br Materials and methods Information related

Materials and methods Information related to Material and methods can be found in the Supplemental information section.
Results
Discussion We previously reported that O-GlcNAcylation is important in hESC commitment towards adipose and ectoderm lineages (Maury et al., 2013). However, O-GlcNAc proteins regulating hESC commitment have yet to be characterized. Here, we aimed at extending our understanding of O-GlcNAc regulation occurring during human stem cell differentiation. Therefore, we revealed for the first time that RING1B, the catalytic core of PRC1, is O-GlcNAcylated in hESC. We revealed that RING1B has at least 2 O-GlcNAcylation sites which are occupied and linked to specific residues: T250/S251 and S278 respectively. Interestingly, the relative percentage of RING1B O-GlcNAcylation on T250/S251 residue decreases during differentiation. We then studied the function of RING1B O-GlcNAcylation using an anti-RING1B ChIP strategy followed by a WGA re-ChIP experiment. Functionally, O-GlcNAcylation on RING1B seemed to modulate RING1B DNA-binding. Non-O-GlcNAc RING1B preferentially bound near genes related to metabolic and Digoxigenin-11-ddUTP Supplier processes. In contrast, O-GlcNAcylated RING1B preferentially bound near genes related to neural differentiation. Several groups have previously shown that PRC1 can switch gene targets leading to specific cell phenotypes (Rajasekhar and Begemann, 2007; Chen et al., 2011). Our data suggest that O-GlcNAc functions might regulate RING1B DNA-binding and potentially RING1B gene targeting. Therefore, change in RING1B O-GlcNAcylation might be one of the mechanisms enabling PRC1 to switch its gene targets. As RING1B O-GlcNAcylation decreases during hESC differentiation, O-GlcNAcylated RING1B-bound genes (related to neuron differentiation) might be de-repressed in favor of non-O-GlcNAcylated RING1B-bound genes (related to metabolism and cell cycle) (Fig. 3F). This hypothetical mechanism might be necessary during differentiation to 1) slow down cell growth, and 2) direct stem cell differentiation towards neuronal lineage. This is in contrast with maintained hESC where the higher level of O-GlcNAcylated RING1B might prevent neuronal differentiation whereas lower level of non-O-GlcNAcylated RING1B might promote the expression of genes related to metabolism and cell cycle. Such mechanism may be necessary in maintained hESC to promote cell growth. We previously showed that O-GlcNAc excess in differentiating hESC leads to an 80% mRNA and protein expression decrease for PAX6 (neuronal lineage marker) (Maury et al., 2013). Interestingly, we demonstrated here that PAX6 is bound by O-GlcNAcylated RING1B. This suggests that O-GlcNAcylation increase on RING1B might be one of the reasons for the previously observed effect of O-GlcNAc excess on hESC differentiation. Mechanistically, O-GlcNAc is probably not directly regulating RING1B binding to chromatin as RING1B is not known to bind to either histone or DNA (Vidal, 2009). Instead, O-GlcNAc might be regulating RING1B binding to PRC1 recruitment subunit (CBX2, 4, 6, 7, 8 and RYBP) which are known to bind to specific gene subsets (Vincenz and Kerppola, 2008; Morey et al., 2013). As RING1B can only bind to one CBXs/RYBP protein (Bezsonova et al., 2009; Wang et al., 2010); each CBXs/RYBP protein will form different PRC1 variants which are thought to target and repress specific gene subsets (Vincenz and Kerppola, 2008; Morey et al., 2013). In addition, RING1B O-GlcNAcylation sites appear in a region (from I248 to P324) previously reported as mediating the exclusive binding of RING1B to CBX7 and RYBP (Bezsonova et al., 2009; Wang et al., 2010). Altogether, these data suggest that RING1B O-GlcNAcylation might regulate RING1B affinity for PRC1 recruitment subunit (CBXs/RYBP) ultimately modulating PRC1 DNA-binding.
Author contributions
Acknowledgments The authors would like to thank the Biomedical Research Council of the Agency for Science Technology and Research (A*STAR) for their generous funding support. We also thank Dr. Ng Jia Hui, Dr. Jimmy Sheng-Hao Chao, Dr. Cheong Nge, Dr. Zhang Peiqing, Ms. Yi Ling Chia, Ms. Qiao Jing Lew, and Ms. Cheryl Chan for their technical assistance. Julien J.P. Maury also thanks the Singapore International Graduate Award (SINGA) for his scholarship.