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.

DNA methylation profiles of CpG islands for cellular differentiation and development in mammals.

DNA methylation has been implicated in mammalian development. Transcription units contain CpG islands, but expression of CpG island associated genes in normal tissues was not believed to be controlled by DNA methylation. There are, however, numerous CpG islands containing tissue-dependent and differentially methylated regions (T-DMR), which are potential methylation sites in normal cells and tissues. Genomic scanning which focused on T-DMRs in CpG islands revealed that the DNA methylation profile of each cell/tissue is more complicated than previously considered. Differentiation of cells is associated with both methylation and demethylation, which occur at multiple loci. The epigenetic system characterized by DNA methylation requires cells to memorize gene expression patterns, thus, standardizing cellular phenotypes.

Similar effects of cocaine and immobilization stress on the levels of heat-shock proteins and stress-activated protein kinases in the rat hippocampus, and on swimming behaviors: the contribution of dopamine and benzodiazepine receptors.

Cocaine (COC) has been reported to cause effects similar to physiological stressors in the brain neuroendocrinal system, including heat-shock protein (HSP) expression, although these effects have not been elucidated in detail. In the present study, we examined the effects of repeated (4 days) treatments with cocaine hydrochloride (35 mg/kg, i.p.) and 10 min immobilization stress (IM) on the distribution of HSP (HSP27, HSP60, HSP70, HSC70) and stress-activated protein kinase (SAPK) (SAPKalpha, SAPKbeta, SAPKgamma) immunoreactive nerve cells (positive cells) in the rat hippocampus. The swimming behaviors of the rats in the forced swimming test were also examined. In both COC and IM groups, an early enhancement (5 h time point) of hippocampal HSP (HSP27, HSP60, HSP70, HSC70) and SAPK (SAPKbeta, SAPKgamma) positive cells was observed, whereas a recovery (SAPKs) or attenuation (HSP60 and HSC70) was observed at the 24 h time point. In both groups, a depression of the swimming behaviors (attenuation in the activity counts and time until immobility) below the control level was observed at the 5 h point, but a recovery was observed at the 24 h time point. At the 48 h time point, all parameters returned to the control level. These alterations in the levels of HSPs and SAPKs, and the swimming behaviors were similar to those observed in the stress (IM) group, and were characteristic in that all of these alterations were attenuated by the benzodiazepine inverse agonist, Ro 15-4513 (5 mg/kg, i.p.), and the dopamine D1 receptor antagonist, SCH23390 (0.5 mg/kg, i.p.), which was not observed in the groups treated with another stressor-like drug (bicuculline).

Effects of heat on embryos and foetuses.

OBJECTIVES: This paper reviews the effects of elevated maternal temperature on embryo and foetal development in experimental animals and in humans. CONCLUSIONS: Hyperthermia during pregnancy can cause embryonic death, abortion, growth retardation and developmental defects. Processes critical to embryonic development, such as cell proliferation, migration, differentiation and programmed cell death (apoptosis) are adversely affected by elevated maternal temperatures, showing some similarity to the effects of ionizing radiation. The development of the central nervous system is especially susceptible: a 2.5 degrees C elevation for 1 h during early neural tube closure in rats resulted in an increased incidence of cranio-facial defects, and a 'spike' temperature elevation of 2-2.5 degrees C in an exposure of 1 h during early neurogenesis in guinea pigs caused an increase in the incidence of microencephaly. However, in general, thresholds and dose-response relationships vary between species and even between different strains of the same species, depending on genotype. This precludes rigorous quantitative extrapolation to humans, although some general principles can be inferred. In humans, epidemiological studies suggest that an elevation of maternal body temperature by 2 degrees C for at least 24 h during fever can cause a range of developmental defects, but there is little information on thresholds for shorter exposures. Further experimental and epidemiological studies are recommended, focusing on stage-specific developmental effects in the central nervous system using a variety of sensitive assays.

In utero exposure to brief hyperthermia interferes with the production and migration of neocortical neurons and induces apoptotic neuronal death in the fetal mouse brain.

To investigate the pathogenetic mechanisms of brain maldevelopment induced by maternal hyperthermia, we exposed pregnant ICR mice to 43 degrees C for 12.5 min on day 13.5 or 14.5 of gestation and examined the proliferation and migration of neuronal precursor cells in the telencephalon of their fetuses. The brain weight was significantly decreased in heat-stressed fetuses when examined at 72 h after treatment. Histological examination revealed that the thickness of the neopallium, especially that of the intermediate (migratory) zone and the cortical plate, was decreased in the heated group. BrdU/anti-BrdU immunohistochemistry showed that cell proliferation in the matrix cell zone was suppressed for up to 8 h after hyperthermia and that the migration of BrdU-labeled neurons from the matrix cell zone to the primordial cortex was decelerated significantly. In addition, apoptotic cell death which is rarely observed in the brain of control animals increased in the brain of heat-stressed fetuses at 8-12 h after treatment. Thus, it seems that brief hyperthermia at critical stages of neuronal differentiation can interfere with the production and migration of neuronal precursor cells and result in abnormal brain development and neurobehavioural disturbances.

Ontogenesis of anti-oxidative enzymes in mouse embryos and fetuses: an immunohistochemical study.

The ontogenesis of two anti-oxidative enzymes, thioredoxin (TRX) and glutaredoxin (GRX), was examined immunohistochemically in mouse embryos and fetuses at various developmental stages. They were found to be localized in various tissues, with some tissue specificity and temporal sequence. Both TRX and GRX began to be expressed in many tissues at embryonic (E) day E10 or E11 and tended to increase as the developmental stage advanced. In the heart and neuroepithelium, however, their immunoreactivity was already positive at E8.5. In some fetal organs like the liver, pancreas and kidney, TRX and GRX showed heterogeneous localization, suggesting that their expression may reflect the variable functional states of the cell. These results suggest that TRX and GRX may be associated with tissue differentiation in embryos and fetuses are involved in the acquisition of the capacity of resistance against oxygen radicals.

Equine follicle-stimulating hormone: molecular cloning of beta subunit and biological role of the asparagine-linked oligosaccharide at asparagine(56) of alpha subunit.

Equine FSH (eFSH) and eCG are members of the glycoprotein hormone family. These proteins are heterodimeric, composed of noncovalently associated alpha and beta subunits. We have previously reported that recombinant eCG has potent LH- and FSH-like activities and that the oligosaccharide at Asn(56) of the alpha subunit plays an indispensable role in expressing LH- but not FSH-like activity. In the present study, we cloned eFSH beta subunit cDNA and expressed wild-type recombinant eFSH and a partially deglycosylated mutant FSH (eFSH alpha56/beta) to investigate the biological role of the oligosaccharide at Asn(56) in FSH activity. The wild-type eFSH and eCG stimulated estradiol production in a dose-dependent manner in the primary cultures of rat granulosa cells, indicating that these equine gonadotropins have FSH activity. Partially deglycosylated eCG (eCG alpha56/beta) also stimulated estradiol production, confirming that the FSH-like activity of eCG is resistant to the removal of the N-linked oligosaccharide. Partially deglycosylated eFSH (eFSH alpha56/beta), however, did not show any FSH activity, indicating that the oligosaccharide at Asn(56) was necessary for eFSH. Thus, FSH-like activities of two gonadotropins, eCG and eFSH, are evoked through the distinct molecular mechanisms regarding the biological role of oligosaccharide at Asn(56) of the alpha subunit.

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.

Carboxy-PTIO increases the tetrahydrobiopterin level in mouse brain microvascular endothelial cells.

The aim of the present study was to characterize the increase in tetrahydrobiopterin (BH4), which is a cofactor for nitric oxide synthase (NOS), by carboxy-PTIO, a scavenger of nitric oxide (NO), in vascular endothelial cells. BH4 level was determined by oxidation under acidic conditions as biopterin. Addition of lipopolysaccharide (LPS) to endothelial cells increased mRNA levels of inducible NOS (iNOS) and GTP-cyclohydrolase I (GTPCH), which is a rate-limiting enzyme for BH4 synthesis, and the biopterin level. NOS inhibitors, NO-donors and L-arginine, a substrate of NOS, did not affect the increase in the biopterin level induced by LPS, suggesting that BH4 synthesis is unlikely to be modulated by NO produced by iNOS during LPS treatment. However, carboxy-PTIO increased the biopterin level in the absence and the presence of LPS. Carboxy-PTIO did not affect the expression of GTPCH mRNA level. Moreover, 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTPCH, inhibited only about 30% of the carboxy-PTIO-induced increase in the biopterin level. Whereas, N-acetylserotonin, an inhibitor of sepiapterin reductase, strongly inhibited the increase in biopterin level. Carboxy-PTIO inhibited the accumulation of pterin, a decomposition product of BH4 in endothelial cells. These findings suggest that carboxy-PTIO accumulates BH4 under basal and LPS-treated conditions in vascular endothelial cells due to both inhibition of the decomposition of BH4 to pterin and activation of the salvage pathway of BH4 synthesis via sepiapterin reductase.

Dynamic expression and essential functions of Hes7 in somite segmentation.

The basic helix-loop-helix (bHLH) gene Hes7, a putative Notch effector, encodes a transcriptional repressor. Here, we found that Hes7 expression oscillates in 2-h cycles in the presomitic mesoderm (PSM). In Hes7-null mice, somites are not properly segmented and their anterior-posterior polarity is disrupted. As a result, the somite derivatives such as vertebrae and ribs are severely disorganized. Although expression of Notch and its ligands is not affected significantly, the oscillator and Notch modulator lunatic fringe is expressed continuously throughout the mutant PSM. These results indicate that Hes7 controls the cyclic expression of lunatic fringe and is essential for coordinated somite segmentation.

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.

PAL31 expression in rat trophoblast giant cells.

PAL31 is a proliferation-related acidic nuclear protein that belongs to the leucine-rich protein family and is expressed cell-cycle-dependently. Trophoblasts differentiate into the trophoblast giant cells (TGCs) through the unusual type of cell cycle, namely endoreduplication. In the present study, we investigated the spatiotemporal pattern of PAL31 expression in rat placenta and Rcho-1 cell line. The PAL31 mRNA concentration varied in different areas of the placenta, and was barely detectable in the TGC layer. In Rcho-1 cells, although the level of PAL31 mRNA decreased dramatically during differentiation, PAL31 was detected even after differentiation. The site of intranuclear localization of PAL31 mostly overlapped with that of PCNA in the undifferentiated Rcho-1 cells, while they were not overlapped in differentiated cells. Thus, the subcellular localization of PAL31 in Rcho-1 cells significantly changed, and loss of cell cycle dependency after differentiation was noted. PAL31 is suggested to play a role in the endoreduplication distinct from the usual DNA duplication.

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.

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.

Intracellular Ca2+ signaling pathway is involved in light-induced phase advance, but may not be in phase delay, of the circadian melatonin rhythm in chick pineal cell.

Chick pineal cells have photoreceptive, circadian clock and melatonin synthetic capacities, and express circadian oscillation of melatonin release in vitro. Light pulses cause phase-dependent phase shift of the melatonin rhythm. The purpose of this study was to address the questions whether intracellular calcium is involved in both light-induced phase advance and delay. Thapsigargin and cyclopiazonic acid, which deplete the intracellular calcium stores, blocked the light-induced phase advance in a dose-dependent manner. The pulses of ryanodine receptor antagonist (dantrolene sodium or ruthenium red) also blocked the light-induced phase advance. Most agents did not cause a significant phase shift by themselves. On the other hand, all the agents used, failed to block the light-induced phase delay, even if the magnitude of phase delay was decreased using low intensity light. An antagonist of nitric oxide synthase blocked neither light-induced phase advance nor phase delay. These results indicate the following possibilities: (1) the mechanism of light-induced phase advance and delay may be different in chick pineal cells, or (2) if intracellular calcium is involved in both light-induced phase advance and delay, the sensitivity to light and/or agents used in this study may differ according to Zeitgeber time.