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  • CCG-1423 molecular In this study we further


    In this study, we further investigate how STAT3 integrate to the core regulatory circuit in ESC pluripotency and differentiation, and identify Mettl8 as a downstream target of STAT3 in mESCs. We discover the role of METTL8 as a negative regulator of JNK signaling in stem cells. Our results provide insights into the crosstalk between STAT3 and JNK signaling during stem cell differentiation.
    Discussion In this study, we identified Mettl8 as a transcriptional target of STAT3 that mediates STAT3’s functions in mESCs. According to previous studies, STAT3 responds to external cytokine LIF and then transcribes critical pluripotent factors in the nucleus, such as Oct4 and Nanog. Nevertheless, it remains undefined if STAT 3 is able to regulate stem cell pluripotency or differentiation by other mechanisms. We report here that METTL8 mediates a role of STAT3 during ESC differentiation. Our results indicate that STAT3, besides modulating Oct4 and Nanog expression, plays diverse roles in maintenance of ESC identity. We found that METTL8 impedes differentiation of mESCs rather than pluripotency. Through RNA-seq and microarray analysis, it is discovered that lineage markers increased in Mettl8-deficient ESCs, while pluripotency markers showed no change. However, we think those elevated lineage genes are not a strong enough driving force for ESC differentiation. As a result, Mettl8 KO CCG-1423 molecular maintain a pluripotent state. Nevertheless, once those Mettl8-deficient cells were deprived of pluripotent conditions they would be more vulnerable to differentiate. However, different to embryo lethality caused by Stat3 deficiency, no abnormal phenotype was observed in Mettl8 KO mice. So it needs further investigation if there is redundancy between METTL family members. One of the METTL family genes, Mettl3, is also reported to bind mRNA transcripts, and inhibits mRNA stability (Geula et al., 2015). We also used an affinity purification approach and subsequent mass spectrometry analysis to explore the interacting proteins with METTL8, and found a number of RNA binding proteins in the candidates list (Table S3). This result suggests that the binding between METTL8 and Mapkbp1 mRNA may be not direct, and that some RNA binding proteins may mediate the interaction, which needs further investigation. In addition, mRNA of Gata6, which is a lineage marker, was also proved to bind to METTL8 by subsequent experiments (data not shown). Coincidently Gata6 mRNA also showed an upregulation in Mettl8 knockdown cells in both microarray and RNA-seq analysis, which suggests that METTL8 may affect differentiation through alternative factors and mechanisms. It is reported that METTL8 causes 3-methylcytidine (m3C) modification on human mRNAs (Xu et al., 2017). METTL8 contains a conserved methytransferase domain and is predicted to have methytransferase activity. Consistently, from the PAR-CLIP data in our study, most candidate METTL8 binding partners are mRNAs. We should further investigate if METTL8 modifies m3C on Mapkbp1 mRNA and then causes translation inhibition. In addition, other METTL family proteins also reported to modify mRNAs. For example, METTL3 and METTL14 catalyze m(6)A RNA methylation (Liu et al., 2014), which is critical for glioblastoma stem cell self-renewal (Cui et al., 2017). MAPKBP1 enhances JNK signaling activated by mitogen-activated protein kinase kinase 1 (MEK1) and transforming growth factor β-activated kinase 1 (Koyano et al., 1999). Given that extracellular signal-regulated kinase (ERK) signaling is a major driving force for mESC differentiation and that MEK 1 also activates ERK signaling, the crosstalk between JNK and ERK signaling is worth being further studied. Moreover, studies showed that JNK signaling is required for lineage-specific differentiation but not stem cell self-renewal (Xu and Davis, 2010), which is consistent with our finding that METTL8 affects differentiation but not the pluripotent state. It is reported that JNK signaling can induce STAT3-Ser727 phosphorylation, which is important for the transactivation potency of STAT3 (Lim and Cao, 1999). Here, we also found that, when the STAT3 pathway was inhibited, JNK phosphorylation showed a higher level. However, this phenotype became subtle in Mettl8 KO cells, indicating that STAT3 has a feedback on JNK signaling in mESCs mediated by METTL8 (Figure S5B).