DNA methylation constitutes an important epigenetic regulation mechanism in many eukaryotes, although the extent of DNA methylation in the regulation of gene expression in the mammalian genome is poorly understood. We developed D-REAM, a genome-wide DNA methylation analysis method for tissue-dependent and differentially methylated region (T-DMR) profiling with restriction tag-mediated amplification in mouse tissues and cells. Using a mouse promoter tiling array covering a region from −6 to 2.5 kb (∼30,000 transcription start sites), we found that over 3000 T-DMRs are hypomethylated in liver compared to cerebrum. The DNA methylation profile of liver was distinct from that of kidney and spleen. This hypomethylation profile marked genes that are specifically expressed in liver, including key transcription factors such as Hnf1a and Hnf4a. Genes with T-DMRs, especially those lacking CpG islands and those with HNF-1A binding motifis in their promoters, showed good correlation between their tissue-specific expression and liver hypomethylation status. T-DMRs located downstream from their transcription start sites also showed tissue-specific gene expression. These data indicate that multilayered regulation of tissue-specific gene function could be elucidated by DNA methylation tissue profiling.

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.

In female rats, apoptotic cell death in the corpus luteum is induced by the prolactin (PRL) surge occurring in the proestrous afternoon during the estrous cycle. We have previously shown that this luteolytic action of PRL is mediated by the Fas/Fas ligand (FasL) system. During pregnancy or pseudopregnancy, apoptosis does not occur in the corpus luteum. Progesterone (P 4 ), a steroid hormone secreted from luteal steroidogenic cells, attenuated PRL-induced apoptosis in cultured luteal cells in a dose-dependent manner. P 4 significantly decreased the expression of mRNA of Fas, but not FasL, in cultured luteal cells prepared from both proestrous and midpseudopregnant rats. These data indicate that P 4 suppresses PRL-induced luteal cell apoptosis via reduction of the expression level of Fas mRNA in the corpus luteum, suggesting that P 4 acts as an important factor that can change the sensitivity of corpus luteum to PRL.z 2000 Federation of European Biochemical Societies.

These results suggest that the transgenic rats, whose GH secretion is suppressed, develop obesity due to early onset of an increase in food intake and a decrease in locomotor activity with leptin resistance resulting from deteriorating leptin transport from peripheral blood to cerebrospinal fluid.

Abstract. 20α-Hydroxysteroid dehydrogenase (20α-HSD) converts progesterone to a biologically inactive steroid, 20α-dihydroprogesterone. To examine a potential role of 20α-HSD in tissues other than corpus luteum (CL) during pregnancy, mouse 20α-HSD cDNA was cloned and was used for the examination of its mRNA localization in the conceptus and uterus at different stages of gestation. Using probes based on the rat sequence, mouse 20α-HSD cDNA clone was isolated from a cDNA library prepared from ovaries of day 12 pseudopregnant mice. The mouse 20α-HSD cDNA was 1193 bp in length and encoded 323 amino acids with an estimated molecular weight of 37 kD. The mouse 20α-HSD was homologous to the rat 20α-HSD in nucleotide and amino acid sequences (both in 93% homology), and to other members of aldo-keto reductase superfamily at the cofactor (NADP(H)) binding site. By in situ hybridization, 20α-HSD mRNA was localized in endometrial epithelial cells on days 10, 15 and 18 of pregnancy, maternal placental endothelial cells on day 10, and fetal epidermal cells on day 15. During pseudopregnancy as well as early pregnancy, however, 20α-HSD mRNA was not detected in endometrial cells. Together, 20α-HSD mRNA was localized in cells that surround fetuses during the mid and late stages of gestation.

Abstract. In the corpus luteum of rats and mice, 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to a biologically inactive metabolite, 20α-dihydroprogesterone (20α-OHP). The reduction of progesterone by 20α-HSD is believed to be important for functional luteolysis in these rodent species. In addition to the corpus luteum, expression of 20α-HSD has been demonstrated in tissues such as the placenta, endometrial epithelia, and fetal skin, although the roles it plays in the latter tissues remain to be determined. To determine the contribution of 20α-HSD to functional luteolysis and to the rodent reproductive system more generally, we generated a strain of mice with targeted disruption of the 20α-HSD gene. In the 20α-HSD -/-mice we obtained, which lacked the genomic region essential for catalytic reaction, neither 20α-HSD activity in the corpus luteum nor an increase in the serum concentrations of 20α-OHP during pseudopregnancy or pregnancy was detected. The durations of the estrous cycle, pseudopregnancy, and pregnancy were significantly prolonged in the 20α-HSD -/-mice, although the serum progesterone levels decreased to levels low enough for delivery of pups at term of pregnancy. In addition, the number of pups, especially live pups, was markedly decreased in the 20α-HSD -/-mice. These findings suggest that the role of 20α-HSD in functional luteolysis is relatively minor but that it is involved in the survival of newborn mice. Key words: 20α-Hydroxysteroid dehydrogenase (20α-HSD), Luteolysis, Mouse, Ovary, Reproduction (J. Reprod. Dev. 53: [499][500][501][502][503][504][505][506][507][508] 2007) he enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) is expressed in the rodent corpus luteum, and it catalyzes the conversion of p r o g e s t e r o n e t o i t s i n a c t i v e f o r m , 2 0 α -dihydroprogesterone (20α-OHP). In the estrous cycle of rats, 20α-HSD is thought to play a crucial role in recurrence of unique short cycles through promotion of functional luteolysis [1,2]. The finding that the transcription factors Sp1 and Sp3 stimulate the promoter activity of mouse 20α-HSD

We recently reported that exogenous DNA injected into testis as a liposome complex can be transferred into the egg via sperm by natural mating and integrated in the genome (testis-mediated gene transfer: TMGT). Here, we studied the efficiency of each of the several liposomes in associating foreign DNA with sperm, the expression of an introduced gene in early embryos, and the presence of the DNA in fetuses and pups at different ages. The CMV/beta-actin/EGFP fusion gene, encapsulated with different liposomes, was injected into rat testis, and spermatozoa in the cauda epididymis were obtained 1, 4, and 14 days after injection. We tested each of the 8 liposomes, and found that only 2, DMRIE-C and SuperFect, led to the detection of foreign DNA on all of the days examined, with relatively higher ratios of rats having positive sperm. By means of TMGT using either of those two liposomes, more than 80% of morula-stage embryos expressed EGFP, as observed by fluorescence microscopy. Then we detected introduced DNA in the progeny by PCR and Southern dot blot, and found that the ratio of animals carrying the foreign DNA decreased as they developed, and that only a part of postpartum progeny were foreign-DNA-positive with high incidence of mosaicism. These results suggest that, although, the success rate is still limited, foreign DNA could be integrated into the genome of the progeny by TMGT at least under specific experimental conditions, the efficiency of which depends largely on the characteristics of the liposome. The results also suggest that TMGT could be applicable to fetal gene therapy as well as to the generation of transgenic animals.

BackgroundMitochondria are semi-autonomous, semi-self-replicating organelles harboring their own DNA (mitochondrial DNA, mtDNA), and their dysregulation is involved in the development of various diseases. While mtDNA does not generally undergo epigenetic modifications, almost all mitochondrial proteins are encoded by nuclear DNA. However, the epigenetic regulation of nuclear-encoded mitochondrial genes (nuclear mt genes) has not been comprehensively analyzed.ResultsWe analyzed the DNA methylation status of 899 nuclear mt genes in the liver, brain, and heart tissues of mouse, and identified 636 nuclear mt genes carrying tissue-dependent and differentially methylated regions (T-DMRs). These nuclar mt genes are involved in various mitochondrial functions and they also include genes related to human diseases. T-DMRs regulate the expression of nuclear mt genes. Nuclear mt genes with tissue-specific hypomethylated T-DMRs were characterized by enrichment of the target genes of specific transcription factors such as FOXA2 in the liver, and CEBPA and STAT1 in the brain.ConclusionsA substantial proportion of nuclear mt genes contained T-DMRs, and the DNA methylation status of numerous T-DMRs should underlie tissue-dependent mitochondrial functions.