Masako Tada

Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells

The resetting of a somatic epigenotype to a totipotential state has been demonstrated by successful animal cloning, via transplantation of somatic nuclei into enucleated oocytes. We have established an experimental system, which reproduces the nuclear reprogramming of somatic cells in vitro by fusing adult thymocytes with embryonic stem (ES) cells. Analysis of the lymphoid-cell-specific V-(D)-J DNA rearrangement of the T cell receptor and immunoglobin genes shows that the ES cells have hybridized with differentiated cells. In these ES cell hybrids, the inactivated X chromosome derived from a female thymocyte adopts some characteristics of an active X chromosome, including early replication timing and unstable Xist transcription. We also found that an Oct4-GFP transgene, which is normally repressed in thymocytes, is reactivated 48 hr after cell fusion. The pluripotency of the ES-thymocyte hybrid cells is shown in vivo, since they contribute to all three primary germ layers of chimeric embryos. The somatic DNA methylation pattern of the imprinted H19 and Igf2r genes is maintained in these hybrids, unlike hybrids between ES and EG (embryonic germ) cells in which the differential methylation is erased. Thus, ES cells have the capacity to reset certain aspects of the epigenotype of somatic cells to those of ES cells.

Embryonic germ cells induce epigenetic reprogramming of somatic nucleus in hybrid cells

Genomic reprogramming of primordial germ cells (PGCs), which includes genome‐wide demethylation, prevents aberrant epigenetic modifications from being transmitted to subsequent generations. This process also ensures that homologous chromosomes first acquire an identical epigenetic status before an appropriate switch in the imprintable loci in the female and male germ lines. Embryonic germ (EG) cells have a similar epigenotype to PGCs from which they are derived. We used EG cells to investigate the mechanism of epigenetic modifications in the germ line by analysing the effects on a somatic nucleus in the EG‐thymic lymphocyte hybrid cells. There were striking changes in methylation of the somatic nucleus, resulting in demethylation of several imprinted and non‐imprinted genes. These epigenetic modifications were heritable and affected gene expression as judged by re‐activation of the silent maternal allele of Peg1/Mest imprinted gene in the somatic nucleus. This remarkable change in the epigenotype of the somatic nucleus is consistent with the observed pluripotency of the EG‐somatic hybrid cells as they differentiated into a variety of tissues in chimeric embryos. The epigenetic modifications observed in EG‐somatic cell hybrids in vitro are comparable to the reprogramming events that occur during germ cell development.

Epigenotype switching of imprintable loci in embryonic germ cells.

Abstract Expression of imprinted genes is dependent on their parental origin. This is reflected in the heritable differential methylation of parental alleles. The gametic imprints are however reversible as they do not endure for more than one generation. To investigate if the epigenetic changes in male and female germ line are similar or not, we derived embryonic germ (EG) cells from primordial germ cells (PGCs) of day 11.5 and 12.5 male and female embryos. The results demonstrate that they have an equivalent epigenotype. First, chimeras made with EG cells derived from both male and female embryos showed comparable fetal overgrowth and skeletal abnormalities, which are similar to but less severe than those induced by androgenetic embryonic stem (ES) cells. Thus, EG cells derived from female embryos resemble androgenetic ES cells more than parthenogenetic cells. Furthermore, the methylation status of both alleles of a number of loci in EG cells was similar to that of the paternal allele in normal somatic cells. Hence, both alleles of Igf2r region 2, Peg1/Mest, Peg3, Nnat were consistently unmethylated in EG cells as well as in the primary embryonic fibroblasts (PEFs) rescued from chimeras. More strikingly, both alleles of p57kip2 that were also unmethylated in EG cells, underwent de novo methylation in PEFs to resemble a paternal allele in somatic cells. The exceptions were the H19 and Igf2 genes that retained the methylation pattern in PEFs as seen in normal somatic tissues. These studies suggest that the initial epigenetic changes in germ cells of male and female embryos are similar.

Mapping of the Fas-associated factor 1 gene, Faf1, to mouse Chromosome 4c6 by FISH

been established [5], the distal end, 21q22.3, is the most gene rich [6]. The most distal =2.5 Mbp are homologous to mouse Chr 10 (Fig. 1), whereas a region of =200 kb, =1000 kb further proximal, is known to harbor two genes, cystathione-beta synthetase (CBS) and alpha crystallin (CRYA), which map to mouse Chr 17 (position 17.4 on the consensus map [1]). The most distal known gene on 21q22 that maps to mouse Chr 16, MX, is about 1000 kb from CRYA and CBS (Fig. 1). Recently, the human intestinal trefoil factor (TFF3) has been mapped to 21q22.3, within 250 kb of BCE-I and the spasmolytic polypeptide (SML1), two other trefoil genes [4]. The trefoil genes code for trefoil peptides that are produced in the gastrointestinal tract, and expression is increased in response to pathological damage [3,4]. Our results map one of these trefoil cluster genes to mouse Chr 17, at position 17.0, close to Crya and Cbs. An anonymous probe from 21q22.3, D21S56, called D17H21S56 in mouse, has previously been mapped to this region at position 17.2 on the consensus map [1]. Our results show that a region of at least 1000 kb is homologous between human 21q22.3 and mouse Chr 17. This homology, including the trefoil gene cluster, has to be taken into account when considering mouse models for Down syndrome. While this work was in progress, we became aware that Chinery and associates [7] have mapped the Tff3 gene by FISH to mouse Chr 17, consistent with our results.

Mapping of the eukaryotic initiation factor eIF-lA gene, Eif1a, to mouse chromosome 12D-E by FISH

Species: Mouse Locus name: Eukaryotic initiation factor 1A (eIF-1A) Locus symbol: Eifla Map position: mouse Chromosome (Chr) 12 band D-E Method of mapping: FISH. The cDNA probe was labeled with biotin-16-dUTP, and the signal was detected by streptavidin-FITC (BRL) and biotinylated anti-avidin D (Vector) as described [1]. Chromosomes were counterstained with propidium iodide and Gbanded with DAPI. Database deposit information: GenBank accession number for sequence is U28419. Mouse Genome Database accession number for mapping is MGD-INEX-33. Molecular reagents used for mapping: D52, a 1.9-kb cDNA probe encoding Eifla was used in FISH. Previously identified homologs: Saccharomyces cerevisiae homolog, TIFll [2], is at YMR260C. Human, rabbit, and wheat homologs [3] have not been mapped yet. Discussion: In FISH analysis for the Eifla gene, doublet signals were located on mouse chromosome band 12D in ten mitotic spreads (Fig. 1) and on 12E in three spreads. In addition, in six spreads we could not discriminate between 12D or 12E. We therefore conclude that Eifla maps close to the D-E boundary of Chr 12. It is unlikely that we were detecting two different loci on Chr 12 since genomic Southern analysis with the same cDNA probe detected a single band in DNA digested with a range of restriction enzymes (data not shown). Experiments using a mouse-hamster somatic cell hybrid panel also indicated Eifla maps to mouse Chr 12 (data not shown). Eukaryotic initiation factor 1A (formerly called eIF-4C) functions in the early steps of translation by promoting the dissociation of 80S ribosomes into 40S and 60S subunits and stabilizing a preinitiation complex composed of initiator methionyl-tRNA (Met-tRNA,), eIF-2, GTP, and 40S ribosomal subunit [4]. Mouse eDNA for Eifla has been cloned recently, and its expression pattern in the early development was analyzed [5]. The transcription of Eifla transiently increases at the two-cell stage, for which the first round of DNA replication is critical. The following decrease of transcription is mediated by histone deacetylation, which was shown by using an inhibitor of histone deacetylase, trapoxin. Thus, the study of transcriptional regulation of Eifla provides insight into the mechanisms of zygotic gene activation in terms of chromatin structure.

Plasticity of Somatic Nucleus by Epigenetic Reprogramming Via Cell Hybridization

Publisher Summary This chapter presents various studies that focus on the ability of pluripotential stem cells to reprogram somatic nuclei in hybrid cells. The pluripotential stem cells such as the embryonic stem (ES) and gEG cells are capable of reprogramming the somatic nuclei by cell fusion, similar to the nuclear reprogramming represented by the transplantation of somatic nuclei into enucleated oocytes in various animal species. This plasticity of the somatic nucleus is induced by trans-acting factors derived from the ES and gEG cells. Nuclear reprogramming of somatic cells in hybrid cells is demonstrated by the reactivation of the inactive X chromosome present in a female somatic cell, contribution of the ES and gEG hybrid cells to the primary germ layers of chimeric embryos, and reactivation of the somatic-cell-derived Oct4–GFP marker gene, which is specifically expressed in pluripotent cells. Apart from the ability of both the ES and gEG cells to confer pluripotency to somatic nuclei, the gEG cells have an additional capacity not found in the ES cells, which is the ability to erase DNA methylation associated with imprinted genes. Nuclear reprogramming of the somatic nuclei in the gEG hybrid cells corresponds to the germ-cell-type reprogramming, and nuclear reprogramming of somatic nuclei in the ES hybrid cells corresponds to the preimplantation embryo type. Nuclear reprogramming of somatic nuclei in the ES hybrid cells is perhaps similar to what occurs during the process of somatic cell cloning via nuclear transplantation.