Ayumu Suzuki

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.

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.

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 GSK3β 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. Stem Cells 2015;33:713–725

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.

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.

Does MAX open up a new avenue for meiotic research

Meiosis is a central event of sexual reproduction. Like somatic cells, germ cells conduct mitosis to increase their cell number, but unlike somatic cells, germ cells switch their cell division mode from mitosis to meiosis at a certain point in gametogenesis. However, the molecular basis of this switch remains elusive. In this review article, we give an overview of the onset of mammalian meiosis, including our recent finding that MYC Associated Factor X (MAX) prevents ectopic and precocious meiosis in embryonic stem cells (ESCs) and germ cells, respectively. We present a hypothetical model of a MAX‐centered molecular network that regulates meiotic entry in mammals and propose that inducible Max knockout ESCs provide an excellent platform for exploring the molecular mechanisms of meiosis initiation, while excluding other aspects of gametogenesis.

Unexpected link between MAX and meiotic onset

Germ cells are uniquely programmed cell lineage that can transfer genetic and epigenetic information to subsequent generations. Similar to somatic cells, germ cells undergo mitosis for substantial durations to increase their numbers. However, germ cells switch their mode of cell division from mitosis to meiosis to convert to haploid cells at appropriate time points; female germ cells undergo meiosis in the genital ridge at midgestation, but male germ cells initiate this switch after birth in the testes of mammals. Although it is known that retinoic acid is crucially involved in this transition, the molecular mechanisms governing the switch remain largely obscure. However, our recent study revealed MAX as a negative regulator of meiosis, suppressing ectopic and precocious meiotic onset in embryonic stem cells (ESCs) and spermatogonial stem cells (SSCs), respectively. Before reaching this conclusion, we first demonstrated that Max expression ablation in ESCs not only activates expression of meiosis-related genes, but also induces cytological changes reminiscent of germ cells at leptotene and zygotene stages of meiosis by immunocytochemical analysis of SYCP3, a major component of the synaptonemal complex. We also showed that these cytological changes are dependent on the retinoic acid-STRA8 axis, underscoring that these changes faithfully recapitulate bona fide meiotic processes. However, intriguingly, we found that Max expression-ablated ESCs appear to directly convert to meiosis-like cells without passing through the primordial germ cell state. Subsequently, we demonstrated that the Max gene shows a transient but significant decline in its expression level during the early stage of meiosis in both male and female germ cells, implying that downregulation of Max expression levels is a physiologically required step for meiosis. Finally, we found that forced reduction of Max expression in SSCs induces meiotic entry with much greater efficiency compared with that observed in ESCs. MAX is generally known as an obligated partner protein of MYC that functions as a transcription factor. Indeed, MYC by itself has almost no intrinsic DNA-binding activity, and MAX confers that activity on MYC. MYC/MAX transcription complexes activate transcription of numerous genes, especially those encoding positive regulators of the cell cycle, such as cyclin D, leading to acceleration of cell proliferation. However, it has recently been shown that MAX is also a subunit of atypical polycomb complex 1 (PRC1.6), and we now know that the Max expression ablation-mediated meiotic onset in ESCs and SSCs reflects disruption of the transcriptional repressing function of PRC1.6. MAX is a unique component of PRC1.6 that not present in any of the other 5 PRC1 complex subtypes (PRC1.1–5). PRC1.6 consists of at least 13 subunits including MAX. Furthermore, similar to Max expression deficiency, knockdown of genes encoding other components of this complex, such as L3MBTL2, augments expression levels of meiosis-related genes in ESCs. However, the magnitude of alteration in the expression levels accompanied by Max expression deficiency is much more conspicuous than that resulting from knockdown of genes encoding other components of the PRC1.6 complex. Notably, our study demonstrating the consequence of Max expression deficiency is the only report showing that transcriptional activation of meiotic genes is accompanied by cytological changes reminiscent of meiosis. One obvious possibility explaining these conspicuous phenotypes associated with Max expression ablation, but not the loss of expression of genes corresponding to other components of the PRC1.6 complex, is the dependence of the complex on MAX for its binding to DNA. However, we assume that loss of MYC activity associated with the loss of MAX, but not the loss of other genes, may also be related to the amplification of the levels of meiotic changes in ESCs and SSCs. MYC plays crucial roles in preserving the stem cell state of both cell types by activating several genes directly linked to cell proliferation, which is apparently contradictory phenomenon against meiosis. Consistent with this notion, our results demonstrated that induction of meiosis-like changes by disruption of the PRC1.6 complex through Max expression ablation becomes much less