Jun Ohgane

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.

Interplay between DNA methylation, histone modification and chromatin remodeling in stem cells and during development

Genes constitute only a small proportion of the mammalian genome, the majority of which is composed of non-genic repetitive elements including interspersed repeats and satellites. A unique feature of the mammalian genome is that there are numerous tissue-dependent, differentially methylated regions (T-DMRs) in the non-repetitive sequences, which include genes and their regulatory elements. The epigenetic status of T-DMRs varies from that of repetitive elements and constitutes the DNA methylation profile genome-wide. Since the DNA methylation profile is specific to each cell and tissue type, much like a fingerprint, it can be used as a means of identification. The formation of DNA methylation profiles is the basis for cell differentiation and development in mammals. The epigenetic status of each T-DMR is regulated by the interplay between DNA methyltransferases, histone modification enzymes, histone subtypes, non-histone nuclear proteins and non-coding RNAs. In this review, we will discuss how these epigenetic factors cooperate to establish cell- and tissue-specific DNA methylation profiles.

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.

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.

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. Developmental Dynamics 230:149–164, 2004.

Potential link between estrogen receptor-α gene hypomethylation and uterine fibroid formation

Uterine leiomyomas are the most common uterine tumors in women. Estrogen receptor-alpha (ER-alpha) is more highly expressed in uterine leiomyomas than in normal myometrium, suggesting a link between uterine leiomyomas and ER-alpha expression. DNA methylation is an epigenetic mechanism of gene regulation and plays important roles in normal embryonic development and in disease progression including cancers. Here, we investigated the DNA methylation status of the ER-alpha promoter region (-1188 to +229 bp) in myometrium and leiomyoma. By sodium bisulfite sequencing, 49 CpG sites in the proximal promoter region of ER-alpha gene were shown to be unmethylated in both leiomyoma and normal myometrium. At seven CpG sites in the distal promoter region of the ER-alpha gene, there was a variation in DNA methylation status in myometrium and leiomyoma. Further analysis of the DNA methylation status by bisulfite restriction mapping among 11 paired samples of myometrium and leiomyoma indicated that CpG sites in the distal region of ER-alpha promoter are hypomethylated in leiomyomas of nine patients. In those patients, ER-alpha mRNA levels tended to be higher in the leiomyoma than in the myometrium. In conclusion, there was an aberrant DNA methylation status in the promoter region of ER-alpha gene in uterine leiomyoma, which may be associated with high ER-alpha mRNA expression.

Oocyte-specific linker histone H1foo is an epigenomic modulator that decondenses chromatin and impairs pluripotency.

Mammalian oocytes contain the histone H1foo, a distinct member with low sequence similarity to other members in the H1 histone family. Oocyte-specific H1foo exists until the second embryonic cell stage. H1foo is essential for oocyte maturation in mice; however, the molecular function of this H1 subtype is unclear. To explore the function of H1foo, we generated embryonic stem (ES) cells ectopically expressing H1foo fused to an EGFP (H1foo-ES). Interestingly, ectopic expression of H1foo prevented normal differentiation into embryoid bodies (EBs). The EB preparations from H1foo-ES cells maintained the expression of pluripotent marker genes, including Nanog, Myc and Klf9, and prevented the shift of the DNA methylation profile. Because the short hairpin RNA-mediated knockdown of H1foo-EGFP recovered the differentiation ability, H1foo was involved in preventing differentiation. Furthermore, ChIP analysis revealed that H1foo-EGFP bound selectively to a set of hypomethylated genomic loci in H1foo-ES, clearly indicating that these loci were targets of H1foo. Finally, nuclease sensitivity assay suggested that H1foo made these target loci decondensed. We concluded that H1foo has an impact on the genome-wide, locus-specific epigenetic status.

Genome-wide DNA methylation profile of tissue-dependent and differentially methylated regions (T-DMRs) residing in mouse pluripotent stem cells

DNA methylation profile, consisting of tissue‐dependent and differentially methylated regions (T‐DMRs), has elucidated tissue‐specific gene function in mouse tissues. Here, we identified and profiled thousands of T‐DMRs in embryonic stem cells (ESCs), embryonic germ cells (EGCs) and induced pluripotent stem cells (iPSCs). T‐DMRs of ESCs compared with somatic tissues well illustrated gene function of ESCs, by hypomethylation at genes associated with CpG islands and nuclear events including transcriptional regulation network of ESCs, and by hypermethylation at genes for tissue‐specific function. These T‐DMRs in EGCs and iPSCs showed DNA methylation similar to ESCs. iPSCs, however, showed hypomethylation at a considerable number of T‐DMRs that were hypermethylated in ESCs, suggesting existence of traceable progenitor epigenetic information. Thus, DNA methylation profile of T‐DMRs contributes to the mechanism of pluripotency, and can be a feasible solution for identification and evaluation of the pluripotent cells.