Masataka Hirasaki

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.

Sirt1, p53, and p38MAPK 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 p38MAPK 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. STEM CELLS2012;30:1634–1644

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.

Protein phosphatase Siw14 controls intracellular localization of Gln3 in cooperation with Npr1 kinase in Saccharomyces cerevisiae.

Saccharomyces cerevisiae Deltasiw14 disruptant exhibits caffeine sensitivity. To understand the function of Siw14, double disruptants for SIW14 and each of 102 viable protein kinases (PKase) genes were constructed and examined for suppression of caffeine sensitivity based on the premise that the sensitivity was caused either by accumulation of an unknown phosphorylated Siw14 substrate(s) or by depletion of an unphosphorylated substrate(s) of Siw14 in the Deltasiw14 disruptant. Among 102 pkase disruptions, only one, Deltanpr1, suppressed the caffeine sensitivity of the Deltasiw14 disruptant. Because Gln3 (a phosphorylated transcriptional activator)-dependent transcription is induced by disruption of NPR1, we further examined the effect of disruption and overexpression of GLN3 on the caffeine sensitivity of the Deltasiw14 disruptant. Disruption of GLN3 was found to partially suppress the caffeine sensitivity of the Deltasiw14 disruptant, while overexpression of GLN3 in wild-type cells caused caffeine sensitivity, providing the first evidence that Siw14 functions in the Gln3 regulatory network. We also found that, unlike in a wild-type background, Gln3 accumulates in the nucleus whether cells are exposed or not to caffeine in the Deltasiw14 disruptant, and that this nuclear localization was abolished by disruption of NPR1. Interestingly, the level of Gln3 phosphorylation in both the Deltasiw14 and Deltanpr1 disruptants decreased relative to wild type, independent of exposure to caffeine. We conclude that Siw14 controls the intracellular localization of Gln3 in combination with Npr1, and one of the causes for the caffeine sensitivity of the Deltasiw14 disruptant was an accumulation of dephosphorylated Gln3 in the nucleus.

Combined Overexpression of JARID2, PRDM14, ESRRB, and SALL4A Dramatically Improves Efficiency and Kinetics of Reprogramming to Induced Pluripotent Stem Cells

Identification of a gene set capable of driving rapid and proper reprogramming to induced pluripotent stem cells (iPSCs) is an important issue. Here we show that the efficiency and kinetics of iPSC reprogramming are dramatically improved by the combined expression of Jarid2 and genes encoding its associated proteins. We demonstrate that forced expression of JARID2 promotes iPSC reprogramming by suppressing the expression of Arf, a known reprogramming barrier, and that the N‐terminal half of JARID2 is sufficient for such promotion. Moreover, JARID2 accelerated silencing of the retroviral Klf4 transgene and demethylation of the Nanog promoter, underpinning the potentiating activity of JARID2 in iPSC reprogramming. We further show that JARID2 physically interacts with ESRRB, SALL4A, and PRDM14, and that these JARID2‐associated proteins synergistically and robustly facilitate iPSC reprogramming in a JARID2‐dependent manner. Our findings provide an insight into the important roles of JARID2 during reprogramming and suggest that the JARID2‐associated protein network contributes to overcoming reprogramming barriers. Stem Cells 2016;34:322–333

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.

Saccharomyces cerevisiae protein phosphatase Ppz1 and protein kinases Sat4 and Hal5 are involved in the control of subcellular localization of Gln3 by likely regulating its phosphorylation state

A Saccharomyces cerevisiae mutant lacking PPZ1, encoding a serine/threonine protein phosphatase (PPase), is caffeine-sensitive. To clarify the function of Ppz1 in resistance to caffeine, we attempted systematically to identify protein kinase (PKase) whose disruption lead to suppression of caffeine sensitive phenotype of the ∆ppz1 disruptant since disruption of PPZ1 might cause caffeine sensitivity by increasing its phosphorylated substrates and we presumed that disruption of genes for PKase sharing the substrate with Ppz1 could restore the resistance through bypassing necessity for dephosphorylation of substrates. Among the 102 viable pkase disruptions, disruption of either SAT4 or HAL5 suppressed the caffeine sensitivity phenotype and increased expression of ENA1, encoding a P-type ATPase of the ∆ppz1 disruptant. Because increased expression of ENA1 in the ∆ppz1 disruptant was found to be suppressed by disruption of GLN3, localization and phosphorylation of Gln3 in the ∆ppz1 disruptant was compared to that in the ∆ppz1∆sat4 and ∆ppz1∆hal5 double disruptants. Gln3 was found to accumulate in the nucleus in the ∆ppz1 disruptant, and this nuclear localization was abolished by disruption of either SAT4 or HAL5. Interestingly, the level of Gln3 phosphorylation in the ∆ppz1∆sat4 and ∆ppz1∆hal5 disruptants decreased relative to wild type independent of caffeine. From these observations, we conclude that Ppz1 controls Gln3 localization by regulating its phosphorylation state in combination with Sat4 and Hal5.

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.

Deciphering cellular functions of protein phosphatases by comparison of gene expression profiles in Saccharomyces cerevisiae

Expression profiles of protein phosphatase (PPase) disruptants were analyzed by use of Pearsons correlation coefficient to find profiles that correlated with those of 316 Reference Gene (RG) disruptants harboring deletions in genes with known functions. Twenty-six Deltappase disruptants exhibited either a positive or negative correlation with 94 RG disruptants when the p value for Pearsons correlation coefficient was >0.2. Some of the predictions that arose from this analysis were tested experimentally and several new Delta ppase phenotypes were found. Notably, Delta sit4 and Delta siw14 disruptants exhibited hygromycin B sensitivity, Delta sit4 and Delta ptc1 disruptants grew slowly on glycerol medium, the Delta ptc1 disruptant was found to be sensitive to calcofluor white and congo red, while the Delta ppg1 disruptant was found to be sensitive to congo red. Because on-going analysis of expression profiles of Saccharomyces cerevisiae disruptants is rapidly generating new data, we suggest that the approach used in the present study to explore PPase function is also applicable to other genes.