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Max Is a Repressor of Germ Cell-Related Gene Expression in Mouse Embryonic Stem Cells

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Max Is a Repressor of Germ Cell-Related Gene Expression in Mouse Embryonic Stem Cells ARTICLE Received 19 Sep 2012 | Accepted 20 Mar 2013 | Published 23 Apr 2013 DOI: 10.1038/ncomms2780 Max is a repressor of germ cell-related gene expression in mouse embryonic stem cells Ikuma Maeda1, Daiji Okamura1,w, Yuko Tokitake1, Makiko Ikeda1, Hiroko Kawaguchi1, Nathan Mise2,w, Kuniya Abe2, Toshiaki Noce3, Akihiko Okuda4 & Yasuhisa Matsui1 Embryonic stem cells and primordial germ cells (PGCs) express many pluripotency-asso- ciated genes, but embryonic stem cells do not normally undergo conversion into primordial germ cells. Thus, we predicted that there is a mechanism that represses primordial germ cell- related gene expression in embryonic stem cells. Here we identify genes involved in this putative mechanism, by using an embryonic stem cell line with a Vasa reporter in an RNA interference screen of transcription factor genes expressed in embryonic stem cells. We identify five genes that result in the expression of Vasa when silenced. Of these, Max is the most striking. Transcriptome analysis reveals that Max knockdown in embryonic stem cells results in selective, global derepression of germ cell-specific genes. Max interacts with histone H3K9 methyltransferases and associates with the germ cell-specific genes in embryonic stem cells. In addition, Max knockdown results in a decrease in histone H3K9 dimethylation at their promoter regions. We propose that Max is part of protein complex that acts as a repressor of germ cell-related genes in embryonic stem cells. 1 Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Miyagi 980-8575, Japan. 2 Mammalian Cellular Dynamics, RIKEN BioResource Center, Tsukuba, Ibaraki 305-0074, Japan. 3 Department of Physiology, School of Medicine, Keio University, Tokyo 113-0021, Japan. 4 Division of Developmental Biology, Research Center for Genomic Medicine, Saitama Medical University, Yamane Hidaka, Saitama 350-1241, Japan. w Present addresses: Gene Expression Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA (D.O.); Department of Environmental Preventive Medicine, Jichi Medical University, Tochigi 329-0498, Japan (N.M.). Correspondence and requests for materials should be addressed to Y.M. (email: [email protected]). NATURE COMMUNICATIONS | 4:1754 | DOI: 10.1038/ncomms2780 | www.nature.com/naturecommunications 1 & 2013 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms2780 mbryonic stem cell (ESC) lines are pluripotent stem cell VR15 cells were cultured in 96-well plates and subjected to lines that are established in culture from early epiblast of lipofection-mediated transfection with short interfering RNAs Epreimplantation embryos1,2, whereas primordial germ cells (siRNAs). Transfected cells were cultured for 1 week, and RFP (PGCs) arise in situ from proximal epiblast of postimplantation fluorescence in each well was recorded daily. We identified wells embryos as a result of inducing signals from extraembryonic that had significantly brighter fluorescence than negative control tissues; PGCs migrate into the indifferent embryonic gonads wells (Fig. 1b). Whole-well fluorescence images were captured (genital ridges) and eventually become the founder cells that give semiautomatically using a microscope equipped with a low- rise to eggs or sperm. Although both ESCs and PGCs express magnification objective lens and a motorized stage. The library pluripotency markers (for example, Oct3/4 and Nanog), these cell used for this screen comprised 864 genes, each of which encoded types differ in important respects. For example, ESCs maintain a transcription factor gene that was expressed in ESCs, as pluripotency and can give rise to virtually all embryonic cell confirmed by transcriptome analysis (Supplementary Data 1). For types; in contrast, PGCs can only give rise to gametes. This each of the 864 genes, two independent siRNAs were selected difference in developmental potential might result, at least in part, from commercially available, predesigned siRNAs; this library of from the expression of germ cell-related genes in PGCs. Indeed, 1,728 siRNAs was assembled into 96-well format. ESCs maintain their identity and do not convert to PGCs when From the 864 genes, five candidates—Max, Brg1 (also known under normal culture conditions; moreover, forced expression of as Smarca4), L3mbtl2, Mga and Atf7ip—were identified in the germ cell-specific DAZ (deleted in azoospermia) family proteins screening (Fig. 1c). KD of Max, Brg1, L3mbtl2 or Atf7ip in VR15 in human ESCs resulted in germ cell differentiation and cells resulted in a gradual time-dependent increase in RFP progression in meiosis in culture3. Therefore, there may be fluorescence; in contrast, Mga KD resulted in a relatively acute mechanisms—such as the repression of germ-cell-related gene increase in fluorescence, which was followed by a subtle decline in expression—that separate the cellular status of ESCs from that of a later stage (Supplementary Fig. S2). The decline in RFP PGCs. fluorescence in Mga KD cells was likely due to substantial cell Here, we performed an RNA interference (RNAi) screen to death because many dead cells were seen floating in the wells after identify genes involved in the repression of germ-cell-related gene 4 days in culture (data not shown). To exclude the possibility that expression in undifferentiated mouse ESCs, and the Max gene the increases in RFP fluorescence were due to off-target effects of was identified as a strong candidate. Max is a well-characterized the candidate siRNAs, we confirmed that, for each of the five partner of Myc family proteins, and is known to be required candidate genes, siRNAs targeting distinct sequences in the for almost all of the functions of Myc family proteins4,5. messenger RNAs caused the same effects on RFP fluorescence as Reportedly, loss of Max in mouse ESCs resulted in loss of the the original candidate siRNAs (Supplementary Fig. S3a–e). In pluripotent state and subsequent apoptosis, and both phenotypes addition, in the case of Max, we simultaneously transfected VR15 were circumvented by either forced expression of Nanog or cells with siRNA for Max and an expression vector of Max cultivation under MAPK/Gsk3 inhibiting (2i) conditions6. Max is lacking its 30-UTR that included the target sequences of the a member of the E2F6 complex; this complex contains histone siRNAs, and found that the expression vector reversed the methyltransferases and is involved in gene repression via histone siRNA-induced Vasa::RFP expression (Supplementary Fig. S3f). H3K9 methylation7–10. We showed that transient knockdown We confirmed that expression of the endogenous Vasa gene was (KD) of the Max gene in ESCs resulted in global and selective clearly upregulated in Vasa::RFP-positive (VR15) or Vasa::Venus- expression of germ-cell-related genes; moreover, the Max- positive (VV3) cells after KD of each candidate gene 5 days or 3 mediated repression of these genes in ESCs seemed to be days after transfection of siRNAs, respectively (Fig. 2a,b), because accomplished via histone methylation. VR15 cells showed slightly slower induction of the Vasa reporter than VV3 cells (data not shown). In this study, we focused on Max. Interestingly, ESCs subjected Results to Max-KD (Max-KD ESCs) retained an undifferentiated ESC-like RNAi screening. To develop a screening system to identify genes appearance (Fig. 1d); this finding was surprising given that ESCs that repress germ-cell-related genes in ESCs, we created two ES lacking all Max reportedly lose the capacity for self-renewal and cell lines that carried either the red fluorescent protein (RFP) or undergo apoptosis6. The reason for this apparent contradiction the Venus reporter driven by the promoter from the mouse may be that KD of Max gene expression by siRNA results in vasa homologue (Vasa, also known as Ddx4); these lines were incomplete and transient loss of Max protein function (Fig. 3a). By designated VR15 and VV3, respectively. Vasa is considered day 1 after siRNA transfection, Max protein was decreased to 12% the best marker of differentiated PGCs11,12. During mouse of that at day 0 in VR15 cells, and was subsequently recovered to embryogenesis, PGCs begin to express Vasa just after they 37% at day 5 (Fig. 3a). Delayed and incomplete recovery of Max colonize the genital ridges, and this expression persists until the protein compared with recovery of Max mRNA suggests post- onset of meiotic prophase; moreover, Vasa is specifically and transcriptional regulation of Max expression (Fig. 3). In the case of exclusively expressed in germ cells throughout development. In VV3 ESCs, Max protein was decreased to 22% at day 3 (data not addition, Vasa is not expressed in undifferentiated pluripotent shown). We confirmed induction of germ-cell-specific genes cells such as ESCs or cells of the inner cell mass (ICM) or instead of apoptosis by transient downregulation of Max by using epiblasts; therefore, Vasa reporters are suitable markers of germ- ESCs in which Max gene was homozygously disrupted, but Max cell differentiation in ESCs. We generated a transgenic (Tg) cDNA was introduced in ROSA26 gene locus together with mouse line using the VR15 (ref. 13) ESCs and confirmed that tetracycline off system6. In this system, we found that presence of expression of the RFP reporter in the Tg mouse embryos was 0.1 mgmlÀ 1 of doxycycline for the first one day in culture resulted restricted to PGCs in genital ridges (Supplementary Fig. S1). By
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