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.

pncRNA-D is an irradiation-induced 602-nt long noncoding RNA transcribed from the promoter region of the cyclin D1 (CCND1) gene. CCND1 expression is predicted to be inhibited through an interplay between pncRNA-D and RNA-binding protein TLS/FUS. Because the pncRNA-D–TLS interaction is essential for pncRNA-D–stimulated CCND1 inhibition, here we studied the possible role of RNA modification in this interaction in HeLa cells. We found that osmotic stress induces pncRNA-D by recruiting RNA polymerase II to its promoter. pncRNA-D was highly m6A-methylated in control cells, but osmotic stress reduced the methylation and also arginine methylation of TLS in the nucleus. Knockdown of the m6A modification enzyme methyltransferase-like 3 (METTL3) prolonged the half-life of pncRNA-D, and among the known m6A recognition proteins, YTH domain-containing 1 (YTHDC1) was responsible for binding m6A of pncRNA-D. Knockdown of METTL3 or YTHDC1 also enhanced the interaction of pncRNA-D with TLS, and results from RNA pulldown assays implicated YTHDC1 in the inhibitory effect on the TLS–pncRNA-D interaction. CRISPR/Cas9-mediated deletion of candidate m6A site decreased the m6A level in pncRNA-D and altered its interaction with the RNA-binding proteins. Of note, a reduction in the m6A modification arrested the cell cycle at the G0/G1 phase, and pncRNA-D knockdown partially reversed this arrest. Moreover, pncRNA-D induction in HeLa cells significantly suppressed cell growth. Collectively, these findings suggest that m6A modification of the long noncoding RNA pncRNA-D plays a role in the regulation of CCND1 gene expression and cell cycle progression.

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.

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 SIGNIFICANCE STATEMENTInduced pluripotent stem cells (iPSCs), which show embryonic stem cell-like properties, are generated by forced expression of transcription factors including OCT4, SOX2, KLF4, and c-MYC (OSKM). The identification of a gene set that can achieve proper reprogramming to iPSCs is essential for understanding the molecular mechanism underlying reprogramming and for cellbased therapies in regenerative medicine. Here, we show that JARID2 promotes iPSC reprogramming via regulation of gene expression and physical interaction with pluripotency factors including PRDM14, ESRRB, and SALL4A. Furthermore, our data suggest that the combined expression of OSKM and these four factors can drive highly efficient and rapid reprogramming to iPSCs.

c-Myc participates in diverse cellular processes including cell cycle control, tumorigenic transformation, and reprogramming of somatic cells to induced pluripotent cells. cMyc 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.