Naka Hattori

Placentomegaly in cloned mouse concepti caused by expansion of the spongiotrophoblast layer

Abstract Hypertrophic placenta, or placentomegaly, has been reported in cloned cattle and mouse concepti, although their placentation processes are quite different from each other. It is therefore tempting to assume that common mechanisms underlie the impact of somatic cell cloning on development of the trophoblast cell lineage that gives rise to the greater part of fetal placenta. To characterize the nature of placentomegaly in cloned mouse concepti, we histologically examined term cloned mouse placentas and assessed expression of a number of genes. A prominent morphological abnormality commonly found among all cloned mouse placentas examined was expansion of the spongiotrophoblast layer, with an increased number of glycogen cells and enlarged spongiotrophoblast cells. Enlargement of trophoblast giant cells and disorganization of the labyrinth layer were also seen. Despite the morphological abnormalities, in situ hybridization analysis of spatiotemporally regulated placenta-specific genes did not reveal any drastic disturbances. Although repression of some imprinted genes was found in Northern hybridization analysis, it was concluded that this was mostly due to the reduced proportion of the labyrinth layer in the entire placenta, not to impaired transcriptional activity. Interestingly, however, cloned mouse fetuses appeared to be smaller than those of litter size-matched controls, suggesting that cloned mouse fetuses were under a latent negative effect on their growth, probably because the placentas are not fully functional. Thus, a major cause of placentomegaly is expansion of the spongiotrophoblast layer, which consequently disturbs the architecture of the layers in the placenta and partially damages its function.

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.

Epigenetic regulation of Nanog gene in embryonic stem and trophoblast stem cells

The Nanog and Oct‐4 genes are essential for maintaining pluripotency of embryonic stem (ES) cells and early embryos. We previously reported that DNA methylation and chromatin remodeling underlie the cell type‐specific mechanism of Oct‐4 gene expression. In the present study, we found that there is a tissue‐dependent and differentially methylated region (T‐DMR) in the Nanog up‐stream region. The T‐DMR is hypomethylated in ES cells, but is heavily methylated in trophoblast stem (TS) cells and NIH/3T3 cells, in which the Nanog gene is repressed. Furthermore, in vitro methylation of T‐DMR suppressed Nanog promoter activity in reporter assay. Chromatin immunoprecipitation assay revealed that histone H3 and H4 are highly acetylated, and H3 lysine (K) 4 is hypermethylated at the Nanog locus in ES cells. Conversely, histone deacetylation and H3‐K4 demethylation occurred in TS cells. Importantly, in TS cells, hypermethylation of H3‐K9 and ‐K27 is found only at the Nanog locus, not the Oct‐4 locus, indicating that the combination of histone modifications associated with the Nanog gene is distinct from that of the Oct‐4 gene. In conclusion, the Nanog gene is regulated by epigenetic mechanisms involving DNA methylation and histone modifications.

Dimethyl Sulfoxide Has an Impact on Epigenetic Profile in Mouse Embryoid Body

Dimethyl sulfoxide (DMSO), an amphipathic molecule, is widely used not only as a solvent for water‐insoluble substances but also as a cryopreservant for various types of cells. Exposure to DMSO sometimes causes unexpected changes in cell fates. Because mammalian development and cellular differentiation are controlled epigenetically by DNA methylation and histone modifications, DMSO likely affects the epigenetic system. The effects of DMSO on transcription of three major DNA methyltransferases (Dnmts) and five well‐studied histone modification enzymes were examined in mouse embryonic stem cells and embryoid bodies (EBs) by reverse transcription‐polymerase chain reaction. Addition of DMSO (0.02%–1.0%) to EBs in culture induced an increase in Dnmt3a mRNA levels with increasing dosage. Increased expression of two subtypes of Dnmt3a in protein levels was confirmed by Western blotting. Southern blot analysis revealed that DMSO caused hypermethylation of two kinds of repetitive sequences in EBs. Furthermore, restriction landmark genomic scanning, by which DNA methylation status can be analyzed on thousands of loci in genic regions, revealed that DMSO affected DNA methylation status at multiple loci, inducing hypomethylation as well as hypermethylation depending on the genomic loci. In conclusion, DMSO has an impact on the epigenetic profile: upregulation of Dnmt3a expression and alteration of genome‐wide DNA methylation profiles with phenotypic changes in EBs.

Regulation of the Expression of Human Organic Anion Transporter 3 by Hepatocyte Nuclear Factor 1α/β and DNA Methylation

Human organic anion transporter 3 (hOAT3/SLC22A8) is predominantly expressed in the proximal tubules of the kidney and plays a major role in the urinary excretion of a variety of organic anions. The promoter region of hOAT3 was characterized to elucidate the mechanism underlying the tissue-specific expression of hOAT3. The minimal promoter of hOAT3 was identified to be located approximately 300 base pairs upstream of the transcriptional start site, where there are canonical TATA and hepatocyte nuclear factor (HNF1) binding motifs, which are conserved in the rodent Oat3 genes. Transactivation assays revealed that HNF1α and HNF1β markedly increased hOAT3 promoter activity, where the transactivation potency of HNF1β was lower than that of HNF1α. Mutations in the HNF1 binding motif prevented the transactivation. Electrophoretic mobility shift assays demonstrated binding of the HNF1α/HNF1α homodimer or HNF1α/HNF1β heterodimer to the hOAT3 promoter. It was also demonstrated that the promoter activity of hOAT3 is repressed by DNA methylation. Moreover, the expression of hOAT3 was activated de novo by forced expression of HNF1α alone or both HNF1α and HNF1β together with the concomitant DNA demethylation in human embryonic kidney 293 cells that lack expression of endogenous HNF1α and HNF1β, whereas forced expression of HNF1β alone could not activate the expression of hOAT3. This suggests a synergistic action of the HNF1α/HNF1α homodimer or HNF1α/HNF1β heterodimer and DNA demethylation for the constitutive expression of hOAT3. These results indicate that the tissue-specific expression of hOAT3 might be regulated by the concerted effect of genetic (HNF1α and HNF1β) and epigenetic (DNA methylation) factors.

Analysis of CpG islands of trophoblast giant cells by restriction landmark genomic scanning

Rat trophoblast giant cells each contain at least 100 times more genomic DNA per nucleus than diploid cells. This unusual phenomenon appears to be of interest in relation to the molecular mechanism of cell differentiation and gene expression in the placenta. In the present study, we analyzed the CpG islands of trophoblast giant cells by restriction landmark genomic scanning (RLGS) using the methylation-sensitive landmark enzymes, Not I and Bss HII. More than 1,000 and 1,900 spots were detected by RLGS using Not I and Bss HII, respectively, in the placental junctional zone, where more than 90% of genomic DNA is present in the cells with higher DNA content. Of these, 97% (1,009 spots) and 99% (1,911 spots) of the spots found in the junctional zone showed an identical pattern and identical intensity with those of diploid cell controls, for which genomic DNA was extracted from the labyrinth zone and maternal kidney. Therefore, the giant cells are basically polyploid. More importantly, 24 tissue-specific spots were detected by RLGS using Not I. Subsequent cloning and sequencing of four typical spots of the genomic DNA confirmed that these DNA fragments contained abundant CpG dinucleotides and showed characteristics of CpG islands. Of these 24 spots, there were ten spots specific for the placenta, and three of them were specific for the junctional zone, indicating that methylation status of CpG islands in the placental tissue differed between the junctional zone and labyrinth zone. These results suggest that multiple rounds of endoreduplication and modification of CpG islands by cytosine methylation occur during the differentiation process of giant cells.

Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cells

In the mammalian genome, numerous CpG‐rich loci define tissue‐dependent and differentially methylated regions (T‐DMRs). Euchromatin from different cell types differs in terms of its tissue‐specific DNA methylation profile as defined by these T‐DMRs. G9a is a euchromatin‐localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3‐K9 and ‐K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG‐rich loci, which are predicted in silico, in G9a−/− embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild‐type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a−/− cells. Virtual‐image RLGS (Vi‐RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3‐K9 and/or ‐K27 dimethylation in the G9a−/− cells. These data indicate that G9a site‐selectively contributes to DNA methylation.

Regulation of tissue-specific expression of the human and mouse urate transporter 1 gene by hepatocyte nuclear factor 1 α/β and DNA methylation

Expression of Urate transporter 1 (URAT1/SLC22A12) is restricted to the proximal tubules in the kidney, where it is responsible for the tubular reabsorption of urate. To elucidate the mechanism underlying its tissue-specific expression, the transcriptional regulation of the hURAT1 and mUrat1 genes was investigated. Hepatocyte nuclear factor 1 α (HNF1α) and HNF1β positively regulate minimal promoter activity of the URAT1 gene as shown by reporter gene assays. Electrophoretic mobility shift assays revealed binding of HNF1α and/or HNF1β to the HNF1 motif in the hURAT1 promoter. Furthermore, the mRNA expression of Urat1 is reduced in the kidneys of Hnf1α-null mice compared with wild-type mice, confirming the indispensable role of HNF1α in the constitutive expression of URAT1 genes. It was also shown that the proximal promoter region of mUrat1 was hypermethylated in the liver and kidney medulla, whereas this region was relatively hypomethylated in the kidney cortex. These methylation profiles are in a good agreement with the proximal tubule-restricted expression of mUrat1 in the kidney cortex. Taken together, these results strongly suggest that tissue-specific expression of the URAT1 genes is coordinately regulated by the transcriptional activation by HNF1α/HNF1β heterodimer and repression by DNA methylation.

Applying whole-genome studies of epigenetic regulation to study human disease

Epigenetics may be broadly defined as the study of processes that produce a heritable phenotype that is not strictly dependent on DNA sequence. The definition has traditionally been restricted to processes that occur in the cells nucleus, with the term “heritable” having a loose meaning that can be applied to either the entire organism or single cells. For example, a process that produces a phenotype only in a specific cell type (for instance, chromatin-mediated maintenance of a differentiated state) is usually considered epigenetic even if it is not directly inherited, but instead must be reestablished or actively maintained at each cell division. Given this definition, the field of epigenetics has long focused on proteins that affect DNA packaging, and thereby affect the utilization of the genetic information encoded in the DNA template. This focus extends to the enzymatic modification of those proteins, and to the enzymatic modification of the DNA template itself, primarily DNA methylation. This review is written in conjunction with the international symposium on Genome-wide Epigenetics 2005, held at the University of Tokyo, Japan on November 8–10, 2005. Over the past decade, the field of epigenetics has undergone an exciting expansion in the number of researchers, techniques available and our understanding of epigenetic phenomena. The purpose of this short review is not to summarize all of these advances, but rather to guide the reader to more detailed sources of information by sketching an outline of the major thrusts in the field, emphasizing mammalian epigenetics in particular.

Cell type‐specific methylation profiles occurring disproportionately in CpG‐less regions that delineate developmental similarity

Our previous studies using restriction landmark genomic scanning (RLGS) defined tissue‐ or cell‐specific DNA methylation profiles. It remains to be determined whether the DNA sequence compositions in the genomic contexts of the NotI loci tested by RLGS influence their tendency to change with differentiation. We carried out 3834 methylation measurements consisting of 213 NotI loci in the mouse genome in 18 different tissues and cell types, using quantitative real‐time PCR based on a Virtual image rlgs database. Loci were categorized as CpG islands or other, and as unique or repetitive sequences, each category being associated with a variety of methylation categories. Strikingly, the tissue‐dependently and differentially methylated regions (T‐DMRs) were disproportionately distributed in the non‐CpG island loci. These loci were located not only in 5′‐upstream regions of genes but also in intronic and non‐genic regions. Hierarchical clustering of the methylation profiles could be used to define developmental similarity and cellular phenotypes. The results show that distinctive tissue‐ and cell type‐specific methylation profiles by RLGS occur mostly at NotI sites located at non‐CpG island sequences, which delineate developmental similarity of different cell types. The finding indicates the power of NotI methylation profiles in evaluating the relatedness of different cell types.