MAX controls meiotic entry in sexually undifferentiated germ cells.

Meiosis is a specialized type of cell division that occurs physiologically only in germ cells. We previously demonstrated that MYC-associated factor X (MAX) blocks the ectopic onset of meiosis in embryonic and germline stem cells in culture systems. Here, we investigated the Max gene's role in mouse primordial germ cells. Although Max is generally ubiquitously expressed, we revealed that sexually undifferentiated male and female germ cells had abundant MAX protein because of their higher Max gene expression than somatic cells. Moreover, our data revealed that this high MAX protein level in female germ cells declined significantly around physiological meiotic onset. Max disruption in sexually undifferentiated germ cells led to ectopic and precocious expression of meiosis-related genes, including Meiosin, the gatekeeper of meiotic onset, in both male and female germ cells. However, Max-null male and female germ cells did not complete the entire meiotic process, but stalled during its early stages and were eventually eliminated by apoptosis. Additionally, our meta-analyses identified a regulatory region that supports the high Max expression in sexually undifferentiated male and female germ cells. These results indicate the strong connection between the Max gene and physiological onset of meiosis in vivo through dynamic alteration of its expression.

Alternative splicing for germ cell-specific Mga transcript can be eliminated without compromising mouse viability or fertility.

The stimulated by retinoic acid gene 8 (STRA8)/MEIOSIN complex and polycomb repressive complex (PRC) 1.6, a PRC1 subtype, are believed to be positive and negative regulators of meiotic onset, respectively. During meiotic initiation, the transcription repressive activity of PRC1.6 must be attenuated so that meiosis-related genes can be effectively activated by the STRA8/MEIOSIN complex. However, the molecular mechanisms that control the impairment of PRC1.6 function remain unclear. We recently demonstrated that the Mga gene, which encodes a scaffolding component of PRC1.6, produces variant mRNA by alternative splicing specifically during meiosis. Furthermore, the anomalous MGA protein encoded by the variant mRNA bears an intrinsic ability to function as a dominant negative regulator against the construction of PRC1.6 and is therefore assumed to be, at least in part, involved in impairment of the complex. Therefore, to unequivocally evaluate the physiological significance of Mga variant mRNA production in gametogenesis, we examined the consequences of a genetic manipulation that renders mice unable to produce Mga variant mRNA. Our data revealed that mutant mice were equivalent to wild-type mice in terms of viability and fertility. Our detailed examination of spermatogenesis also revealed that this genetic alteration is not associated with any apparent abnormalities in testis size, spermatogenic cycle, timing of meiotic onset, or marker gene expression of spermatogonia and spermatocytes. Taken together, these data indicate that the production of germ cell-specific Mga variant mRNA is dispensable not only for viability but also for gametogenesis.

Two DNA binding domains of MGA act in combination to suppress ectopic activation of meiosis-related genes in mouse embryonic stem cells.

Although the physiological meaning of the high potential of mouse embryonic stem cells (ESCs) for meiotic entry is not understood, a rigid safeguarding system is required to prevent ectopic onset of meiosis. PRC1.6, a non-canonical PRC1, is known for its suppression of precocious and ectopic meiotic onset in germ cells and ESCs, respectively. MGA, a scaffolding component of PRC1.6, bears two distinct DNA-binding domains termed bHLHZ and T-box. However, it is unclear how this feature contributes to the functions of PRC1.6. Here, we demonstrated that both domains repress distinct sets of genes in murine ESCs, but substantial numbers of meiosis-related genes are included in both gene sets. In addition, our data demonstrated that bHLHZ is crucially involved in repressing the expression of Meiosin, which plays essential roles in meiotic entry with Stra8, revealing at least part of the molecular mechanisms that link negative and positive regulation of meiotic onset.

Identification of germ cell-specific Mga variant mRNA that promotes meiosis via impediment of a non-canonical PRC1.

A non-canonical PRC1 (PRC1.6) prevents precocious meiotic onset. Germ cells alleviate its negative effect by reducing their amount of MAX, a component of PRC1.6, as a prerequisite for their bona fide meiosis. Here, we found that germ cells produced Mga variant mRNA bearing a premature termination codon (PTC) during meiosis as an additional mechanism to impede the function of PRC1.6. The variant mRNA encodes an anomalous MGA protein that lacks the bHLHZ domain and thus functions as a dominant negative regulator of PRC1.6. Notwithstanding the presence of PTC, the Mga variant mRNA are rather stably present in spermatocytes and spermatids due to their intrinsic inefficient background of nonsense-mediated mRNA decay. Thus, our data indicate that meiosis is controlled in a multi-layered manner in which both MAX and MGA, which constitute the core of PRC1.6, are at least used as targets to deteriorate the integrity of the complex to ensure progression of meiosis.

Transformation of normal cells by aberrant activation of YAP via cMyc with TEAD.

YAP (also known as YAP1 or YAP65) is a transcriptional coactivator that interacts with a number of transcription factors including RUNX and TEAD and plays a pivotal role in controlling cell growth. YAP is classified as a proto-oncogene. However, the mechanism by which activated YAP induces cancerous changes is not well known. Here we demonstrate that overexpression of YAP in NIH3T3 cells was sufficient for inducing tumorigenic transformation of cells. Mechanistically, YAP exerts its function in cooperation with the TEAD transcription factor. Our data also show that cMYC is a critical factor that acts downstream of the YAP/TEAD complex. Furthermore, we also found that aberrant activation of YAP is sufficient to drive tumorigenic transformation of non-immortalized mouse embryonic fibroblasts. Together our data indicate that YAP can be categorized as a new type of proto-oncogene distinct from typical oncogenes, such as H-RAS, whose expression in non-immortalized cells is tightly linked to senescence.

Identification of the Coiled-Coil Domain as an Essential Methyl-CpG-Binding Domain Protein 3 Element for Preserving Lineage Commitment Potential of Embryonic Stem Cells.

Embryonic stem cells (ESCs) exhibit two salient features beneficial for regenerative medicine: unlimited self-renewal and pluripotency. Methyl-CpG-binding domain protein 3 (Mbd3), a scaffolding component of the nucleosome remodeling deacetylase complex, is a specific regulator of pluripotency, as ESCs lacking Mbd3 are defective for lineage commitment potential but retain normal self-renewal properties. However, functional similarities and dissimilarities among the three Mbd3 isoforms (a, b, and c) have not been intensively explored. Herein, we demonstrated that Mbd3c, which lacks an entire portion of the MBD domain, exerted equivalent activity for counteracting the defective lineage commitment potential of Mbd3-knockout ESCs. Our analyses also revealed that the coiled-coil domain common to all three MBD3 isoforms, but not the MBD domain, plays a crucial role in this activity. Mechanistically, our data demonstrate that the activity of the coiled-coil domain is exerted, at least in part, through recruitment of polycomb repressive complex 2 to a subset of genes linked to development and organogenesis, thus establishing stable transcriptional repression. Stem Cells 2018;36:1355-1367.

Link between embryonic stem cell pluripotency and homologous allelic pairing of Oct4 loci.

The Oct4 gene is a master regulator of the pluripotent properties of embryonic stem cells (ESCs). Recently, Oct4 loci were shown to frequently localize in close proximity to one another during the early stage of cellular differentiation, implicating this event as an important prerequisite step for ESCs to exert their full differentiation potential. Although the differentiation capacity of embryonal carcinoma cells (ECCs), such as F9 and P19 ECC lines, is severely restricted compared with ESCs, ECCs bear a highly similar expression profile to that of ESCs including expression of Oct4 and other pluripotency marker genes. Therefore, we examined whether allelic pairing of Oct4 loci also occurs during differentiation of F9 and P19 ECCs. Our data clearly demonstrate that this event is only observed within ESCs, but not ECCs, subjected to induction of differentiation, indicating transient allelic pairing of Oct4 loci as a specific feature of pluripotent ESCs. Moreover, our data revealed that this pairing did not occur broadly across chromosome 17, which carries the Oct4 gene, but occurred locally between Oct4 loci, suggesting that Oct4 loci somehow exert a driving force for their allelic pairing.

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

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.

Forced expression of Nanog or Esrrb preserves the ESC status in the absence of nucleostemin expression.

Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in a plethora of functions including ribosomal biogenesis and maintenance of telomere integrity. In addition to its expression in cancerous cells, the NS gene is expressed in stem cells including embryonic stem cells (ESCs). Previous knockdown and knockout studies have demonstrated that NS is important to preserve the self-renewality and high expression levels of pluripotency marker genes in ESCs. Here, we found that forced expression of Nanog or Esrrb, but not other pluripotency factors, resulted in the dispensability of NS expression in ESCs. However, the detrimental phenotypes of ESCs associated with ablation of NS expression were not mitigated by forced expression of Rad51 or a nucleolar localization-defective NS mutant that counteracts the damage associated with loss of NS expression in other NS-expressing cells such as neural stem/progenitor cells. Thus, our results indicate that NS participates in preservation of the viability and integrity of ESCs, which is distinct from that in other NS-expressing cells.

Functional compensation between Myc and PI3K signaling supports self-renewal of embryonic stem cells.

c-Myc and phosphatidylinositol 3-OH kinase (PI3K) both participate in diverse cellular processes, including cell cycle control and tumorigenic transformation. They also contribute to preserving embryonic stem cell (ESC) characteristics. However, in spite of the vast knowledge, the molecular relationship between c-Myc and PI3K in ESCs is not known. Herein, we demonstrate that c-Myc and PI3K function cooperatively but independently to support ESC self-renewal when murine ESCs are cultured under conventional culture condition. Interestingly, culture of ESCs in 2i-condition including a GSK3ß and MEK inhibitor renders both PI3K and Myc signaling dispensable for the maintenance of pluripotent properties. These results suggest that the requirement for an oncogenic proliferation-dependent mechanism sustained by Myc and PI3K is context dependent and that the 2i-condition liberates ESCs from the dependence of this mechanism.

Identification of Ccr4-not complex components as regulators of transition from partial to genuine induced pluripotent stem cells.

Somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs) by defined factors. However, substantial cell numbers subjected to iPSC induction stray from the main reprogramming route and are immortalized as partial iPSCs. These partial iPSCs can become genuine iPSCs by exposure to the ground state condition. However, such conversion is only possible for mouse partial iPSCs, and it is not applicable to human cells. Moreover, the molecular basis of this conversion is completely unknown. Therefore, we performed genome-wide screening with a piggyBac vector to identify genes involved in conversion from partial to genuine iPSCs. This screening led to identification of Cnot2, one of the core components of the Ccr4-Not complex. Subsequent analyses revealed that other core components, Cnot1 and Cnot3, also contributed to the conversion. Thus, our data have uncovered a novel role of core components of the Ccr4-Not complex as regulators of transition from partial to genuine iPSCs.

In vivo function and evolution of the eutherian-specific pluripotency marker UTF1.

Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. UTF1 is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the UTF1 gene during mouse development. We found that homozygous UTF1 mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of UTF1 expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of UTF1 expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the UTF1 gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the UTF1 genes.

Striking similarity in the gene expression levels of individual Myc module members among ESCs, EpiSCs, and partial iPSCs.

Predominant transcriptional subnetworks called Core, Myc, and PRC modules have been shown to participate in preservation of the pluripotency and self-renewality of embryonic stem cells (ESCs). Epiblast stem cells (EpiSCs) are another cell type that possesses pluripotency and self-renewality. However, the roles of these modules in EpiSCs have not been systematically examined to date. Here, we compared the average expression levels of Core, Myc, and PRC module genes between ESCs and EpiSCs. EpiSCs showed substantially higher and lower expression levels of PRC and Core module genes, respectively, compared with those in ESCs, while Myc module members showed almost equivalent levels of average gene expression. Subsequent analyses revealed that the similarity in gene expression levels of the Myc module between these two cell types was not just overall, but striking similarities were evident even when comparing the expression of individual genes. We also observed equivalent levels of similarity in the expression of individual Myc module genes between induced pluripotent stem cells (iPSCs) and partial iPSCs that are an unwanted byproduct generated during iPSC induction. Moreover, our data demonstrate that partial iPSCs depend on a high level of c-Myc expression for their self-renewal properties.

Sirt1, p53, and p38(MAPK) are crucial regulators of detrimental phenotypes of embryonic stem cells with Max expression ablation.

c-Myc participates in diverse cellular processes including cell cycle control, tumorigenic transformation, and reprogramming of somatic cells to induced pluripotent cells. c-Myc is also an important regulator of self-renewal and pluripotency of embryonic stem cells (ESCs). We recently demonstrated that loss of the Max gene, encoding the best characterized partner for all Myc family proteins, causes loss of the pluripotent state and extensive cell death in ESCs strictly in this order. However, the mechanisms and molecules that are responsible for these phenotypes remain largely obscure. Here, we show that Sirt1, p53, and p38(MAPK) are crucially involved in the detrimental phenotype of Max-null ESCs. Moreover, our analyses revealed that these proteins are involved at varying levels to one another in the hierarchy of the pathway leading to cell death in Max-null ESCs.

Indefinite self-renewal of ESCs through Myc/Max transcriptional complex-independent mechanisms.

Embryonic stem cells (ESCs) can self-renew indefinitely under the governance of ESC-specific transcriptional circuitries in which each transcriptional factor regulates distinct or overlapping sets of genes with other factors. c-Myc is a key player that is crucially involved in maintaining the undifferentiated state and the self-renewal of ESCs. However, the mechanism by which c-Myc helps preserve the ESC status is still poorly understood. Here we addressed this question by performing loss-of-function studies with the Max gene, which encodes the best-characterized partner protein for all Myc family proteins. Although Myc/Max complexes are widely regarded as crucial regulators of the ESC status, our data revealed that ESCs do not absolutely require these complexes in certain contexts and that this requirement is restricted to empirical ESC culture conditions without a MAPK inhibitor.

Differential requirement for nucleostemin in embryonic stem cell and neural stem cell viability.

Stem cells have the remarkable ability to self-renew and to generate multiple cell types. Nucleostemin is one of proteins that are enriched in many types of stem cells. Targeted deletion of nucleostemin in the mouse results in developmental arrest at the implantation stage, indicating that nucleostemin is crucial for early embryogenesis. However, the molecular basis of nucleostemin function in early mouse embryos remains largely unknown, and the role of nucleostemin in tissue stem cells has not been examined by gene targeting analyses due to the early embryonic lethality of nucleostemin null animals. To address these questions, we generated inducible nucleostemin null embryonic stem (ES) cells in which both alleles of nucleostemin are disrupted, but nucleostemin cDNA under the control of a tetracycline-responsive transcriptional activator is introduced into the Rosa26 locus. We show that loss of nucleostemin results in reduced cell proliferation and increased apoptosis in both ES cells and ES cell-derived neural stem/progenitor cells. The reduction in cell viability is much more profound in ES cells than in neural stem/progenitor cells, an effect that is mediated at least in part by increased induction and accumulation of p53 and/or activated caspase-3 in ES cells than in neural stem/progenitor cells.

Identification of an ES cell pluripotent state-specific DUSP6 enhancer.

To identify genes with pluripotent state-specific expression in embryonic stem (ES) cells, we compared gene expression profiles between undifferentiated and differentiated mouse ES cells using DNA microarrays. Among the numerous genes identified, we focused on dual specificity phosphatase 6 (DUSP6), which had previously been shown to be expressed in undifferentiated human ES cells. We have identified and characterized a regulatory enhancer that we have termed PEDRE that controls pluripotent state-specific expression of DUSP6. This 82-base pair enhancer overlaps with, but is distinct from, a recently identified regulatory element that is regulated by the FGF-ERK pathway. The sequence of PEDRE is 100% identical between mouse and human DUSP6, suggesting that the molecular basis of DUSP6 gene expression in undifferentiated state of ES cells is highly conserved during evolution.

Consequence of the loss of Sox2 in the developing brain of the mouse.

The transcription factor Sox2 is expressed at high levels in neural stem and progenitor cells. Here, we inactivated Sox2 specifically in the developing brain by using Cre-loxP system. Although mutant animals did not survive after birth, analysis of late gestation embryos revealed that loss of Sox2 causes enlargement of the lateral ventricles and a decrease in the number of neurosphere-forming cells. However, although their neurogenic potential is attenuated, Sox2-deficient neural stem cells retain their multipotency and self-renewal capacity. We found that expression level of Sox3 is elevated in Sox2 null developing brain, probably mitigating the effects of loss of Sox2.

Putative "stemness" gene jam-B is not required for maintenance of stem cell state in embryonic, neural, or hematopoietic stem cells.

Many genes have been identified that are specifically expressed in multiple types of stem cells in their undifferentiated state. It is generally assumed that at least some of these putative "stemness" genes are involved in maintaining properties that are common to all stem cells. We compared gene expression profiles between undifferentiated and differentiated embryonic stem cells (ESCs) using DNA microarrays. We identified several genes with much greater signal in undifferentiated ESCs than in their differentiated derivatives, among them the putative stemness gene encoding junctional adhesion molecule B (Jam-B gene). However, in spite of the specific expression in undifferentiated ESCs, Jam-B mutant ESCs had normal morphology and pluripotency. Furthermore, Jam-B homozygous mutant mice are fertile and have no overt developmental defects. Moreover, we found that neural and hematopoietic stem cells recovered from Jam-B mutant mice are not impaired in their ability to self-renew and differentiate. These results demonstrate that Jam-B is dispensable for normal mouse development and stem cell identity in embryonic, neural, and hematopoietic stem cells.

The Sox2 regulatory region 2 functions as a neural stem cell-specific enhancer in the telencephalon.

Sox2 is expressed at high levels in neuroepithelial stem cells and persists in neural stem/progenitor cells throughout adulthood. We showed previously that the Sox2 regulatory region 2 (SRR2) drives strong expression in these cells. Here we generated transgenic mouse strains with the beta-geo reporter gene under the control of the SRR2 in order to examine the spatiotemporal function of this regulatory region. We show that the SRR2 functions specifically in neural stem/progenitor cells. However, unlike Nestin 2nd intronic enhancer, the SRR2 shows strong regional specificity functioning only in restricted areas of the telencephalon but not in any other portions of the central nervous system such as the spinal cord. We also show by in vitro clonogenic assay that at least some of these SRR2-functioning cells possess the hallmark properties of neural stem cells. In adult brains, we could detect strong beta-geo expression in the subventricular zone of the lateral ventricle and along the rostral migrating stream where actively dividing cells reside. Chromatin immunoprecipitation assays reveal interactions of POU and Sox factors with SRR2 in neural stem/progenitor cells. Our data also suggest that the specific recruitment of these proteins to the SRR2 in the telencephalon defines the spatiotemporal activity of the enhancer in the developing nervous system.