Mitsuko Hirosawa

Epigenetic Switching by the Metabolism-sensing Factors in the Generation of Orexin Neurons from Mouse Embryonic Stem Cells

Background: Orexin plays a central role in the integration of sleep/wake states and feeding behaviors. Results: Orexin neurons were induced from pluripotent stem cells by supplementation of ManNAc. Conclusion: ManNAc induced switching of epigenetic factors from Sirt1/Ogt to Mgea5 at Hcrt gene locus. Significance: This study will be useful to investigate molecular mechanism in the orexin system and development of regenerative medicine. The orexin system plays a central role in the integration of sleep/wake and feeding behaviors in a broad spectrum of neural-metabolic physiology. Orexin-A and orexin-B are produced by the cleavage of prepro-orexin, which is encoded on the Hcrt gene. To date, methods for generating other peptide neurons could not induce orexin neurons from pluripotent stem cells. Considering that the metabolic status affects orexin expression, we supplemented the culture medium with a nutrient factor, ManNAc, and succeeded in generating functional orexin neurons from mouse ES cells. Because DNA methylation inhibitors and histone deacetylase inhibitors could induce Hcrt expression in mouse ES cells, the epigenetic mechanism may be involved in this orexin neurogenesis. DNA methylation analysis showed the presence of a tissue-dependent differentially methylated region (T-DMR) around the transcription start site of the Hcrt gene. In the orexin neurons induced by supplementation of ManNAc, the T-DMR of the Hcrt gene was hypomethylated in association with higher H3/H4 acetylation. Concomitantly, the histone acetyltransferases p300, CREB-binding protein (CBP), and Mgea5 (also called O-GlcNAcase) were localized to the T-DMR in the orexin neurons. In non-orexin-expressing cells, H3/H4 hypoacetylation and hyper-O-GlcNAc modification were observed at the T-DMRs occupied by O-GlcNAc transferase and Sirt1. Therefore, the results of the present study suggest that the glucose metabolite, ManNAc, induces switching from the inactive state by Ogt-Sirt1 to the active state by Mgea5, p300, and CBP at the Hcrt gene locus.

Effect of protein nutrition on the mRNA content of insulin-like growth factor-binding protein-1 in liver and kidney of rats

Effect of quantity and nutritional quality of dietary proteins on the content of mRNA of insulin-like growth factor-binding protein-1 (IGFBP-1) was studied in rat liver and kidney. IGFBP-1 mRNA content per unit RNA increased in liver and kidney of rats fed on a protein-free diet and in those of fasted rats compared with that in the rats fed on a casein diet. When rats were given a gluten diet for 7 d, IGFBP-1 mRNA content in liver did not change significantly but that in kidney increased considerably compared with that in those organs of the rats fed on the casein diet. Because IGFBP-1 mRNA has been demonstrated both in liver parenchymal and non-parenchymal cells (Takenaka et al. 1991), the effect of the protein-free diet on these two types of cells has been studied. An increase in IGFBP-1 mRNA content under protein deprivation was observed in both liver parenchymal and non-parenchymal cells, suggesting that these two types of cells are regulated in a similar mode as far as IGFBP-1 mRNA content is concerned. The physiological and nutritional significance of the previously stated results on protein anabolism are discussed when considered together with our previous observations on the plasma concentrations of IGF-1 (Takahashi et al. 1990) and IGFBP (Umezawa et al. 1991) and insulin-like growth factor-1 mRNA content in liver (Miura et al. 1991).

Novel O-GlcNAcylation on ser 40 of canonical H2A isoforms specific to viviparity

We report here newly discovered O-linked-N-acetylglucosamine (O-GlcNAc) modification of histone H2A at Ser40 (H2AS40Gc). The mouse genome contains 18 H2A isoforms, of which 13 have Ser40 and the other five have Ala40. The combination of production of monoclonal antibody and mass spectrometric analyses with reverse-phase (RP)-high performance liquid chromatography (HPLC) fractionation indicated that the O-GlcNAcylation is specific to the Ser40 isoforms. The H2AS40Gc site is in the L1 loop structure where two H2A molecules interact in the nucleosome. Targets of H2AS40Gc are distributed genome-wide and are dramatically changed during the process of differentiation in mouse trophoblast stem cells. In addition to the mouse, H2AS40Gc was also detected in humans, macaques and cows, whereas non-mammalian species possessing only the Ala40 isoforms, such as silkworms, zebrafish and Xenopus showed no signal. Genome database surveys revealed that Ser40 isoforms of H2A emerged in Marsupialia and persisted thereafter in mammals. We propose that the emergence of H2A Ser40 and its O-GlcNAcylation linked a genetic event to genome-wide epigenetic events that correlate with the evolution of placental animals.

Bridging sequence diversity and tissue-specific expression by DNA methylation in genes of the mouse prolactin superfamily

Much of the DNA in genomes is organized within gene families and hierarchies of gene superfamilies. DNA methylation is the main epigenetic event involved in gene silencing and genome stability. In the present study, we analyzed the DNA methylation status of the prolactin (PRL) superfamily to obtain insight into its tissue-specific expression and the evolution of its sequence diversity. The PRL superfamily in mice consists of two dozen members, which are expressed in a tissue-specific manner. The genes in this family have CpG-less sequences, and they are located within a 1-Mb region as a gene cluster on chromosome 13. We tentatively grouped the family into several gene clusters, depending on location and gene orientation. We found that all the members had tissue-dependent differentially methylated regions (T-DMRs) around the transcription start site. The T-DMRs are hypermethylated in nonexpressing tissues and hypomethylated in expressing cells, supporting the idea that the expression of the PRL superfamily genes is subject to epigenetic regulation. Interestingly, the DNA methylation patterns of T-DMRs are shared within a cluster, while the patterns are different among the clusters. Finally, we reconstituted the nucleotide sequences of T-DMRs by converting TpG to CpG based on the consideration of a possible conversion of 5-methylcytosine to thymine by spontaneous deamination during the evolutionary process. On the phylogenic tree, the reconstituted sequences were well matched with the DNA methylation pattern of T-DMR and orientation. Our study suggests that DNA methylation is involved in tissue-specific expression and sequence diversity during evolution.

An epigenetic regulatory element of the Nodal gene in the mouse and human genomes

Nodal signaling plays critical roles during embryonic development. The Nodal gene is not expressed in adult tissues but is frequently activated in cancer cells, contributing to progression toward malignancy. Although several regulatory elements of the Nodal gene have been identified, the epigenetic mechanisms by which Nodal expression is regulated over the long term remain unclear. We found a region exhibiting dynamic changes in DNA methylation at approximately -3.0 kb to -0.4 kb upstream from the transcriptional start site (TSS) that we termed the epigenetic regulatory element (ERE). The ERE was unmethylated in mouse embryonic stem cells (mESCs) but became increasingly methylated in differentiated cells and tissues, concomitant with the downregulation of Nodal mRNA expression. In vitro reporter assays identified an Oct3/4 binding motif within the ERE, indicating that the ERE is responsible for the activation of Nodal in mESCs. Furthermore, the ERE was a target of differentiation-associated Polycomb silencing, and the chromatin condensed when mESCs differentiated to embryoid bodies (EBs). Pharmacological inhibition of PRC2 led to the reactivation of Nodal expression in EBs and mouse embryonic fibroblasts (MEFs). The ERE was also targeted by PRC2 in normal human cells. In NODAL-expressing human cancer cells, accumulation of EZH2 and trimethylation of H3K27 at the ERE were diminished. In conclusion, Nodal is epigenetically controlled through the ERE in the mouse embryo and human cells.

Molecular diversity of rat placental lactogens

Summary A major function of the placenta is the production of placental lactogens (Pls). Progesterone is essential for pregnancy in all mammals and is secreted by the ovary and placenta, depending on the animal species. In the rat, the main source of progesterone throughout pregnancy is the ovary, and 20α-hydroxysteroid dehydrogenase (20α-HSD) is a key enzyme controlling ovarian progesterone secretion. The primary action of prolactin (PRL) in the maintenance of ovarian progesterone secretion is suppression of the activity of ovarian 20α-HSD. In this review, the sequence homologies between cDNAs for PLs and PRL and the intimate functional relationship between the ovary and placenta are discussed in order to speculate how and why the molecular diversity of rat PLs has developed.

Putative Epimutagens in Maternal Peripheral and Cord Blood Samples Identified Using Human Induced Pluripotent Stem Cells

The regulation of transcription and genome stability by epigenetic systems are crucial for the proper development of mammalian embryos. Chemicals that disturb epigenetic systems are termed epimutagens. We previously performed chemical screening that focused on heterochromatin formation and DNA methylation status in mouse embryonic stem cells and identified five epimutagens: diethyl phosphate (DEP), mercury (Hg), cotinine, selenium (Se), and octachlorodipropyl ether (S-421). Here, we used human induced pluripotent stem cells (hiPSCs) to confirm the effects of 20 chemicals, including the five epimutagens, detected at low concentrations in maternal peripheral and cord blood samples. Of note, these individual chemicals did not exhibit epimutagenic activity in hiPSCs. However, because the fetal environment contains various chemicals, we evaluated the effects of combined exposure to chemicals (DEP, Hg, cotinine, Se, and S-421) on hiPSCs. The combined exposure caused a decrease in the number of heterochromatin signals and aberrant DNA methylation status at multiple gene loci in hiPSCs. The combined exposure also affected embryoid body formation and neural differentiation from hiPSCs. Therefore, DEP, Hg, cotinine, Se, and S-421 were defined as an “epimutagen combination” that is effective at low concentrations as detected in maternal peripheral and cord blood.

A Method for Obtaining Epigenomic Data

Chapter Summary To obtain epigenomic data for the cells of interest, the target genomic deoxyribonucleic acid (DNA) and/or the posttranslational histone modification site in the chromatin should be selected. Our protocol is summarized in Figure 1 . In this section, we describe two basic steps in epigenomic analyses: bisulfite reaction and chromatin immunoprecipitation.

Epigenetics of Placental Development and Function

Chapter Summary Epigenetics plays a key role in the development and maintenance of cellular phenotypes and functions. The term epigenome refers to genome-wide epigenetic information. Over the past decade, epigenetics has expanded into numerous research fields, including mammalian reproduction. DNA methylation is a major epigenetic event associated with histone modifications, causing gene silencing and genome stability. Placentas, as well as other tissues and cell types, have unique DNA methylation profiles comprising several tissue-dependent differentially methylated regions (T-DMRs), in which epigenetic changes control cellular differentiation and development. Epigenetic alterations at T-DMRs of specific gene loci or disruption of the epigenome have the potential to produce aberrant cells and tissues with prolonged abnormal phenotypes, suggesting that orchestrated epigenetic profile of trophoblast cells is essential for placental development and function.