable individuals, they can often undergo embryonic arrest at any stage of embryogenesis, leading to diverse morphological abnormalities. In an effort to gain further insights into reprogramming and the properties of SECNT embryos, we performed a large-scale gene expression profiling of 87 single blastocysts using GeneChip microarrays. Sertoli cells, cumulus cells, and embryonic stem cells were used as donor cells. The gene expression profiles of 87 blastocysts were subjected to microarray analysis. Using principal component analysis and hierarchical clustering, the gene expression profiles were clearly classified into 3 clusters corresponding to the type of donor cell. The results revealed that each type of SECNT embryo had a unique gene expression profile that was strictly dependent upon the type of donor cells, although there was considerable variation among the individual profiles within each group. This suggests that the reprogramming process is distinct for embryos cloned from different types of donor cells. Furthermore, on the basis of the results of comparison analysis, we identified 35 genes that were inappropriately reprogrammed in most of the SECNT embryos; our findings demonstrated that some of these genes, such as Asz1, Xlr3a and App, were appropriately reprogrammed only in the embryos with a transcriptional profile that was the closest to that of the controls. Our findings provide a framework to further understand the reprogramming in SECNT embryos.

Spatiotemporal expression of transcription factors is crucial for genomic reprogramming. Pou5f1 (Oct4) is an essential transcription factor for reprogramming. A recent study reported that OCT4A, which is crucial for establishment and maintenance of pluripotent cells, is expressed in oocytes, but maternal OCT4A is dispensable for totipotency induction. Whereas another study reported that OCT4B, which is not related to pluripotency, is predominantly expressed instead of OCT4A during early preimplantation phases in mice. To determine the expression states of OCT4 in murine preimplantation embryos, we conducted in-depth expression and functional analyses. We found that pluripotency-related OCT4 mainly localizes to the cytoplasm in early preimplantation phases, with no major nuclear localization until the 8–16-cell stage despite high expression in both oocytes and early embryos. RNA-sequencing analysis using oocytes and early preimplantation embryos could not identify the splice variants creating alternative forms of OCT4 protein. Forced expression of OCT4 in zygotes by the injection of polyadenylated mRNA clearly showed nuclear localization of OCT4 protein around 3–5-fold greater than physiological levels and impaired developmental competency in a dose-dependent manner. Embryos with modest overexpression of OCT4 could develop to the 16-cell stage; however, more than 50% of the embryos were arrested at this stage, similar to the results for OCT4 depletion. In contrast, extensive overexpression of OCT4 resulted in complete arrest at the 2-cell stage accompanied by downregulation of zygotically activated genes and repetitive elements related to the totipotent state. These results demonstrated that OCT4 protein localization was spatiotemporally altered during preimplantation development, and strict control of Oct4 protein levels was essential for proper totipotential reprogramming.