Epigenetics: the DNA methylation profile of tissue-dependent and differentially methylated regions in cells.

Methylation of DNA, which occurs at cytosines of CpG sequences, is a unique chemical modification of the vertebrate genome. Methylation patterns can be copied to daughter DNA after mitosis; thus DNA methylation has been suggested to act as a "cellular memory of the genome function". Genome-wide analysis of DNA methylation revealed that there are numerous tissue-dependent differentially methylated regions (T-DMRs) in unique sequences of the mammalian genome. There are T-DMRs in both CpG-rich and -poor sequences. Methylation of T-DMRs is responsible for gene-silencing and chromatin structure change. Each tissue/cell type has a unique DNA methylation profile that consists of methylation patterns of numerous loci in the genome. DNA methylation profiles are not associated with bulk DNA, which is mainly comprised of repetitive sequences. Disruption of DNA methylation profiles putatively produce abnormal cells and tissues. Cloned mice produced by somatic nuclear transfer are associated with aberrant DNA methylation profiles. Tissue/cell type-specific DNA methylation profiles can provide a novel viewpoint for understanding normal and aberrant development, in terms of both differentiation and reproduction.

Molecular cloning of rat leukemia inhibitory factor receptor alpha-chain gene and its expression during pregnancy.

Leukemia inhibitory factor (LIF) is a secreted glycoprotein and a pluripotent growth factor that acts on diverse cell systems. LIF transmits its effects via binding to transmembrane receptors, of which both high- and low-affinity forms have been identified. In this study, we analyzed the structure and expression of rat LIF receptor alpha-chain (rLIFR alpha) cDNA. A full-length clone of the cDNA encoding the membrane-bound form of rLIFR alpha protein was prepared by a combination of LA-PCR and 5' RACE using DNA reverse-transcribed from total RNA isolated from the livers of day-12 and day-14 pregnant rats as templates. The nucleotide sequence of a full-length clone was determined and further confirmed by analysis of shorter DNA fragment prepared by PCR using pfu polymerase. The gene for rLIFR alpha encodes a 1093 amino acid residue protein. The rLIFR alpha protein shows a high degree of similarity to mouse and human LIF receptor alpha-chain protein (89% and 76% amino acid sequence identities, respectively). Only one molecular species of mRNA for the rLIFR alpha gene was detected in the liver and placenta. rLIFR alpha was expressed in liver of both non-pregnant and pregnant rats. The level of mRNA for the rLIFR alpha gene in placenta was maximum on day 16 of pregnancy.

DNA methylation regulates placental lactogen I gene expression.

Expression of rat placental lactogen I is specific to the placenta and never expressed in other tissues. To obtain insight into the mechanism of tissue-specific gene expression, we investigated the methylation status in 3.4 kb of the 5'-flanking region of the rat placental lactogen I gene. We found that the distal promoter region of the rat placental lactogen I gene had more potent promoter activity than that of the proximal area alone, which contains several possible cis-elements. Although there are only 17 CpGs in the promoter region, in vitro methylation of the reporter constructs caused severe suppression of reporter activity, and CpG sites in the placenta were more hypomethylated than other tissues. Coexpression of methyl-CpG-binding protein with reporter constructs elicited further suppression of the reporter activity, whereas treatment with trichostatin A, an inhibitor of histone deacetylase, reversed the suppression caused by methylation. Furthermore, treatment of rat placental lactogen I nonexpressing BRL cells with 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation, or trichostatin A resulted in the de novo expression of rat placental lactogen I. These results provide evidence that change in DNA methylation is the fundamental mechanism regulating the tissue-specific expression of the rat placental lactogen I gene.

Nutritional regulation of gene expression of insulin-like growth factor-binding proteins and the acid-labile subunit in various tissues of rats.

The plasma concentration and liver mRNA content of IGF-I are regulated by the quantity and quality of dietary proteins. To determine whether the synthesis of IGF-binding proteins (BPs) is also affected by protein nutrition, we assessed plasma concentration, tissue mRNA content and liver transcription rate of each BP after rats were fed either a 12% casein or a protein-free diet for 1 week. Protein deprivation reduced the plasma concentration of IGFBP-3 and IGFBP-4 and increased that of IGFBP-1 and IGFBP-2. The mRNA content in tissues and liver transcription rates of IGFBP-3 and IGFBP-4 did not change in response to protein deprivation although their plasma concentrations decreased. The increased plasma IGFBP-1 and IGFBP-2 concentrations were explained by the increased mRNA content and transcription rate of their genes in the liver. Although IGFBP-1 mRNA was increased by protein deprivation not only in liver but also in kidney, IGFBP-2 mRNA was increased only in liver and did not increase in any other tissue examined. In addition, the liver mRNA content of the acid-labile subunit, which can form a ternary complex with IGFs and IGFBP-3, was not affected by protein deprivation. These results show that tissue-specific synthesis of each BP is regulated in a distinct way in response to protein deprivation.

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.

CpG island of rat sphingosine kinase-1 gene: tissue-dependent DNA methylation status and multiple alternative first exons.

It is generally recognized that CpG islands are not methylated in normal tissues. SPHK1 is a key enzyme catalyzing the production of sphingosine 1-phosphate, a novel signaling molecule for the proliferation and differentiation of various cells, including neural cells. Sequencing of genomic DNA and cDNA reveals that rat Sphk1a consists of six exons encoding 383 amino acids. Furthermore, we identified six alternative first exons for mRNA subtypes (Sphk1a, -b, -c, -d, -e, and -f) within a 3.7-kb CpG island. The CpG island contains a tissue-dependent, differentially methylated region (T-DMR; approximately 200 bp), which is located - 800 bp upstream of the first exon of Sphk1a. T-DMR is hypomethylated in the adult brain where Sphk1a is expressed, whereas it is hypermethylated in the adult heart where the gene is not expressed. In fetal tissues, hypomethylation of T-DMR is not associated with expression of Sphk1a, which suggests that differential availability of transcription factors is also likely to be involved in the mechanism of its expression. Here, we identify rat Sphk1, using multiple alternative first exons for the subtypes, and demonstrate that there is a CpG island bearing T-DMR.

Retinoic acid promotes differentiation of trophoblast stem cells to a giant cell fate.

Trophoblast stem cell (TS cell) lines have the ability to differentiate into trophoblast subtypes in vitro and contribute to the formation of placenta in chimeras. In order to investigate the possible role of retinoic acid (RA) in placentation, we analyzed the effects of exogenous RA on TS cells in vitro and the developing ectoplacental cone in vivo. TS cells expressed all subtypes of the retinoid receptor family, with the exception of RARbeta, whose expression was stimulated in response to RA. TS cells treated with RA were compromised in their ability to proliferate and exhibited properties of differentiation into trophoblast giant cells. During TS cell differentiation into trophoblast subtypes induced by withdrawal of FGF4, RA treatment further illustrated its role in the specification of cell fate by the promotion of differentiation into giant cells and the suppression of spongiotrophoblast formation. Moreover, administration of RA during pregnancy resulted in the overabundance of giant cells at the expense of spongiotrophoblast cells. RA hereby acts as an extracellular signal whose potential function can be linked to specification events mediating trophoblast cell fate. Taken together with the spatial patterns of giant-cell formation and RA synthesis in vivo, these findings implicate a function for RA in giant-cell formation during placentation.

DNA methylation variation in cloned mice.

Mammalian cloning has been accomplished in several mammalian species by nuclear transfer. However, the production rate of cloned animals is quite low, and many cloned offspring die or show abnormal symptoms. A possible cause of the low success rate of cloning and abnormal symptoms in many cloned animals is the incomplete reestablishment of DNA methylation after nuclear transfer. We first analyzed tissue-specific methylation patterns in the placenta, skin, and kidney of normal B6D2F1 mice. There were seven spots/CpG islands (0.5% of the total CpG islands detected) methylated differently in the three different tissues examined. In the placenta and skin of two cloned fetuses, a total of four CpG islands were aberrantly methylated or unmethylated. Interestingly, three of these four loci corresponded to the tissue-specific loci in the normal control fetuses. The extent of aberrant methylation of genomic DNA varied between the cloned animals. In cloned animals, aberrant methylation occurred mainly at tissue-specific methylated loci. Individual cloned animals have different methylation aberrations. In other words, cloned animals are by no means perfect copies of the original animals as far as the methylation status of genomic DNA is concerned.

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

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.