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Hao Y, Li X, Qin K, Shi Y, He Y, Zhang C, Cheng B, Zhang X, Hu G, Liang S, Tang Q, Chen X. Chemoproteomic and Transcriptomic Analysis Reveals that O-GlcNAc Regulates Mouse Embryonic Stem Cell Fate through the Pluripotency Network. Angewandte Chemie (International ed. in English) 2023 36852467
Abstract:
Self-renewal and differentiation of embryonic stem cells (ESCs) are influenced by protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification, but the underlying mechanism remains incompletely understood. Herein, we report the identification of 979 O-GlcNAcylated proteins and 1340 modification sites in mouse ESCs (mESCs) by using a chemoproteomics method. In addition to OCT4 and SOX2, the third core pluripotency transcription factor (PTF) NANOG was found to be modified and functionally regulated by O-GlcNAc. Upon differentiation along the neuronal lineage, the O-GlcNAc stoichiometry at 123 sites of 83 proteins-several of which were PTFs-was found to decline. Transcriptomic profiling reveals 2456 differentially expressed genes responsive to OGT inhibition during differentiation, of which 901 are target genes of core PTFs. By acting on the core PTF network, suppression of O-GlcNAcylation upregulates neuron-related genes, thus contributing to mESC fate determination.
O-GlcNAc proteins:
AMRA1, SETX, SKT, BCORL, AGRIN, MGAP, ARI1A, KANL3, CHD6, PHRF1, ZCH24, EP300, KIF7, KI67, CE350, ANR11, NUMA1, TPR, MORC3, TAF4B, KMT2B, EMD, AKAP1, TCOF, DCTN1, MNT, NCOA3, ATN1, ECP3, DPOD2, CTND2, PIAS3, AF10, ACK1, GET3, DSG2, ESS2, ATX2, PDLI1, ULK1, BARD1, KDM6A, ZN106, NSD1, ZFR, HIPK1, SETB1, LAMC1, MYCN, GCR, EGR1, RC3H2, ATX1L, DERPC, K2C8, HSPB1, JUND, FGFR1, G3P, ATF2, COF1, HEXB, VIME, PO5F1, CBL, CCNB1, PO2F1, RS2, NFKB1, MAX, PABP1, NEDD1, PTN12, FMR1, ELK1, FOXK1, STAT3, SOX15, PLIN2, CBP, NEDD4, YAP1, RFX1, SOX2, LMNA, ROA1, S1PR2, ARNT, RD23A, PLTP, KMT2A, KLF16, FOXP1, TB182, GMEB2, SENP1, YTHD1, MRTFB, DOCK4, STIM1, TBX3, NCOA1, ERF, SIAE, NACAM, ATF1, WNK1, G3BP2, DNLI3, G3BP1, RLA2, GABPA, S30BP, ZEP1, ENAH, SOX13, CAPR2, APLP2, CLUS, TLE3, GATA4, MITF, CHD8, ZCH18, TANC1, CDK12, SAP25, LIN41, MLXIP, HROB, VRTN, CO039, PDLI7, SMCA4, PRC2C, MILK2, MIDN, YETS2, PBIP1, FUBP2, TFPT, SRBP2, GSE1, F117B, ZN865, WDR62, QRIC1, FOXK2, RREB1, TNR6C, DAB2P, TNR6A, RHG17, PKHA7, COBL1, FCHO2, TET1, ARMX5, GARL3, TET2, CDV3, PHAR4, C2CD3, LIN54, NPA1P, TAB3, TASO2, RESF1, NUFP2, UNKL, COBL, KDM6B, PRSR1, SMG7, RBM27, PHF12, ZDBF2, PUR4, SYNRG, UIMC1, SIN3A, NFAC2, SRC8, SKIL, ELF1, KLF4, NCOR1, KLF3, NCOA2, FOXD3, PAPOA, HCFC1, P3C2A, SIX4, ZFHX3, TOB1, AP180, GLI3, ATRX, MAFK, NPM, M3K7, DAG1, SPTB2, TAF6, TIF1B, SPT6H, SH3G1, ARI3A, TLE1, TLE4, IF4G2, MINT, ZIC3, ZYX, NUP62, PHC1, TFE3, TIF1A, SF01, DAZL, RBL1, KNL1, BCL9L, SBNO1, SLAI1, PKP4, CDK13, SH3R1, JHD2C, HECD1, ARMX2, LAR4B, RHG21, HELZ, SCAF8, UTF1, PKHG2, NIPBL, CCD66, F135A, RPRD2, WWC2, ZN532, KRBA1, TAF9B, RBM26, INT1, BCR, AHDC1, PTN23, PAPD7, KDM3A, KMT2D, CHD4, RN220, NUP98, NFRKB, GGYF2, LCOR, TEX2, PF21A, KDM3B, FNBP4, CNOT1, LARP1, RHG26, NU188, CNDD3, PICAL, SPAG5, HUWE1, SMAP2, CPEB3, MYCB2, PRC2B, PRR14, MACOI, ATX2L, CKP2L, PRC2A, MCAF1, SI1L2, KANL1, ERBIN, R3HD2, RERE, PUM2, PUM1, NU214, WNK4, TCAM1, SAS6, CAMP3, UBN2, TNC18, AGFG2, UBP2L, WNK3, ZN598, CTIP, SHAN2, NANOG, DDX42, RHG32, VGLU3, LPP, TET3, MYPT2, IF4B, CNO10, MISSL, TB10B, CARF, TGO1, ZN879, SP130, ZC3HE, ZNT6, SUN2, TNR6B, ARI5B, EMSY, BNC2, KAT6B, KMT2C, CLAP2, CNOT4, SRRM2, TOX4, GEPH, SYP2L, LARP4, KANK2, SALL4, YTHD3, TOIP2, KAT6A, ASXL2, POGZ, SREK1, TAF5, ZHX2, EPC2, SI1L1, CND2, RBM14, SUCO, CNOT2, DIDO1, SMAG1, LENG8, CDAN1, DPPA4, LRIF1, VCIP1, MB214, TAB1, SCYL2, ASPP2, LS14B, SYEP, F193A, BCOR, OGT1, SUGP1, NAV1, SYNJ1, ADNP2, RPGF2, BICRL, EP400, PHC3, VP37A, EPN2, P66A, PDLI5, ELYS, ZBT20, ANLN, AGFG1, MATR3, CASC3, I2BPL, PO121, ALMS1, SF3A1, GRHL2, ATF7, CACL1, DC1L1, MTSS1, SPART, TDIF2, HBP1, NUP58, RFIP5, BRD8, WIPI1, CDK8, CS047, ATX7, NUP35, LUZP1, RPAP2, NDC1, MAVS, AMOT, CSKI2, P66B, TAF9, IPO4, ZCH14, UBAP2, NCOA5, FUBP1, RBM47, AJUBA, VPS36, DCP1A, EGLN2, YTHD2, SRGP2, GRHL1, BCL7B, P4R3B, PLRG1, CIC, WAC, TRPS1, MED1, ACATN, NRBP, RP25L, NONO, TAB2, RBM10, EPN4, DDAH2, NOG2, ZN281, HGS, NASP, ARIP4, ANR17, ZN318, TRI33, MZT2, ZWINT, ECD, YIF1B, ROA0, DHRS7, TPD54, SSBP3, PSRC1, SARNP, BCL9, SP2, NOP56, SH24A, FIP1, PLIN3, MYPT1, KC1D, TCF20, TOR3A, SALL1, ZN704, RBP2, UBE4B, TBX20, AFF4, RBCC1, 4ET, PALLD, ELF2, TSSC4, NUDT3, HAKAI, ADRM1, NCOA6, FANCA, GIT2, BAG3, TOB2, ZN207, SON, TBL1X, PLEC, MACF1, GOGA5, QKI, GAB1, DMRT1, YLPM1, PCM1, RHG07, TAF7, FOXO1, ADA23, AKA12, UXT, MAN1, NCOR2, AKT3, COR1B, TNIP1, GANP, DEMA, CARM1, RGAP1, ITSN2, ZO2, KLF5, ADNP, ARI3B, BCL3, SE1L1, E41L1, ZN292
Species: Mus musculus
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Burt RA, Dejanovic B, Peckham HJ, Lee KA, Li X, Ounadjela JR, Rao A, Malaker SA, Carr SA, Myers SA. Novel Antibodies for the Simple and Efficient Enrichment of Native O-GlcNAc Modified Peptides. Molecular & cellular proteomics : MCP 2021 20 34678516
Abstract:
Antibodies against posttranslational modifications (PTMs) such as lysine acetylation, ubiquitin remnants, or phosphotyrosine have resulted in significant advances in our understanding of the fundamental roles of these PTMs in biology. However, the roles of a number of PTMs remain largely unexplored due to the lack of robust enrichment reagents. The addition of N-acetylglucosamine to serine and threonine residues (O-GlcNAc) by the O-GlcNAc transferase (OGT) is a PTM implicated in numerous biological processes and disease states but with limited techniques for its study. Here, we evaluate a new mixture of anti-O-GlcNAc monoclonal antibodies for the immunoprecipitation of native O-GlcNAcylated peptides from cells and tissues. The anti-O-GlcNAc antibodies display good sensitivity and high specificity toward O-GlcNAc-modified peptides and do not recognize O-GalNAc or GlcNAc in extended glycans. Applying this antibody-based enrichment strategy to synaptosomes from mouse brain tissue samples, we identified over 1300 unique O-GlcNAc-modified peptides and over 1000 sites using just a fraction of sample preparation and instrument time required in other landmark investigations of O-GlcNAcylation. Our rapid and robust method greatly simplifies the analysis of O-GlcNAc signaling and will help to elucidate the role of this challenging PTM in health and disease.