Cell Resource Center for Biomedical Research
|Director, Professor||Yasuhisa MATSUI|
|Assistant Professor||Kentarou MOCHIZUKI|
|Assistant Professor(Additional)||Daisuke SAITO|
|Assistant Professor||Yohe HAYASHI|
|Assistant Professor||Kei OHTUSKA|
|Assistant Professor||Hiromitsu OHTA|
|Technical Staff||Ikue AIHARA|
|Technical Staff||Yumi MATSUOKA|
|Technical Assistant||Yuko TOKITAKE|
|Technical Assistant||Fujimi KOIZUMI|
1. Cell Bank
The purpose of the Cell Resource Center for Biomedical Research is the collection, establishment, quality control, distribution of useful cell lines, and construction of database for researchers. The cell lines include transplantable animal cell lines, such as Yoshida sarcoma and rat ascites hepatoma (AH series) cell lines as well as human, murine cell lines and hybridoma cells. The Cell Line Catalog is available on the web site (http://www.idac.tohoku.ac.jp/dep/ccr/).
2. Research Interest
Our goal is to elucidate the molecular mechanisms of mouse primordial germ cell (PGC) formation and their subsequent development. These studies are meaningful not only for basic research interests for germ cell development, but also for furthering our understanding the possible applications of germ cells and pluripotent stem cells in practical science.
3. Recent topics
1) Maeda et al., Max is a repressor of germ-cell-related gene expression in mouse embryonic stem cells. Nature Communications 4, 1754 (2013).
It is of interest that embryonic stem cells (ESCs) and primordial germ cells (PGCs) share similar transcriptome profiles including the expression of the pluripotency markers such as Oct3/4 and Nanog, although ESCs maintain pluripotency, whereas PGCs can only contribute to gametes. Thus, we predict that there may be a mechanism that separates cellular status of ESCs from that of PGCs. To identify genes involved in this putative mechanism, we used an ESC line with Vasa reporter gene for an RNAi screen of transcription factor genes expressed in ESCs; we identified 5 genes that resulted in the expression of Vasa when silenced. Of these, Max was the most striking; it encodes a partner for myc-family proteins. Transcriptome analysis revealed that Max knockdown (KD) in ESCs resulted in selective, global derepression of germ-cell-specific genes. Max interacted with histone H3K9 methyltransferases and associated with the promoter regions of germ-cell-specific genes in ESCs. In addition, Max-KD resulted in a decrease in histone H3K9 dimethylation at those promoter regions. We propose that Max is an epigenetic global repressor of germ-cell-related genes in ESCs.
2) Mochizuki, K. et al. Implication of DNA demethylation and bivalent histone modification for selective gene regulation in mouse primordial germ cells. PLoS ONE 7, e46036 (2012).
We focused on epigenetic mechanisms that regulate PGC-specific gene expression, especially DNA demethylation of regulatory regions. We first found that regulatory regions of three representative PGC-specific genes, mil-1, Blimp1, and Stella, underwent progressive DNA demethylation that was coincident with their upregulation during PGC development. We next examined involvement of a maintenance DNA methyltransferase, Dnmt1 in the expression of the PGC-specific genes by using siRNA to inhibit its expression in embryonic stem (ES) cells, and found that the flanking regions of all three genes became hypomethylated and that their expression was upregulated. Furthermore, in epiblasts of Dnmt1-knockout embryos, flanking regions of the three genes were hypomethylated, and those genes were expressed prematurely. Together, these results demonstrated that DNA demethylation of the regulatory regions of the PGC-specific genes resulted in upregulation of their expression (Figure). Interestingly, however, we also found that repression of two representative somatic genes, Hoxa1 and Hoxb1, in PGCs was not dependent on DNA methylation; their flanking regions were consistently hypomethylated during PGC development, but induction of their expression was not observed in PGCs. Moreover, the promoter regions of those genes exhibited methylation of both lysine 27 and lysine 4 of histone H3 in PGCs, so that this kind of epigenetic modification may be involved in repression of their expression in PGCs (Figure).
3) Okamura, D. et al. Cell-cycle gene-specific control of transcription has a critical role in proliferation of primordial germ cells. Genes & Development 26, 2477-2482 (2012).
We identified Larp7 as genes preferentially expressed in PGCs by a differential cDNA screening, and found that Larp7 played critical roles in proliferation of PGCs. Larp7 is a member of 7SKsnRNP that inhibits P-TEFb, a transcription activator. The Larp7-mutant embryos showed severe reduction of PGCs number, and PGCs were arrested in the G1-phase of cell-cycle. On the other hand, PGC survival was normal and remaining PGCs normally entered meiotic prophase in embryonic ovary, and the expression of the pluripotency-associated genes was not affected in the mutant PGC, suggesting that Larp7 specifically functions to cell-cycle in PGCs. We found that Larp7 negatively controlled the expression of CDK-inhibitors, p15 and p27, suggesting that Larp7 enhances the G1-S transition by specifically inhibiting their transcription in PGCs (Figure).
4) Matsui, Y. et al. The majority of early primordial germ cells acquire pluripotency by Akt activation. Development 141, 4457-4467 (2014).
Primordial germ cells (PGCs) are undifferentiated germ cells in embryos whose fate is to become gametes; however, mouse PGCs can easily be reprogrammed into pluripotential embryonic germ (EG) cells in culture in the presence of particular extra-cellular factors such as combinations of Steel factor, LIF and bFGF. Early PGCs form EG cells more readily than do later PGCs, and PGCs lose the ability to form EG cells by E15.5. Here, we examined the effects of Akt activation in PGCs during EG cell formation; notably, Akt activation, in collaboration with LIF and bFGF, enhanced EG cell formation and caused about 60% of E10.5 PGCs to become EG cells. The results indicated 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 EG cell formation. Those findings indicated that Akt signal enhanced and/or collaborated with signaling pathways of bFGF and of LIF in PGCs for acquisition of pluripotency in PGCs.
4. Recent publications
- Matsui, Y., and Okamura, D. Mechanisms of germ cell specification in mouse embryos. BioEssays 27, 136-143 (2005).
- Seki, Y., Hayashi, K., Itoh, K., Mizugaki, M., Saitou, M., and Matsui, Y. Extensive and orderly reprogramming of genome-wide chromatin modification associated with specification and early development of germ cells in mice. Dev. Biol. 278, 440-458 (2005).
- Hayashi, K., Yoshida, K., and Matsui, Y. A histone H3 methyltransferase controls epigenetic events required for meiotic prophase. Nature 438, 374-378 (2005).
- Sasaki, H. and Matsui, Y. Epigenetic events in mammalian germ cell development: reprogramming and beyond. Nat.Rev.Genet. 9, 129-140 (2008).
- Okamura, D., Tokitake, Y., Niwa, H., Matsui, Y. Requirement of Oct3/4 for germ cell specification. Dev. Biol. 317, 576-584 (2008).
- Morita-Fujimura, Y., Tokitake, Y., and Matsui, Y. Heterogeneity of mouse primordial germ cells reflecting the distinct status of their differentiation, proliferation and apoptosis can be classified by the expression of cell surface proteins integrin a6 and c-Kit. Dev. Growth Differ. 51, 567-584 (2009).
- Matsui, Y., and Tokitake, Y. Primordial germ cells contain subpopulations that have greater ability to develop into pluripotential stem cells. Dev.Growth Differ. 51, 657-667 (2009).
- Maeda, I. and Matsui, Y. In vitro assay system for primordial germ cell development. Cell Res. 19, 1125-1126 (2009).
- Matsui, Y. The molecular mechanisms regulating germ cell development and potential. J. Androl. 31, 61-65 (2010).
- Mochizuki, K. and Matsui, Y. Epigenetic profiles in primordial germ cells: global and fine tuning of the epigenome for acquisition of totipotency. Dev. Growth Differ. 52, 517-525 (2010).
- Mochizuki K., Tachibana, M., Saitou, M., Tokitake, Y., and Matsui, Y. Implication of DNA demethylation and bivalent histone modification for selective gene regulation in mouse primordial germ cells. PLoS ONE 7, e46036 (2012).
- Okamura, D., Mochizuki, K., Taniguchi, H., Tokitake, Y., Ikeda, M., Yamada, Y., Tournier, C., Yamaguchi, S., Tada, T., Scholer, H.R. and Matsui, Y. REST and its downstream molecule Mek5 regulate survival of primordial germ cells. Developmental Biology 372, 190-202 (2012).
- Okamura, D., Maeda, I., Taniguchi, H., Tokitake, Y., Ikeda, M., Ozato, K., Mise, N., Abe, K., Noce, T., Izpisua Belmonte, J. C. and Matsui, Y. Cell-cycle gene-specific control of transcription has a critical role in proliferation of primordial germ cells. Genes & Development 26, 2477-2482 (2012).
- Maeda, I., Okamura, D., Tokitake, Y., Ikeda, M, Kawaguchi, H., Mise, N., Abe, K., Noce, T., Okuda, A, Matsui, Y. Max is a repressor of germ-cell-related gene expression in mouse embryonic stem cells. Nature Communications 4, 1754 (2013).
- Matsui, Y. and Mochizuki, K. A current view of the epigenome in mouse primordial germ cells. Molecular Reproduction and Development 81, 160-170 (2014).
- Leitch, H.G., Okamura, D., Durcova-Hills, G., Stewart, C.L., Gardner, R.L., Matsui, Y., Papaioannou, V.E. On the fate of primordial germ cells injected into early mouse embryos. Developmental Biology 385, 155-159 (2014).
- Matsui, Y., Takehara, A., Tokitake, Y., Ikeda, M., Obara, Y., Morita-Fujimura, Y., Kimura, T., and Nakano, T. The majority of early primordial germ cells acquire pluripotency by Akt activation. Development 141, 4457-4467 (2014).