Kentaro Mochizuki

Epigenetic profiles in primordial germ cells: Global modulation and fine tuning of the epigenome for acquisition of totipotency

Germ cells, after fate determination as primordial germ cells (PGCs) in early embryos, undergo various unique changes in epigenetic status during their development, and these changes differ from the epigenetic changes occurring in any other somatic cells. For example, PGCs undergo demethylation of DNA and change histone modification states on a genome‐wide scale. Although the full physiological significance of these epigenetic alterations is still unclear, we can now discuss some of their mechanisms due to recent experimental evidence demonstrating the expression of candidate molecules involved in the processes of epigenetic change. On the other hand, DNA demethylation associated with PGC‐specific gene expression, reprogramming of imprinted genes and regulation of retrotransposons in PGCs differentially occur from the genome‐wide DNA demethylation. The tendency of epigenetic changes to appear on the whole genome, as well as more precise changes in the epigenetic status of particular parts of the genome, may play important roles in establishing the properties of PGCs required for acquiring totipotency.

Implication of DNA demethylation and bivalent histone modification for selective gene regulation in mouse primordial germ cells.

Primordial germ cells (PGCs) sequentially induce specific genes required for their development. We focused on epigenetic changes that regulate PGC-specific gene expression. mil-1, Blimp1, and Stella are preferentially expressed in PGCs, and their expression is upregulated during PGC differentiation. Here, we first determined DNA methylation status of mil-1, Blimp1, and Stella regulatory regions in epiblast and in PGCs, and found that they were hypomethylated in differentiating PGCs after E9.0, in which those genes were highly expressed. We used siRNA to inhibit a maintenance DNA methyltransferase, Dnmt1, in embryonic stem (ES) cells and found that the flanking regions of all three genes became hypomethylated and that expression of each gene increased 1.5- to 3-fold. In addition, we also found 1.5- to 5-fold increase of the PGC genes in the PGCLCs (PGC-like cells) induced form ES cells by knockdown of Dnmt1. We also obtained evidence showing that methylation of the regulatory region of mil-1 resulted in 2.5-fold decrease in expression in a reporter assay. Together, these results suggested that DNA demethylation does not play a major role on initial activation of the PGC genes in the nascent PGCs but contributed to enhancement of their expression in PGCs after E9.0. However, we also found that repression of representative somatic genes, Hoxa1 and Hoxb1, and a tissue-specific gene, Gfap, in PGCs was not dependent on DNA methylation; their flanking regions were hypomethylated, but their expression was not observed in PGCs at E13.5. Their promoter regions showed the bivalent histone modification in PGCs, that may be involved in repression of their expression. Our results indicated that epigenetic status of PGC genes and of somatic genes in PGCs were distinct, and suggested contribution of epigenetic mechanisms in regulation of the expression of a specific gene set in PGCs.

A current view of the epigenome in mouse primordial germ cells.

Primordial germ cells (PGCs) are undifferentiated germ line cells in embryos that emerge at early stages of embryonic development, and then differentiate into eggs or sperm in gonads to give rise to individuals of successive generations. During germ cell development, several dynamic changes in epigenetic modifications including DNA methylation and histone modifications occur, and these changes are thought to be reprogramming processes that are required for germ cells to confer totipotency to the zygote. Initially, the epigenetic status of particular gene loci in PGCs was studied, but more recently, genome‐wide studies have provided more comprehensive views of the PGC epigenome. Mouse PGCs undergo global DNA demethylation that starts shortly after PGC specification in early embryos. Although the functional importance of global DNA demethylation is not fully understood, demethylation of imprinted genes is crucial for erasure of methylation‐based imprinting, and demethylation of PGC‐specific genes is crucial for proper transcriptional regulation. PGCs also have unique patterns of histone modification, such as hypomethylation of H3K9 and hypermethylation of H3K27, and experimental evidence suggests that the unique epigenetic modifications of histones are important to the proper development of PGCs. Mol. Reprod. Dev. 81: 160–170, 2014.

REST and its downstream molecule Mek5 regulate survival of primordial germ cells.

In mouse embryos, some primordial germ cells (PGCs) are eliminated by apoptosis, but the molecular pathways that lead to PGC survival versus apoptosis have not been fully characterized. Here, we found that REST (repressor element 1-silencing transcription factor), a transcription factor that binds a conserved regulatory element, NRSE/RE1, played a role in PGC survival. REST expression was higher in PGCs than in surrounding somatic cells. Moreover, in mouse embryos with a PGC-specific conditional REST mutation, the PGC population experienced more apoptosis and was significantly smaller than that in control embryos; these findings indicated that REST functioned in a cell-autonomous fashion that was critical for PGC survival. Several anti-apoptotic genes were among the previously identified REST-target gene candidates; moreover, some of these genes were downregulated in the REST-deficient PGCs. Mek5, which encodes a component in the a MAP kinase cascade, was one of these downregulated REST-target gene candidates, and a Mek5 mutation, like the REST mutation, caused an increase in PGC apoptosis; these finding suggested that REST promoted PGC survival via regulation of the Mek5 expression. Importantly, there were a normal number of PGCs in the REST mutants at birth, and both the male and female REST-mutant adults were fertile; these final observations revealed that the PGC population was very robust and could recover from a genetically induced reduction in cell number.

Induction of Neurite Outgrowth in PC12 Cells Treated with Temperature-Controlled Repeated Thermal Stimulation

To promote the functional restoration of the nervous system following injury, it is necessary to provide optimal extracellular signals that can induce neuronal regenerative activities, particularly neurite formation. This study aimed to examine the regulation of neuritogenesis by temperature-controlled repeated thermal stimulation (TRTS) in rat PC12 pheochromocytoma cells, which can be induced by neurotrophic factors to differentiate into neuron-like cells with elongated neurites. A heating plate was used to apply thermal stimulation, and the correlation of culture medium temperature with varying surface temperature of the heating plate was monitored. Plated PC12 cells were exposed to TRTS at two different temperatures via heating plate (preset surface temperature of the heating plate, 39.5°C or 42°C) in growth or differentiating medium for up to 18 h per day. We then measured the extent of growth, neuritogenesis, or acetylcholine esterase (AChE) activity (a neuronal marker). To analyze the mechanisms underlying the effects of TRTS on these cells, we examined changes in intracellular signaling using the following: tropomyosin-related kinase A inhibitor GW441756; p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580; and MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126 with its inactive analog, U0124, as a control. While a TRTS of 39.5°C did not decrease the growth rate of cells in the cell growth assay, it did increase the number of neurite-bearing PC12 cells and AChE activity without the addition of other neuritogenesis inducers. Furthermore, U0126, and SB203580, but not U0124 and GW441756, considerably inhibited TRTS-induced neuritogenesis. These results suggest that TRTS can induce neuritogenesis and that participation of both the ERK1/2 and p38 MAPK signaling pathways is required for TRTS-dependent neuritogenesis in PC12 cells. Thus, TRTS may be an effective technique for regenerative neuromedicine.

Dnd1-mediated epigenetic control of teratoma formation in mouse

ABSTRACT Spontaneous testicular teratoma develops from primordial germ cells (PGCs) in embryos; however, the molecular mechanisms underlying teratoma formation are not fully understood. Mutation of the dead-end 1 (Dnd1) gene, which encodes an RNA-binding protein, drastically enhances teratoma formation in the 129/Sv mouse strain. To elucidate the mechanism of Dnd1 mutation-induced teratoma formation, we focused on histone H3 lysine 27 (H3K27) trimethylation (me3), and found that the levels of H3K27me3 and its responsible methyltransferase, enhancer of zeste homolog 2 (Ezh2), were decreased in the teratoma-forming cells of Dnd1 mutant embryos. We also showed that Dnd1 suppressed miR-26a-mediated inhibition of Ezh2 expression, and that Dnd1 deficiency resulted in decreased H3K27me3 of a cell-cycle regulator gene, Ccnd1. In addition, Ezh2 expression or Ccnd1 deficiency repressed the reprogramming of PGCs into pluripotent stem cells, which mimicked the conversion of embryonic germ cells into teratoma-forming cells. These results revealed an epigenetic molecular linkage between Dnd1 and the suppression of testicular teratoma formation. Summary: Teratoma development from primordial germ cells is repressed by downregulation of Ccnd1 via histone H3 lysine 27 methylation controlled by Dnd1-mediated maintenance of Ezh2.

Pax6-dependent regulation of the rat Fabp7 promoter activity

Fabp7 gene encodes a brain‐specific fatty acid‐binding protein that is widely used as a marker for neural stem cells. Here, we report that the activity of rat Fabp7 promoter was regulated directly by a transcription factor, Pax6. Deletion analyses identified an essential region (−837 to −64 from transcription start site) in the rat Fabp7 promoter. This region controls promoter activity in rat embryos and in the mouse cultured cell line MEB5. Over‐expressing wild‐type Pax6 or a dominant‐negative Pax6 mutant enhanced and suppressed, respectively, the promoter activity. Pax6 can bind the region directly, although the region contains no clear binding motif for Pax6. The rat Fabp7 promoter also contains conserved binding sites for Pbx/POU (−384 to −377) and CBF1 (−270 to −262). However, specific deletion of the sites showed no significant reduction in the promoter activity, although a gel mobility shift assay confirmed that CBF1 binds the conserved sequence. Taken together, these results suggest that the rat Fabp7 promoter is mainly regulated by Pax6. The Pax6‐dependent regulation of the rat Fabp7 expression might have an evolutionary aspect between rat and mouse; the former may need to efficiently use fatty acids to make the brain bigger than the latter.

DNA methylation of the Fthl17 5’-upstream region regulates differential Fthl17 expression in lung cancer cells and germline stem cells

The Ferritin heavy polypeptide-like 17 (Fthl17) gene is a member of the cancer/testis antigen gene family, and is preferentially expressed in cancer cells and in testis. Although DNA methylation has been linked to the regulation of human FTHL17 gene expression, detailed epigenetic regulation of its expression has not been investigated. To address this, we assessed the epigenetic regulation of murine Fthl17 gene expression in cancer cells and germ cells. Fthl17 was more highly expressed in testis, a murine lung cancer cell line, KLN205, and in germline stem cells (GSCs) than in normal lung tissues. Furthermore, the Fthl17 expression level in GSCs was significantly higher than in KLN205 cells. We performed bisulfite-sequencing and luciferase (luc) reporter assays to examine the role of DNA methylation of the Fthl17 promoter in the regulation of Fthl17 expression. In KLN205 cells, testis, and GSCs, the Fthl17 5’-upstream region was hypo-methylated compared with normal lung tissues. Luc reporter assays indicated that hypo-methylation of the -0.6 kb to 0 kb region upstream from the transcription start site (TSS) was involved in the up-regulation of Fthl17 expression in KLN205 cells and GSCs. Because the -0.6 kb to -0.3 kb or the -0.3 kb to 0 kb region were relatively more hypo-methylated in KLN205 cells and in GSCs, respectively, compared with other regions between -0.6 kb to 0 kb, those regions may contribute to Fthl17 up-regulation in each cell type. Following treatment with 5-Azacytidine, the -0.3 kb to 0 kb region became hypo-methylated, and Fthl17 expression was up-regulated in KLN205 cells to a level comparable to that in GSCs. Together, the results suggest that hypo-methylation of different but adjacent regions immediately upstream of the Fthl17 gene contribute to differential expression levels in lung cancer cells and GSCs, and hypo-methylation of the TSS-proximal region may be critical for high level expression.

PS4.66Fthl17 gene expression is regulated by DNA methylation state of its 5’-upstream region in cancer cells and germline stem cells

aldehyde metabolic enzymes that would normally convert retinol (Vitamin A) to retinoic acid (RA) and reduced RA levels during critical developmental times in early gastrulation underlie the later malformations associated with FAS. Methods: To biochemically mimic the alcohol-induced RA deficiency in vivo, we genetically engineered a mouse expressing Cyp26A1-eGFP from the endogenous Gsc promoter. The Gsc promoter dictates spatial-temporal expression to the Spemann Mangold Organizer at the start of gastrulation. Cyp26A1 degrades endogenous RA in these cells, mimicking the reduced RA levels induced by acute alcohol exposure and dysregulating the induction of neural crest cells. Results:Gsc:Cyp26A1micewere derived by germline transmission; F1 mice are born with a Mendelian ratio of 0.75:1 (het:wt, n=208). This loss of mutant embryo viability may reflect the prevalent miscarriages observed in human pregnancies with PAE. Gsc: Cyp26A1xRARE-LacZ E8.5 embryos show reduction in RA activity in the frontonasal prominence region (LacZ expression in early face and forebrain). Mutant embryos also demonstrate body-axis developmental variation, indicating early developmental perturbation of RA pathways in FASD relevant tissues (n=48). E18.5 embryos were next examined using scanning EM to demonstrate mutant embryos have sentinel FASD craniofacial malformations: larger philtrum-tophiltrum-lip length ratio, smaller bigonial line width, and smaller whisker pad area (n=66). Gsc:Cyp26A1mice also develop craniofacial malocclusions at significantly higher rates thanWT littermates (12.5% vs 0.04%; n=208 and 3711, respectively). Conclusion: Our data provide in vivo evidence that strongly supports RA-deficiency as a major molecular etiology of craniofacial malformations associated with FASD. The finding suggests Vitamin A supplementation may significantly reduce or prevent FASD outcomes in children with PAE.