Biomedical subjects

Jun Ohgane

Publications and source records attributed to Jun Ohgane.

11 recordsLinked to original sources

PubMed2006

Neonatal exposure to diethylstilbestrol alters the expression of DNA methyltransferases and methylation of genomic DNA in the epididymis of mice.

Fetal and neonatal exposure to diethylstilbestrol (DES) is known to cause many abnormalities, such as cancer, in the male and female reproductive tracts later in life, and epigenetic mechanisms, such as DNA methylation, may be involved in these processes. In the present study, newborn C57BL/6 male mice were exposed to 3 mug of DES from postnatal days 1 to 5. Subsequently, the expression levels of the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b and the transcription factors Sp1 and Sp3, which have been reported to regulate the expression of Dnmts, were examined at days 5, 14 and 30. Furthermore, restriction landmark genomic scanning (RLGS), which can analyze genome-wide DNA methylation, was performed to clarify whether or not aberrant DNA methylation was present in the epididymis of the DES-treated mice at day 30. Increased expression of Dnmt3b was observed at days 5 and 14, followed by increased expression of Dnmt1 and Dnmt3a at day 30, as evaluated by real-time RT-PCR. The expression of Sp1 was also increased at day 30. The RLGS analysis revealed that 7 loci of the genomic DNA were demethylated and 1 locus was methylated in the epididymis of the DES-treated mice. Four of these loci specifically demethylated in DES-treated mice were cloned, and all were found to be located within CpG islands near genes. In conclusion, our results indicated the possibility that DES-induced abnormalities of reproductive organs are associated with altered expression levels of DNA methyltransferases and DNA methylation.

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PubMed2006

Methylation in embryonic stem cells in vitro.

Stem cells raise the possibility of regenerating failing body parts with new tissue. Before stem cells can safely fulfill their promise, many technical problems, including understanding the stem cell phenotype, must be overcome. DNA methylation, which is responsible for gene silencing and is associated with chromatin remodeling, is an epigenetic system that determines the specific characteristic of a variety of cells, including stem cells. Each cell type has a unique DNA methylation profile produced by varied loci-specific methylation. Investigation of such DNA methylation profiles provides a way of identifying pluripotent stem cells. Further, it is likely that analysis of the epigenetic status of stem cells may provide novel information regarding "sternness" within these populations.

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PubMed2005

Analysis of tissue-specific DNA methylation during development.

The establishment of a cell identification method more accurate than the conventional morphological and cell-type-specific marker analyses has been desired. DNA methylation is related to the gene activity including gene-silencing and is a key mechanism of epigenetics underling cellular differentiaion and development in mammals. Recent studies indicated that there exist unique genomic DNA methylation profiles specific to the cell type. DNA methylation profiles a mechanism for memorizing the set of genes inherent in individual type of cells. In this chapter, we present the methods to analyze DNA methylation status for identifying cells or tissues.

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PubMed2004

Non-coding RNA directed DNA demethylation of Sphk1 CpG island.

The formation of DNA methylation patterns is one of the epigenetic events that underlie mammalian development. The Sphk1 CpG island is a target for tissue-dependent DNA methylation as well as a template for generating multiple subtypes. The number of mammalian non-coding RNA genes is rapidly expanding. In this study, we found endogenous antisense transcripts, Khps1 subtypes with different sizes (600-20,000nt). A subtype, Khps1a, was a 1290-bp, non-coding, 5'-capped and 3'-polyadenylated RNA that originated from the CpG island and overlapped with a tissue-dependent differentially methylated region (T-DMR) of Sphk1. Intriguingly, overexpression of two fragments of Khps1 caused demethylation of CG sites in the T-DMR. Furthermore, this RNA-directed demethylation was associated with DNA methylation at three CC(A/T)GG sites in the T-DMR. The link between the RNA-directed CG demethylation and non-CG methylation provides a novel mechanism of epigenetic regulation and potential tool for epigenetic manipulation of mammalian cells.

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PubMed2004

Skewed X-inactivation in cloned mice.

In female mammals, dosage compensation for X-linked genes is accomplished by inactivation of one of two X chromosomes. The X-inactivation ratio (a percentage of the cells with inactivated maternal X chromosomes in the whole cells) is skewed as a consequence of various genetic mutations, and has been observed in a number of X-linked disorders. We previously reported that phenotypically normal full-term cloned mouse fetuses had loci with inappropriate DNA methylation. Thus, cloned mice are excellent models to study abnormal epigenetic events in mammalian development. In the present study, we analyzed X-inactivation ratios in adult female cloned mice (B6C3F1). Kidneys of eight naturally produced controls and 11 cloned mice were analyzed. Although variations in X-inactivation ratio among the mice were observed in both groups, the distributions were significantly different (Ansary-Bradley test, P<0.01). In particular, 2 of 11 cloned mice showed skewed X-inactivation ratios (19.2% and 86.8%). Similarly, in intestine, 1 of 10 cloned mice had a skewed ratio (75.7%). Skewed X-inactivation was observed to various degrees in different tissues of different individuals, suggesting that skewed X-inactivation in cloned mice is the result of secondary cell selection in combination with stochastic distortion of primary choice. The present study is the first demonstration that skewed X-inactivation occurs in cloned animals. This finding is important for understanding both nuclear transfer technology and etiology of X-linked disorders.

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PubMed2004

Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells.

The first cell differentiation event in mammalian embryogenesis segregates inner cell mass lineage from the trophectoderm at the blastocyst stage. Oct-4, a member of the POU family of transcription factors, is necessary for the pluripotency of the inner cell mass lineage. Embryonic stem (ES) cells, which contribute to all of embryonic lineages, express the Oct-4 gene. Trophoblast stem (TS) cells, which have the ability to differentiate into trophoblast lineage in vitro, never contribute to embryonic proper tissues in chimeras and differentiate only into trophoblastic cells in the placenta. Expression of the Oct-4 gene was undetectable and severely repressed in trophoblastic lineage, including the stem cells. We found that the culture of TS cells with 5-aza-2'-deoxycytidine or trichostatin A caused the activation of the Oct-4 gene. Analysis of the DNA methylation status of mouse Oct-4 gene upstream region revealed that Oct-4 enhancer/promoter region was hypomethylated in ES cells but hypermethylated in TS cells. Furthermore, in vitro methylation suppressed Oct-4 enhancer/promoter activity in reporter assay. In the placenta of Dnmt1(n/n) mutant mice, most of the CpGs in the enhancer/promoter region were unmethylated, and Oct-4 gene expression was aberrantly detected. Chromatin immunoprecipitation assay revealed that Oct-4 enhancer/promoter region was hyperacetylated in ES cells compared with TS cells, thus demonstrating that DNA methylation status is closely linked to the chromatin structure of the Oct-4 gene. Here we propose that the epigenetic mechanism, consisting of DNA methylation and chromatin remodeling, underlies the developmental stage- and cell type-specific mechanism of Oct-4 gene expression.

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PubMed2004

Different molecular mechanisms underlie placental overgrowth phenotypes caused by interspecies hybridization, cloning, and Esx1 mutation.

To obtain a deeper insight into the genes and gene networks involved in the development of placentopathies, we have assessed global gene expression in three different models of placental hyperplasia caused by interspecies hybridization (IHPD), cloning by nuclear transfer, and mutation of the Esx1 gene, respectively. Comparison of gene expression profiles of approximately 13,000 expressed sequence tags (ESTs) identified specific subsets of genes with changed expression levels in IHPD, cloned, and Esx1 mutant placentas. Of interest, only one gene of known function and one EST of unknown function were found common to all three placentopathies; however, a significant number of ESTs were common to IHPD and cloned placentas. In contrast, only one gene was shared between IHPD and Esx1 mutant, and cloned and Esx1 mutant placentas, respectively. These genes common to different abnormal placental growth genotypes are likely to be important in the occurrence of placentopathy.

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PubMed2004

The Sall3 locus is an epigenetic hotspot of aberrant DNA methylation associated with placentomegaly of cloned mice.

DNA methylation controls various developmental processes by silencing, switching and stabilizing genes as well as remodeling chromatin. Among various symptoms in cloned animals, placental hypertrophy is commonly observed. We identified the Spalt-like gene3 (Sall3) locus as a hypermethylated region in the placental genome of cloned mice. The Sall3 locus has a CpG island containing a tissue-dependent differentially methylated region (T-DMR) specific to the trophoblast cell lineage. The T-DMR sequence is also conserved in the human genome at the SALL3 locus of chromosome 18q23, which has been suggested to be involved in the 18q deletion syndrome. Intriguingly, larger placentas were more heavily methylated at the Sall3 locus in cloned mice. This epigenetic error was found in all cloned mice examined regardless of sex, mouse strain and the type of donor cells. In contrast, the placentas of in vitro fertilized (IVF) and intracytoplasmic sperm injected (ICSI) mice did not show such hypermethylation, suggesting that aberrant hypermethylation at the Sall3 locus is associated with abnormal placental development caused by nuclear transfer of somatic cells. We concluded that the Sall3 locus is the area with frequent epigenetic errors in cloned mice. These data suggest that there exists at least genetic locus that is highly susceptible to epigenetic error caused by nuclear transfer.

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PubMed2003

Genome-wide analysis of DNA methylation status of CpG islands in embryoid bodies, teratomas, and fetuses.

Differentiation of embryonic stem (ES) cells into embryoid bodies (EBs) provides an in vitro system for the study of early lineage determination during mammalian development. We have previously reported that there are 247 CpG islands that potentially have tissue-dependent and differentially methylated regions (T-DMRs). This provided evidence that the formation of DNA methylation patterns at CpG islands is a crucial epigenetic event underlying mammalian development. Here we present an analysis by the restriction landmark genomic scanning (RLGS) using NotI as a landmark enzyme of the genome-wide methylation status of CpG islands of ES cells and EBs and of teratomas produced from ES cells. These results are considered in relation to the methylation status of CpG islands of genomic DNA from normal fetus (10.5dpc) and adult tissues. We have prepared a DNA methylation panel that consists of 259 T-DMRs and includes novel T-DMRs that are distinctly methylated or unmethylated in the teratomas. The DNA methylation pattern was complex and differed for the ES cells, EBs, and teratomas, providing evidence that differentiation of cells involves both de novo DNA methylation as well as demethylation. Comparison of the numbers of T-DMRs, that were differentially methylated or unmethylated among the cells and tissue types studied, revealed that the teratomas were the most epigenetically different from ES cells. Thus, analysis of the DNA methylation profiles prepared in this study provides new insights into the differentiation of ES cells and development of fetus, EB, teratoma, and somatic tissues.

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PubMed2002

Differentiation of trophoblast lineage is associated with DNA methylation and demethylation.

Our previous study has shown that the placenta and kidney had different genomic methylation patterns regarding CpG island loci detected by restriction landmark genomic scanning (RLGS). To investigate whether differentiation involves changes in DNA methylation, we analyzed the rat Rcho-1 cell line, which retains trophoblast cell features and differentiates from stem cells into trophoblast giant cells in vitro. By RLGS, a total of 1,232 spots were identified in the Rcho-1 stem and differentiated giant cells. Four spots (0.3%) were detected only in giant cells, implying that the loci were originally methylated, but became demethylated during differentiation. Another four spots (0.3%) were detected only in stem cells, implying that these loci, originally unmethylated, became methylated during differentiation. DNAs from three loci that became methylated during differentiation were cloned and sequenced. All showed high homologies with expressed sequence tags (ESTs) or with genomic DNA of other species, suggesting that these loci are biologically important. Thus, the eight differentially methylated loci should be good tools to study epigenetic modification specific to differentiation of trophoblast giant cells.

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PubMed2002

Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice.

BACKGROUND: DNA methylation is involved in many gene functions such as gene-silencing, X-inactivation, imprinting and stability of the gene. We recently found that some CpG islands had a tissue-dependent and differentially methylated region (T-DMR) in normal tissues, raising the possibility that there may be more CpG islands capable of differential methylation. RESULTS: We investigated the genome-wide DNA methylation pattern of CpG islands by restriction landmark genomic scanning (RLGS) in mouse stem cells (ES, EG and trophoblast stem) before and after differentiation, and sperm as well as somatic tissues. A total of 247 spots out of 1500 (16%) showed differences in the appearance of their RLGS profiles, indicating that CpG islands having T-DMR were numerous and widespread. The methylation pattern was specific, and varied in a precise manner according to cell lineage, tissue type and during cell differentiation. CONCLUSIONS: Genomic loci with altered methylation status seem to be more common than has hitherto been realized. The formation of DNA methylation patterns at CpG islands is one of the epigenetic events which underlies the production of various cell types in the body. These findings should have implications for the use of embryonic stem cells and cells derived from them therapeutically, and also for the cloning of animals by the transfer of somatic cell nuclei.

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