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2015-Maury-Et-Al-Stem Cell Res RING1B O-GlcNAcylation regulates gene targeting of polycomb repressive complex 1 in human embryonic stem cells Julien Jean Pierre Maury, Chadi Farran, Daniel Ng, Yuin-Han Loh, Xuezhi Bi, Muriel Bardor, Andre Boon-Hwa Choo To cite this version: Julien Jean Pierre Maury, Chadi Farran, Daniel Ng, Yuin-Han Loh, Xuezhi Bi, et al.. RING1B O- GlcNAcylation regulates gene targeting of polycomb repressive complex 1 in human embryonic stem cells. Stem Cell Research, Elsevier, 2015, 15 (1), pp.182 - 189. 10.1016/j.scr.2015.06.007. hal- 01841067 HAL Id: hal-01841067 https://hal-normandie-univ.archives-ouvertes.fr/hal-01841067 Submitted on 17 Jul 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Stem Cell Research (2015) 15, 182–189 Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/scr SHORT REPORT RING1B O-GlcNAcylation regulates gene targeting of polycomb repressive complex 1 in human embryonic stem cells Julien Jean Pierre Maury a,b,c,⁎, Chadi A. EL Farran c,d, Daniel Ng a, Yuin-Han Loh c,d, Xuezhi Bi a, Muriel Bardor a,e,1, Andre Boon-Hwa Choo a,b,⁎⁎,1 a Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, 138668 Singapore b Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, 9 Engineering Drive 1, 117575 Singapore c Institute of Molecular and Cell Biology, Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, #08-01 Proteos, 138673 Singapore d Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, 117543 Singapore e Université de Rouen, Normandie Université, Laboratoire Glycobiologie et Matrice Extracellulaire Végétale (Glyco-MEV) EA 4358, IRIB, VASI, Faculté des Sciences et Techniques, Mont-Saint-Aignan Cédex 76821, France Received 2 March 2015; received in revised form 25 May 2015; accepted 11 June 2015 Available online 17 June 2015 Abstract O-linked-N-acetylglucosamine (O-GlcNAc) post-translationally modifies and regulates thousands of proteins involved in various cellular mechanisms. Recently, O-GlcNAc has been linked to human embryonic stem cells (hESC) differentiation, however the identity and function of O-GlcNAc proteins regulating hESC remain unknown. Here, we firstly identified O-GlcNAc modified human stem cell regulators such as hnRNP K, HP1γ, and especially RING1B/RNF2. Thereafter, we focused our work on RING1B which is the catalytic subunit of the polycomb repressive complex 1 (PRC1) a major epigenetic repressor essential for pluripotency maintenance and differentiation. By point-mutation, we show that T250/S251 and S278 RING1B residues are bearing O-GlcNAc, and that T250/S251 O-GlcNAcylation decreases during differentiation. O-GlcNAc seems to regulate RING1B-DNA binding as suggested by our ChIP-sequencing results. Non-O-GlcNAcylated RING1B is found to be enriched near cell cycle genes whereas O-GlcNAcylated RING1B seems preferentially enriched near neuronal genes. Our data suggest that during hESC differentiation, the decrease of RING1B O-GlcNAcylation might enable PRC1 to switch its target to induce neuron ⁎ Correspondence to: J.J.P. Maury, Institute of Molecular and Cell Biology, 61 Biopolis Drive, #08-01 Proteos, 138673 Singapore. Tel.: +65 8403 2622. ⁎⁎ Correspondence to: A.B.H. Choo, Stem Cell Group, Bioprocessing Technology Institute, 20 Biopolis Way, #06-01 Centros, 138668 Singapore. Tel.: +65 6408 0856; fax: +65 6478 9561. E-mail addresses: [email protected] (J.J.P. Maury), [email protected] (A.B.-H. Choo). 1 The last authors contributed equally to this work. http://dx.doi.org/10.1016/j.scr.2015.06.007 1873-5061/© 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). RING1B O-GlcNAcylation regulates gene targeting of PRC1 in human embryonic stem cells 183 differentiation. Overall, we demonstrate that O-GlcNAc modifies and regulates an essential epigenetic tool, RING1B, which may contribute to hESC pluripotency maintenance and differentiation. © 2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Introduction cardiac-differentiation was also shown to be controlled by global levels of O-GlcNAc (Kim et al., 2009). Moreover, we O-linked N-acetylglucosamine (O-GlcNAc) post-translationally demonstrated that O-GlcNAc excess increased adipose commit- modifies specific serine and threonine residues from nucleo- ment and decreased ectoderm commitment of differentiating cytoplasmic proteins (Torres and Hart, 1984). O-GlcNAc is hESC suggesting that O-GlcNAcylated proteins are regulating dynamically added and removed by the O-GlcNAc transferase specific hESC differentiation pathways (Maury et al., 2013). and hydrolase respectively to regulate protein–protein inter- Polycomb repressive complex 1 (PRC1) is an epigenetic action, protein–DNA interaction, and protein stability (Ozcan repressor essential for regulating pluripotency maintenance et al., 2010). Even though thousands of nucleocytoplasmic and differentiation (Rajasekhar and Begemann, 2007). PRC1 proteins have already been reported (Hart and Akimoto, 2009), catalytic activity subunit, RING1B/RNF2 protein, mono- only few studies have recently started to establish a link ubiquitinylates histone 2A (H2AK119ub) which in turn repress between O-GlcNAcylation and stem cell pluripotency. O-GlcNAc the expression of genes targeted by PRC1 (Vidal, 2009). transferase was shown to be essential for mouse embryonic Interestingly, Gambetta et al., 2009 showed in Drosophila stem cell (mESC) viability (Shafi et al., 2000)andforzebrafish embryos that PRC1 Ring (human RINGs ortholog) was enriched proper morphogenesis (Webster et al., 2009). In mESC, cell through its O-GlcNAc modification (supporting material, Fig. S5 pluripotency maintenance and somatic cell reprogramming Gambetta et al., 2009); suggesting that human RING1B protein is regulated by Oct4 O-GlcNAcylation (a pluripotency might be O-GlcNAcylated. Interestingly, Speakman C., et al. transcription factor) (Jang et al., 2012). In addition, mESC also reported recently that some polycomb-target genes in Figure 1 hnRNP K, HP1γ, and RING1B are O-GlcNAcylated in hESC. (A–D) Western and Eastern blot analyses of immunoprecipitated (IP) O-GlcNAcylated proteins; hnRNP K; HP1γ, and RING1B proteins from hESC treated without (−) or with (+) 100 μM of PUGNAc for 24 h. Of note, in (D) input and IP solutions were run under non-reducing conditions because HP1γ and antibody light chain co-elute and are difficult to distinguish under reducing conditions. Thus, HP1γ apparent molecular weight is changed from 25 kDa (reducing condition) to 40 kDa (non-reducing condition). CTD110.6 antibody was used for the immunoprecipitation and Eastern blots of O-GlcNAc. 184 J.J.P. Maury et al. mESC had an altered expression following O-GlcNAc excess Results (Speakman et al., 2014), suggesting that O-GlcNAc might directly or indirectly regulate the function of polycomb RING1B is O-GlcNAcylated in hESC proteins. Here, we started by investigating RING1B O-GlcNAcylation in We started by investigating the O-GlcNAcylation of RING1B hESC. Through various strategies (immunoprecipitation, point- by immunoprecipitation in hESC. As a positive control, we mutations and mass spectrometry), we demonstrated that also tested two other stem cell regulators previously RING1B is O-GlcNAcylated on specific residues T250/S251 and reported as being O-GlcNAcylated: hnRNP K and HP1γ S278 and that RING1B O-GlcNAcylation decrease during (Drougat et al., 2012; Gambetta et al., 2009). hnRNP K is a hESC differentiation. Finally, chromatin immunoprecipita- RNA-binding protein regulating neuronal (Cao et al., 2012) tion experiment suggests that O-GlcNAcylation of RING1B and erythroid differentiations (Naarmann-de Vries et al., 2013). might regulate PRC1 DNA-binding. HP1γ is a heterochromatin-associated protein involved in gene repression and differentiation (Morikawa et al., 2013). Materials and methods In our experiments, hnRNP K, HP1γ, and RING1B were all detected after IP of O-GlcNAcylated proteins (Fig. 1A). In Information related to Material and methods can be found in addition, a higher amount of these proteins was immuno- the Supplemental information section. precipitated from cells treated with PUGNAc (O-GlcNAc Figure 2 RING1B is O-GlcNAcylated at S250/T251 and S278 and RING1B O-GlcNAcylation decreases during hESC differentiation. (A) Overview of RING1B treatment before mass spectrometry analysis. Briefly, immunoprecipitated RING1B protein was digested and subject to phosphatase treatment to remove phosphate groups. Then the peptides were subjected to BEMAD reaction in order to substitute O-GlcNAc group by DTT. Finally, the DTT-modified peptides were enriched on thiopropyl sepharose and analyzed by MS. (B–C) MS2 analysis of RING1B DTT-modified peptides [(TS)-OGlcNAc]GNATVDHLSK
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