Publications by authors named "Yasuhisa Matsui"

72 Publications

Abnormal early folliculogenesis due to impeded pyruvate metabolism in mouse oocytes.

Biol Reprod 2021 Apr 6. Epub 2021 Apr 6.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan.

Fetal ovarian germ cells show characteristic energy metabolism status, such as enhanced mitochondrial metabolism as well as glycolysis, but their roles in early folliculogenesis are unclear. We show here that inhibition of pyruvate uptake to mitochondria by UK5099 in organ cultures of fetal mouse ovaries resulted in repressed early folliculogenesis without affecting energy production, survival of oocytes, or meiosis. In addition, the abnormal folliculogenesis by UK5099 was partially rescued by α-ketoglutarate and succinate, intermediate metabolites in the TCA cycle, suggesting the importance of those metabolites. The expression of TGFβ-related genes Gdf9 and Bmp15 in ovarian germ cells, which are crucial for folliculogenesis, was downregulated by UK5099, and the addition of recombinant GDF9 partially rescued the abnormal folliculogenesis induced by UK5099. We also found that early folliculogenesis was similarly repressed, as in the culture, in the ovaries of a germ cell-specific knockout of Mpc2, which encodes a mitochondria pyruvate carrier that is targeted by UK5099. These results suggest that insufficient Gdf9 expression induced by abnormal pyruvate metabolism in oocytes results in early follicular dysgenesis, which is a possible cause of defective folliculogenesis in humans.
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http://dx.doi.org/10.1093/biolre/ioab064DOI Listing
April 2021

Paternal age affects offspring via an epigenetic mechanism involving REST/NRSF.

EMBO Rep 2021 Feb 5;22(2):e51524. Epub 2021 Jan 5.

Department of Developmental Neuroscience, Tohoku University Graduate School of Medicine, Sendai, Japan.

Advanced paternal age can have deleterious effects on various traits in the next generation. Here, we establish a paternal-aging model in mice to understand the molecular mechanisms of transgenerational epigenetics. Whole-genome target DNA methylome analyses of sperm from aged mice reveal more hypo-methylated genomic regions enriched in REST/NRSF binding motifs. Gene set enrichment analyses also reveal the upregulation of REST/NRSF target genes in the forebrain of embryos from aged fathers. Offspring derived from young mice administrated with a DNA de-methylation drug phenocopy the abnormal vocal communication of pups derived from aged fathers. In conclusion, hypo-methylation of sperm DNA can be a key molecular feature modulating neurodevelopmental programs in offspring by causing fluctuations in the expression of REST/NRSF target genes.
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http://dx.doi.org/10.15252/embr.202051524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7857438PMC
February 2021

Proteomic and metabolomic analyses uncover sex-specific regulatory pathways in mouse fetal germline differentiation†.

Biol Reprod 2020 10;103(4):717-735

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan.

Regulatory mechanisms of germline differentiation have generally been explained via the function of signaling pathways, transcription factors, and epigenetic regulation; however, little is known regarding proteomic and metabolomic regulation and their contribution to germ cell development. Here, we conducted integrated proteomic and metabolomic analyses of fetal germ cells in mice on embryonic day (E)13.5 and E18.5 and demonstrate sex- and developmental stage-dependent changes in these processes. In male germ cells, RNA processing, translation, oxidative phosphorylation, and nucleotide synthesis are dominant in E13.5 and then decline until E18.5, which corresponds to the prolonged cell division and more enhanced hyper-transcription/translation in male primordial germ cells and their subsequent repression. Tricarboxylic acid cycle and one-carbon pathway are consistently upregulated in fetal male germ cells, suggesting their involvement in epigenetic changes preceding in males. Increased protein stability and oxidative phosphorylation during female germ cell differentiation suggests an upregulation of aerobic energy metabolism, which likely contributes to the proteostasis required for oocyte maturation in subsequent stages. The features elucidated in this study shed light on the unrevealed mechanisms of germ cell development.
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http://dx.doi.org/10.1093/biolre/ioaa115DOI Listing
October 2020

Author Correction: Transcriptomic analysis reveals differences in the regulation of amino acid metabolism in asexual and sexual planarians.

Sci Rep 2019 Aug 8;9(1):11799. Epub 2019 Aug 8.

Department of Biology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori, 036-8561, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-019-48084-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687728PMC
August 2019

Corrigendum to "Derivation of pluripotent stem cells from nascent undifferentiated teratoma" [Dev. Biol. 446 (2019) 43-55].

Dev Biol 2019 Sep 3;453(1):110. Epub 2019 Jul 3.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan; Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan; The Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Chuo-ku, Tokyo, Japan; Center for Regulatory Epigenome and Diseases, Tohoku University School of Medicine, Sendai, Miyagi, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.ydbio.2019.06.021DOI Listing
September 2019

Comprehensive Analysis of Mouse Cancer/Testis Antigen Functions in Cancer Cells and Roles of TEKT5 in Cancer Cells and Testicular Germ Cells.

Mol Cell Biol 2019 09 12;39(17). Epub 2019 Aug 12.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi, Japan

The cancer/testis antigen (CTA) genes were identified as human genes preferentially expressed in cancer cells and testis, but the contribution of CTAs to cancer and male germ cell development is unclear. In this study, we comprehensively examined mouse CTA functions and found that the majority of CTAs are involved in growth and/or survival of cancer cells. We focused on one mouse CTA gene, , for its detailed functional analysis. knockdown (KD) in ovarian cancer cells caused G arrest and apoptosis, and p27 was concomitantly upregulated. KD also resulted in decreased levels of acetylated α-tubulin and subsequent fragmentation of β-III-tubulin, upregulation of HDAC6 that deacetylates α-tubulin, and nuclear accumulation of SMAD3 that induces p27 expression. Because depolymerization of tubulin is known to cause translocation of SMAD3 to the nucleus, these results together suggested that TEKT5 negatively regulates expression and consequently maintains cell cycle via stabilization of tubulin. We also found that the number of spermatids was significantly decreased and acetylated α-tubulin levels were decreased by KD of in testis. Because acetylated α-tubulin is required for sperm morphogenesis, these results suggest that TEKT5 is necessary for spermiogenesis via maintenance of acetylated α-tubulin levels.
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http://dx.doi.org/10.1128/MCB.00154-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692122PMC
September 2019

Shortened G1 phase of cell cycle and decreased histone H3K27 methylation are associated with AKT-induced enhancement of primordial germ cell reprogramming.

Dev Growth Differ 2019 08 14;61(6):357-364. Epub 2019 Jun 14.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Japan.

Primordial germ cells (PGCs) are reprogrammed into pluripotent embryonic germ cells (EGCs) under specific culture conditions, but the detailed mechanisms of PGC reprogramming have not yet been fully clarified. Previous studies have demonstrated that AKT, an important intracellular signaling molecule, promotes reprogramming of PGCs into EGCs. Because AKT likely inhibits p53 functions to enhance PGC reprogramming, and p53 negatively regulates cell cycle progression, we analyzed cell cycle changes in PGCs following AKT activation and found that the ratio of PGCs in the G1/G0 phase was decreased while that of PGCs in the G2/M phase was increased after AKT activation. We also showed that the expression of the CDK inhibitor p27kip1, which prevents the G1‐S transition and is transcriptionally activated by p53, was significantly downregulated by AKT activation. The results suggested that the characteristic cell cycle changes of PGCs by AKT activation are, at least in part, due to decreased expression of p27 . We also investigated changes in histone H3K27 tri-methylation (H3K27me3) by AKT activation in PGCs, because we previously found that decreased H3K27me3 was involved in PGC reprogramming via upregulation of cyclin D1. We observed that AKT activation in PGCs resulted in H3K27 hypomethylation. In addition, DZNeP, an inhibitor of the H3K27 trimethyl transferase Ezh2, stimulated EGC formation. These results together suggested that AKT activation promotes G1-S transition and downregulates H3K27me3 to enhance PGC reprogramming.
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http://dx.doi.org/10.1111/dgd.12621DOI Listing
August 2019

Transcriptomic analysis reveals differences in the regulation of amino acid metabolism in asexual and sexual planarians.

Sci Rep 2019 04 16;9(1):6132. Epub 2019 Apr 16.

Department of Biology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori, 036-8561, Japan.

Many flatworms can alternate between asexual and sexual reproduction. This is a powerful reproductive strategy enabling them to benefit from the features of the two reproductive modes, namely, rapid multiplication and genetic shuffling. The two reproductive modes are enabled by the presence of pluripotent adult stem cells (neoblasts), by generating any type of tissue in the asexual mode, and producing and maintaining germ cells in the sexual mode. In the current study, RNA sequencing (RNA-seq) was used to compare the transcriptomes of two phenotypes of the planarian Dugesia ryukyuensis: an asexual OH strain and an experimentally sexualized OH strain. Pathway enrichment analysis revealed striking differences in amino acid metabolism in the two worm types. Further, the analysis identified serotonin as a new bioactive substance that induced the planarian ovary de novo in a postembryonic manner. These findings suggest that different metabolic states and physiological conditions evoked by sex-inducing substances likely modulate stem cell behavior, depending on their different function in the asexual and sexual reproductive modes. The combination of RNA-seq and a feeding assay in D. ryukyuensis is a powerful tool for studying the alternation of reproductive modes, disentangling the relationship between gene expression and chemical signaling molecules.
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http://dx.doi.org/10.1038/s41598-019-42025-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6467871PMC
April 2019

Identification of the X-linked germ cell specific miRNAs (XmiRs) and their functions.

PLoS One 2019 1;14(2):e0211739. Epub 2019 Feb 1.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi, Japan.

MicroRNAs (miRNAs) play a critical role in multiple aspects of biology. Dicer, an RNase III endonuclease, is essential for the biogenesis of miRNAs, and the germ cell-specific Dicer1 knockout mouse shows severe defects in gametogenesis. How miRNAs regulate germ cell development is still not fully understood. In this study, we identified germ cell-specific miRNAs (miR-741-3p, miR-871-3p, miR-880-3p) by analyzing published RNA-seq data of mouse. These miRNA genes are contiguously located on the X chromosome near other miRNA genes. We named them X chromosome-linked miRNAs (XmiRs). To elucidate the functions of XmiRs, we generated knockout mice of these miRNA genes using the CRISPR/Cas9-mediated genome editing system. Although no histological abnormalities were observed in testes of F0 mice in which each miRNA gene was disrupted, a deletion covering miR-871 and miR-880 or covering all XmiRs (ΔXmiRs) resulted in arrested spermatogenesis in meiosis in a few seminiferous tubules, indicating their redundant functions in spermatogenesis. Among candidate targets of XmiRs, we found increased expression of a gene encoding a WNT receptor, FZD4, in ΔXmiRs testis compared with that in wildtype testis. miR-871-3p and miR-880-3p repressed the expression of Fzd4 via the 3'-untranslated region of its mRNA. In addition, downstream genes of the WNT/β-catenin pathway were upregulated in ΔXmiRs testis. We also found that miR-871, miR-880, and Fzd4 were expressed in spermatogonia, spermatocytes and spermatids, and overexpression of miR-871 and miR-880 in germ stem cells in culture repressed their increase in number and Fzd4 expression. Previous studies indicated that the WNT/β-catenin pathway enhances and represses proliferation and differentiation of spermatogonia, respectively, and our results consistently showed that stable β-catenin enhanced GSC number. In addition, stable β-catenin partially rescued reduced GSC number by overexpression of miR-871 and miR-880. The results together suggest that miR-871 and miR-880 cooperatively regulate the WNT/β-catenin pathway during testicular germ cell development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0211739PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6358104PMC
November 2019

Sex-specific histone modifications in mouse fetal and neonatal germ cells.

Epigenomics 2019 04 22;11(5):543-561. Epub 2019 Jan 22.

Department of Bioscience, Tokyo University of Agriculture, Setagaya, Tokyo, Japan.

Aims: Epigenetic signatures of germline cells are dynamically reprogrammed to induce appropriate differentiation, development and sex specification. We investigated sex-specific epigenetic changes in mouse fetal germ cells (FGCs) and neonatal germ cells.

Materials & Methods: Six histone marks in mouse E13.5 FGCs and P1 neonatal germ cells were analyzed by chromatin immunoprecipitation and sequencing. These datasets were compared with transposase-accessible chromatin sites, DNA methylation and transcriptome.

Results: Different patterns of each histone mark were detected in female and male FGCs, and H3K4me3/H3K27me3 bivalent marks were enriched in different chromosomal regions of female and male FGCs.

Conclusion: Our results suggest that histone modifications may affect FGC gene expression following DNA methylation erasure, contributing to the differentiation into female and male germ cells.
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http://dx.doi.org/10.2217/epi-2018-0193DOI Listing
April 2019

Derivation of pluripotent stem cells from nascent undifferentiated teratoma.

Dev Biol 2019 02 5;446(1):43-55. Epub 2018 Dec 5.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan; Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi, Japan; The Japan Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST), Chuo-ku, Tokyo, Japan; Center for Regulatory Epigenome and Diseases, Tohoku University School of Medicine, Sendai, Miyagi, Japan. Electronic address:

Teratomas are tumors consisting of components of the three germ layers that differentiate from pluripotent stem cells derived from germ cells. In the normal mouse testis, teratomas rarely form, but a deficiency in Dead-end1 (Dnd1) in mice with a 129/Sv genetic background greatly enhances teratoma formation. Thus, DND1 is crucial for suppression of teratoma development from germ cells. In the Dnd1 mutant testis, nascent teratoma cells emerge at E15.5. To understand the nature of early teratoma cells, we established cell lines in the presence of serum and leukemia inhibitory factor (LIF) from teratoma-forming cells in neonatal Dnd1 mutant testis. These cells, which we designated cultured Dnd1 mutant germ cells (CDGCs), were morphologically similar to embryonic stem cells (ESCs) and could be maintained in the naïve pluripotent condition. In addition, the cells expressed pluripotency genes including Oct4, Nanog, and Sox2; differentiated into cells of the three germ layers in culture; and contributed to chimeric mice. The expression levels of pluripotency genes and global transcriptomes in CDGCs as well as these cells' adaption to culture conditions for primed pluripotency suggested that their pluripotent status is intermediate between naïve and primed pluripotency. In addition, the teratoma-forming cells in the neonatal testis from which CDGCs were derived also showed gene expression profiles intermediate between naïve and primed pluripotency. The results suggested that germ cells in embryonic testes of Dnd1 mutants acquire the intermediate pluripotent status during the course of conversion into teratoma cells.
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http://dx.doi.org/10.1016/j.ydbio.2018.11.020DOI Listing
February 2019

SETDB1 is essential for mouse primordial germ cell fate determination by ensuring BMP signaling.

Development 2018 12 3;145(23). Epub 2018 Dec 3.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi 980-8575, Japan

In mouse embryos, primordial germ cells (PGCs) are fate-determined from epiblast cells. Signaling pathways involved in PGC formation have been identified, but their epigenetic mechanisms remain poorly understood. Here, we show that the histone methyltransferase SETDB1 is an epigenetic regulator of PGC fate determination. -deficient embryos exhibit drastic reduction of nascent PGCs. , and are de-repressed whereas mesoderm development-related genes, including BMP4 signaling-related genes, are downregulated by knockdown during PGC-like cell (PGCLC) induction. In addition, binding of SETDB1 is observed at the flanking regions of , and in cell aggregates containing PGCLCs, and trimethylation of lysine 9 of histone H3 is reduced by knockdown at those regions. Furthermore, DPPA2, OTX2 and UTF1 binding is increased in genes encoding BMP4 signaling-related proteins, including SMAD1. Finally, overexpression of , and in cell aggregates containing PGCLCs results in the repression of BMP4 signaling-related genes and PGC determinant genes. We propose that the localization of SETDB1 to , and , and subsequent repression of their expression, are crucial for PGC determination by ensuring BMP4 signaling.
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http://dx.doi.org/10.1242/dev.164160DOI Listing
December 2018

DNMTs and SETDB1 function as co-repressors in MAX-mediated repression of germ cell-related genes in mouse embryonic stem cells.

PLoS One 2018 7;13(11):e0205969. Epub 2018 Nov 7.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan.

In embryonic stem cells (ESCs), the expression of development-related genes, including germ cell-related genes, is globally repressed. The transcription factor MAX represses germ cell-related gene expression in ESCs via PCGF6-polycomb repressive complex 1 (PRC1), which consists of several epigenetic factors. However, we predicted that MAX represses germ cell-related gene expression through several additional mechanisms because PCGF6-PRC1 regulates the expression of only a subset of genes repressed by MAX. Here, we report that MAX associated with DNA methyltransferases (DNMTs) and the histone methyltransferase SETDB1 cooperatively control germ cell-related gene expression in ESCs. Both DNA methylation and histone H3 lysine 9 tri-methylation of the promoter regions of several germ cell-related genes were not affected by knockout of the PRC1 components, indicating that the MAX-DNMT and MAX-SETDB1 pathways are independent of the PCGF6-PRC1 pathway. Our findings provide insights into our understanding of MAX-based repressive mechanisms of germ cell-related genes in ESCs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205969PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221296PMC
April 2019

Identification of KLF9 and BCL3 as transcription factors that enhance reprogramming of primordial germ cells.

PLoS One 2018 4;13(10):e0205004. Epub 2018 Oct 4.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan.

Primordial germ cells (PGCs) are precursors of eggs and sperm. Although PGCs are unipotent cells in vivo, they are reprogrammed into pluripotent stem cells (PSCs), also known as embryonic germ cells (EGCs), in the presence of leukemia inhibitory factor and basic fibroblast growth factor (bFGF) in vitro. However, the molecular mechanisms responsible for their reprogramming are not fully understood. Here we show identification of transcription factors that mediate PGC reprogramming. We selected genes encoding transcription factors or epigenetic regulatory factors whose expression was significantly different between PGCs and PSCs with in silico analysis and RT-qPCR. Among the candidate genes, over-expression (OE) of Bcl3 or Klf9 significantly enhanced PGC reprogramming. Notably, EGC formation was stimulated by Klf9-OE even without bFGF. G-protein-coupled receptor signaling-related pathways, which are involved in PGC reprogramming, were enriched among genes down-regulated by Klf9-OE, and forskolin which activate adenylate cyclase, rescued repressed EGC formation by knock-down of Klf9, suggesting a molecular linkage between KLF9 and such signaling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0205004PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6171932PMC
March 2019

Repression of Somatic Genes by Selective Recruitment of HDAC3 by BLIMP1 Is Essential for Mouse Primordial Germ Cell Fate Determination.

Cell Rep 2018 09;24(10):2682-2693.e6

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi 980-8575, Japan; Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8577, Japan; Center for Regulatory Epigenome and Diseases, Tohoku University School of Medicine, Sendai, Miyagi 980-8575, Japan. Electronic address:

Primordial germ cells (PGCs) are fate determined from pluripotent epiblasts. Signaling pathways and transcriptional regulators involved in PGC formation have been identified, but detailed molecular mechanisms of PGC fate determination remains poorly understood. Using RNAi screening, we identified histone deacetylase 3 (HDAC3) as a regulator of PGC formation. Hdac3 deficiency resulted in decreased nascent PGCs in vitro and in vivo, and somatic developmental genes were de-repressed by Hdac3 knockdown during PGC induction. We also demonstrated BLIMP1-dependent enrichment of HDAC3 and deacetylation of H3 and H4 histones in the somatic developmental genes in epiblast-like cells. In addition, the HDAC3/BLIMP1-targeted somatic gene products were enriched in PGC determinant genes; overexpression of these gene products in PGC-like cells in culture resulted in repression of PGC determinant genes. We propose that selective recruitment of HDAC3 to somatic genes by BLIMP1 and subsequent repression of these somatic genes are crucial for PGC fate determination.
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http://dx.doi.org/10.1016/j.celrep.2018.07.108DOI Listing
September 2018

Metabolomic and Proteomic Analyses of Mouse Primordial Germ Cells.

Methods Mol Biol 2019 ;2045:259-269

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan.

Primordial germ cells (PGCs), the precursors of gametes, are the only cells capable of acquiring totipotency upon fertilization, but the molecular mechanisms regulating germ cell characteristics have not been fully elucidated. Although intracellular metabolic status and regulation are responsible for the control of cell function and differentiation, little is known about the metabolic features of PGCs. Here, we describe use of an integrated metabolomic, proteomic, and energy metabolic analysis method to comprehensively elucidate the metabolic characteristics of PGCs using mass spectrometry.
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http://dx.doi.org/10.1007/7651_2018_164DOI Listing
June 2020

Dnd1-mediated epigenetic control of teratoma formation in mouse.

Biol Open 2018 Jan 29;7(1). Epub 2018 Jan 29.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan

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 () gene, which encodes an RNA-binding protein, drastically enhances teratoma formation in the 129/Sv mouse strain. To elucidate the mechanism of 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 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, 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.
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http://dx.doi.org/10.1242/bio.032318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829515PMC
January 2018

Distinct requirements for energy metabolism in mouse primordial germ cells and their reprogramming to embryonic germ cells.

Proc Natl Acad Sci U S A 2017 08 17;114(31):8289-8294. Epub 2017 Jul 17.

Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University, Sendai, Miyagi 980-8575, Japan;

Primordial germ cells (PGCs), undifferentiated embryonic germ cells, are the only cells that have the ability to become gametes and to reacquire totipotency upon fertilization. It is generally understood that the development of PGCs proceeds through the expression of germ cell-specific transcription factors and characteristic epigenomic changes. However, little is known about the properties of PGCs at the metabolite and protein levels, which are directly responsible for the control of cell function. Here, we report the distinct energy metabolism of PGCs compared with that of embryonic stem cells. Specifically, we observed remarkably enhanced oxidative phosphorylation (OXPHOS) and decreased glycolysis in embryonic day 13.5 (E13.5) PGCs, a pattern that was gradually established during PGC differentiation. We also demonstrate that glycolysis and OXPHOS are important for the control of PGC reprogramming and specification of pluripotent stem cells (PSCs) into PGCs in culture. Our findings about the unique metabolic property of PGCs provide insights into our understanding of the importance of distinct facets of energy metabolism for switching PGC and PSC status.
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http://dx.doi.org/10.1073/pnas.1620915114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5547595PMC
August 2017

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

PLoS One 2017 16;12(2):e0172219. Epub 2017 Feb 16.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, Sendai, Miyagi, Japan.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0172219PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5312872PMC
August 2017

Targeted disruption of the mouse protein phosphatase ppm1l gene leads to structural abnormalities in the brain.

FEBS Lett 2016 Oct 29;590(20):3606-3615. Epub 2016 Sep 29.

Department of Biochemistry, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.

PPM1L, a member of the metal-dependent protein phosphatase (PPM) family, is involved in regulating the stress-activated protein kinase pathway and ceramide trafficking. However, the physiological function of PPM1L in the brain is unclear. In this study, we generated and analyzed ppm1l-deficient mice in order to investigate PPM1L functions in the brain. Our results indicate that ppm1l is highly expressed in the central nervous system during mouse development and that ppm1l mice display impaired motor performance and morphological abnormalities in the forebrain. Electron microscopic and immunohistochemical analyses suggest that these abnormalities are due to impaired axonal tract formation. Our novel findings suggest an important role for PPM1L in brain development.
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http://dx.doi.org/10.1002/1873-3468.12429DOI Listing
October 2016

Selective de-repression of germ cell-specific genes in mouse embryonic fibroblasts in a permissive epigenetic environment.

Sci Rep 2016 09 9;6:32932. Epub 2016 Sep 9.

Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer (IDAC), Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8575, Japan.

Epigenetic modifications play crucial roles on establishment of tissue-specific transcription profiles and cellular characteristics. Direct conversions of fibroblasts into differentiated tissue cells by over-expression of critical transcription factors have been reported, but the epigenetic mechanisms underlying these conversions are still not fully understood. In addition, conversion of somatic cells into germ cells has not yet been achieved. To understand epigenetic mechanisms that underlie germ cell characteristics, we attempted to use defined epigenetic factors to directly convert mouse embryonic fibroblasts (MEFs) into germ cells. Here, we successfully induced germ cell-specific genes by inhibiting repressive epigenetic modifications via RNAi or small-molecule compounds. Under these conditions, some tissue-specific genes and stimulus-inducible genes were also induced. Meanwhile, the treatments did not result in genome-wide transcriptional activation. These results suggested that a permissive epigenetic environment resulted in selective de-repression of stimulus- and differentiation-inducible genes including germ cell-specific genes in MEFs.
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http://dx.doi.org/10.1038/srep32932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016969PMC
September 2016

Transgenic expression of Telomerase reverse transcriptase (Tert) improves cell proliferation of primary cells and enhances reprogramming efficiency into the induced pluripotent stem cell.

Biosci Biotechnol Biochem 2016 Oct 13;80(10):1925-33. Epub 2016 Jun 13.

a Graduate School of Agricultural Science , Tohoku University , Sendai , Japan.

The enzymatic activity of telomerase is important for the extension of the telomere repeat sequence and overcoming cellular senescence. We generated a conditional transgenic mouse line, carrying the telomerase reverse transcriptase (Tert) expression cassette, controlled by the Cre-loxP-mediated recombination. In our study, Cre recombinase expression efficiently activated Tert expression, resulting in its increased enzymatic activity, which extended the period of cellular proliferation until the keratinocytes entered senescence. This suggests that transgenic Tert expression is effective in enhancing primary cell proliferation. Notably, Tert expression increased colony formation of induced pluripotent stem (iPS) cells after the introduction of four reprogramming factors, Oct-4, klf4, SOX-2, and c-Myc into the transgenic fibroblasts. To the best of our knowledge, this is the first study to show that the transgenic Tert expression enhances reprogramming efficiency of iPS cells, which indicates a critical role for Tert in the reprogramming process.
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http://dx.doi.org/10.1080/09168451.2016.1191330DOI Listing
October 2016

Loss of MAX results in meiotic entry in mouse embryonic and germline stem cells.

Nat Commun 2016 Mar 30;7:11056. Epub 2016 Mar 30.

Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical University, Yamane Hidaka, Saitama 350-1241, Japan.

Meiosis is a unique process that allows the generation of reproductive cells. It remains largely unknown how meiosis is initiated in germ cells and why non-germline cells do not undergo meiosis. We previously demonstrated that knockdown of Max expression, a gene encoding a partner of MYC family proteins, strongly activates expression of germ cell-related genes in ESCs. Here we find that complete ablation of Max expression in ESCs results in profound cytological changes reminiscent of cells undergoing meiotic cell division. Furthermore, our analyses uncovers that Max expression is transiently attenuated in germ cells undergoing meiosis in vivo and its forced reduction induces meiosis-like cytological changes in cultured germline stem cells. Mechanistically, Max depletion alterations are, in part, due to impairment of the function of an atypical PRC1 complex (PRC1.6), in which MAX is one of the components. Our data highlight MAX as a new regulator of meiotic onset.
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http://dx.doi.org/10.1038/ncomms11056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4820925PMC
March 2016

The protein phosphatase 6 catalytic subunit (Ppp6c) is indispensable for proper post-implantation embryogenesis.

Mech Dev 2016 Feb 8;139:1-9. Epub 2016 Feb 8.

Division of Cancer Chemotherapy, Miyagi Cancer Center Research Institute, Japan. Electronic address:

Ppp6c, which encodes the catalytic subunit of phosphoprotein phosphatase 6 (PP6), is conserved among eukaryotes from yeast to humans. In mammalian cells, PP6 targets IκBε for degradation, activates DNA-dependent protein kinase to trigger DNA repair, and is reportedly required for normal mitosis. Recently, Ppp6c mutations were identified as candidate drivers of melanoma and skin cancer. Nonetheless, little is known about the physiological role of Ppp6c. To investigate this function in vivo, we established mice lacking the Ppp6c phosphatase domain by crossing heterozygous mutants. No viable homozygous pups were born, indicative of a lethal mutation. Ppp6c homozygous mutant embryos were identified among blastocysts, which exhibited a normal appearance, but embryos degenerated by E7.5 and showed clear developmental defects at E8.5, suggesting that mutant embryos die after implantation. Accordingly, homozygous blastocysts showed significant growth failure of the inner cell mass (ICM) in in vitro blastocyst culture, and primary Ppp6c exon4-deficient MEFs showed greatly reduced proliferation. These results establish for the first time that the Ppp6c phosphatase domain is indispensable for mouse embryogenesis after implantation.
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http://dx.doi.org/10.1016/j.mod.2016.02.001DOI Listing
February 2016

Sex Specification and Heterogeneity of Primordial Germ Cells in Mice.

PLoS One 2015 23;10(12):e0144836. Epub 2015 Dec 23.

Department of Bioscience, Tokyo University of Agriculture, Setagaya, Tokyo, Japan.

In mice, primordial germ cells migrate into the genital ridges by embryonic day 13.5 (E13.5), where they are then subjected to a sex-specific fate with female and male primordial germ cells undergoing mitotic arrest and meiosis, respectively. However, the sex-specific basis of primordial germ cell differentiation is poorly understood. The aim of this study was to investigate the sex-specific features of mouse primordial germ cells. We performed RNA-sequencing (seq) of E13.5 female and male mouse primordial germ cells using next-generation sequencing. We identified 651 and 428 differentially expressed transcripts (>2-fold, P < 0.05) in female and male primordial germ cells, respectively. Of these, many transcription factors were identified. Gene ontology and network analysis revealed differing functions of the identified female- and male-specific genes that were associated with primordial germ cell acquisition of sex-specific properties required for differentiation into germ cells. Furthermore, DNA methylation and ChIP-seq analysis of histone modifications showed that hypomethylated gene promoter regions were bound with H3K4me3 and H3K27me3. Our global transcriptome data showed that in mice, primordial germ cells are decisively assigned to a sex-specific differentiation program by E13.5, which is necessary for the development of vital germ cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0144836PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4689518PMC
June 2016

Mice doubly-deficient in the Arf GAPs SMAP1 and SMAP2 exhibit embryonic lethality.

FEBS Lett 2015 Sep 18;589(19 Pt B):2754-62. Epub 2015 Aug 18.

Department of Biological Science, Graduate School of Humanities and Sciences, Nara Women's University, Nara, Japan. Electronic address:

In mammals, the small Arf GTPase-activating protein (SMAP) subfamily of Arf GTPase-activating proteins consists of closely related members, SMAP1 and SMAP2. These factors reportedly exert distinct functions in membrane trafficking, as manifested by different phenotypes seen in single knockout mice. The present study investigated whether SMAP proteins interact genetically. We report for the first time that simultaneous loss of SMAP1 and SMAP2 promotes apoptosis in the distal region of E7.5 mouse embryos, likely resulting in embryonic lethality. Thus, at least one SMAP gene, either SMAP1 or SMAP2, is required for proper embryogenesis.
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http://dx.doi.org/10.1016/j.febslet.2015.07.050DOI Listing
September 2015

Novel sex-dependent differentially methylated regions are demethylated in adult male mouse livers.

Biochem Biophys Res Commun 2015 Jul 8;462(4):332-8. Epub 2015 May 8.

Department of Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, Tokyo, Japan. Electronic address:

In mammalian livers, sexual dimorphisms are observed in tissue-specific functions and diseases such as hepatocellular carcinoma. We identified sex-dependent differentially methylated regions (S-DMRs) which had been previously been characterized as growth hormone- STAT5 dependent. In this study, we performed genome-wide screening and identified ten additional hypomethylated S-DMR gene regions in male livers. Of these S-DMRs, Uggt2 and Sarnp were hypomethylated in both male and female livers compared to brain and embryonic stem (ES) cells. Similarly, Adam2, Uggt2, and Scp2 were hypomethylated in female embryonic germ (EG) cells and not in male EG cells, indicating that these S-DMRs are liver-specific male hypo-S-DMRs. Interestingly, the five S-DMRs were free from STAT5 chromatin immunoprecipitation (ChIP) signals, suggesting that S-DMRs are independent of the growth hormone-STAT5-pathway. Instead, the DNA methylation statuses of the S-DMRs of Adam2, Snx29, Uggt2, Sarnp, and Rnpc3 genes were under the control of testosterone. Importantly, the hypomethylated S-DMRs of the Adam2 and Snx29 regions showed chromatin decondensation. Epigenetic factors could be responsible for the sexual dimorphisms in DNA methylation status and chromatin structure, as the expression of Dnmt1, Dnmt3b, and Tet2 genes was lower in male mice compared to female mice and TET2 expression recovered following orchidectomy by testosterone treatment. In conclusion, we identified novel male-specific hypomethylated S-DMRs that contribute to chromatin decondensation in the liver. S-DMRs were tissue-specific and the hypomethylation is testosterone-dependent.
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http://dx.doi.org/10.1016/j.bbrc.2015.04.137DOI Listing
July 2015

The majority of early primordial germ cells acquire pluripotency by AKT activation.

Development 2014 Dec 30;141(23):4457-67. Epub 2014 Oct 30.

CREST, JST, Kawaguchi, Saitama 332-0012, Japan Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan.

Primordial germ cells (PGCs) are undifferentiated germ cells in embryos, the fate of which is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotent embryonic germ cells (EGCs) in culture in the presence of particular extracellular factors, such as combinations of Steel factor (KITL), LIF and bFGF (FGF2). Early PGCs form EGCs more readily than do later PGCs, and PGCs lose the ability to form EGCs by embryonic day (E) 15.5. Here, we examined the effects of activation of the serine/threonine kinase AKT in PGCs during EGC formation; notably, AKT activation, in combination with LIF and bFGF, enhanced EGC formation and caused ∼60% of E10.5 PGCs to become EGCs. The results indicate that the majority of PGCs at E10.5 could acquire pluripotency with an activated AKT signaling pathway. Importantly, AKT activation did not fully substitute for bFGF and LIF, and AKT activation without both LIF and bFGF did not result in EGC formation. These findings indicate that AKT signal enhances and/or collaborates with signaling pathways of bFGF and of LIF in PGCs for the acquisition of pluripotency.
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http://dx.doi.org/10.1242/dev.113779DOI Listing
December 2014

The ADP-ribosylation factor 1 gene is indispensable for mouse embryonic development after implantation.

Biochem Biophys Res Commun 2014 Oct 12;453(4):748-53. Epub 2014 Oct 12.

Department of Biological Science, Graduate School of Humanities and Sciences, Nara Women's University, Nara, Japan. Electronic address:

ADP-ribosylation factor (Arf) 1 is thought to affect the morphologies of organelles, such as the Golgi apparatus, and regulate protein trafficking pathways. Mice have six Arf isoforms. In knockdown experiments with HeLa cells, no single Arf isoform among Arf1-5 is required for organelle morphologies or any membrane trafficking step. This suggests that the cooperation of two or more Arfs is a general feature. Although many cell biological and biochemical analyses have proven the importance of Arf1, the physiological roles of Arf1 in mice remain unknown. To investigate the activity of Arf1 in vivo, we established Arf1-deficient mice. Arf(-/-) blastocysts were identified at the expected Mendelian ratio. The appearance of these blastocysts was indistinguishable from that of wild-type and Arf(+/-) blastocysts, and they grew normally in an in vitro culture system. However, Arf(-/-) embryos were degenerated at E5.5, and none survived to E12.5, suggesting that they died soon after implantation. These data establish for the first time that the Arf1 gene is indispensable for mouse embryonic development after implantation.
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http://dx.doi.org/10.1016/j.bbrc.2014.10.014DOI Listing
October 2014