Site-specific O-GlcNAcylation of Psme3 maintains mouse stem cell pluripotency by impairing P-body homeostasis -- Soka University

The research group of Professor Shoko Nishihara at Soka University (Director of the Glycan & Life System Integration Center, Soka University), in collaboration with Professor Kazuo Yamamoto's research group at the University of Tokyo, showed that O-GlcNAc sugar chain modification inhibits the formation of P-body, thus maintaining mESC pluripotency.

In recent years, it has become increasingly clear that glycans play an important role in determining the properties of stem cells. In this study, we revealed that O-GlcNAc of Ser111 on the proteasome activation subunit 3 (Psme3) is an important modification that maintains pluripotency in mouse embryonic stem cells (mESCs). O-GlcNAc on Ser111 of Psme3 promotes the degradation of DEAD box polypeptide 6 (Ddx6) required for P-body assembly, consequently reducing P-body amount. As a result, mRNAs encoding core transcription factors involved in the maintenance of pluripotency, such as Klf4 and Klf2, are translated and the pluripotency state is maintained. Conversely, when a mutation is introduced into Ser111 to inhibit O-GlcNAcylation of Psme3, Ddx6 is stabilized and the P body amount increases. Thus, the protein level of the pluripotent core transcription factor decreases, and mESCs are primed for differentiation. These findings establish O-GlcNAcylation at Ser111 of Psme3 as a critical switch that regulates mESC pluripotency via control of P-body homeostasis.

The results of this study were published in the scientific journal Cell Reports on July 13th at 11:00 AM (EST).

Part of this research was supported by JSPS Grant for Scientific Research 18K06139 and Joint Research Fund of the Glycan & Life System Integration Center, Soka University.

<Results Significance>

P-bodies are membrane-less cytoplasmic organelles that regulate stem cell identity. P-bodies are produced by liquid-liquid phase separation and require the RNA helicase DEAD Box Polypeptide 6 (Ddx6) for their formation. On the other hand, proteasome-activating subunit 3 (Psme3) has been known to regulate the homeostasis of membrane-less cytoplasmic organelles formed by liquid-liquid phase separation, such as the Cajal body and nuclear speckle. The function of O-GlcNAc, which is the sole sugar chain modification present in the cytoplasm, has become gradually clearer. However, the connection between these three elements is rather surprising. In this study, we revealed that Psme3 interacted with Ddx6 and that O-GlcNAc of Ser111 of Psme3 mediated Ddx6 degradation in mESCs. These findings established a relationship between O-GlcNAc, proteasome, and P-bodies homeostasis in mESCs. The results of this study provide new findings related to the role of proteasome and O-GlcNAc in P-body biology and pluripotency networks, and provide a valuable groundwork for future investigations in developmental biology and stem cell biology.

<Research Background>

The pluripotency of mESCs is tightly controlled by a complex network of exogenous and endogenous factors. O-GlcNAc is the sole sugar chain modification found in cytoplasmic and nucleoproteins and plays a vital role in the regulation of basic cellular processes. O-GlcNAc is added to the protein Ser or Thr by O-GlcNAc transferase and removed by O-GlcNAcase. So far, several research groups, including our, have reported its functions in mESCs. However, due to the variety of O-GlcNAcylated proteins, the role of O-GlcNAc in the pluripotency network is still poorly understood. In the present study, we exploited the highly sensitive O-GlcNAc modification detection method developed by our collaborator Yamamoto et al. to identify O-GlcNAcylated proteins in mESCs and mouse epiblast-like cells (mEpiLCs). Interestingly, mEpiLCs, which are slightly more differentiated but retain their pluripotency, differed from mESCs in their pattern of O-GlcNAcylated proteins. Since the regulation of the proteasome by O-GlcNAc and the pluripotency network of mESCs was still unknown, we selected the proteasome activation subunit 3 (Psme3) from the detected proteins and proceeded with the analysis.

Psme3 interactome was analyzed by expressing a Psme3-FLAG tagged protein and immunoprecipitation followed by mass spectrometry. As a result, we observed that Psme3 interacts with DEAD box polypeptide 6 (Ddx6), Grbp2, Fxr1, Rpl7, etc... which have all been reported to interact with the P bodies. Overexpression of Psme3 resulted in degradation of Ddx6 and reduced P-bodies. Mass spectrometry and immunoprecipitation revealed that Ser111 of Psme3 was O-GlcNAcylated. Using a site-specific mutagenesis methodology, we introduced a mutation in the Ser111 and expressed in mESCs for analysis. In the Ser111 mutants, the O-GlcNAc modification and the interaction with Ddx6 was significantly reduced, indicating that the O-GlcNAc modification of Ser111 in Psme3 regulated the interaction with Ddx6 and its subsequent degradation. mESCs overexpressing Ser111 mutants, Ddx6 degradation was suppressed, the amount of P-body increased, and the protein level of pluripotent core transcription factors, such as Klf4 and Klf2 decreased, resulting in exit from the pluripotent state. These findings revealed that O-GlcNAc on Ser111 in Psme3 acts as a pivotal switch that regulates mESCs pluripotency through regulation of P-body homeostasis.

Publication：Scientific journal “Cell reports ７/13 issue”

Title：Site-specific O-GlcNAcylation of Psme3 maintains mouse stem cell pluripotency by impairing P-body homeostasis

Authors： Federico Pecori, Nanako Kondo, Chika Ogura, Taichi Miura, Masahiko Kume, Youhei Minamijima, Kazuo Yamamoto, Shoko Nishihara

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